Principles of Cancer Biotherapy

Principles of Cancer Biotherapy 5th Edition

Principles of Cancer Biotherapy 5th Edition

Edited by Robert K. Oldham Hematology-Oncology Associates of Southeast Missouri Hospital Southeast Medical Plaza Cape Girardeau, MO USA

Robert O. Dillman Hoag Cancer Center Newport Beach, CA USA University of California Irvine, CA USA

Robert K. Oldham Hematology-Oncology Associates of Southeast Missouri Hospital Southeast Medical Plaza Cape Girardeau, MO USA

Robert O. Dillman Hoag Cancer Center Newport Beach, CA USA University of California Irvine, CA USA

e-ISBN 978-90-481-2289-9 ISBN 978-90-481-2277-6 DOI 10.1007/978-90-481-229-9 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009929387 First edition published 1987 by Raven Press Second edition published 1993 by Williams & Wilkins Third and Fourth edition published 1998 Kluwer Academic Publishers © Springer Science+Business Media B.V. 2009 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

The idea for the first edition of Principles of Cancer Biotherapy was formulated in the early 1980s. As the founding director of the Biological Response Modifiers Program for the National Cancer Institute from 19801984, one of us (rko) envisioned a textbook that would embody the principles of the then fledgling fourth modality of cancer treatment - biotherapy. Contributing authors were solicited in 1985, and the first edition came off the presses in 1987. Principles represented the first comprehensive textbook on the use of cancer biotherapy and summarized the work done in this field through 1986. The second edition of Principles was published in 1991 about the time biotherapy was more broadly recognized as the fourth major cancer treatment modality. Subsequent textbooks by DeVita, Hellman and Rosenberg [1] in 1991, Mitchell [2] in 1993 and Rosenberg [3] in 2000 validated the importance of this modality in cancer care. This third edition was published in 1998 and confirmed the tremendous progress that had been made in the previous five years using biologicals in cancer treatment. It was generally agreed that biopharmaceuticals were producing major opportunities for new cancer therapies. Cancer biotherapy was emerging as a more specific and targeted form of systemic cancer treatment. Cancer growth control was also becoming an effective method of treatment complementing cancer destruction as mechanisms of cancer treatment and “cure”. The fourth edition of Principles was published in 2003. It was apparent that biotherapy and the use of biopharmaceuticals had not only become recognized as the fourth modality of cancer treatment, it was clear that biopharmaceuticals had become the dominant form of new cancer therapeutics which in the future will replace less selective, more toxic forms of therapy. For years, the chemical manipulation of small molecules has been pursued in drug development. We now have all the tools for the biological manipulation of natural substances for therapeutic use. In fact, as we better understand the interaction between biological molecules and their receptors, it is clear that biological manipulation and chemical manipulation are coming together to bring molecular medicine to the bedside. Many biological molecules are large and have functions other than

those mediated by their active sites. There is increasing evidence that drug development will focus on the interaction between the smaller active regions of these large biological molecules and their receptors. This opens up a broad field of molecular design for extending and improving the therapeutic activities of natural biological molecules. This has recently been extended to small molecules interacting with DNA/RNA (anti-sense), with enzymes such as tyrosine kinase and with growth and vascular factor receptors. The last decade has been extraordinarily productive in the development of new anticancer drugs through chemical and biological manipulation of these natural molecules. Thus, the body itself has become the “medicine cabinet” of the future. In the 1990s and beyond into this millennium, medicine will face extraordinary demands. While technology brings us tremendous opportunity, it also highlights problems in our medical care system. Most new technology is expensive and, as it comes from the laboratory to the clinic, is by its very nature untried and unproven. Our medical care system involves a private and government insurance reimbursement system that favors paying for marginally effective medical care of the past rather than innovative medical treatments of the future. Such a system is inhibitory to the development of effective new anti-cancer medicines. To more rapidly and efficiently exploit the opportunities in cancer biotherapy in the future, patients, employers, insurers, universities, and government must come together and redefine the system of reimbursement to maximize the patient’s opportunity for access to new and potentially effective cancer therapies. To simply reimburse for old ineffective or marginally effective treatment is not the answer. Provisions must be made to fund clinical research and afford these new approaches broader use at the bedside. We must develop methods to allow our patients access to the opportunities of the future, while maintaining solid support for effective therapies of the past. No longer is it acceptable to pay only for medical care that utilizes old technology, such as chemotherapy, that is approved but only marginally effective. Across the broad spectrum of human malignancies, most chemotherapeutic drugs are toxic and of limited medical value. We must support clinical research

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vi in its efforts to bring newer methods of cancer treatment to the clinic, methods that are less toxic and more effective. We believe cancer biotherapy will ultimately replace much of what we utilize today in cancer treatment. In light of this view, we want to thank all the authors for their dedication to purpose in writing this fifth edition of Principles. This book summarizes an evolving science and a rapidly changing medical practice. As we progress into the millennium, it now becomes possible to envision a much more diversified system of cancer research and treatment that will afford greater opportunities for our patients. As indicated in some of the chapters in Principles, there is increasing evidence that our historical “kill and cure” outlook in cancer treatment is in need of modification. Some forms of cancer biotherapy use the strategy of tumor growth stabilization and control through continued biological therapy over a longer period of time, akin to the use of insulin in the treatment of diabetes.

Preface These chapters illustrate some of these new methods of thinking and illustrate new strategies for the treatment and control of cancer. It is always difficult to move from past dogmas to future opportunities, but this fifth edition of Principles of Cancer Biotherapy illustrates why it is so important for researchers, regulators and clinicians to explore and apply these new opportunities in cancer biotherapy to the benefit of our patients. Robert K. Oldham, M.D.

Robert O. Dillman MD

References 1. DeVita VT Jr, Hellman S, Rosenberg SA. Biologic Therapy of Cancer. J.B. Lippincott, 1991. 2. Mitchell MS. Biological Approaches to Cancer Treatment. McGraw-Hill, 1993. 3. Rosenberg SA. Principles and Practices of the Biologic Therapy of Cancer. J.B. Lippincott, 2000.

Contents

Preface Robert K. Oldham

v

Contributors

ix

1. Cancer biotherapy: general principles Robert K. Oldham

1

2. The pathogenesis of cancer metastasis: relevance to therapy Sun-Jin Kim, Cheryl Hunt Baker, Yasuhiko Kitadai, Toru Nakamura, Toshio Kuwai, Takamitsu Sasaki, Robert Langley, and Isaiah J. Fidler

17

3. Developmental therapeutics and the design of clinical trials Robert K. Oldham

41

4. Recombinant proteins and genomics in cancer therapy Kapil Mehta, Bulent Ozpolat, Kishorchandra Gohil, and Bharat B. Aggarwal

53

5. Current concepts in immunology Robert K. Oldham

85

6. Therapeutic approaches to cancer-associated immune suppression Robert K. Oldham

101

7. Cancer vaccines Kenneth A. Foon and Malek M. Safa

147

8. Cytokines Walter M. Lewko and Robert K. Oldham

155

9. Interferons: therapy for cancer David Goldstein, Robert Jones, Richard V. Smalley, and Ernest C. Borden

277

10. Monoclonal antibody therapy Robert O. Dillman

303

11. Immunotoxins Arthur E. Frankel, Jung-Hee Woo, and David M. Neville

407

12. Drug Immunoconjugates Malek Safa, Kenneth A. Foon, and Robert K. Oldham

451

13. Targeted radionuclide therapy of cancer John M. Pagel, Otto C. Boerman, Hazel B. Breitz, and Ruby Meredith

463

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viii

Contents

14. Stem cell/bone marrow transplantation as biotherapy Robert K. Oldham

497

15. Cellular immunotherapy (CI), where have we been and where are we going? John R. Yannelli

505

16. Growth and differentiation factors as cancer therapeutics Kapil Mehta, Robert K. Oldham, and Bulent Ozpolat

527

17. Granulocyte colony-stimulating factor: biology and clinical potential MaryAnn Foote and George Morstyn

569

18. Granulocyte-macrophage colony-stimulating factor Maryann Foote and George Morstyn

581

19. Cancer gene therapy Donald J. Buchsbaum, C. Ryan Miller, Lacey R. Mcnally, and Sergey A. Kaliberov

589

20. Regulatory process for approval of biologicals for cancer therapy Antonio J. Grillo-López

613

21.1. Cancer biotherapy: 2009 disease-related activity Robert K. Oldham and Robert O. Dillman

631

21.2. Biological therapy of melanoma Robert K. Oldham

633

21.3. Biological therapy of genitourinary cancer Robert K. Oldham

645

21.4. Biological therapy of colon cancer Robert O. Dillman

659

21.5. Biological therapy of breast cancer Robert O. Dillman

669

21.6. Biological therapy of lung cancer Robert O. Dillman

679

21.7. Biological therapy of B and T cell lymphoproliferative disorders Robert O. Dillman

693

21.8. Biological therapy of multiple myeloma Robert K. Oldham

711

21.9. Biological therapy of squamous cell cancers of the head and neck Robert O. Dillman

713

21.10. Biological therapy of glioblastoma Robert O. Dillman

723

22. Speculations for 2009 and beyond Robert K. Oldham

733

Index

739

Contributors

Robert K. Oldham Hematology-Oncology Associates of Southeast Missouri Hospital Southeast Medical Plaza Cape Girardeau, MO USA

Toshio Kuwai Department of Cancer Biology M. D. Anderson Cancer Center The University of Texas Houston, TX USA

Robert O. Dillman Hoag Cancer Center Newport Beach, CA USA University of California Irvine, CA USA

Takamitsu Sasaki Department of Cancer Biology M. D. Anderson Cancer Center The University of Texas Houston, TX USA

Sun-Jin Kim Department of Cancer Biology M. D. Anderson Cancer Center The University of Texas Houston, TX USA Cheryl Hunt Baker Department of Cancer Biology M. D. Anderson Cancer Center The University of Texas Houston, TX USA Yasuhiko Kitadai Department of Cancer Biology M. D. Anderson Cancer Center The University of Texas Houston, TX USA Toru Nakamura Department of Cancer Biology M. D. Anderson Cancer Center The University of Texas Houston, TX USA

Robert Langley Department of Cancer Biology M. D. Anderson Cancer Center The University of Texas Houston, TX USA Isaiah J. Fidler Department of Cancer Biology M. D. Anderson Cancer Center The University of Texas Houston, TX USA Kapil Mehta Department of Experimental Therapeutics M. D. Anderson Cancer Center The University of Texas Houston, TX USA Bulent Ozpolat Department of Experimental Therapeutics M. D. Anderson Cancer Center The University of Texas Houston, TX USA

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x Kishorchandra Gohil Department of Internal Medicine The University of California Davis, CA USA Bharat B. Aggarwal Department of Experimental Therapeutics M. D. Anderson Cancer Center The University of Texas Houston, TX USA Kenneth A. Foon Department of Hematological Malignancies Nevada Cancer Institute Las Vegas, NV USA Malek M. Safa Division of Hematology / Oncology University of Cincinnati Cincinnati, OH USA Walter M. Lewko Cancer Cellular Therapeutics, Inc. Cape Girardeau, MO USA David Goldstein Department of Medical Oncology Prince of Wales Hospital Randwick, Sydney Australia Robert Jones Centre for Oncology and Applied Pharmacology CRUK Beatson Laboratories Glasgow, Scotland UK Richard V. Smalley (deceased) Synertron Inc. Madison, WI USA Arthur E. Frankel Cancer Research Institute of Scott &White Temple, TX USA

Contributors Jung-Hee Woo Cancer Research Institute of Scott &White Temple, TX USA David M. Neville Angimmune LLC, Bethesda, MD USA John M. Pagel The Fred Hutchinson Cancer Research Center Seattle, WA USA Otto C. Boerman University Medical Center Nijmegen The Netherlands Hazel B. Breitz Poniard Pharmaceuticals, Inc. Seattle, WA USA Ruby F. Meredith Department of Radiation Oncology University of Alabama Birmingham Birmingham, Alabama USA John R. Yannelli Department of Microbiology, Immunology and Molecular Genetics Markey Cancer Center University of Kentucky, School of Medicine Lexington, Kentucky USA MaryAnn Foote Thousand Oaks, CA USA Dr George Morstyn Department of Microbiology Monash University Victoria Australia

Contributors

xi

Donald J. Buchsbaum Department of Radiation Oncology University of Alabama at Birmingham Birmingham, AL USA

C. Ryan Miller Department of Pathology University of North Carolina Chapel Hill, NC USA

Lacey R. McNally Department of Radiology University of Alabama at Birmingham Birmingham, AL USA

Antonio J. Grillo-López Rancho Santa Fé, CA USA

Sergey A. Kaliberov Department of Radiation Oncology University of Alabama at Birmingham Birmingham, AL USA

1

Cancer biotherapy: general principles ROBERT K. OLDHAM

The term biotherapy encompasses the therapeutic use of any biological substance, but more specifically, it connotes the use of products of the mammalian genome. With modern techniques of genetic engineering, the mammalian genome represents the new “medicine cabinet.” Biological response modifiers (BRM) are agents and approaches whose mechanisms of action involve the individual’s own biological responses. Biologicals and BRM work through diverse mechanisms in the biotherapy of cancer. They may (a) augment the host’s defenses through the administration of cells, natural biologicals, or the synthetic derivatives thereof as effectors or mediators (direct or indirect) of an antitumor response; (b) increase the individual’s antitumor responses through augmentation or restoration of effector mechanisms, or decrease a component of the host’s reaction that is deleterious; (c) augment the individual’s responses using modified tumor cells or vaccines to stimulate a greater response, or increase tumor cell sensitivity to an existing biological response; (d) decrease transformation and/or increase differentiation or maturation of tumor cells; (e) interfere with growth-promoting factors and angiogenesis inducing factors produced by tumor cells; (f) decrease or arrest the tendency of tumor cells to metastasize to other sites; (g) increase the ability of the patient to tolerate damage by cytotoxic modalities of cancer treatment; and/or (h) use biological molecules to target and bind to cancer cells and induce more effective cytostatic or cytocidial antitumor activity. While several of these approaches involve the augmentation or use of biological responses, an understanding of the biological properties of immune response molecules, growth and maturation factors, and other biological substances will assist in the development of specific molecular entities that can act on biological responses and/or act directly on tumor cells. Thus, one can visualize the development of biological approaches with response-modifying as well as direct cytolytic, cytostatic, growth-inhibiting (antiproliferative), or maturational effects on tumor cells. Biotherapy is the fourth modality of cancer therapy and can be effective alone or in association with surgery, radiotherapy, and chemotherapy. To put biotherapy into perspective, it is important to dispel a historical miscon-

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

ception associated with immunotherapy: biotherapy can be effective against clinically apparent, even bulky, cancer, and treatment should not be restricted to situations where the tumor cell mass is imperceptible [64, 71]. Thus, the clinical trial designs for biotherapy can be similar to those used previously for other modalities of cancer treatment, as long as one measures both pharmacokinetics and the biological responses affected by these approaches [65]. Testing is continuing for biotherapy using the interferons, lymphokines, chemokines and cytokines, growth and maturation factors, angiostatic factors, monoclonal antibodies and their immunoconjugates, vaccines, and cellular therapy [68].

Historical Perspectives The use of chemical and biological compounds to modulate biological responses has been under active investigation for over 30 years. Although various chemicals, bacterial extracts, and viruses have been found to modulate immune responses in experimental animals, and, to a more limited extent, in humans, these “nonspecific” immunomodulators have not been highly effective in clinical trials [71]. Molecular biologists have recently developed techniques for the isolation of genes and their subsequent translation into appropriate production systems. These methods make available virtually unlimited quantities of highly purified biological compounds for experimental and therapeutic use. As a result, several classes of biologicals are being evaluated in preclinical models and clinical trials (Table 1). The continued investigation of nonspecific immunomodulators and adjuvants, as well as the recent advent of genetically engineered biologicals, makes the need for predictive preclinical assays of biological activity and efficacy apparent [33]. In vitro assays of biological activity (bioassays) are generally used to define and quantitate the activity of a given biological substance. Subsequently, flow cytometry, immunoperoxidase staining, enzyme-linked immunosorbent assays (ELISA), tetramer assays, radioimmunoassays (RIA), and variations of these methods allow the precise determination of levels of these molecules and activities in appropriate fluids and tissues.

1

2 Table 1. Biologicals and BRM Immunomodulators (chemicals, bacterial extracts, viruses, etc.) Lymphokines/cytokines α, β, γ-interferon; IL-1–32, tumor necrosis factor (TNF); etc. Growth/maturation factors (CSF, IL-2, EPO, etc.) Effector cells (cytotoxic and helper T cells, NK, and LAK cells, gene-engineered cells, etc.) Tumor-associated antigens and gene-engineered cellular vaccines Monoclonal antibodies Immunoconjugates

Finally, there is the need to assess the in vivo activity of these materials in preclinical models to develop predictive assays for clinical efficacy and provide information useful in the rational selection of agents and the design of clinical trials [33, 54]. Given the variability in the biological behavior of cancer and its interface with the human outbred host, it is not surprising that trials of nonspecific and specific immunotherapy, as translated from artificially constructed animal models, have not been uniformly successful in cancer treatment [4, 33]. Naturally occurring cancers arise in a particular organ from one cell or a few cells under some carcinogenic stimulus. In humans, these initial foci of cancer cells may grow – and sometimes lie dormant – over very long periods of time (from 1% to 30% of the human life span) before there is clinical evidence of cancer. Dissemination of cells from the initial focus may occur at any time during the development of the primary tumor. Subsequently, growth and metastasis occur over periods of months to years from primary and secondary foci, causing complex biological interactions to occur. In contrast, experimentally induced cancer is an artificial (even artifactual) situation. A short-duration, highdose carcinogen may be used to induce cancer quickly so that experiments can proceed rapidly. In transplantable models, the tumor cells are injected into young, normal, syngeneic animals, thereby circumventing the influences of environmental or genetic factors that may be operative in the natural host during tumor development. Many of the experimental models are transplantable tumors that have been maintained for decades and have only the most remote relevance to cancer in humans. The injection represents a single, instantaneous point source for a defined tumor load that has been manipulated in vitro. Regardless of whether that tumor load is 10 or 106 cells, it is being placed artificially in a single site and allowed to grow and metastasize from that selected and artificial, single site. Thus, these transplantable cancers are simply not analogous to clinical

Cancer biotherapy: general principles cancer and the conclusions drawn from them are unlikely to be broadly applicable to human cancers [33]. The modern era of cancer treatment began in the 1950s with the recognition that most cancers were systemic problems. It became obvious that lymphatic and blood-borne metastases often occurred simultaneously with local growth and regional spread. The early success of chemotherapy in leukemia and lymphoma prompted a massive and enthusiastic search for chemicals that might have cytolytic or cytostatic effects on cancer cells. Millions of compounds have been “screened” for antitumor activity [11], and this effort has given clinicians less than 75 “approved and active” anticancer chemotherapy drugs [84]. In addition, there are about 15 approved hormonal agents, ten targeted small molecules, and 25 biological drugs. There is now widespread recognition that only a few drugs in cancer treatment can effectively palliate and sometimes cure [19]. The development of three modalities (surgery, radiotherapy and chemotherapy) and their subsequent integration into what is now multimodal cancer treatment has been summarized [18, 20]. Between 1975 and 1990 a plateau was reached in cancer treatment. New surgical techniques (e.g., debulking, intra-operative methods for radiotherapy, and catheter isolation/infusion for chemotherapy) and new methods of radiotherapy (e.g., neutrons, protons, interstitial therapy, isotopes) continue to be developed, but these two modalities are primarily useful in local and regional cancer treatment. There has been continued, but slow, progress in the treatment of highly replicative, drug-sensitive malignancies over the past 15 years. It is now apparent that further progress with chemotherapy will depend on a greater understanding of the metabolic and enzymatic processes of cancer cells and the differences between these and normal cells. In addition, there are the problems of drug resistance, selectivity of action, and drug delivery. Cancer cells are more like than unlike normal cells with respect to chemotherapy sensitivity but they are more likely to become chemotherapy resistant with drug exposure. Many chemotherapeutic agents are highly cytolytic, but the problems of normal tissue toxicity, drug delivery, and tumor-cell resistance remain [19, 46]. Thus, cancer remains a systemic problem, and further systemic but more selective approaches are required for more effective treatment [20]. In addition to standard chemotherapy drugs that are approved and in clinical use, there are approximately 15 hormonal drugs available. These are primarily used in breast cancer and prostate cancer, and many are variations on the theme of blocking hormonal receptors. The evolution in this area has been primarily testing new

Robert K. Oldham hormonal agents for superiority in therapy and for lesser degrees of toxicity when used in large-scale trials against hormonally sensitive tumors. Then newest area of drug development is in targeted therapy. Here, small molecules are tested for the ability to block a particular receptor, enzyme, or target which may be involved in cell replication or cell metabolism. Eight targeted agents are currently available in the clinic, and a very large number of these small molecules are currently in clinical trials, most of which are variations on the theme of blocking a particular receptor or enzyme system with many of these drugs being multifunctional in that they can block more than one receptor or system. The scientific basis for biotherapy as the fourth modality of cancer treatment is now firm [21–23, 68, 70, 81, 82, 94, 97]. Historically, there was an attempt to establish immunotherapy in this role. Whereas immunotherapy was reproducible under specific experimental protocols, it was not strikingly effective in animals bearing palpable tumors and did not translate well to patients. Given the observation that immunotherapy was more effective with small tumor burdens, investigators began to study both “specific” and “nonspecific” immunotherapy as treatment for minimal residual disease. Although it became widely accepted that the treatment of animals with minimal residual disease was analogous to the postsurgical treatment of cancer in humans, this analogy was often stretched beyond reason. Immunotherapy in young, normal, syngeneic animals was often begun on the day of the tumor transplant (or within 1 or 2 days), using a transplant of a very small number of tumor cells (1–1,000) to a single site. In many of these studies, and in studies in which the tumor was surgically resected and no evident disease remained, the effects of immunotherapy were reasonably reproducible; the therapy was most beneficial when the tumor mass was less than 106 cells. These experimental results produced a dogma that immunological manipulation or immunotherapy could work only when the tumor cell mass was imperceptible [4, 71]. This posed real problems for clinical immunotherapy, since the tumor cell mass at clinical diagnosis or after surgery is usually three orders of magnitude (or more) greater than 106 cells. Despite the obvious difficulties with the experimental models and the translation to humans, clinicians began larger scale immunotherapy trials in the 1970s. The results of initial, small, uncontrolled trials were often reported as positive. Larger, randomized, controlled studies were done to confirm the efficacy of a particular immunotherapeutic regimen in a particular type of cancer. Although some of the controlled studies were positive, most yielded marginal or

3 negative results. Thus, immunotherapy had a poor image by the end of the 1970s [71, 109]. Immunotherapy failed to establish itself as a major mode of cancer treatment for several reasons. One important factor was the lack of definition and purity of immunotherapeutic agents. Many of the nonspecific approaches involved the use of complex chemicals, bacteria, viruses, and poorly defined extracts in an attempt to “stimulate” the immune response. Thus, molecular definition of the actual stimulating entity was not available. Given the lack of analogy between model systems and humans, the poorly characterized reagents, and the problems of variability in experimental procedures, the lack of demonstrable clinical efficacy was predictable [71]. Immunotherapy is not an appropriate term for the modern use of biologicals and BRM in medicine. Biological control mechanisms should be envisioned on a much broader basis than the immune system. While immunotherapy remains a subcategory of biotherapy, growth and differentiation factors, chemokines and angiostatic factors, the use of synthetically derived molecular analogs, and the pharmacological exploitation of biological molecules is much broader than immunotherapy (Table 1) [68]. Certain specific developments led to biotherapy becoming the fourth modality of cancer therapy. Advances in molecular biology have given scientists the capability to clone individual genes and produce significant quantities of highly purified genomic products as medicines. Unlike extracted and purified biological molecules, available in small quantities as semi-purified mixtures, the products of cloned genes have a level of purity on a par with drugs. They can be analyzed alone or in combination as to their effects in cancer biology. In addition, progress in nucleic acid sequencing and translation, protein sequencing and synthesis, the isolation and purification of biological products, and mass cell culture has given the scientific community the opportunity to alter nucleic acids and proteins at the nucleotide or amino acid level to manipulate then optimize their biological activity [42]. The elucidation of the human genome and the encoded products broadened the opportunities for advancements in biotherapy. As a result of gene cloning, a major approach in cancer treatment has evolved. Interleukin-2 can be used to stimulate the growth of a broad range of lymphocytes (T, NK, and LAK cells). This has given clinicians the ability to have large quantities of specific subclasses of effector cells for cancer treatment. Emerging evidence suggests that these effector cells can be helpful in the regional and systemic treatment of advanced, bulky

4 cancer. It was the availability of Interleukin-2 that allowed this technology to prosper [83]. In addition, IL-2 is now being used as the T cell growth factor for gene engineered lymphocytes containing new genes to enhance their cancer treatment capacity [80, 103]. Another major technical advance was the discovery of hybridomas. A major limitation on the use of antibodies had been the inability to make reproducible hightiter, specific antisera and to define these preparations on a molecular basis. Immunoglobulin reagents can now be produced with the same level of molecular purity as cloned gene products and drugs. Processes to easily “humanize” antibodies have produced therapeutic antibodies with excellent specificity, low immunogenicity and optimal pharmacokinetics. These antibodies are also powerful tools in the isolation and purification of tumor-associated antigens, lymphokines/cytokines, and other biologicals, which can then be used in biotherapy. The advances in molecular biology and hybridoma technology have eclipsed previous techniques for the isolation and purification of biological molecules [63, 77]. Technical advances in instrumentation, computers, and computer software have been critically important in the isolation and purification of biological molecules. The construction of nucleotide or amino acid sequences to fit any biological message is now possible. While this synthetic capability is currently limited to smaller gene products, techniques by which analysis and construction of nucleotide sequences will occur in an automated way, making enormously complex molecules possible to synthesize and manufacture, are rapidly becoming available.

Cancer biotherapy: general principles neoplasms [33]. There may exist in animals and in humans specific or nonspecific defects important in the development of their autochthonous tumors. Corrections of such defects may require a form of biotherapy totally different from that required to assist the normal host in controlling a transplanted cancer.

Model Screening Criteria Theoretically, an ideal procedure for screening new biologicals should employ a system of sequential and progressively more demanding protocols designed to select a maximum number of effective agents. The term screening denotes a series of sequential assays through which promising agents are tested for therapeutic potential. For some biotherapies, a general screening procedure may be inappropriate. For example, the activity of a monoclonal antibody with antitumor specificity would not be detected by use of the general screen of biological activity. Design considerations for general screening in biotherapy have been extensively reviewed [33, 54, 56, 61]. A step-by-step approach to the screening of potential BRM was developed to define their effects on T-cell, B-cell, NK-cell, and macrophage functions. A progressive in vitro and then in vivo sequence allows the variables of dose, schedule, route, duration and maintenance of activity, adjuvanticity, and synergistic potential to be explored in an orderly fashion [33]. Unfortunately, the screening program was used only briefly by the NCI and no replacement program is now in use.

Evaluation of Screening Programs

Preclinical Models Biological Activity in Preclinical Models Central to the identification of biotherapy that might be useful in cancer patients is the recognition that, in the main, the challenge in humans is the eradication of metastases. Metastases can result from the dissemination of different subpopulations of cells within the primary neoplasm [32]. This may explain the observation that cells within a metastasis can be antigenically distinct from those that predominate in the parental tumor [32]. Metastases may also emanate from other metastases. The implications of cellular heterogeneity as it relates to the outcome of the specific immunotherapy are obvious. In addition, normal animals are not comparable to animals or humans bearing autochthonous

Screening programs for chemotherapeutic agents were initiated in the mid-1950s, and attempts have been made to randomly examine thousands of compounds for antitumor activity [11]. Such large screening programs are empirically rather than rationally based, and are no longer appropriate [4, 5, 20, 33, 71]. Whether induced or transplantable animal tumor systems are valid models for testing therapeutic approaches for human cancer has been a controversial issue [33]. In patients, therapy successful against one type of cancer may not be successful against another type, or even for another patient with the same histological type of cancer. Unlike the model systems, in which treatment can be given with precise timing relative to the metastatic phase of a resected tumor or injected tumor cells, cancer diagnosis in humans is generally late, and micrometastases (and often macro-metastases) have become

Robert K. Oldham well established before treatment can be initiated. Thus, screening programs can only provide tentative indications on agents and approaches of interest.

Biotherapy: Specific Agents and Approaches Nonspecific Immunomodulators Since the early 1900s, immunotherapy with bacterial or viral products has been utilized with the hope of “nonspecifically” stimulating the host’s immune response [71]. These agents had been useful as adjuvants and as nonspecific stimulants in animal tumor models, but human trials have been disappointing. Perhaps purified viruses or specific chemicals will lead to the development of more effective adjuvants or stimu-

5 lants of the immune response. Bacillus Calmette-Guerin (BCG) and other whole organisms were used early in immunotherapy. The use of a purified derivative of bacterial components, such as muramyl di- or tripeptide, “packaged” in liposomes as a method to stimulate macrophages to greater anticancer activity has also been tried. Such adjuvants may prove useful with genetically engineered or synthetic tumor-associated antigens, active specific immunotherapy, or immunoprophylaxis. Multiple agents that appear capable of augmenting one or more immune functions already exist. Several of these have been associated with prolongation of survival in prospectively randomized trials involving patients with a wide variety of malignancies (Table 2). Although some of these agents have produced modest clinical benefits, and do represent a potential method of immune augmentation, it is doubtful that they will have a major role in future cancer therapy. Great problems

Table 2. Biologicals and biological response modifiers Immunomodulating agents Alkyl lysophospholipids (ALP) Azimexon BCG Bestatin Brucella abortus Cornyebacterium parvum Cimetidine Sodium diethyldithiocarbamate (DTC) Endotoxin Glucan ‘Immune’ RNAs Krestin Lentinan T-cell growth factors (‘TCGF’ – interleukin 2 [IL-2]) Levamisole Muramyldipeptide (MDP), tripeptide (MTP) Maleic anhydride-divinyl ether (MVE-2) Mixed bacterial vaccines (MBV) Nocardia rubra cell wall skeletons (CWS) Picibanil (OK432) Prostaglandin inhibitors (aspirin, indomethacin) Thiobendazole Tuftsin Interferons and interferon inducers Interferons (α, β, γ) Poly IC-LC Poly A-U GE-132 Brucella abortus Tilorone Viruses Pyrimidinones Thymosins Thymosin alpha-1 Thymosin fraction 5 Other thymic factors

Lymphokines, cytokines, growth/maturation factors Antigrowth factors Chalones Colony-stimulating factors (CSF) Growth factors (transforming growth factor, TGF) Lymphocyte activation factor (LAF-interleukin 1 [IL-1]) Lymphotoxins (TNF, α, β, LT) Macrophage activation factors (MAF) Macrophage chemotactic factor Macrophage cytotoxic factor (MCF) Macrophage growth factor (MGF) Migration inhibitory factor (MIF) Maturation factors Interleukin 3–18, etc. Thymocyte mitogenic factor (TMF) Transfer factor Transforming growth factor (TFR, α, β) Antigens Tumor-associated antigens Molecular vaccines Cell-engineered cellular vaccines Effector cells Macrophages NK cells Cytotoxic T cells LAK cells T helper cells Miscellaneous approaches Allogeneic immunization Liposome-encapsulated biologicals Bone-marrow transplantation and reconstitution Plasmapheresis and ex vivo treatments (activation columns, immunoabsorbents, and ultrafiltration) Virus infection of cells (oncolysates)

6 exist for most of the agents, including lack of chemical definition, low purity, and poor reproducibility from one lot to another. An additional problem has been the inability to define clearly a mechanism of action for these agents in humans. The preclinical screening established by the Biological Response Modifiers Program (BRMP) of the National Cancer Institute was one mechanism to do this [31]. Data from this type of screening with subsequent phase I and phase II clinical data could have provided interesting insights and correlations [98, 104, 105]. Unfortunately, this approach has been abandoned. The ability to specifically control and manipulate immune responses with highly purified, defined molecules obtained by genetic engineering is the future. Thus, it seems probable that nonspecific immunotherapy as a sole modality has become obsolete, although as adjuvants some may find a role in active specific immunotherapy (Chapter 6).

Active Specific Immunotherapy There has been a substantial effort to produce active immunization of autochthonous or syngeneic hosts with irradiated or chemically modified tumor cells in an attempt to use active specific immunotherapy (AST) [46]. Inherent is the assumption that tumor cells express immunogenic tumor-associated antigens (TAA). Treatment of tumor cells with a variety of unrelated agents, such as irradiation, mitomycin, lipophilic agents, neurominidase, viruses, or admixtures of cells with bacterial or chemical adjuvants, has produced non-tumoroigenic tumor cell preparations that are immunogenic upon injection into syngeneic hosts (Table 3). AST using BCG-tumor cell (“antigens”) vaccines has been reevaluated using a syngeneic guinea pig hepatocarcinoma. The definition of several variables of vaccine preparation, such as a ratio of bacterial organisms to viable, metabolically active tumor cells, the procedures of tumor cell dissociation and cryobiologic preservation, and the irradiation attenuation of cells, has resulted in the development of an effective non-tumorigenic vaccine. It has proven effective in both micro- and macro-metastatic disease [47]. The nature of the anatomic alteration in metastatic nodules after AST was explored using a specific monoclonal antibody to assess vascular permeability within these tumor nodules [50, 51]. Immunohistologic analysis demonstrated significantly more antibody in tumors from vaccinated animals than in comparable tumors from unvaccinated guinea pigs. These data support the hypothesis that the anatomic characteristics of tumor foci restrict drug and antibody access, thus pro-

Cancer biotherapy: general principles Table 3. Studies of immunotherapy with random designs Specific cancer and type of immunotherapy Leukemia BCG/AML BCG/Cells/AML MER/AML CP/Cells/AML All/Hodgkin’s NHL/MM Lev/ALL BDG/NHL Lev/MM Lung cancer IT BCG IP BCG IP BCG & Lev IP CP Lev Thy Fr V BCG/Cells TAA/Freund’s Adjuvant Breast cancer Poly A/Poly U BCG Lev Colon cancer BCG MER CP Lev Melanoma IL/BCG BCG/BCG + Cells BCG CP Lev

Positive studies

+ + + +

+

+ + + + +

+ + +

+ + +

+ +

+ + + +

+

+ + + + + + +

Genitourinary cancer IC BCG/bladder BCG/prostate

+ +

Gynecological cancer CP/cervix CP/ovary BCG/ovary

+ +

Other cancers Lev/H & N CP/H & N BCG/Cells/Sarcoma MER/Neuroblastoma

Equivocal Negative studies studies

+ + + +

+

+ + + +

tecting tumors not only from immunotherapy but from other forms of treatment as well [45].

Robert K. Oldham The regulation of the blood supply to neoplastic tissue may be unique in comparison to normal tissue. Tumor metastatic nodules may have a vascular “barrier,” which contributes to a limitation in delivery for chemotherapeutic agents, monoclonal antibodies, and immune effector cells. Such vascular barriers may provide an environment in which some tumor cells survive blood-borne chemotherapeutic and biologic agents. Thus, solid tumor nodules may serve as “pharmacologic sanctuaries,” allowing even drug-sensitive tumor cells to continue to grow [45–47]. Hanna and co-workers [47] demonstrated that strategically timed chemotherapy subsequent to immunotherapy can effectively double the number of survivors attainable with immunotherapy alone. This effect was not drug specific. These results suggest a new basis for AST in the treatment of solid tumors. Inflammatory disruption of anatomic barriers of metastatic nodules combined with strategically administered chemotherapy or biotherapy may prove useful in the design of future biotherapy trials in humans. Another approach used more recently involves the delivery of lymphokines and cytokines, such as interleukins, colony stimulating factors, tumor necrosis factor, lymphotoxins, macrophage cytotoxic factors, and activated complexes (such as those generated by plasma perfusion over protein A columns) to the tumor and its vascular bed. These substances are known to have powerful effects on tumor vasculature and cellular infiltrations, something leading to tumor necrosis. This approach may, in addition, increase the access of antibody, immunoconjugates, drugs, and activated cells to the cancer nodule [45]. A more recent method to influence the blood supply of tumor nodules has been the use of a monoclonal antibody, Bevacizumab, which targets the vascular endothelial growth factor (VEGF) receptor. Bevacizumab is primarily used with chemotherapy, and it may be there are similar mechanisms at play with regard to increasing chemotherapy drug access to tumor nodules by the use of a monoclonal antibody targeting the VEGF receptor [38, 49, 52]. A major limitation of AST has been the availability of purified TAA. Whereas whole cell vaccine preparations contain viable, non-tumorigenic cells prepared from individual tumors, purified TAA is now prompting largescale immunization. TAA purification and characterization followed by genetic engineering of the antigen for vaccine production is underway and several purified antigens are in clinical trials, especially in melanoma. Alternatively, synthetic peptide sequences of the active portion of TAA may prove useful in the near future. Even the combining site of antibody to TAA has recently been suggested as a potential vaccine. These technologies

7 are now undergoing extensive preclinical and clinical evaluation (see Chapter 7).

Thymic Factors It has been known for years that thymic extracts have immunological activity [40, 41]. Thymosin fraction 5 and thymosin alpha-1 have received the most attention in the laboratory and the clinic. Thymosin fraction 5 is an extract containing a variety of thymic polypeptides, and alpha-1 is a synthetic polypeptide component present in many thymic extracts. Thymic preparations have been shown to enhance and suppress immune responses in both intact and thymectomized animals. Many investigators have reported that the thymosins can correct selected immunodeficiency states, both natural and experimentally induced. There have also been reports that thymic factors can augment suppressed or depressed T-cell responses in patients with cancer. Studies in preclinical screening have demonstrated stimulation of T-cell activity [99], but clinical studies have not shown striking effects [25, 98, 99], and none have been approved for clinical use.

Recombinant DNA Technology Recombinant DNA technology, commonly referred to as genetic engineering, has provided us with the tool for the biosynthesis and subsequent mass production of a significant number of biologicals [8, 29, 39]. This is highly relevant to lymphokines/cytokines as well as growth and maturation factors, and should revolutionize the treatment of cancer over the next 10 years. The process involves the incorporation (recombination) of a segment of a DNA molecule containing a desired gene into a vector, usually a plasmid, which in turn is inserted into a host organism, usually an Escherichia coli, although other bacteria, yeasts, insects, and mammalian cells have been utilized. The cells are cloned and the cells producing the desired protein or polypeptide are selected. This clone is mass produced using fermentation techniques, and the protein molecule is harvested and purified. The resultant product is a highly purified (95–99%) protein solution, and has a high specific activity (i.e., biological activity per weight of protein). The relevant DNA can be obtained by a variety of methods. Once messenger RNA (mRNA) is isolated, complementary DNA (cDNA) can be produced through the use of reverse transcriptase. Alternatively, an artificial DNA molecule can be constructed once the nucleotide or amino acid sequence is known. This can be used to isolate the complementary sequence, which is then isolated and cloned. RNA molecules can also be used in cell-free systems

8 Table 4. Clinical lymphokines/cytokines Colony-stimulating factors Erythropoietin Interferons: α, β, γ Interleukins 1–32, etc. Lymphotoxincs: α, β (TNF) Macrophage-activating factors Thymosins Transfer factor(s)

to produce these biologicals. There are over 200 restriction enzymes that can cut desired fragments of DNA and lead to their isolation. These enzymes can uniquely cleave a DNA molecule at specific, predictable sites relative to the nucleotide sequence. These fragments are then incorporated into the plasmid, and combine with plasmid DNA. Plasmids have a symbiotic relationship with selected bacteria inducing resistance to a variety of antibiotics, which allows for selection of engineered clones. A number of alpha interferons, as well as beta and gamma interferon, have been genetically engineered [24, 39, 106, 111]; multiple interleukins, colony-stimulating factors, and tumor necrosis factor have been cloned [107]. The number of cloned biological products increases yearly (see Chapter 4). These biological products and their receptors (Table 4) are rapidly being translated into highquality pharmaceuticals for clinical testing and approval for general use.

Lymphokines/Cytokines Lymphokines and cytokines are molecules secreted by a variety of cells (Table 4). They provide one means through which the cells involved in the immune process communicate with one another and direct the overall process [40]. Lymphokines/cytokines with specific effects on cell proliferation have been identified and may prove useful as anticancer agents. Interleukins (IL) 1–32 are among the multiple lymphokines that appear to be involved in a cascade phenomenon leading to the induction of a variety of immune responses [86]. Other examples include multiple subclasses of colony-stimulating factors (CSF) chemokines and ligands. The list of lymphokines/cytokines is long, and the potential for therapeutic manipulation is great [73, 74]. The identification of these biological activities is the start of a process that should ultimately provide us with the knowledge and the tools to identify more accurately and control a number of immune responses. Physicians may then be able to manipulate the immune system (Fig. 1) intelligently, in favor of the host. Further, by selective activation

Cancer biotherapy: general principles and subsequent cloning in vitro, T-cell lines with specific cytotoxic and helper capabilities can be obtained and utilized in autologous and allogeneic adoptive immunotherapy [16, 27, 93, 103, 112]. Many investigators have held the rather simplistic view that the immune system (Fig. 1) might be manipulated in vivo and corrected to better deal with the cancer problem. Evidence to date suggests that the pharmacologic use of biologicals, in rather high doses, is a more effective method for cancer treatment, with immunoactivation and immunomodulation, playing the dominant roles [69, 70]. Another specific use of lymphokines may be in the pharmacological regulation of tumors of the lymphoid system. Although many of these tumors are considered to be generally unresponsive to normal growth-controlling mechanisms mediated by lymphokines, it is possible that large quantities of pure lymphokines administered as medicinals, or the use of certain molecular analogs of these naturally occurring lymphokines, may be useful in the treatment of lymphoid malignancies. The use of certain lymphokines/cytokines has been extended to other cancers, in that in vitro observations suggest an antiproliferative activity in some solid tumors. These antiproliferative effects might be maximized by testing the tumor cells of each patient to “custom tailor” the treatment rather than giving these biologicals as general treatment, as has been done with anticancer drugs [70]. IL-1, originally known as lymphocyte activating factor, is a macrophage-derived cytokine that has an enhancing effect on murine thymocyte proliferation. Both IL-1 and viable macrophages are necessary for the initial step in activation of IL-2 (Fig. 2). Cloning of IL-1 and IL-2 has made large quantities of highly purified materials available for clinical studies. Preclinical studies with IL-2 have been oriented around in vitro cell production protocols and induction or maintenance of antitumor T-cell effects in vivo [15, 16, 67, 70]. IL-2 has been used to activate peripheral blood cells and initially stimulated much interest [93, 104]. These activated cells are generally more cytotoxic against cancer cells than normal cells; however, their lineage can be T cell or NK cell (LAK cells) depending on the technique employed. This approach, though expensive and technically demanding, illustrates that cellular therapy is feasible and can be effective [67, 70, 85, 103]. A lymphotoxic product of antigen/mitogen-stimulated leukocytes was called lymphotoxin [92]. Lymphotoxin may be a principle effector of delayed hypersensitivity and, although conflicting data have been reported, may also be involved in the cytoxic reactions of T-cell-mediated

Robert K. Oldham

9

Figure 1. Immune response

lysis and NK- or K-cell lysis. Depending upon the type of tumor cell involved, the in vitro effect of lymphotoxin may be either cytolytic or cytostatic. Mouse tumor cells are frequently killed by homologous and heterologous lymphotoxins, whereas in other species reversible inhibition of tumor cell proliferation is more common [48]. Human lymphotoxin is of at least two species, alpha and beta [30, 37, 60, 88, 108]. Alpha lymphotoxin is tumor necrosis factor (TNF). Both have been cloned, and TNF has undergone rather extensive clinical trials [31, 91, 101]. While some antitumor responses have been seen, the lymphotoxins have not been highly efficacious as single agents in the treatment of advanced human tumors and the toxicity of systemic administration has been unacceptable. Continued trials are underway combining lymphotoxins such as TNF with other lymphokine/cytokines and with chemotherapy. Targeted delivery of these molecules may prove more efficacious since they have high toxicity administered intravenously with what is probably minimal delivery to the tumor cell

site [60, 28]. Thus, intratumoral and regional perfusion studies with TNF have yielded positive results in patients with melanoma and sarcoma [17, 53, 59, 60, 90]. There is now evidence that the combined use of various lymphokines may produce enhanced antiproliferative effects. Selective assays for lymphokine antiproliferative cocktails may prove useful in “tailoring” such preparations for individual patients [6, 95, 97, 102, 100, 110]. More than 100 biological molecules have already been described and named as lymphokines/cytokines (Table 5). Biologicals such as the interferons, lymphotoxins, TNF, CSF, IL-1–22 are now under evaluation (see Chapter 8). The studies require quantities of material obtained through genetic engineering, using sensitive and rapid assay procedures to monitor production, purification, and bioavailability.

Interferons Interferons are small, biologically active proteins with antiviral, antiproliferative, and immunomodulatory

10

Cancer biotherapy: general principles Monocytic Macrophage

Ag Stimulated T Cell

? CSF

Activated Macrophage

IL-1

Activated T cells

Ag Stimulated T cell

IL -2

Effector T cell Precursor

Mature Effector T Cells

Figure 2. Model for interleukin stimulation of T-cell immune responses

activities (see Chapter 9). Each interferon has distinctive capabilities in altering a variety of immunological and biological responses. As a class, the interferons appear to have some growth-regulating capacity in that antiproliferative effects are measurable with in vitro assays and in animal model systems. The relative efficacy of the mixtures of natural interferons that occur after virus stimulation as compared to the cloned interferons remains to be precisely determined. There are more than 20 interferon molecules (and theoretically hundreds of recombinant hybrids there from), and efforts are underway altering individual interferon molecules in specific ways, so the range of biological activities of the interferons as antiviral agents, as immunomodulating agents, and as antiproliferative agents may be very broad [66, 69]. In addition to antiviral and antiproliferative activity, the interferons have profound effects on the immune system. Low doses enhance antibody formation and lymphocyte blastogenesis, while higher doses inhibit

these functions. Low to moderate doses may inhibit delayed hypersensitivity while enhancing macrophage phagocytosis and cytotoxicity, natural killer activity, and surface antigen expression. Interferons prolong and inhibit cell division (both transformed and normal cells). In addition, interferons stimulate the induction of several intracellular enzyme systems with resultant profound effect on macromolecular activities and protein synthesis. We can draw some preliminary conclusions about interferon therapy for human cancer [9, 36, 43, 44, 57, 69, 96] (Tables 6 and 7). One is that the Cantell, lymphoblastoid, and recombinant alpha-interferons are surprisingly similar, both quantitatively and qualitatively, in their toxicity, antitumor efficacy, and other biologic effects. Second, objectively defined antitumor responses in phase I alpha-interferon trials (mostly involving lymphoma, myeloma, Kaposi’s sarcoma, melanoma, and renal cancer) were observed in approximately 10% of all patients treated. That level of activity may not seem impressive, but it does exceed the average response rate of 1–2% in phase I trials of chemotherapeutic agents. We should also note that very few responses in patients with tumors of the breast, colon, lung, or lower genitourinary system have been seen with alpha-interferon as a single agent. Overall, even though interferons have toxicities, they were more tolerable and less permanent than those observed in early-phase chemotherapy testing. A third impression, suggested by increased response rates with higher alpha-interferon doses, is that interferons may produce their acute antitumor effect by a direct cytostatic action, rather than an indirect immunomodulatory mechanism [12, 23, 57, 66, 69, 72, 98]. Finally, clinical experience with beta- and gammainterferons indicated that both produce response rates and response patterns similar to those obtained with alpha-interferon, although beta-interferon is only approved for multiple sclerosis and gamma only for chronic granulomatous disease. What is clear from the current preclinical and clinical studies is that the interferons have antitumor activity even in bulky, drug-resistant cancers [102]. Clinical activity has been seen most reproducibly in several lymphomas and leukemias (Table 6), but responses in many other tumor types with approval in melanoma and renal cancer [36].

Growth and Maturation Factors Using technology similar to that employed for lymphokine/cytokines, scientists have cloned and produced a variety of growth and maturation factors. The

Robert K. Oldham

11

Table 5. Antigen-nonspecific mediators,a unrestricted by MHC Helper factors LAF (lymphocyte-activating factor, IL-1) NMF (normal macrophage factor) BAF (B-cell-activating factor) TRF (T-cell-replacing factor) MP (mitogenic protein) TDF (thymus differentiation factor) Transferrin MF (mitogenic [blastogenic] factor) NSF (nonspecific factor) TDEF (T-cell-derived enhancing factor) TEF (thymus extracted factor) Complement components DSRF (deficient serum-restoring factor) Suppressor factors Inhibitor(s) of DNA synthesis AIM (antibody-inhibitory material) IDA (inhibitor of DNA synthesis) LTF (lymphoblastogenesis inhibition factor) FIF (feedback inhibition factor) MIFIF (Mif inhibition factor) SIRS (soluble immune response suppressor) IRF (immunoregulatory gamma-globulin) Chalones IF (interferons) AFP (alpha-fetoprotein) LDL (low-density lipoproteins) CRP (C-reactive protein) Fibrinogen degradation products NIP (normal immunosuppressive protein) LMWS (low-molecular-weight suppressor) HSF (histamine-induced suppressor factor) TCSF (T-cell suppressive factor) Factors acting on inflammatory cells MIF (migration inhibitory factor) MCF (macrophage chemotactic factor) MSF (macrophage slowing factor) MEF (migration enhancement factor) MAF (macrophage activating factor) MFF (macrophage fusion factor) PRS (pyrogen-releasing substance) LIF (leukocyte inhibition factor) NCF (neutrophil chemotactic factor) PAR (products of antigenic recognition) BCF (basophil chemotactic factor) ECF (eosinophil chemotactic factor) a

ESP (eosinophil stimulation factor) LCF (lymphocyte chemotactic factor) LTF (lymphocyte trapping factor) Factors acting on vascular endothelium SRF (skin reactive factor) TPF (thymic permeability factor) LNPF (lymph node permeability factor) LNAF (lymph node activating factor) AIPF (anaphylactoid inflammation promoting factor) IVPF (increased vascular permeability factor) Factors acting on other cells Interferons TMIF (tumor cell migration inhibition factor) OAF (osteoclast activating factor) Fibroblast chemotactic factor Pyrogens FAF (fibroblast activating factor) Growth-stimulating factors BCGF (B-cell growth factor) BCDF (B-cell differentiation factor) MGF (macrophage growth factor) MF (mitogeni [blastogenic] factor) LIAF (lymphocyte-induced angiogenesis factor) CSF (colony-stimulating factor) TDF (thymus differentiation factor) Thymopoietin, thymosin TCGF (T-cell growth factor, IL-2) IL-3 (interleukin 3) EGF (epidermal growth factor) Direct-acting factors Lysosomal enzymes CTF (cytotoxic factors) MTF or MCF (macrophage toxic [cytotoxic] factor) SMC (specific macrophage cytotoxin) MCF (macrophage cytolytic factor) ACT (adherent cell toxin) Chromosomal breakage factors Microbicidal factors LT (lymphotoxin) PIF (proliferation inhibitory factor) CIF (cloning inhibition factor) IDS (inhibitor of DNA synthesis) Transforming factors TNF (tumor necrosis factor)

These names/factors are based on biological activity. Several may represent the activity of a single molecule.

clinical trials have focused mainly on erythropoietin and the colony-stimulating factors. The former is a drug now approved for use in refractory anemia, and GM-CSF and GCSF are approved for the treatment of bone marrow dysplasia and chemotherapy-induced marrow suppression. Oprelvekim to stimulate platelet production is now approved. These factors are reviewed in later chapters, but it should be noted that they represent only the early beginnings of the very broad field of growth and matu-

ration factors (See Chapter 16). It is now clear that a variety of biological substances up- and down-regulate growth of both normal and neoplastic cells. These substances may stimulate or inhibit growth and may change the maturation cycle of various normal and neoplastic cells. Contained within this broad category of factors are the tumor growth factors, colony-stimulating factors, and a variety of still to be defined factors important in the regulation of cell growth and maturation. Future

12

Cancer biotherapy: general principles

Table 6. Apha-interferon activitya Active Hairy-cell leukemia Chronic myelogenous leukemia Myeloproliferative disorders Non-Hodgkin’s lymphoma Cutaneous T-cell lymphoma Kaposi’s sarcoma Multiple myeloma Melanoma Renal cell carcinoma Bladder cancer (intravesical) Inactive Breast cancer Colon cancer Lung cancer Prostate cancer Acute myelogenous leukemia a

As a single agent.

Table 7. Malignancies: Summary of responses to alphainterferon with date of FDA approval Tumor type

Response rate (%)

Multiple myeloma Hairy cell leukemia Low-grade lymphoma High-grade lymphoma Kaposi’s sarcoma Chronic myelogenous leukemia Melanoma Renal cancer

18–27 80–90 38–73 0–10 25–40 80–90 20–30 10–20

Approved 1986 1997 1988 1995 1995

therapeutic use of these factors may regulate growth and spread of cancer. Such a chronic growth restraining strategy may not “cure” cancer; rather treatment may be more analogous to using insulin in diabetes. This is a field that is undergoing explosive growth and should be watched carefully over the coming decade for molecules with therapeutic activity.

Monoclonal Antibodies The advent of hybridoma technology in the late 1970s made available an important tool for the production of monoclonal antibodies for therapeutic trials [10] (see Chapter 10). These antibodies are now being produced in huge quantities and in highly purified form for cancer treatment. The humanization of murine antibody combining sites has yielded a whole new class of therapeutic antibodies [84]. They will undoubtedly define a new range of

antigens on the cell surfaces, which will improve our understanding of cell differentiation and of cancer biology. Major problems in understanding the biology of the cancer cell have been the difficulties of isolating, purifying, and characterizing tumor-associated antigens (TAA). The use of monoclonal antibody technology will improve the definition of the neoplastic cell surface and identify its differences from the normal counterpart. This will be of great value in cancer diagnosis and histopathologic classification, and will be useful in the imaging of tumor cell masses and in the therapy of cancer [1, 7, 13, 14, 32, 33, 34, 35, 50, 51, 55, 63, 75, 84, 89]. Finally, antibodies may be a useful reagent in treating certain immune deficiencies and in altering immune responses. The removal of T cells from bone marrow to improve bone marrow transplantation techniques is an example of using antibody as a BRM [63]. A more recent example is the use of anti-CTLA-4 antibodies to regulate the immune response. Two monoclonal antibodies are in development that have influence on T-regulatory cells, which influence the immune response to cancer. It is already clear that this class of monoclonal antibodies can influence T-regulatory cells in a yet undefined manner, such that regression of bulky melanoma can occur. At the same time, they give a range of interesting toxicities with regard to uveitis, colitis, and depigmentation syndromes relative to their use in melanoma [26]. In spite of encouraging data from the use of antibodies and, especially, immunoconjugates to target toxic substances to cancer, the heterogeneity of cancer is an important consideration [2, 71–78, 87]. If a single antibody or a fixed combination of a few antibodies covering only a portion of the tumor cells is used, and if that preparation does not eliminate the true replicating cell population (stem cell) from the patient’s tumor population, eventual outgrowth of viable, perhaps resistant cells is inevitable. Therefore, it seems logical to proceed with attempts to type human tumors and to deliver toxic substances to them utilizing “cocktails” of antibodies sufficient to cover all the tumor cells suspected of replication in each patient. This type of approach may require a considerable amount of testing for each patient and a “typing” of one or more tumors from each patient [3, 58, 62, 78, 79, 87]. Such approaches may be more individually designed than is easily approachable through the product development paradigm that has been used in the development of new anticancer drugs. If however, the spectrum of human tumor heterogeneity is great, the goal of the ideal antibody conjugated to the ideal toxic agent may not be achievable.

Robert K. Oldham

Future Perspectives How rapidly will biotherapy develop and what role will it have in cancer treatment in the next decade? It is certain that we now have much more powerful tools for improving cancer therapy in the future. We now have the techniques to decipher the major problems in cancer biology down to the genetic level. These techniques, along with the recognition that biotherapy can provide increased selectivity in cancer treatment, support the belief that new and highly effective approaches are near. Biotherapy provides an additional technique, which may work effectively in combination with surgery or radiotherapy (to decrease the local and regional tumor) or with chemotherapy (to reduce the systemic tumor burden). It may work very effectively via antibodies in directing radioisotopes selectively to the tumor site, and with chemotherapy, toxins, and other cytostatic or cytotoxic molecules in directing the agent to the tumor bed, enhancing activity and selectivity. The use of biotherapy is at an early stage. We have already seen that highly purified biologicals can be effective in patients with clinically apparent, even advanced, bulky cancer. Clinical studies with alpha-interferon have demonstrated the responsiveness of radiation- and drugresistant lymphoma, melanoma, and renal carcinoma. IL-2 with effector cells or alone (in high doses) produces partial and complete remissions in melanoma and kidney cancer. These results, along with the clinical results using monoclonal antibody alone and conjugated to toxic substances in selected cancers (lymphoma, melanoma, gastrointestinal, leukemia and breast cancer), confirm the concept that we need not think of biotherapy as a tool that can be used only in patients with undetectable and minimal residual tumor burdens [84]. While this modality may work best with minimal tumor burdens (a situation that is also true for chemotherapy), biotherapy can be used as a single modality in clinically apparent disease. It may be even more effective in multimodality treatment regimens. Biotherapy offers the hope for selective treatment to enhance the therapeutic/toxic ratio and lessen the problem of nonspecific toxicity, a major impediment to the development of more effective cancer treatment. This decade will provide many opportunities to pursue new approaches in cancer treatment. These approaches will employ new techniques in the laboratory and clinic, requiring special training and expertise. The medical oncologist trained in the administration of chemotherapy drugs, is not necessarily well prepared to give biologicals for cancer treatment. Biotherapy uses biological substances that are often active on, or work in

13 association with, the immune system. The tremendous diversity of this system is best understood by clinical immunologists and cell biologists who are well suited to assist in the translation of biotherapeutic approaches to the clinic. Given these new techniques and new approaches, we must redesign many of the mechanisms for developmental therapeutics [59]. It may well be that the specificity of biologicals will require that biotherapy be developed in an individualistic fashion and applied to each patient in a specific, personalized way. This concept was conceptualized in 1977 by the Nobel laureate Sir Peter Medawar: The cure of cancer is never going to be found. It is far more likely that each tumor in each patient is going to present a unique research problem for which laboratory workers and clinicians between them will have to work out a unique solution.

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2

The pathogenesis of cancer metastasis: relevance to therapy SUN-JIN KIM, CHERYL HUNT BAKER, YASUHIKO KITADAI, TORU NAKAMURA, TOSHIO KUWAI, TAKAMITSU SASAKI, ROBERT LANGLEY, AND ISAIAH J. FIDLER

Introduction Metastasis, the spread of malignant tumor cells from a primary neoplasm to distant parts of the body where they multiply to form new growths, is a major cause of death from cancer. The treatment of cancer poses a major problem to clinical oncologists, because by the time many cancers are diagnosed, metastasis may already have occurred, and the presence of multiple metastases makes complete eradication by surgery, radiation, drugs, or biotherapy nearly impossible. Metastases can be located in different organs and in different locations within the same organ. These aspects significantly influence the response of tumor cells to therapy and the efficiency of anticancer drugs, which must be delivered to tumor lesions to destroy cells without leading to undesirable side effects. Similarly, immune effector cells of current biotherapeutic regimens may not reach or localize in metastases with different organs. Exacerbating the problems of treating metastatic disease is the fact that tumor cells in different metastases and in some instances even in different regions within a single metastasis may respond differently to treatment. Tumor cell resistance to current therapeutic modalities is the single most important reason for the lack success in treating many types of solid neoplasms. In part, the emergence of treatment-resistant tumor cells is due to the heterogeneous nature of malignant neoplasms. This diversity, which permits selected variants to develop from the parent tumor, implies not only that the primary tumor and metastases can differ in their response to treatment, but also that individual metastases can differ markedly from one another. Insight into the molecular mechanisms that regulate the pathogenesis of cancer metastasis as well as a better understanding of the interaction between metastatic cells and the host microenvironment should provide a foundation for the design of new therapeutic approaches. Moreover, the development of adequate models that allow for the isolation and characterization of cells possessing metastatic potential will be invaluable in the

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

design of more effective therapeutic modalities. In this chapter, we discuss the biology of cancer metastasis with special emphasis on recent data demonstrating that the microenvironment of different organs can influence the biological behavior of tumor cells at different steps of the metastatic process and the development of biologic diversity in malignant neoplasms. These findings have obvious implications for the therapy of neoplasms in general and metastases in particular.

The Pathogenesis of Cancer Metastasis The process of cancer metastasis is dynamic, complex and consists of multiple, sequential, and interrelated steps shown schematically in Fig. 1. To produce a clinically relevant lesion, metastatic cells must survive all the steps of the process. If the disseminating tumor cell fails to complete any one of these steps, it will fail to produce a metastasis. Thus, the successful metastatic cell has been likened to a decathlon champion who must be proficient in all ten events to be successful, not just a few [58]. The outcome of this process depends on both the intrinsic properties of the tumor cells and their interactions with host factors [48, 56, 57, 99]. The essential steps in the formation of a metastasis are: (1) After the initial transforming event, either unicellular or multicellular, growth of neoplastic cells must be progressive, with nutrients for the expanding tumor mass initially supplied by simple diffusion. (2) Extensive vascularization must occur if a tumor mass is to exceed 1–2 mm in diameter. The synthesis and secretion of proangiogenic angiogenesis factor probably plays a key role in establishing a neocapillary network from the surrounding host tissue. (3) Local invasion of the host stroma by some tumor cells could occur by several nonmutually exclusive mechanisms. (4) Thin-walled venules, like lymphatic channels, offer very little resistance to penetration by tumor cells and provide the most

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The pathogenesis of cancer metastasis: relevance to therapy

THE PATHOGENESIS OF A METASTASIS TRANSFORMATION

ANGIOGENESIS

MOTILITY & INVASION

Capillaries, Venules, lymphatic vessels

ARREST IN CAPILLARY BEDS

EMBOLISM & CIRCULATION

TRANSPORT

ADHERENCE

EXTRAVASATION INTO ORGAN PARENCHYMA

MULTICELL AGGREGATES (lymphocytes,platelets)

RESPONSE TO MICROENVIRONMENT

METASTASIS OF METASTASES TUMOR CELL PROLIFERATION & ANGIOGENESIS

METASTASES

Figure 1. The pathogenesis of cancer metastasis

common pathways for tumor cell entry into the circulation. Although clinical observations have suggested that carcinomas frequently metastasize and grow via the lymphatic system, whereas malignant tumors of mesenchymal origin more often spread by the hematogenous route, the presence of numerous venolymphatic anastomoses invalidates this concept. (5) Detachment and embolization of small tumor cell aggregates occurs next, the vast majority of circulating tumor cells being rapidly destroyed. (6) Once the tumor cells have survived the circulation, they must (7) arrest in the capillary beds of organs, either by adhering to capillary endothelial cells or by adhering to subendothelial basement membrane, which may be exposed. (8) Extravasation occurs next, probably by the same mechanisms that influence initial invasion. (9) Proliferation within the organ parenchyma completes the metastatic process (10). To continue

growing, the micrometastases must develop a vascular network and continue to evade the host immune system. Moreover, the cells can invade, penetrate blood vessels, and enter the circulation to produce additional metastases (Fig. 1).

NeovascularizationAngiogenesis Oxygen can diffuse from capillaries for only 150–200 μm. When distances of cells from a blood supply exceed this, cell death follows [80]. Thus, the expansion of tumor masses beyond 1 mm in diameter depends on neovascularization, i.e., angiogenesis [78, 79]. The formation of new vasculature consists of multiple, interdependent steps. It begins with local degradation of the basement

Sun-Jin Kim et al. membrane surrounding capillaries, followed by invasion of the surrounding stroma and migration of endothelial cells in the direction of the angiogenic stimulus. Proliferation of endothelial cells occurs at the leading edge of the migrating column, and the endothelial cells begin to organize into three-dimensional structures to form new capillary tubes. Differences in cellular composition, vascular permeability, blood vessel stability, and growth regulation distinguish vessels in neoplasms from those in normal tissue [1, 3, 79, 156]. The onset of angiogenesis involves a change in the local equilibrium between proangiogenic and antiangiogenic molecules [55]. Some of the common proangiogenic factors include bFGF, which induces the proliferation of a variety of cells and has also been shown to stimulate endothelial cells to migrate, to increase production of proteases, and to undergo morphogenesis [80]. Likewise, VEGF/VPF has been shown to induce the proliferation of endothelial cells, to increase vascular permeability, and to induce production of urokinase plasminogen activator by endothelial cells [46]. Additional proangiogenic factors include IL-8 [268], platelet-derived endothelial cell growth factor, which has been shown to stimulate endothelial cell DNA synthesis and to induce production of FGF, hepatocyte growth factor (HGF), or scatter factor, that increases endothelial cell migration, invasion, and the production of proteases, and platelet-derived growth factor (PDGF). The production of bFGF, VEGF, and IL-8 by tumor or host cells or the release of proangiogenic molecules from the extracellular matrix is known to induce the growth of endothelial cells and formation of blood vessels. Further, the organ microenvironment can directly contribute to the induction and maintenance of the proangiogenic factors bFGF [226, 227] and IL-8 [228]. The production of angiogenic molecules, e.g., VEGF, bFGF, and IL-8 by melanoma cells is regulated by complex interactions with keratinocytes in the skin [106]. Reports from our laboratory showed that IL-8 is an important molecule in melanoma growth and progression. Constitutive expression of IL-8 directly correlated with the metastatic potential of human melanoma cells. Further, IL-8 induced proliferation, migration, and invasion of endothelial cells and, hence, neovascularization [97]. Several organ-derived cytokines (produced by inflammatory cells) are known to induce expression of IL-8 in normal and transformed cells [106]. Since IL-8 expression in melanocytes and melanoma cells can be induced by inflammatory signals, the question of whether specific organ microenvironments could influence the expression of IL-8 was analyzed. Melanoma cells were implanted into the subcutis, the spleen (to produce liver metastasis),

19 and intravenously (to produce lung metastasis) of athymic nude mice. Subcutaneous tumors, lung lesions, and liver lesions expressed high, intermediate, and no IL-8, respectively, at both the mRNA and protein levels. Melanoma cells established from the tumors growing in vivo exhibited similar levels of IL-8 mRNA transcripts as continuously cultured cells, thus demonstrating that the differential expression of IL-8 was not due to the selection of a subpopulation of cells [97]. IL-8 expression can be upregulated by coculturing melanoma cells with keratinocytes (skin) and inhibited by coculturing melanoma cells with hepatocytes (liver). We also investigated the effects of two cytokines produced by keratinocytes (IL-1, IFN-β) and two cytokines produced by hepatocytes (TGF-α, TGF-β) on the regulation of IL-8 in human melanoma cells. IL-1 upregulated the expression of IL-8 in human melanoma cells at both the mRNA and protein levels in a dose- and timedependent manner in the presence of de novo protein synthesis. IFN-β did not affect constitutive IL-8 mRNA and protein production in human melanoma cells, but it did block the induction of IL-8 by IL-1. TGF-β inhibited the expression of IL-8, while TGF-α had no effect on IL-8 expression [226]. Patients with renal cell carcinoma exhibit high levels of bFGF in the serum or the urine that inversely correlates with survival [181, 184]. Human renal cancer cells implanted into the kidney of nude mice were highly metastatic to the lung, whereas renal cancer cells implanted subcutaneously remain local [227]. The subcutaneous or intramuscular tumors expressed a lower level of mRNA transcripts for bFGF than did tumor cells growing in culture, whereas the renal tumors had 20 times higher levels of bFGF mRNA and protein levels as compared with the cultured cells. A histopathologic examination of the tumors demonstrated that the subcutaneous tumors had few blood vessels, whereas the renal tumors had many [227, 228]. Direct correlation between the level of bFGF and advanced disease was also reported for patients diagnosed with colon carcinoma. Patients with Duke’s C or D tumors had markedly higher levels of bFGF in the blood than patients with Duke’s B. In situ hybridization analysis revealed that bFGF was overexpressed at the tumor periphery associated with cell division. Northern blot analysis detected no mRNA transcripts for bFGF [129]. In a follow-up study of patients with colon cancer, bFGF expression was found to be highest in the primary tumors of patients who presented with metastatic disease and therefore identify a cohort of patients who appeared to be free of metastatic disease at the time of surgery (low bFGF expression) as compared to another cohort of patients who did develop metastatic disease (high bFGF) [130].

20

Tumor Cell Invasion To reach blood vessels or lymphatics, tumor cells must penetrate host stroma that includes basement membrane. The interaction with the basement membrane consists of attachment, matrix dissolution, motility and penetration [154]. At least three nonmutually excluding mechanisms can be involved in tumor cell invasion of tissues. First, mechanical pressure produced by rapidly proliferating neoplasms may force cords of tumor cells along tissue planes of least resistance [154, 155]. Second, increased cell motility can contribute to tumor cell invasion. Most tumor cells possess the necessary cytoplasmic machinery for active locomotion and increased tumor cell motility is preceded by a loss of cell-to-cell cohesive forces. In epithelial cells, the loss of cell-to-cell contact is associated with downregulation of the expression of E-cadherin, a cell surface glycoprotein involved in calcium-dependent homotypic cell-to-cell cohesion. Reduced levels of E-cadherin are associated with a decrease in cellular/ tissue differentiation and increased grade in carcinomas [120]. Many differentiated carcinomas express higher levels of E-cadherin mRNA, as do adjacent normal epithelial cells, whereas poorly differentiated carcinomas do not. Mutations in the E-cadherin gene and abnormalities of α-catenin, which is an E-cadherin-associated protein, have been associated with the transition of cells from the noninvasive to the invasive phenotype [261]. Third, invasive tumor cells secrete enzymes capable of degrading basement membranes, which constitute a barrier between epithelial cells and the stroma. Epithelial cells and stromal cells produce a complex mixture of collagens, proteoglycans, and other molecules, which contains ligands for adhesion receptors and is permeable to molecules but not to cells [230]. To invade the basement membrane, a tumor cell must first attach to extracellular matrix (ECM) components by a receptor-ligand interaction. One group of such cell surface receptors are the integrins which specifically bind cells to laminin, collagen, or fibronectin [218]. Many integrins that bind to different components of the ECM are expressed on the surface of human carcinoma cells. Tumor progression has been associated with a gradual decrease of integrin expression suggesting that the loss of integrins, coupled with the loss of E-cadherin, may facilitate detachment from a primary neoplasm. Subsequent to binding, tumor cells can degrade connective-tissue ECM and basement membrane components [177]. The production of enzymes such as type IV collagenase (gelatinase, matrix metalloproteinase) and heparinase in metastatic tumor cells correlates with invasive capacity of human carcinoma cells. Type IV collagenolytic

The pathogenesis of cancer metastasis: relevance to therapy metalloproteinases with apparent molecular masses of 98, 92, 80, 68, and 64-kDa have been detected in highly metastatic cells. Poorly metastatic cells, on the other hand, appear to secrete very low amounts of only the 92-kDa metalloproteinase [171, 172].

Lymphatic Metastasis Early clinical observations led to the impression that carcinomas spread mainly by the lymphatic route and mesenchymal tumors spread mainly by means of the bloodstream. We now know, however, that the lymphatic and vascular systems have numerous connections and that disseminating tumor cells may pass from one system to the other [27]. For these reasons, the division of metastasis into lymphatic spread and hematogenous spread is arbitrary. During invasion, tumor cells can easily penetrate small lymphatic vessels and be passively transported in the lymph. Tumor emboli may be trapped in the first lymph node encountered on their route, or they may bypass regional draining lymph nodes to form distant nodal metastases (“skip metastasis”). Although this phenomenon was recognized in the late 1800s [193], its implications for treatment were frequently ignored in the development of surgical approaches to treat cancers [263, 264]. Regional lymph nodes (RLN) in the area of a primary neoplasm may become enlarged as a result of hyperplasia or growth of tumor cells in the node. Although the use of morphologic criteria for assessing prognoses based on lymph node appearance is debatable, lymphocytedepleted lymph nodes are believed to indicate a less favorable prognosis than those demonstrating reactive morphologic characteristics [16]. Hyperplastic responses could indicate reactivity to autochthonous tumors, and this could benefit the host. Whether the RLN can retain tumor cells and serve as a temporary barrier for cell dissemination is controversial. In most experimental animal systems used to investigate this question, normal lymph nodes were subjected to a sudden challenge with a large number of tumor cells, a situation that may not be analogous to RLN at the early stages of cancer spread in humans, when small numbers of cancer cells continuously enter the lymphatics. This issue is important because of practical considerations for surgical management of such neoplasms as cutaneous melanoma. It raises the question, is elective prophylactic lymph node dissection appropriate for the treatment of micrometastases? The biologic justification for elective lymph node dissection in patients with melanoma presumes that metastasis of some cutaneous melanomas

Sun-Jin Kim et al. occurs first in the RLN, and that only at a later time do tumor cells gain access to the circulation to reach distant organs. If this is the case, and RLN can act as a temporary barrier to the spread of cancer, removing the RLN with micrometastases could clearly increase the cure rate in subgroups of patients with melanoma. Some evidence exists that patients with melanomas of intermediate thickness (1–4 mm) do in fact have an improved survival rate subsequent to elective lymph node dissection. Similarly, some data suggest that an improved survival rate can be achieved for selected patients with head and neck cancers by elective lymph node dissection or local treatment with x-irradiation [25]. Recent advances in mapping of the lymphatics draining cutaneous melanoma (by the use of dyes or radioactive tracers) have allowed surgeons to identify the lymph node draining the tumor site (i.e., the sentinel lymph node) [36]. The presence of melanoma micrometastases in sentinel lymph nodes is correlated with poor prognosis and hence indicates wide field dissection. A series of more than 500 melanoma cases with longer than 4 years’ median clinical follow-up concluded that absence of disease in sentinel lymph node correlates with increased disease-free status (in other nodes) and few or no skip metastases [119, 175]. These data suggest that elective lymph node dissection when metastatic cells are present in sentinel lymph nodes produces beneficial results in patients with melanoma.

Hematogenous Metastasis During blood-borne metastasis, tumor cells must survive transport in the circulation, adhere to small blood vessels or capillaries, and invade the vessel wall. The mere presence of tumor cells in the circulation does not in itself constitute metastasis, since most cells released into the bloodstream are eliminated rapidly [60, 263, 264]. Using radiolabeled tumor cells, we found that by 24 h after entry into the circulation, less than 1% of the cells are still viable, and less than 0.1% of tumor cells placed into the circulation eventually survive to produce metastases [60]. Although most tumor cells are destroyed in the bloodstream, it seems that the greater the number of cells released by a primary tumor, the greater the probability that some cells will survive to form metastases. The number of tumor emboli in the circulation appears to correlate well with the size and clinical duration of the primary tumor, and the development of necrotic and hemorrhagic areas in large tumors facilitates this process by providing tumor cells easy access to the circulation [263].

21 To a large degree, the rapid death of most circulating tumor cells is probably due to such simple mechanical factors as blood turbulence. Tumor cell survival can be increased by aggregation. Tumor cells can aggregate with each other or with host cells, such as platelets and lymphocytes [67]. Once metastatic cells reach the microcirculation, they interact with cells of the vascular endothelium. These interactions include nonspecific mechanical lodgment of tumor cell emboli as well as formation of stable adhesions between tumor cells and small-vessel endothelial cells. The organ distribution of metastatic foci is believed to depend, in part, on the ability of blood-borne malignant cells to adhere to specific endothelium and produce endothelial cell retraction [185, 186]. The formation of fibrin clots at sites of tumor cell arrest in the microcirculation can result in blood vessel damage [45, 46]. In some tumor systems, fibrin formation is not essential for tumor cell implantation or metastasis formation. The increased coagulability often observed in patients with cancer may be related to the high levels of thromboplastin found in certain tumors or to production of high levels of procoagulant-A activity, which can directly activate factor X in the clotting process. Since reduced blood flow could lead to increased trapping of circulating tumor cells and perhaps to their increased survival, the use of anticoagulants in the treatment or control of metastasis has been tried, albeit to a limited success. The adhesion of tumor cells to the vascular endothelium is regulated by mechanisms similar to those used by leukocytes. The initial attachment of leukocytes to vascular endothelial cells is regulated by the selectin family of adhesion molecules which consists of three closely related cell surface molecules. E-selectin, which is expressed by endothelial cells, mediates initial attachment of lymphocytes (and tumor cells) by interaction with specific carbohydrate ligands that contain sialylated fucosylated lactosamines. The expression of mucin-type carbohydrates on the surface of human colon carcinoma has been correlated with their metastatic potential [161], perhaps through differential interaction with E-selectins expressed on specific endothelial cells. The development of firm adhesion requires the interaction of other adhesion molecules, another selective process in metastasis. Several classes of cell-to-cell adhesion molecules regulate this adhesion. These include the hyaluronate receptor CD44 and its splice-variants [183], the integrins α5β1, α6β1, and α6β4, and the galactoside-binding galectin-3 [218]. The arrest of tumor cells in capillary beds leads to the retraction of endothelial cells and the exposure of the tumor cells to the ECM. The adhesion of metastatic cells

22 to components of the ECM, such as fibronectin, laminin, and thrombospondin, facilitates metastasis to specific tissues, and peptides containing sequences of these components of the ECM can reduce formation of hematogenous metastases [246]. Extravasation of arrested tumor cells is believed to operate by mechanisms similar to those responsible for local invasion. Tumor cells can grow and destroy the surrounding vessel, invade by penetrating the endothelial basement membrane, or they can follow migrating white blood cells [50]. The ability of malignant cells to extravasate into surrounding tissues of particular organs seems to arise, in part, from their selective adherence to and invasion of certain tissues [187]. Malignant cells frequently penetrate thin-walled capillaries but rarely invade arteries or arteriole walls, which are rich in elastin fibers. This resistance to invasion is not necessarily mediated by mechanical strength alone. Connective tissues have been shown to produce protease inhibitors, and these may block enzyme-dependent processes of invasion. The invasion, survival, and growth of malignant cells at particular secondary sites also involve their responses to tissue or organ factors. Tumor cells can recognize tissue-specific motility factors that direct their movement and invasion [196]. After tumor cells invade organ parenchyma, they must also respond to organ-specific factors that influence their growth [187].

Metastasis of Metastases The tumor cells proliferating within metastases can invade host stroma, penetrate blood vessels, and enter the circulation to produce secondary metastases, the so-called “metastasis of metastases” [73, 235, 236]. Hart and Fidler used the preferential growth of B16 melanoma metastases in specific organs. Following the intravenous injection of B16 melanoma cells into syngeneic mice, tumor growths developed in the lungs and in fragments of lung or ovarian tissue implanted intramuscularly into the quadriceps femoris but not in renal tissue implanted as a control [101]. Tumor growth in the specific transplanted organ could have been caused by the arrest and growth of tumor cells immediately following intravenous injection, i.e., “initial metastases.” Alternatively, tumor cells injected intravenously could have been arrested in the lungs, where they developed; once metastases were established, tumor cells could enter the circulation to be arrested at other organs and produce “secondary metastases” [235]. To distinguish between these possibilities, Nicolson and Fidler performed

The pathogenesis of cancer metastasis: relevance to therapy several experiments [73]: Two weeks after normal, tumor-free mice were joined parabiotically to metastasisbearing animals, there was no evidence of any tumor growth in the “guest” animals. However, when the parabiont animals were allowed to survive for 4 weeks after separation from the metastasis-bearing animals, 40% developed lung metastases. Since the host mice did not have primary tumors at the time of parabiosis, the metastases in the guest mice could have only arisen as metastasis from metastases [101].

The Biologic and Metastatic Heterogeneity of Neoplasms Only a few tumor cells that enter the circulation can produce metastases. In fact, since less than 0.01% of circulating cells are likely to produce a secondary growth, the development of metastases could represent the fortuitous survival of a few tumor cells or the selection from the parent tumor of a subpopulation of metastatic cells endowed with properties that enhance their survival [65, 66, 71]. Data generated by our research group and many others prove that neoplasms are biologically heterogeneous and that metastasis is indeed a selective process. The first experimental proof of metastatic heterogeneity of neoplasms was provided by Fidler and Kripke in 1977 working with the murine B16 melanoma [71]. Using the modified fluctuation assay of Luria and Delbruck [157], they showed that different tumor cell clones, each derived from an individual cell isolated from the parent tumor, varied dramatically in their ability to produce pulmonary nodules after intravenous inoculation into syngeneic recipient mice. Control subcloning procedures demonstrated that the observed diversity was not a consequence of the cloning procedure [71]. The finding that preexisting tumor cell subpopulations proliferating in the same tumor exhibit heterogeneous metastatic potential has since been confirmed in many laboratories with a wide range of experimental animal tumors of different histories and histologic origins [reviewed in 58, 63, 64, 208, 209]. In addition, studies using young nude mice as models for metastasis of human neoplasms have shown that several human tumor lines and freshly isolated tumors such as colon carcinoma, renal carcinoma, and prostate cancer also contain subpopulations of cells with widely differing metastatic properties [64]. We studied the biologic and metastatic heterogeneity in a mouse melanoma induced in C3H mice by chronic exposure to ultraviolet B radiation and painting with

Sun-Jin Kim et al. croton oil [134, 135]. One mouse thus treated developed a melanoma designated by Kripke as K-1735 [134]. The original K-1735 melanoma was established in culture and immediately cloned [69]. In an experiment similar in design to the one described for the B16 melanoma, the clones differed greatly from each other and from the parent tumor in their ability to produce lung metastases. In addition to differences in number of metastases, we also found significant variability in the size and pigmentation of the metastases. Metastases to the brain, heart, liver and skin were found as well; those growing in the brain were uniformly pigmented, whereas those growing in the lymph nodes, heart, liver, or skin generally had no pigment. To determine whether the absence of metastasis production by some (but not all) clones of the K-1735 was a consequence of their immunologic rejection by the normal host [134–136], we examined their metastatic behavior in young nude mice. In addition to the lack of a functional T-cell system, unstressed 3-week-old nude mice are also deficient in natural killer cell activity. In such recipients, the immunologic barrier to metastatic cells that also may be highly immunogenic is removed, and they may thus successfully complete the process. This was true for cells of two clones that did not produce metastases in normal syngeneic mice but produced tumor foci in the young nude recipients. Most of the nonmetastatic clones were nonmetastatic in both normal syngeneic and in the nude recipients. Therefore, the clones’ failure to metastasize in syngeneic mice probably was not caused by their immunologic rejection by the host but by their inability to complete one or another step in the complex metastatic process.

Enhanced Metastatic Potential of Tumor Cells Harvested from Metastases Our studies and most data reported by others have led us to conclude that metastasis is a highly selective process regulated by a number of as yet imperfectly understood mechanisms. This belief is contrary to the once widely accepted notion that neoplastic dissemination is the ultimate expression of cellular anarchy. In fact, suggesting that cancer metastasis is a selective process is a more optimistic view in terms of cancer therapy than one that postulates that tumor dissemination is an entirely random event. Belief that certain rules govern the spread of neoplastic disease implies that elucidation and understanding of these rules will lead to better therapeutic interventions.

23 We addressed the question of whether the cells that survive to form metastases possess a greater metastatic capacity than most cells in the parent neoplasm [243]. Some support for this possibility comes from the initial in vivo selection experiments of the highly metastatic B16-F10 cell line derived from the parent B16 melanoma [66]. Comparable results have been obtained with the K-1735 tumor. When cells derived from the parent tumor were injected intramuscularly into the hind foot pads of syngeneic mice, the resulting skin tumors produced spontaneous pulmonary metastases. Four cell lines were established from four individual lung nodules harvested from four different mice. The finding that all the lines derived from metastatic deposits produced significantly more metastases than cells of the parent line was good evidence for the hypothesis that metastasis is a selective process, that is, cells populating metastases have an increased metastatic capacity [243]. Our studies demonstrate that the K-1735 melanoma is heterogeneous and contains both nonmetastatic and metastatic cells. In contrast, individual metastases (spontaneous pulmonary metastases) are more uniform. This suggests that metastatic foci could develop by a clonal expansion of a few surviving metastatic cells. Moreover, it explains the observations showing that tumor cells in primary and metastatic lesions differ in their antigenic properties, biochemical characteristics, and response to cytotoxic drugs [reviewed in 61–63].

Clonal Origin of Cancer Metastases Multiple metastases proliferating in a host, even in the same organ, often exhibit diverse biological characteristics of, for example, hormone receptors, antigenicity or immunogenicity, and response to various chemotherapeutic agents. This diversity may result from the nature of the pathogenesis of metastasis, the process of tumor evolution and progression, or both. Pathologists have long been aware that neoplasms frequently exhibit different morphological appearances in different areas. For this reason, the malignant or benign nature of a tumor cannot be determined with confidence unless multiple sections obtained from all parts of the tumor are examined. The zonal differences in tumors are not restricted to morphology alone but include biological characteristics such as growth rates, sensitivity to cytotoxic drugs, antigenicity, and pigmentation [74]. Since primary tumors are not uniform, it is possible that tumor cell aggregates entering the circulation from one

24 zone of the tumor may be different from those entering from another zone. If an embolic aggregate originates from a primary tumor’s homogeneous zone, regardless of whether only one cell or several cells survived to proliferate in distant organs, the resulting metastasis would be like a primary tumor of unicellular origin. If a mixed embolus derived from an area of zonal junctions enters the circulation, the unicellular or multicellular origin of the metastasis would depend on whether a single cell or multiple cells survived to proliferate. To determine whether individual metastases are clonal and whether different metastases can be produced by different progenitor cells, Talmadge et al. [244] performed a series of experiments using the fact that x-irradiation of tumor cells induces random chromosome breaks and rearrangements. Analyzing the karyotype composition of 21 individual melanoma lung metastases after cultivating cells from individual lesions, this research group found unique karyotypic patterns of abnormal, marker chromosomes in most of the lines established from metastases, which suggested that each metastasis originated from a single progenitor cell. Similar results have been obtained in other rodent tumor systems. These studies revealed that the majority of metastases are of clonal origin. Moreover, variant clones with diverse phenotypes are formed, rapidly resulting in the generation of significant cellular diversity within individual metastases [244]. Cancer metastases of a clonal origin can be produced by two different mechanisms, proliferation of a single cell or of many cells. In the case of the second possibility, the cell aggregate at the metastatic site must have a homogeneous composition. To determine which of these possibilities is responsible for the generation of clonal K-1735 melanoma metastases, we injected C3H mice intravenously with aggregates of K-1735 cells consisting of two distinct subpopulations [75]. Cells of line K-1735-M2 are highly metastatic and exhibit a stable normal karyotype. Cells of the X-met-21 line are also highly metastatic but exhibit a stable, submetacentric chromosomal marker. After mixed aggregates (>20 cells) of these two cell types were injected, individual lung metastases were recovered and cultured, and each metastatic line was subjected to chromosome analysis. We reasoned that if an experimental metastasis originated from a single proliferating cell, all tumor cells within the metastatic focus should express either the K-1735-M2 or the X-met-21 chromosomal profile. This indeed was the case. Analysis of the distribution and fate of circulating tumor emboli has demonstrated that multicellular aggregates are more likely to give rise to a metastasis than a single-tumor-cell embolus. This is probably so because tumor cells not on the periphery of circulating emboli

The pathogenesis of cancer metastasis: relevance to therapy can be protected from destruction in the circulation, and a large aggregate of cells can more readily arrest in the capillary bed of an organ. Since the aggregates we injected were large, each containing more than 20 cells, the results suggest that the melanoma lung metastases resulted from the proliferation of a single viable cell within the embolus. Thus, regardless of whether an embolus is initially homogeneous or heterogeneous, metastases can be unicellular in origin. Collectively, these observations indicate that different metastases arise from different progenitor cells and account for the well-documented differences in behavior of different metastases. Among individual metastases of proved clonal origins, however, heterogeneity can develop rapidly to create significant intralesional heterogeneity.

Development of Biological Diversity within and Among Metastases Clinical and histologic observations of neoplasms have suggested that tumors undergo a series of changes during the course of the disease. A tumor initially diagnosed as benign, for example, can evolve over a period of many months into a malignant tumor. This can best be demonstrated in the case of human cutaneous melanoma where the transformation of normal melanocytes and their conversion into metastatic cells has been studied in detail by Clark and coworkers [32, 33]. This progression is gradual and consists of a series of discrete irreversible steps [107]. To explain the process of tumor evolution and progression as originally defined by Foulds in 1954 [81], Nowell [190] suggested that acquired genetic variability within developing clones of tumors, coupled with host selection pressures, can result in the emergence of new tumor cell variants that exhibit increasing growth autonomy or malignancy. Nowell’s hypothesis [190] predicted that accelerating tumor progression toward malignancy can be accompanied by increasing genetic instability of the evolving cells. To test this hypothesis, we have examined the metastatic stability and rates of mutation of paired metastatic and nonmetastatic cloned lines isolated from four different mouse neoplasms. We found that highly metastatic cells were phenotypically less stable than their nonmetastatic counterparts. Moreover, in highly metastatic clones, the rate of spontaneous mutation was found to be severalfold higher than in low-metastatic clones. These results are in accord with the hypothesis that tumor progression occurs as a result of acquired genetic alterations.

Sun-Jin Kim et al. Similar data have been reported for other neoplasms [reviewed in 62, 65]. Evidence that genetic mechanisms can be responsible for tumor progression comes from mutagenesis experiments using nitrosoguanidine. The finding that metastatic cells exhibit higher mutation rates than nonmetastatic cells [31], and that heterogeneity develops more rapidly in tumors containing few subpopulations of cells [186, 208, 210] suggest that accelerated tumor evolution and progression will result in the rapid development of biologic diversity in metastases, especially when such lesions are of clonal origin.

Intratumoral Heterogeneity for Expression of Tyrosine Kinase Growth Factor Receptors It is well recognized that tumor progression, angiogenesis, and metastasis are regulated by the interaction of tumor cells with the host organ microenvironment [70]. The expression of growth factors and their receptors varies within different zones of any given organ and may also differ among different cells within a tumor [62, 63]. Overexpression of transforming growth factor-alpha (TGF-α), epidermal growth factor (EGF), and the EGF receptor (EGFR) has been reported to be associated with poor prognosis in different neoplasms [2, 111, 170, 253, 271]. Expression of vascular endothelial growth factor (VEGF) is associated with increased vascular permeability, cell proliferation, and survival of endothelial cells [85, 86, 148, 272]. The expression of VEGF has also been correlated with microvessel density of neoplasms [52, 242] metastasis, and, hence, poor prognosis [53, 116, 140, 234, 262]. The expression of PDGF and its receptor (PDGFR) by tumor cells, tumor-associated endothelial cells [126, 251], and pericytes and myofibroblasts [127, 131, 151, 269, 270] is common to many neoplasms. In clinical specimens of human colon carcinomas, while PDGF is produced by tumor cells, the expression of PDGFR is restricted to stromal cells, including tumorassociated endothelial cells [131], i.e., tumor-associated stromal cells generate a microenvironment favorable to the survival and progressive growth of tumor cells [213]. Specifically, PDGFR signaling is related to recruitment of pericytes and control of interstitial fluid pressure [14, 192, 202, 213], which are favorable to the survival and progressive growth of neoplasms [155]. The expression of TGF-α, EGF, VEGF, and PDGF-β and their respective receptors was examined in 12 human colon cancer surgical specimens and in orthotopic tumors in nude mice produced by two distinct human colon

25 carcinoma cell lines. Inter- and intratumoral heterogeneity in expression of these growth factors and their receptors was reported. Different proteins produced by tumor cells can regulate the interaction between tumor cells and the organ microenvironment [57]. Many protein tyrosine kinase receptors on tumor cells and tumor-associated endothelial cells can be induced or upregulated by ligands produced by tumor cells via autocrine and paracrine pathways [55]. In many clinical trials, the presence of targets in patients was not confirmed, and the response rate was unpredictable [247]. For inhibition of phosphorylated EGFR, the presence of mutated receptors was reported to be a predictable variable [158], but significant therapeutic responses of cancer cells with wild-type receptors has also been reported [23, 269, 270]. These confusing criteria for selection of patients for treatment strongly indicate the need for better methodologies. The data demonstrating inter- and intratumoral heterogeneity for expression of EGFR, VEGFR2, and PDGFR-β in different human colon cancer specimens of different stages suggest that targeting a single tyrosine kinase receptor is not likely to provide significant therapeutic results. In other words, targeted therapy is effective only against its target and eliminating tumor cells that are dependent on one pathway, e.g., EGFR, is not likely to prevent the proliferation of tumor cells that are independent of this pathway. These results agree with a recent report demonstrating biological heterogeneity of tyrosine kinase receptors in other cancers [30, 173]. Indeed, simultaneous blocking of two protein tyrosine kinase pathways produce more efficient therapeutic effects in prostate [128] and pancreatic [269] carcinoma than did blocking of a single such pathway. Therapeutic efficacy was further increased when three protein tyrosine kinase pathways were inhibited [270]. Cancers are biologically heterogeneous for multiple properties, including antigenicity, sensitivity to chemotherapeutic agents, invasion, and metastasis [76, 77]. The progressive growth, metastasis, and survival of tumor cells depend on their interaction with the organ microenvironment. The expression of multiple tyrosine kinase receptors by different tumor cells within a single neoplasm indicates that targeting a single tyrosine kinase may not produce eradication of the disease.

Zonal Heterogeneity for Gene Expression A better understanding of the cross-talk between cancer cells and the organ microenvironment involving multiple genes first requires the identification of gene expression

26 profiles within the tumor. Among current methods for establishing these profiles, microarray analysis is the most powerful, and several such studies have been undertaken to predict disease outcome for individual patients [9, 10, 29, 115, 207, 216, 258, 259] and to identify cancer patients who should receive chemotherapy [17]. For example, pretherapeutic gene expression profiling has been undertaken to identify breast cancer patients who should receive specific chemotherapy [17], to predict response of rectal cancer patients to preoperative chemoradiotherapy [89], and to predict response of breast cancer patients to docetaxel [28], and a specific gene expression pattern was correlated with recurrence in Dukes’ B colon carcinomas [265]. None of these studies, however, accounted for the biologic heterogeneity of gene expression within a single tumor. Tumor cells depend on multiple and redundant pathways for growth, survival, and adaptation to the host microenvironment [70, 76, 77, 155]. In malignant neoplasms, genetic instability of cancer cells frequently yields extensive intratumoral heterogeneity in the pattern of structural chromosome aberrations [90, 92, 93, 95]. Three dimensional tumor growth and uneven fractional division of tumor cells within a single tumor mass can give rise to biologically different central and peripheral zones. The microenvironment of the central zone differs significantly from that of the peripheral zone, and zonal heterogeneity for several molecules in different tumor systems has been reported [102, 105, 129, 130, 138, 139, 191, 225]. Among these, bFGF, matrix metalloproteinase-2 (MMP-2), and MMP-9 have been shown to be highly expressed at the invasive edge of tumors [105, 130, 131, 138, 139], whereas the cell-to-cell cohesion molecule, E-cadherin, was downregulated at the periphery of the tumors [105, 130, 131, 138, 139, 191]. In fact, the ratio of expression of MMP-2 and MMP-9 to E-cadherin (MMP/E-cadherin ratio) at the periphery of the tumors correlated with metastatic potential and recurrent disease [138, 139]. Affymetrix HG-U133-Plus 2.0 array and laser capture microdissection techniques, demonstrated that different zones of the same pancreatic tumor exhibit differential expression of genes [179, 180]. The study of 1,222 genes demonstrated statistically significant differences. Bioinformatic functional analysis revealed that 346 upregulated genes in the peripheral zone were related to cytoskeleton organization and biogenesis, cell cycle, cell adhesion, cell motility, DNA replication, localization, integrinmediated signaling pathway, development, morphogenesis, and IκB kinase/NF-κB cascade. In the central zone, 876 upregulated genes were related to the regulation of cell proliferation, transcription, transmembrane

The pathogenesis of cancer metastasis: relevance to therapy receptor protein tyrosine kinase signaling pathways, response to stress, small GTPase-mediated signal transduction, hexose metabolism, cell death, response to external stimulus, and carbohydrate metabolism. These data clearly demonstrate zonal heterogeneity for gene expression profiles within single tumors and suggest that characterization of zonal gene expression profiles is essential if microarray analyses of genetic profiles are to produce reproducible data, predict disease prognosis, and allow design of specific therapeutics [179, 180].

Tumor-Organ Interaction: The “Seed and Soil” Hypothesis Clinical observations of cancer patients and studies with experimental rodent tumors have led cancer biologists to conclude that the metastatic pattern of certain tumors is organ-specific and independent of vascular anatomy, rate of blood flow, and number of tumor cells delivered to each organ [reviewed in 62, 65, 263, 264]. Indeed, the distribution and fate of hematogenously disseminated, radiolabeled melanoma cells in experimental animals conclusively demonstrated that tumor cells can reach the microvasculature of many organs, but growth in the organ parenchyma occurs in only specific organs [60]. These findings, however, were not new. More than 100 years earlier, Paget reached a similar conclusion. In 1889, he asked, “What is it that decides what organs shall suffer in a case of disseminated cancer?” Paget’s study was motivated by the discrepancy between considerations of blood flow and the frequency of metastases in different organs. He examined the autopsy records of 735 women who died of breast cancer and many other patients with different neoplasms and noticed the high frequency of breast cancer metastasis to the ovaries and the variations in incidence of skeletal metastases produced by different primary tumors. These findings were not compatible with the view that metastatic spread was due to “a matter of chance” or that tissues “played a passive role” in the process. Paget [193] concluded that metastasis occurred only when certain favored tumor cells (the “seed”) had a special affinity for the growth milieu provided by certain specific organs (the “soil”). The formation of metastasis required the interaction of the right cells with the compatible organ environment. In 1928, Ewing [50] challenged Paget’s seed and soil theory and hypothesized that metastatic dissemination occurs purely by mechanical factors that are a result of the anatomical structure of the vascular system. Both of these explanations have been evoked separately or

Sun-Jin Kim et al. together in order to explain the metastatic site preference of certain types of neoplasms. In a review of clinical studies on site preferences of metastases produced by different human neoplasms, Sugarbaker [235] concluded that common regional metastatic involvements could be attributed to anatomical or mechanical considerations, such as efferent venous circulation or lymphatic drainage to regional lymph nodes, but that metastasis in distant organs from numerous types of cancers were indeed sitespecific. Experimental data supporting Paget’s 1889 seed and soil hypothesis were provided a century later by Hart and Fidler [101], who studied the preferential growth of B16 melanoma metastases in specific organs. Following the intravenous (i.v.) injection of B16 melanoma cells into syngeneic C57BL/6 mice, tumor growths developed in the natural lungs and in grafts of pulmonary or ovarian tissue implanted either subcutaneously (s.c.) or intramuscularly (i.m.). In contrast, neoplastic lesions failed to develop in control grafts of similarly implanted renal tissue or at the site of surgical trauma. Parabiosis experiments suggested that the growth of the B16 melanoma in ectopic lung or ovarian tissue was due to the immediate arrest of circulating neoplastic cells and not to shedding of malignant cells from foci growing in the natural lungs. Quantitative analysis of tumor cell arrest and distribution using cells labeled with [125I]-5-iodo-2´-deoxyuridine indicated that the growth of tumors in the implanted organs was not due to an enhanced initial arrest of B16 cells. No significant differences in immediate tumor cell arrest were detected between implanted fragments of lungs (tumorpositive) and kidney (tumor-negative) or between organbearing and contralateral control limbs. These data demonstrated that the outcome of metastasis is dependent on both tumor cell properties and host factors and supported the seed and soil hypothesis as an explanation of the nonrandom pattern of cancer metastasis. The introduction of peritoneovenous shunts for palliation of malignant ascites provided an opportunity to study some of the factors affecting metastatic spread in humans. Tarin et al. [245] described the outcome in patients with malignant ascites draining into the venous circulation, with the resulting entry of viable tumor cells into the jugular veins. Good palliation with minimal complications was reported for 29 patients with various neoplasms. The autopsy findings in 15 patients substantiated the clinical observations that the shunts did not significantly increase the risk of metastasis. In fact, despite continuous entry of millions of tumor cells into the circulation, metastases in the lung (the first capillary bed encountered) were rare. These results provide compelling verification of the seed and soil hypothesis.

27 A clear demonstration of organ-site specific metastasis comes from studies of experimental brain metastasis. Two murine melanomas were injected into the carotid artery to simulate the hematogenous spread of tumor emboli to the brain. The K-1735 melanoma generated lesions only in the brain parenchyma, whereas the B16 melanoma produced only meningeal growths [219]. Similarly, different human melanomas [220] injected into the internal carotid artery of nude mice also produced unique patterns of brain metastasis. Distribution analysis of radiolabeled melanoma cells injected into the internal carotid artery ruled out the possibility that the patterns of initial cell arrest in the microvasculature of the brain predicted the eventual sites of growth. Rather, the different sites of tumor growth in the brain involved interactions between the metastatic cells and brain endothelial cells, and the response of tumor cells to local growth factors. In other words, site-specific metastases were produced by tumor cells that are receptive to their new environment.

Regulation of Tumor Cell Gene Expression by the Organ Microenvironment Tumor cells with different metastatic capabilities have been shown to differ in expression of proteins, such as collagenases, E-cadherin, IL-8, bFGF, and many others [65]. In most cases, however, these differential expressions were most evident in tumor cells growing at anatomically correct sites. Many biologic investigations of solid tumor cells often use cell lines growing in vitro as monolayer cultures. While easy to accomplish, monolayer cultures are not subjected to any cross talk, e.g., paracrine signaling pathways associated with growth in vivo [37]. Clinical observations of cancer patients and studies in rodent models of cancers have concluded that certain tumors tend to metastasize to certain organs [264]. The concept that metastasis results only when certain tumor cells interact with a specific organ microenvironment was originally proposed in Paget’s venerable “seed and soil” hypothesis [193]. Indeed, spontaneous metastasis is produced by tumors growing at orthotopic sites, whereas the same tumor cells implanted into ectopic sites fail to produce metastasis [124]. To determine the influence of the microenvironment on changes in gene expression, microarray analyses were done on three variant lines of a human pancreatic cancer with different metastatic potentials [141, 179].

28 The variant lines were grown in tissue culture in the subcutis (ectopic) or pancreas (orthotopic) of nude mice. Compared with tissue culture, the number of genes whose expression was affected by the microenvironment was upregulated in tumors growing in the subcutis and pancreas. In addition, highly metastatic pancreatic carcinoma cells growing in the pancreas expressed significantly higher levels of 226 genes than did the low metastatic variant cells. Growth of the tumor cells in the subcutis did not yield similar results, indicating that the orthotopic microenvironment significantly influences gene expression in pancreatic cancer cells [141, 179].

Regulation of the Invasive Phenotype by the Microenvironment The metastatic capacity of human colon cancer cells growing in orthotopic tissues of nude mice directly correlates with the level of collagenase type IV activity [233]. Histological examination of the human colon carcinomas growing in the subcutis, wall of the colon, or kidney of nude mice revealed a thick pseudocapsule around the subcutaneous but not cecal or kidney tumors [61, 70, 76]. These differences suggested that the organ environment could influence the ability of metastatic cells to invade host stroma. Significant differences were found in the levels of secreted type IV collagenases between human colon cancer cells growing subcutaneously or in the cecum of nude mice. In the medium conditioned by human colon cancer cells derived from subcutaneous implants, we detected only a latent form of the 92-kDa type IV collagenase. In contrast, both latent and active forms of the 92-kDa type IV collagenase were found in culture medium conditioned by tumor cells harvested from cecal tumors. Moreover, cancer cells grown in the cecum secreted more than twice as much enzymes as the subcutaneous tumors [178]. The invasive ability of human colon cancer cells is directly influenced by organ-specific fibroblasts. Primary cultures of nude mouse fibroblasts from skin, lung, and colon were established, and invasive and metastatic human colon cancer cells were cultured alone or with the fibroblasts. The cancer cells grew on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts [51]. Cancer cells growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of type IV collagenase, whereas colon cancer cells cocultivated with nude mouse skin fibroblasts did not. One possible explanation is that fibroblasts from the skin produce IFN-β, whereas those

The pathogenesis of cancer metastasis: relevance to therapy from the kidney or colon do not. The incubation of human colon cancer cells [51] and human renal cancer cells [91] with IFN-β significantly reduced the expression and activity of collagenase type independently of antiproliferative activity.

Regulation of Angiogenesis by the Organ Microenvironment The intensity of the angiogenic response varies considerably among different types of tumors and within different zones of a single tumor [48]. The rate of tumor cell division is still several orders of magnitude greater than the rate of neovascularization. As tumors expand, their microenvironment is often hypoxic [260]. Hypoxia is often associated with the activation of the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) to initiate the transcription of genes, e.g., VEGF/VPF [54]. VEGF/VPF increases the permeability of blood vessels by stimulating the functional activity of vesicular-vacuolar organelles, clusters of cytoplasmic vesicles and vacuoles located in microvascular endothelial cells [45–47]. VEGF also induces migration, protease production, and endothelial cell proliferation [54]. In addition, VEGF/ VPF regulates endothelial cell survival by activating the phosphatidylinositol-3 kinase/Akt signal transduction pathway and stimulating expression of the antiapoptotic proteins Bcl-2 and A1 [110]. HIF-1α signaling also encodes the polypeptide chains of PDGF [100]. The functional activity of PDGF is, to a large extent, determined by the anatomical location of a specific tumor. For example, in pancreatic tumors, PDGF has been shown to stabilize developing vascular networks by recruiting pericytes to support the immature blood vessel walls [13, 14]. In tumors of the central nervous system, PDGF stimulates the release of VEGF from the tumor-associated endothelium [96]. In contrast, tumors in the skin rely on PDGF signaling to regulate the level of interstitial fluid pressure in the tumor [199]. In prostate cancer bone metastasis (see next section), PDGF functions as a survival factor for tumor endothelial cells by activating the intracellular effectors MAPK and Akt [142, 143]. The vascular endothelium is regarded as structurally and functionally heterogeneous [88]. To examine this diversity, we generated a broad panel of microvascular endothelial cells from various organs of H-2Kb-tsA58 transgenic mice [142]. cDNA expression profiles generated on the endothelial cells predicted significant organspecific differences in expression levels of tyrosine kinase

Sun-Jin Kim et al. receptors, chemokine receptors, and proteins that regulate the efflux of toxic substrates; these were confirmed at the protein level. Endothelial cells derived from the mouse brain expressed measurable levels of PDGF-Rβ, the chemokine receptor CXCR-2, and P-glycoprotein, whereas endothelial cells from the pulmonary circulation did not express detectable levels of these proteins. The organ-derived endothelial cells also exhibited vast differences in response to stimulation with endothelial cell mitogens. Endothelial cells originating from the brain and liver showed the greatest increase in cell division in response to basic fibroblast growth factor, while EGF was the most potent mitogen for endothelial cells derived from the lung and uterus.

Influence of Lymphoid Cells on Angiogenesis The regulation of angiogenesis by T lymphocytes, macrophages, and mast cells, is well established [55, 59, 68, 83, 160, 164, 169, 204, 223, 240]. For example, invasive cutaneous melanoma is often associated with a local inflammatory reaction involving T lymphocytes and macrophages, a condition often associated with an increased risk of metastasis [19, 217]. The relatively slow growth of tumors in aging mice has been closely linked with decreased vascularization [133] associated with a diminished immunological response [38, 92]. The role of tumor vascularization and its effect on tumor growth in immunosuppressed mice was investigated and it was concluded that the growth of the immunogenic B16 melanoma was delayed in myelosuppressed mice as compared to control mice [98]. Similarly, tumor growth in mice pretreated with doxorubicin (DXR) and then injected with normal splenocytes 1 day before tumor challenge was comparable to that in the control mice, implicating myelosuppression as a cause of retardation of tumor growth and vascularization. Similar results were obtained with athymic nude mice [98]. Many other studies have recognized the importance of macrophages in tumor angiogenesis [5, 204–206, 239]. Macrophages can produce more than 20 molecules that influence endothelial cell proliferation, migration, and differentiation [204, 205]. Macrophages may modify the extracellular matrix, thereby modulating angiogenesis either through direct production of extracellular matrix components or proteases that alter the structure and composition of the extracellular matrix [240]. Macrophages have also been shown to produce antiangiogenic molecules, such as thrombospondin-1 [40, 152, 153, 205].

29 Macrophage-derived metalloelastase has been shown to be responsible for the generation of angiostatin in Lewis lung carcinoma [42], and the addition of plasminogen to 3LL Lewis lung carcinoma cells cultured in vitro did not result in the generation of angiostatin. However, its addition to co-cultured macrophages and carcinoma cells did [42], suggesting that elastase activity in macrophages was significantly enhanced by the cytokine GM-CSF which was secreted by the tumor cells [137], leading to the generation of plasminogen.

Regulation of Response to Chemotherapy by the Organ Microenvironment Clinical observations suggest that the organ environment can influence the response of tumors to chemotherapy. For example, in women with breast cancer, lymph node and skin metastases are more sensitive to chemotherapeutic agents than are metastases residing in either the lung or bone [41]. Several intrinsic properties of tumor cells can render them resistant to chemotherapeutic drugs, including increased expression of the mdr genes, leading to overproduction of the transmembrane transport protein P-glycoprotein (P-gp) [18, 249]. Expression of P-gp often parallels increases in dose or duration of chemotherapy. Indeed, increased levels of P-gp can be induced by selecting tumor cells for resistance to natural product amphiphilic anticancer drugs. Nevertheless, elevated expression of P-gp accompanied by development of the multidrug resistance (MDR) phenotype has also been found in many solid tumors of the colon, kidney, and liver that had not been previously exposed to chemotherapy [249]. One of the most striking examples of site-specific variations in therapeutic response was observed following implantation of colon carcinoma cells into different anatomic locations of nude mice (using the highly metastatic KM12L4 human colon carcinoma cell line) or syngeneic BALB/c mice (using the CT-26 murine colon carcinoma). Mice received injections of the KM12L4a cells into either the subcutis (ectopic site), spleen (leading to experimental liver metastasis), or cecum (growth at the orthotopic site). Tumor-bearing mice were given doxorubicin and subsequently evaluated for response to treatment. Up to 80% growth of subcutaneous tumors was inhibited by two i.v. injections of doxorubicin as compared to about 40% inhibition of the intracecal tumors and less than 10% inhibition of lesions in the liver [267]. Similar results were obtained with murine

30 colon cancer cells. Subcutaneous tumors were sensitive to treatment with doxorubicin; lung metastases were insensitive [42]. However, the tumor cells at both of these sites were equally sensitive to 5-FU, a drug whose activity is not influenced by expression of the MDR phenotype. Northern blot analysis showed that the relative expression of the mdr1 and mdr3 genes was greatest in the cecum, liver, and lungs of mice, and this expression correlated with the relative resistance of tumor cells. Indeed, this expression of mdr was transient and the subsequent culture of cells from a liver metastasis for 7–10 days resulted in a decrease of mdr expression to the level in tumor cells maintained in culture [43]. These events resulted from organ-specific modulation of tumor cell properties. These findings are not restricted to experimental systems. In patients with colon carcinoma, elevated P-gp expression is found on the invasive edge of the primary tumor (growing in the colon) and in metastases located in lymph node, lung, and liver [129]. Whether this finding is due to selection or adaptation is unclear.

The pathogenesis of cancer metastasis: relevance to therapy to up-regulate the growth factor receptors and the activation of these receptors on tumor cells (autocrine effect) and on tumor-associated endothelial cells (paracrine effect). These effects can lead to downstream signaling that activate mitogen-activated protein kinases and phosphatidylinositol-3 kinase pathways and lead to downregulation of the apoptotic proteins caspases 3 and 8 [8, 142, 143, 229]. Activation of growth factor receptors on tumor cells and on the tumor-associated endothelial cells is likely to increase the expression of antiapoptotic proteins, causing endothelial cell resistance to chemotherapy, even though these cells divide every 30–40 days. Inhibition of this growth factor receptor activation by receptor antagonists in tumor-associated endothelial cells (and tumor cells) can lead to BAX induction, activation of caspase-8, and down-regulation of BCL-2 and NF-κB [1, 24], thereby increasing the susceptibility of tumors to chemotherapy. If the tumorassociated endothelial cells continue to cycle, the use of receptor antagonists [6, 7, 125–128] combined with anticycling drugs can cause the destruction of the vasculature within neoplasms, leading to the apoptosis of adjacent tumor cells [20, 21, 193, 232].

Targeting the Vasculature Because systemic antitumor therapy fails primarily because of the genetic instability and biologic heterogeneity of neoplasms, therapeutic agents that target a tumor’s vasculature, a genetically stable and essential component of tumors, have been explored as an alternative to conventional therapy. The structure and architecture of tumor vasculature can differ dramatically from those of normal organs [48, 182, 189]. Modern techniques, such as phage display targeting, have defined “vascular addresses” that may be distinct for different organs and for tumors in those organs [194]. In normal human tissues, for example, endothelial cells are long lived and recycle every 3–5 years, whereas tumor-associated endothelial cells can recycle every 30–40 days. The rate of endothelial cell turnover has been reported to be <0.1% in normal human tissues, whereas in malignant human tumors, it is 2–9.9% [48, 49]. Endothelial cells that line blood vessels in different organs are also phenotypically distinct. The expression of various cytokines and growth factors and their relevant receptors on both tumor cells and organ-specific endothelial cells differs in different organ microenvironments, and the interaction of these growth factor ligands (i.e., EGF, TGF-α, VEGF, and PDGF) with their relevant receptors is essential for endothelial cell survival and growth [6, 7, 128, 142, 143]. For example, tumor cells that produce growth factor ligands are likely

Targeting the Expression of Platelet-Derived Growth Factor Receptor by Reactive Stroma Colon carcinoma cells produce various growth factors and cytokines that contribute to progressive growth and metastasis [117]. One example is the family of PDGFs, members of a family of dimeric disulfide-bonded growth factors exerting their biological effects through activation of two structurally related tyrosine kinase receptors, the PDGF-α- and -β receptors [104]. PDGF consists of dimeric forms, including PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD [15, 144, 149]. The α-receptor binds all possible forms of PDGF except PDGF-DD, whereas the PDGF-β receptor preferentially binds PDGF-BB. PDGF-Rβ is expressed on many tumor types, and PDGF-BB is an important autocrine growth factor for many cell types, including gliomas, sarcomas, pancreatic carcinoma, and prostate cancer [103]. PDGF-R signaling has also been reported to stimulate angiogenesis [213], recruit pericytes [14, 192], and control the interstitial fluid pressure in stroma, thereby affecting transvascular transport of chemotherapeutic agents [200–203]. A recent study of human colon cancer clinical specimens revealed that PDGF-Rβ and phosphorylated PDGF-Rβ are not expressed on tumor cells but, rather, are predominantly

Sun-Jin Kim et al. expressed in stromal cells and in pericytes surrounding the tumor microvessels [131, 132, 238, 241]. Imatinib, a derivative of 2-phenylaminopyrimidine, was originally developed as a competitor for an ATPbinding site of the Abl protein tyrosine kinase [44]; however, it is also a potent tyrosine kinase inhibitor of c-Kit and PDGF-R [22]. We have reported that imatinib can slow both the progressive growth of human pancreatic carcinoma in nude mice [114, 270] and the growth of experimental bone metastasis of human prostate cancer [127, 251]. To examine the role of PDGFR on colon cancer stromal cells, we examined the therapeutic effect of imatinib administered as a single agent or in combination with the chemotherapeutic irinotecan against human colon carcinoma cells growing in orthotopic (cecum and liver) and ectopic (subcutis) organs of nude mice. Blockade of PDGF-Rβ signaling by oral administration of imatinib or imatinib combined with irinotecan significantly inhibited the growth of orthotopic tumors and the incidence of lymph node metastasis in nude mice. Histopathological analysis of human colon carcinoma growing in the cecum and liver of nude mice treated with imatinib alone or with imatinib combined with irinotecan demonstrated decreased stromal reaction and inhibition of phosphorylated PDGF-Rβ in the tumor-associated stromal cells. These effects were associated with the inhibition of tumor cell proliferation, an increase of apoptosis in stromal cells, and a decrease in the number of pericytes surrounding the tumor-associated microvessels. In contrast, treatment of mice with imatinib alone or imatinib combined with irinotecan did not affect the growth of colon carcinoma cells in the subcutaneous space, demonstrating once again that the biology of tumors differs with the organ microenvironment [58, 76]. In general, tumor cells in a neoplasm are biologically heterogeneous and their phenotype can be modified by the organ microenvironment [63, 74]. Histologically, human carcinoma tissues are composed of both parenchyma and stroma. Tumor stroma consists of fibroblasts, smooth muscle cells, inflammatory cells, microvessels, and abundant extracellular matrix (ECM) [99]. Tumorassociated stroma at both primary sites and metastatic sites generate a favorable microenvironment for the survival of cancer cells [155]. Fibroblasts are activated by various growth factors and cytokines that are released by cancer cells, for example TGF-β, PDGF, and bFGF. Activated fibroblasts express αSMA, leading to the term ‘myofibroblasts’ [45]. Stromal reaction (desmoplasia), such as myodifferentiation of fibroblasts [109, 145, 215] clearly alters the stromal phenotype. In the liver, hepatic stellate cells are the only mesenchymal cells present in

31 the parenchyma. These cells are activated by various stimuli from tumor cells and undergo transformation into myofibroblasts, which are characterized by expression of αSMA [211]. In general, tumor-associated stroma is an abundant source of tumor-promoting growth factors and cytokines, and stromal cells activated by cancer cells can in turn regulate the growth and progression of carcinoma cells [112, 165, 222, 250]. The progressive growth of colorectal tumors in experimental animals has been correlated with proliferation of myofibroblasts, whereas regression of colon tumors has been linked to a fibrous capsule, suggesting that the presence of reactive myofibroblasts may inhibit growth of tumor cells [150]. Wounding has been associated with tumorpromoting effects [224] and, functionally, tumor-associated stromal cells are similar to stromal cells in healing wounds insofar as expression of myodifferentiation markers, production of ECM, expression of growth factors and cytokines, and neovascularization [121–123]. The combination of imatinib and irinotecan completely inhibited tumor cell growth at the abdominal wound healing site induced at the time of tumor cell injection into the cecum [131]. Imatinib may inhibit stromal reaction at the orthotopic primary site and the surgical wound. PDGF-R signaling pathway also plays an important role in increasing tumor interstitial hypertension, which may block the accumulation of antitumor drugs [201–203]. The microvasculature in both tumor tissue and normal colon mucosa consists of endothelial cells, pericytes (mural cells or smooth muscle cells), and basement membranes. All of these components are thought to be abnormal in tumor vessels. Pericytes are key cells in vascular development, stabilization, maturation, and remodeling [12, 87]. Functional-blocking antibodies that target PDGF-Rβ block pericyte recruitment during vascular development [252]. In our study, desmin-positive pericytes were found on microvessels in both normal organs and colon cancers, albeit with different morphological characteristics. Specifically, pericytes in tumor-associated vessels, but not those in vessels of normal colon mucosa, were enlarged and overexpressed PDGF-Rβ and p-PDGF-Rβ [131]. In agreement with earlier reports [14, 269, 270], we found that treatment with imatinib decreased pericyte coverage on tumor-associated endothelial cells. The inhibition of PDGF-R signaling by a protein tyrosine kinase inhibitor decreased pericyte recruitment and attachment to endothelial cells and destabilized tumor vasculature by killing pericytes. In the absence of decreased vessel density in tumors treated with imatinib combined with irinotecan, functional rather than quantitative changes of the tumor vasculature by imatinib may diminish tumor growth [84].

32 Recently, targeting the VEGF pathway to inhibit tumor angiogenesis is attracting attention as a novel cancer therapy [113]; however, it has been suggested that VEGFtargeting therapies are mostly active against immature vessels [13], i.e., normalization of the tumor vasculature [118]. Because imatinib inhibits pericyte coverage on tumor vasculature, it will be very interesting to determine if the combined inhibition of PDGF-R and VEGF-R signaling may produce synergistic antivascular effects [45, 118, 200]. Multi-target tyrosine kinase inhibitors are under current investigation in clinical trials. Sorafenib and sunitinib target not only multiple VEGF-Rs but also PDGF-Rβ, and phase II studies show promising activity of these molecules in renal cell carcinoma [180], likely due to inhibition of angiogenesis [266].

Anti-Vascular Therapy of Prostate Cancer Bone Metastasis In clinical specimens of human prostate cancer bone metastasis and in experimental models of orthotopic human prostate cancers in the long bones of nude mice, the expression of PDGF and PDGF-R correlates with the growth of metastatic tumor cells in the bone parenchyma. Specifically, prostate cancer cells growing adjacent to bone tissue express high levels of the PDGF protein and the PDGF-R, which is phosphorylated, and tumor-associated endothelial cells express high levels of phosphorylated PDGF-R [127, 128]. Oral administration of the PDGF-R tyrosine kinase inhibitor imatinib (STI571) to nude mice blocks the PDGF signaling pathway by inhibiting phosphorylation of its receptors [251]. Oral administration of STI571 or STI571 plus injectable taxol to male nude mice reduced the incidence and size of primary tumors and bone lesions and prevented bone lysis as measured by digital radiography. Immunohistochemical analysis of control, untreated bone lesions, demonstrated that human prostate cancer cells growing adjacent to the bone expressed high levels of PDGF and activated (phosphorylated) PDGF-R, whereas tumor cells growing in the adjacent musculature following lysis of the bone did not. Treatment with STI571 and more so with STI571 plus taxol significantly inhibited phosphorylation of PDGF-R on tumor cells and endothelial cells, decreased tumor cell proliferation, and induced significant apoptosis in tumor cells and tumor-associated endothelial cells. These data indicate that targeting PDGF-R phosphorylation can produce significant therapeutic effects against prostate cancer bone metastasis [125–128].

The pathogenesis of cancer metastasis: relevance to therapy To determine whether the PDGF-R expressed on tumor-associated endothelial cells could be the primary target of imatinib, we selected a multidrug-resistant variant of the human PC-3MM2 prostate cancer. These cells were implanted into the tibia of nude mice, and when the lesions were growing progressively, the mice were randomized to different treatment regimens. The bone tumors were resistant to taxol, whereas treatment with imatinib and taxol produced a significant decrease in tumor incidence, bone lysis, and incidence of lymph node metastasis. Initially, this treatment produced apoptosis of tumor-associated endothelial cells (but not tumor cells), followed 1 week later by apoptosis of the tumor cells. Collectively, these studies demonstrated that the primary targets for imatinib treatment are the tumor-associated endothelial cells, i.e., an anti-vascular therapy [126].

Targeting Platelet-Derived Growth Factor Receptor on Endothelial Cells of Multidrug Resistant Prostate Cancer The major cause of death from prostate cancer is due to metastases that are resistant to conventional therapies. Genetic instability of tumor cells leading to biological heterogeneity is largely responsible for the development of hormone-refractory prostate cancer cells [10, 11, 56, 197, 198, 254–257], and selection pressures by chemotherapeutic agents results in the emergence of cells that are resistant to chemotherapy [122, 147, 159, 198, 221, 231]. These resistant cells often express the MDR1 gene and its product, P-glycoprotein (P-gp) [94, 248, 257]. Methods to overcome multidrug resistant activity by blocking transmembrane drug efflux pump action [82, 166–168, 176], liposomal encapsulation of cytotoxic drugs [18], immunotherapeutic monoclonal antibody targeting P-glycoprotein [214] or protein toxins selectively killing cells expressing P-glycoprotein on their surfaces [168] are under clinical investigation. Multidrug resistance-associated protein (MRP) was found in multidrug resistant prostate cancer cell lines that do not express P-glycoprotein [34]. MRP1 [237, 256] and MRP2 [255] have been reported to be expressed in prostate cancer cell lines, but there are very few compounds with proven MRP1-associated multidrug resistant-modulating capacity at present. Glutathione and glutathione-S-transferase (GST) detoxification systems are another drug-resistance mechanism reported to

Sun-Jin Kim et al. protect cancer cells against the lethal effects of chemotherapy [174]. GST-π inactivation is related to early steps of prostate carcinogenesis [146, 237], whereas the hormone-independent disseminated prostate cancers clearly expressed GST-π [256]. However, reversal of multidrug resistance in prostate cancer by challenging the glutathione pathway has been observed in vitro [212]. Many changes in the regulatory processes of apoptosis apparently contributed to the malignant phenotype of prostate cancer cells [35, 39, 188, 256], and methods to stimulate apoptosis are currently under development. However, despite progress in our understanding of the biology of MDR, therapeutic approaches that directly target tumor cells have not been successful. To overcome multidrug resistance of prostate cancer, one may wish to affect the host factors in the organ microenvironment, such as the vasculature [26, 63, 72, 78–80, 122]. Because all cells in the body depend on an adequate supply of oxygen and nutrients and the ability to remove toxic molecules, therapy directed against tumor-associated endothelial cells can destroy tumor cells, regardless of their biological heterogeneity [77]. Recent reports have demonstrated statistically significant therapeutic results in experimental human prostate cancer bone metastasis in nude mice treated with a combination of paclitaxel and various tyrosine kinase inhibitors directed toward PDGFR or EGFR [125–128, 251]. These experimental data have been successfully translated to clinical trials [45, 46]. Since both tumor cells and tumorassociated endothelial cells can express these activated growth factor receptors, it has not been clear whether the combination treatment targeted the tumor cells or the endothelial cells or both. We reported that multidrug resistant PC-3MM2-MDR human prostate cancer cells growing in the prostate of nude mice are resistant to systemic administration of paclitaxel. Targeting the phosphorylated PDGF-R on tumor-associated endothelial cells leads to regression of the multidrug resistant prostate cancer and inhibition of lymph node metastasis. As stated previously, regardless of sensitivity or resistance to hormones or chemotherapeutic drugs, all tumor cells are dependent on a viable vasculature for growth and survival [56, 62, 123]. Tumor cells interact with host factors in the microenvironment to induce growth and expansion of vasculature [70, 72, 121]. We have reported [126–128] that PDGF produced by prostate cancer cells growing adjacent to bone can increases and activate the expression of PDGF-R on tumor-associated endothelial cells by a paracrine mechanism. The systemic administration of imatinib inhibited the phosphorylation of PDGF-R (but not EGF-R) [128], and the combination of imatinib and paclitaxel induced apoptosis of

33 tumor-associated endothelial cells as well as PDGF-Rexpressing tumor cells. The expression of phosphorylated PDGF-R is known to activate anti-apoptotic pathways involving Akt, PI3K, MAPK, and Bcl-2 [142, 143], and treatment with imatinib can inhibit this effect. These data suggest that a major target for imatinib and paclitaxel therapy could well be the tumor-associated endothelial cell. We tested this possibility by using MDR human prostate cancer cells. The acquisition of multidrug resistance by leukemia cells [162], hepatocellular carcinoma [273], and human lung carcinoma [163] has been shown to decrease tumorigenicity and prolong tumor cell doubling time. In agreement with these reports, the in vivo growth of the human prostate cancer PC-3MM2 selected for multidrug resistance (PC-3MM2-MDR) was slower than the parental tumor cells. The ‘seed and soil’ theory [63, 193] held true regardless of the MDR phenotype. Similar to parental cells, MDR tumor cells growing in the bone (but not in the muscle) expressed IL-8, bFGF, EGF, EGF-R, PDGF, and PDGF-R. Endothelial cells of tumor-associated vessels in bone lesions also expressed PDGF-R on their surface, and treatment with imatinib and paclitaxel inhibited the phosphorylation of the PDGF-R on both tumor cells and endothelial cells. The PC-3MM2-MDR bone lesions responded to systemic administration of imatinib and paclitaxel (but not to paclitaxel administered alone), raising the possibility that imatinib could have sensitized the tumor cells to paclitaxel. Immunohistochemical analyses revealed that early apoptosis (after 2–3 weeks of chemotherapy) was mostly limited to tumor-associated endothelial cells. The multidrug resistant tumor cells were still treatment-resistant. In contrast, parental cells underwent extensive apoptosis in mice treated with imatinib and paclitaxel. The PDGF-R has been shown to regulate the interstitial hypertension and transcapillary transport of molecules [108], and the administration of imatinib to mice bearing tumors can increase the concentration of a systemically administered chemotherapeutic drug within a tumor by twofold to threefold [201–203]. Since the PC-3MM2-MDR cells were >50-fold more resistant to paclitaxel even in the presence of imatinib, the in vivo therapeutic response of the PC-3MM2-MDR bone tumors to imatinib and more so to imatinib plus paclitaxel could not have been due to changes in interstitial fluid pressure. Endothelial cells in normal tissues rarely divide, whereas 2–3% of endothelial cells in prostate cancer divide daily [4, 48]. These dividing endothelial cells should be sensitive to anticycling drugs such as paclitaxel. As stated above, the first wave of apoptosis in bone tumors from mice treated with imatinib and

34

The pathogenesis of cancer metastasis: relevance to therapy

paclitaxel for only 2 weeks occurred in tumor-associated endothelial cells, followed by apoptosis of tumor cells and ultimately necrosis. By the fourth week of treatment with imatinib and paclitaxel or imatinib alone, concurrent apoptosis of tumor cells and tumor-associated endothelial cells was observed. Without paclitaxel, imatinib may produce therapeutic effects by the blockade of PDGF-R, which serves as a survival factor [142, 143]. Therefore, targeting the PDGF-R on tumor-associated endothelial cells by imatinib and paclitaxel can produce therapeutic results in multidrug resistant human prostate cancer experimental bone metastasis.

using homogeneous therapy. In other words, it is unlikely that therapy of cancer in general and metastasis in particular can be accomplished using a single modality. Second, cancer is a chronic disease. A chronic disease must be treated chronically, i.e., by management. Third, cancer is the disease of the “seed” and the “soil”. Since the outcome of metastasis depends on multiple interactions of metastatic cells with homeostatic mechanisms in the organ microenvironment, therapy for metastasis should be targeted not only against tumor cells, but also against the homeostatic factors that helps metastatic cells grow and survive.

Conclusions

References

A current definition of the seed and soil hypothesis consists of three principles. First, neoplasms are biologically heterogeneous and contain subpopulations of cells with different properties. Second, the process of metastasis is selective for cells that must succeed in all the steps of this complex process. Although some of the steps in this process contain stochastic elements, as a whole, metastasis favors the survival and growth of a few subpopulations of cells that preexist within the parent neoplasm. Thus, metastases can have a clonal origin, and different metastases can originate from the proliferation of different single cells. Third, the outcome of metastasis depends on multiple interactions (“cross-talk”) of metastatic cells with homeostatic mechanisms, which the tumor cells can exploit. A convincing example of this principle is the establishment of tumor-associated vasculature. Tumor growth and metastasis depend on an adequate blood supply. The extent of angiogenesis within and around tumors is regulated by the balance between proangiogenic and antiangiogenic molecules. The crosstalk between tumor cells, normal cells, leukocytes, and stromal cells occurs through growth factors, growth factor receptors, and cytokine signaling, all of which are released and act on cells within the tumor microenvironment. These interactions enhance the establishment of angiogenesis that in turn regulates the proliferation and survival of metastatic cells. Interruption of one or more of these interactions can lead to the inhibition or regression of metastasis. Because the growth of primary tumors is often controlled with surgery and/or irradiation, antiangiogenic agents may be most beneficial to prevent widespread and metastatic disease. To succeed, several principles must be considered. First, it is essential to remember that all neoplasms are biologically heterogeneous. One cannot treat a heterogeneous disease

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3

Developmental therapeutics and the design of clinical trials ROBERT K. OLDHAM

New techniques in biotechnology and the use of biologicals in cancer treatment have made it apparent that developmental therapeutics for biotherapy are different from standard drug development. Millions of chemicals have been screened as anticancer agents, but less than 75 have reached the clinic as commercial pharmaceuticals. Perhaps 20 of these drugs can be classed as moderately effective; the rest are only marginally useful and all can be highly toxic. With the discovery of monoclonal antibodies and conjugates thereof, the exploitation of bioengineering to produce purified, characterized lymphokines/ cytokines and other biologicals, and further information on the mechanism of action of these natural molecules, the rate of development for biological therapeutics has risen dramatically. Because of their selectivity and with the implicit biological diversity of cancer, new approaches are needed to efficiently bring biotherapy to the clinic. Previously, there have always been fewer promising agents (drugs) to test than patients who needed new approaches. In fact, the drug development paradigm has been a slow and laborious, with each drug taking some 8–12 years to gain commercial approval, at a cost of more than US$500 million per drug. Taxol, a drug approved for general use, entered clinical trials in the late 1980s and only became generally available in 1998; taking more than a decade to pass through our current system of drug development. On average, fewer than four new chemotherapy drugs per year have been approved for general use by oncologists. This expensive and slow paradigm reflects both the toxicity and marginal effectiveness of chemotherapeutic drugs as well as a bureaucratic regulatory system more fearful of criticism over toxicity than a willingness to pursue opportunities for seriously ill patients [33, 70, 71, 83]. More recently, a large number of biological substances and targeted therapies have been making their way to the clinic, increasing the difficulty of decisions as to the order and amount of preclinical and clinical testing. Hundreds of biologic agents are in clinical testing with more agents to test than there are easily available patients for testing. The current system for drug development is not sufficiently flexible, and needs major changes in direction and technique to optimize clinical testing and

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

speed the translation of new approaches to patients [6, 8, 17, 21, 22, 24, 30, 32, 33, 41, 55, 57, 61, 76–82, 90, 91, 94, 98, 99, 103]. Although the concept of biotherapy is not new, the use of recombinant genetics to produce highly purified biologicals as medicinals dates from about 1980 [31]. A member of the alpha-interferon family was the first biological produced by recombinant methods to be used as an anticancer medicine in humans [103, 105]. In the 25 years since the first alpha-interferon molecule was prepared by recombinant methods, a large number of recombinant molecules (lymphokines, cytokines, monoclonal antibodies, growth and differentiation factors, angiogenesis factors and cell receptors) have become available or are being prepared for testing in the clinic (see Chapter 8). Historical aspects in the development of immunotherapy have been reviewed [12, 75]. Before the 1980s, the term immunotherapy was considered to be synonymous with biological therapy by most investigators. However, it is now clear that there are many biological approaches that may affect cancer growth and metastases, yet are not within the immune system. Thus, biotherapy now refers to agents derived from biological sources and/or the use of agents that affect biological responses. The term “biologicals” describes agents extracted from or produced from biological materials. With biotechno-logy, this involves the use of recombinant genetics to isolate the gene, transfect it into an appropriate producer organism, and then the isolation and purification of the protein product. Genomics and proteonomics are processes to yield the “code” to eventually prepare biological therapy by chemical synthesis. The types of materials that alter biological responses for the benefit of the patient have been called biological response modifiers (BRM). The use of biologicals and BRM in the treatment of cancer and other diseases is biotherapy (a term I initially used in 1984 to describe this fourth modality of cancer treatment) [84, 85]. As is always the case, nomenclature can be confusing and terms may be variously defined by different individuals. In the broadest sense, biotherapy includes blood products, transplanted organs, bone

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42 marrow/stem cell transplants, antibiotics (often derived by extraction from biological organisms and later synthesized), and a variety of other agents and approaches. However, in this chapter, the focus will primarily be on BRM, recombinant biologicals and gene engineered or activated cells being developed as medicinals. Immunotherapy has had a checkered past. Kari Cantell said it well when describing some of the early interferon research: “Much second class research was carried out with third class preparations slightly contaminated with interferon” [5]. In addition, there have been questionable approaches (“alternative medicine”) used by certain practitioners, and even frank quackery by others who purported to deliver “immunotherapy” for cancer patients. Even for the very dedicated scientists who have explored immunotherapy over the past several decades, there have been many pitfalls relating to the purity of their preparations, the source of the materials, and the assays and techniques by which their measurements were made. Huge expenditures by the National Institute of Health (NIH) in the area of molecular, biological and viral oncology over the last 40 years led, in large part, to the current technology of “genetic engineering.” In contrast to previous attempts to develop biological therapy, we now have techniques available that can isolate a single gene and use it in a bio-manufacturing process to produce absolutely pure proteins identical to those found in the body. It is with these techniques that thousands of biological compounds and their synthetic analogs and components are being developed. Many of these biologicals will be candidates for use as medicinals. A major value of the research done thus far with interferon is that it may be viewed as a model for the development of other biological substances as medicinals [61, 76]. Interferon research in particular and biotherapy in general must be seen as a new challenge in developmental therapeutics, with agents to be tested appearing at a still accelerating rate. No longer can the historical drug development paradigm be used [55]. The methods used by the Food and Drug Administration [11], National Institutes of Health, and the pharmaceutical industry in the development of drugs must be drastically changed to a new paradigm that will accommodate the realities relevant to biologicals and their use in medicine [33, 55, 71, 82, 86, 87, 90].

Drug Development The current process of drug development involves a very long and costly set of procedures [33, 55, 80]. This includes the initial concept, extraction or synthesis,

Development therapeutics and the design of clinical trials formulation, documentation of biological activity and purity, early studies in the laboratory and in experimental animals to determine the mechanism of action and toxicity, and compilation of all the preclinical data into an investigational new drug application (INDA). It costs millions of dollars for a pharmaceutical company to file a single INDA. During preclinical development, companies make projections about the potential market size and profitability as justification for the investment. These projections are difficult, and are most accurate when related to an existing drug in a known market. It follows that predicting the market size for a totally new agent or new approach is much more difficult. Subsequent to the INDA, further preclinical work on the mechanism of action and preclinical toxicology of a new drug is done. In addition, early-phase clinical studies are begun to determine biological activity in humans. Although the process is not uniform for all classes of pharmaceuticals, studies are termed “phase I” when the dose of the drug is escalated to determine its biological activity and its toxicity. Based on the preclinical toxicology information in small animals and sometimes in primates, projections are made for the starting dose in humans. A low starting dose is selected to avoid severe toxicity in the initial patients in a phase I clinical trial. The rate of dose escalation in these trials and the acceptability of toxic side effects vary with the population at risk and are related to the seriousness of the disorder and the treatment alternatives for the patient. Because the starting doses are very low and because of phase I trial design, using different patients at each dose level (to avoid cumulative toxicity), the chance of a therapeutic effect for the initial patients receiving a new agent is nearly zero. It is axiomatic that the phase I trials are designed to accumulate the maximum amount of clinical information with the least toxicity. Generally, the end point for phase I trials is the achievement of a maximum tolerated dose (MTD), the dose at which side effects are unacceptable to the physician and the patient within the design of that clinical trial. These trials may require 100–500 patients to reach agreement on the MTD in two to four schedules and routes of administration. In addition, pharmacokinetic and metabolism studies are done as part of Phase I. Once the MTD is established, subsequent investigators can be reasonably assured that the upper limit for the dose to be administered is well defined and that therapeutic activity will not be missed in later phase trials because of sub-therapeutic doses. Phase II studies are then conducted to determine the therapeutic activity of the new drug in various types of cancer. Patients with specific cancers are selected in order that these trials can be conducted in reasonably

Robert K. Oldham uniform patient groups. Based on the preclinical information and the clinical toxicology studies, as well as pharmacokinetic and metabolic considerations, therapeutic doses are selected that represent the investigator’s “best guess” as to the therapeutically active dose range. In addition, schedule and route of administration must be considered. In phase II trials, it is often necessary to administer an agent at different doses by different routes (e.g., intramuscular, intravenous, subcutaneous, oral) and on different schedules (e.g., once a day, three times a day, 24-h infusion, etc.). Phase II trials are considered complete when a substantial body of data exists concerning the therapeutic activity of a new drug with reference to best dose, route of administration, and schedule for therapeutic efficacy. If one accepts classical criteria for cancer subtypes, over 100 histologic types of cancer exist. If one assumes a standard statistical criterion of at least 14 patients treated to look for one response so as not to miss a 20% response rate, then a new drug would need to be tested in a minimum of 1,400 patients by each schedule and route to assure clinicians of its inactivity in each tumor type; and if one assumes three routes and five schedules must be tested, over 21,000 patients would need to participate just to prove a specific drug ineffective. Such studies demonstrating inactivity sacrifice patients “for the good of the system.” Such FDA-mandated testing stretches ethical standards to the limit. Following the completion of adequate phase I and phase II trials, phase III trials to compare a new agent with standard treatment are conducted. The extent of phase III trials depends on the treatment alternatives available. In the case of diseases for which other therapies are effective, such trials may need to be extensive, controlled, and employ random designs, sometimes with blinding of the study both to the physician and to the patient. Such phase III trials have produced an enormous literature on the subject of ethics, trial design, end points, and the assessment of efficacy [9, 23, 28, 38, 71, 82, 91, 92, 95, 106, 107, 110]. If, in such phase III trials, a new agent proves therapeutically superior without unacceptable side effects, in comparison with or in addition to “standard” therapy, the new agent is very likely to receive FDA approval. Since “standard therapy” is often only marginally effective, large randomized trials proving a new treatment is significantly (usually marginally) better means most patients derive little or no real therapeutic benefit in the trials. Where efficacious therapy with an approved drug is not available, fewer phase III data may be needed to gain approval, and patients with advanced cancer are often randomized against best supportive care or a placebo. This latter practice also stretches ethical practice to the limit [47, 71, 95].

43 Most drugs fail at the phase I/II level, being either too toxic or inactive. Drugs that complete phase II trials with acceptable toxicity and activity in at least one cancer have a greater than 50% chance of successfully passing phase III with eventual approval by the FDA. For this reason, it has been suggested that drugs should be approved for general use at the end of phase II to speed up the process of bringing new drugs to the clinic [47, 83]. The final step in the commercial development of a new drug requires the filing of a new drug application (NDA). All the information available under the INDA and all of the data from the phase I, II, and III testing are made available to the FDA for review and consideration. Only the FDA is authorized to approve a new drug for sale in the United States; this usually involves defining the drug dose, route of administration, schedule, toxicity, and therapeutic activity in specific diseases (indications). After FDA approval, the company may begin to advertise the agent for the approved use only. Uses outside the specific FDA approval indication(s) are termed “off label” uses. Perhaps 50% of all cancer drug therapy is “off label” use by physicians but the pharmaceutical industry is specifically prohibited by the FDA from providing clinical trial information to clinicians on “off label” drug activity. The long time and enormous expenditure in the current drug development system have been justified as being in the public interest inasmuch as the FDA requires extensive testing so active agents with reasonable toxicities (safe drugs) can be made available as medicinals. The definitions of reasonable toxicity and of therapeutic efficacy have been the subject of considerable debate, both in the general sense and relative to specific drugs. Often, the long-term effects and/or toxicities of approved drugs have prompted secondary revisions in the regulatory process, further lengthening and expanding the steps necessary for new drugs to be approved. This process would seem to be totally in the public interest. However, when viewed in another context, the process clearly has some exclusionary aspects [47, 66, 71, 83]. The FDA defines a single standard for drug development in the United States. Marked differences exist between countries as to a regulatory body’s role in drug development, and there are marked differences in the number of drugs available to patients in different countries. The regulatory agency of each country has its own view of what is ethical and in the public interest (different standards, different ethics). Thus, there can be reasonable debate on which rules are the best for the development of new drugs. Finally, it is obvious that the long development times and the huge expenditures required effectively

44 restrict competition in the area of drug development. Such restriction of competition gives major pharmaceutical firms a virtual monopoly on drug development. Although most pharmaceutical firms would undoubtedly say that they would prefer a lower cost for drug development, one may view the process as being in their best interests (barrier to entry), since it restricts competition from smaller firms that cannot marshal the resources to carry out these extensive and expensive studies [47, 55, 71, 79, 80, 82, 83]. Thus, our drug development paradigm is highly restrictive. It has worked reasonably well over the past 50 years only because of public acceptance of a conservative regulatory structure and because of the small number of relatively toxic drugs that actually were available for clinical testing. The advent of biologicals has placed great pressure on this paradigm. Indeed, the effective development of biologicals will require changes in the regulations and policies for the development of new pharmaceuticals [47, 55, 71, 78, 82, 90, 91].

Biologicals and BRM Development A process of developmental therapeutics similar to that described for chemicals (drugs) is now being applied to biopharmaceuticals. Historically, for new biologicals, the information on composition, formulation, and purity was often imprecise. In contrast to drugs, which are generally small, synthesized molecules with a chemical definition that is quite straightforward, biologicals have had a more variable developmental process. Preparing a biological for testing has involved extraction from a microorganism, from a fraction of a cell culture, or from a tissue (complex chemical mixture). Such extractions yielded complex mixtures with defined biological activity, where the precise chemical composition was incomplete or unknown [75]. Biotechnology is now making available a range of biologicals (lymphokines/cytokines, monoclonal antibodies, antigens, growth and maturation factors) that are pure and as well defined as small molecule drugs [55, 57]. Market size, the potential for profit, the long development period of preclinical and clinical testing, and the large investment necessary to develop new therapeutic agents have defined the scope of drug development. The development and testing of new biologicals will require extensive procedural and regulatory changes if we are to be successful in bringing these agents to the clinic quickly and efficiently [47, 55, 83, 92].

Development therapeutics and the design of clinical trials Perhaps the most cogent example relates to the development of monoclonal antibodies. For years there have been sufficient data to indicate that monoclonal antibodies are eventually going to be very useful both diagnostically and therapeutically [10, 26, 36, 40, 51, 52, 54, 62, 73, 94, 102, 103]. Now there are therapeutic antibodies approved for colon and breast cancer, lymphoma and leukemia with hundreds more in testing [8, 19, 21, 41, 94, 103]. The problems of developing new monoclonal antibodies for therapy is quite different from those previously encountered with drugs. The most striking of these problems relate to market size. Generally speaking, one looks at the population afflicted with a particular disease and makes the presumption that a new drug will be active in a certain percentage of patients with that disease. The percentage is often reasonably high and allows the market forecast to be applicable to a substantial number of patients. For example, the number of patients at risk per year with lung cancer is reasonably well defined, and once some evidence of clinical activity of a new drug in lung cancer is available, the market size for that drug can easily be calculated. For monoclonal antibodies, the situation is very different. At the extreme is anti-idiotypic monoclonal antibody therapy. It has been demonstrated in preclinical models and, to a more limited extent, in humans that anti-idiotypic antibody can be made to the specific tumor antigen (idiotype) of the neoplastic cell [11, 29, 39, 51, 65]. Such antiidiotypic antibodies or idiotype vaccines derived therefrom have proven useful in controlling the growth of the B-cell lymphomas. However, a critical problem for the development of these reagents is market size; here, it is the individual patient with that particular neoplasm. Thus, the market size might be as small as one. Even with some cross-reactivity, many of these antibodies or vaccines are expected to apply only to very small populations, much smaller than even the “orphan drugs” envisioned by FDA policy. Obviously, most pharmaceutical companies will never develop these kinds of “individualized or personalized medicines” under existing FDA guidelines [37, 71, 93, 94]. A less extreme example relates to the development of monoclonal antibodies for tumor-associated antigens that may be restricted to subpopulations of cancer cells [19, 20]. Considerable heterogeneity exists within any one histologic type of cancer (between patients) and even within a single patient’s cancer [54, 64]. It may be that for any one cancer only a small portion of the patient population will have a particular antigen or array of antigens on the cancer cell surface. Therefore, a monoclonal antibody might be applicable only to 1%, 5%, or 10% of the patients with a particular type of neoplasm.

Robert K. Oldham Because of clonal heterogeneity, both between patients and in different clones within individual patients, there may be the need to use multiple antibodies (“cocktails”) in treatment [1, 46, 69, 90]. Given the over 100 histologic types of cancer and the heterogeneity within each cancer type, market calculations may define very narrow applications [55, 63]. These considerations restrict market size to a level unapproachable given drug development costs under the current drug development paradigm. Perhaps less obvious, but equally problematic, will be the development of other biologicals for treatment. For lymphokines and cytokines, there is already evidence of both antigen-specific and non-antigen-specific signals that may have growth regulatory effects (See Chapter 8). One can visualize, with an antigen-specific lymphokine, how the signal may relate to the specific antigen and be applicable in a single patient, or a very few patients (e.g., transfer factor, IgE suppressor factors). Less restricted, but still highly restricted compared to standard drug development, is the development of non-antigen-specific lymphokines (e.g., interferons, lymphotoxins, interleukins), which are active against certain classes of cells, thus making them clinically applicable only in selected populations [55, 57].

Interferons: the Early Model Interferons represented early models for new biological approaches in cancer treatment [57, 61, 76]. “Natural” extracted interferons from stimulated white blood cells were used in initial clinical trials. The low purity, lotto-lot variation, and expense of stimulating leukocytes to produce interferon, along with the difficulties in the extraction and purification methods, limited the clinical use of these materials. These preparations were typical of early forms of nonspecific immunotherapy [5, 75]. With the advent of increased interferon availability through recombinant genetics, and with the very high purity (greater than 99%) of these preparations, extensive trials were completed for alpha-interferon preparations which led to the approval of alpha-interferon as the first genetically engineered anticancer biopharmaceutical [42, 76]. The design of phase I biotherapy trials should differ markedly from those for drugs. The dose-response curve for these agents may be very broad (and sometimes multiphasic), with peak effects at different doses for each system responding to the biopharmaceutical. For example, the immunomodulatory activity of alpha-interferon can be seen at very low doses, whereas the antiproliferative activity appears to be more reproducible at higher doses. In biotherapy, there is a need to measure biological

45 responses in the context of the clinical trials [37, 48, 50, 62, 105]. Since one may be administering the biological to stimulate a particular biological response, for which the dose-response curve may not be known a priori, one must perform studies with pharmacokinetics to assay serum availability, and also measure the desired biological effects to determine the optimal dose at which it might alter a particular biological response (optimal biological response modification, OBRM). Finally, schedule and route of administration have already proven important [42, 76]. The pharmacokinetics after intravenous and intramuscular administration differ for the alpha interferons [43], and there has been a variable lack of absorption of intramuscularly administered beta- and gamma-interferons [34, 74]. Thus, the proper design of phase I biotherapy trials must take into account appropriate measurements of bioavailability, pharmacokinetics, biological response modification, and toxicity, all in the context of escalating doses, to determine the dose– response curves for each of these properties. Responses to biologicals vary substantially between patients, and escalating doses in individual patients can yield valuable data without subjecting patients to “tests” with no therapeutic opportunity. Like most biologicals, the interferons may act through a diverse set of biological mechanisms. Cell surface receptors for their activity exist, and responses to the administration of biologicals are somewhat predetermined by the condition and biological receptor repertoire of the patient. This situation is in direct contradistinction to that of drugs, in which a totally new chemical is often administered to a patient in whom no standard biological response mechanism existed a priori. Thus, biologicals may be viewed in their physiologic role of correcting immunodeficiency states, as well as in the pharmacologic role of augmenting host responses and perhaps having direct antitumor effects. Clearly, it was rational to test chemical drugs to MTD and to treat just below this dose in a “kill or cure” approach to cancer therapy. Current evidence suggests the OBRM dose and the MTD dose should be determined in cancer biotherapy to properly design effective therapeutic trials. Once determined, the design of therapeutic trials for biotherapy may differ greatly from classical chemotherapy studies.

Biotherapy Trial Strategies Many strategies exist or can be envisioned for conducting clinical trials with new biologicals. Some of these strategies have already been utilized in the early-phase

46 testing of the interferons [4, 27, 59, 60, 103]. Two underlying principles are apparent wherein biologicals and drugs differ: when biologicals are administered, patients already have physiologic mechanisms and receptors that respond to them; and biologicals are derivatives of natural products of the mammalian or human genome, and may be expected to have less acute and chronic toxicity than drugs at similar biologically effective doses. However, when high doses are used to exploit a certain action of a biological, acute and chronic toxicities may appear. These two considerations will not necessarily apply to those BRM that are well-defined chemical entities and behave more in the manner of drugs with regard to toxicity.

Empirical Clinical Testing In many early interferon trials a set dose of a “natural” interferon was given to a variety of patients with neoplastic or viral disorders [57, 62, 76]. The chosen dose was the one expected, based on preclinical information or other clinical data, to be relatively nontoxic and yet to have sufficient biological effects to be therapeutically active. In this context, most early clinical trials with leukocyte interferon preparations were conducted at doses under five million units per day, although it became apparent later that doses up to 60 million units per day could be tolerated by the patients for a certain number of days. These trials were conducted as preliminary feasibility trials or pilot phase II trials to gain some information on the biological effects of the interferon preparations. Much of the information derived from them was anecdotal, but they yielded preliminary information about the biological effects and toxicities of alpha-interferons. Now that the development of biotherapy is proceeding more rapidly, other strategies are to be preferred.

Escalating dose Trials Using Different Groups of Patients Phase I trials for biologicals have been modeled after the standard phase I trial design used for drugs. Groups of patients (usually three to five) are treated with a particular dose of a new biological with a single route of administration [103]. These trials are begun with very low (sub-therapeutic) doses based on preclinical information, and the schedule is designed to increase the dose gradually to levels where toxicity occurs [55, 74]. Generally, new patients are entered at each higher dose level after all the patients have been entered on the previous dose and have received at least several treatments. In this way, each patient group provides toxicity

Development therapeutics and the design of clinical trials information before the higher dose is initiated in new patients. By utilizing different groups of patients at each dose level, cumulative toxicity for any one patient is avoided. Patients receive a predetermined number of doses at each dose level and then are followed. During this type of study, biological response modification and clinical toxicity are assessed [4, 48–50, 53]. Pharmacokinetics are also done in selected patients, so that the bioavailability can be determined [103]. The dose-escalation scheme can be a modified Fibonacci series, or some variation of this classical doseescalation method. The considerations involved in doseescalation methods dictated by drug toxicities do not necessarily apply to biologicals. Thus, some inves-tigators have used much faster dose escalations for biologicals. Often, the dose escalation schedule is rather empirical, the one selected being based on the best guess of the investigators involved. This phase I drug development strategy is based on historical data using new drugs and has several potential disadvantages. Since each patient receives only a particular dose for a defined period of time, it is highly likely that a large percentage of the patients will receive sub-therapeutic doses if antitumor effects are observed only at higher dose levels. Early clinical trials with biologicals have not produced severe cumulative toxicity. This clinical trial strategy, which was designed to avoid the cumulative toxic effects seen with drugs, is inappropriate for biotherapy. Since tolerance (tachyphylaxis) may develop for some effects (fever), entering different patients on progressively higher doses may expose patients to avoidable acute toxicities. Finally, the patients who are exposed to only one dose level are not individually assessed for biological response or for antitumor response over a wide dosage range. This could give them a greater opportunity for optimization of biological and therapeutic response [59]. This may be particularly true in the very-early-phase trials in which biological effects and antitumor effects are totally unknown.

Escalating-dose Trials within Individual Patients An alternative clinical trial strategy is the use of an escalating-dose trial within individual patients [58, 59, 74, 94]. There are several variations of this theme, but each involves starting patients at a low dose and escalating the dose in each patient to determine the biological response modifying effects and toxicities over a broad dose range. This clinical trial strategy is very conservative of patients, in that studies in small numbers of patients can give a large amount of information over a

Robert K. Oldham broad dose range in each biotherapy trial. In the context of this dose escalation, pharmacokinetics and biological response modification can be measured in each patient. It is important in such a clinical trial strategy that bioavailability studies be done and the information be available concomitantly with the study. Appropriate “wash-out” periods between doses can be utilized so that the administered biological does not circulate in increasing quantities as the trial continues. For example, with the interferon trials, one can determine the serum level after each dose and administer the next dose when interferon is no longer detectable. This allows the patient to avoid the possibility of severe acute toxic effects based on cumulative serum levels. Obviously, biological and therapeutic effects can still be cumulative and must be monitored by appropriate clinical and biological measurements throughout this type of trial. This strategy offers the possibility of low-, medium-, and high-dose therapy for the individual patient in the context of a single clinical trial. It maximizes the opportunity for the investigator to learn the optimal biological response modifying dose and the toxic dose, and perhaps to gain therapeutic information, all in the context of a single trial. Theoretically, rational maintenance regimens could be designed based on the observations made during the escalating dose trial for each patient. For monoclonal antibody, this strategy may be particularly important, in that the delivery of the monoclonal antibody to the tumor site may be the most important consideration in developmental therapeutics [25, 36, 54, 63, 94]. Giving a low dose and then progressively higher doses to the same patient, with subsequent determination of antibody localization, is a useful way of determining the correct dose for delivery of the antibody and/or its conjugates to the appropriate target organ in the context of a toxicity study. This trial design rationally ties targe-ting (delivery), toxicity, and therapeutic effects together in combined phase I/II studies in a manner perfectly appropriate for antibody and conjugates, in direct contradistinction to drug development [2, 3, 17, 18, 59, 96, 98]. In studies that employ escalating doses, a useful variation is to enter individual patients for a limited number of doses. With this trial design, the initial three or four patients may enter at the lowest dose level and progress through dose level 4 or 5, at which time the second group of patients may be entered at dose 3 and escalated upward in a manner that allows the second group to follow the first group in dosage escalation toward the MTD. A third group may be entered at dose level 6 and so on. This strategy allows one to avoid administering the full dose range to all patients, avoids most cumulative toxicity, and helps avoid sub-therapeutic doses.

47 It increases the patient’s therapeutic opportunity without undue risk of toxicity. This strategy is ethically preferable, most acceptable to patients and quite easy to describe in the informed consent [71].

Schedule In the initial clinical trials, the schedule of administration is generally empirical or is based on preclinical observations. Once there is information on bioavailability, different schedules of administration can be designed rationally. Both the bioavailability of the molecule and its biological effects are relevant. There are some biologicals that have a very short serum half-life (measured in a few seconds to minutes) but may produce much longer lasting biological effects. It may be useful to compare the biological effects and toxicities of biologicals when administered under conditions of rather constant exposure as against intermittent exposure in order to gain a preliminary sense of which schedule is most relevant [35, 74, 108]. Once these data are available, schedules of administration for phase II studies can be more rationally designed.

Route The route of administration and delivery of biotherapy to selected target organs is critical. There are data indicating that certain biologicals are inactivated, or poorly absorbed, when given intramuscularly or subcutaneously. For such biologicals intravenous administration is quite important. When data on bioavailability are not known from preclinical models, it is probably important to conduct early clinical trials with the intravenous route, since such trials can provide early data on serum pharmacokinetics and provide the best opportunity for broad biodistribution of the administered biological. It is important in phase I biotherapy trials to determine if serum levels are measurable and if biological effects are seen in the presence or absence of serum levels for agents given by any route of administration. There may be instances in which second mediators are involved, with useful biological effects occurring without apparent serum bioavailability. Effusions may contain both tumor cells and reactive immunological elements, in which case the administration of a biological into a restricted space such as the thoracic or peritoneal cavity might be appropriate. There are indications that certain immunomodulators may be effective in this setting [56]. Patients with tumors that remain confined to a single compartment for a prolonged period of time may offer unique opportunities for biotherapy [72, 88]. Ovarian cancer, with its propensity for

48 remaining localized in the abdominal peritoneum, may be ideal for evaluating the antitumor activity of cells [72], biologicals, and BRM in a relatively closed space. Interleukin-2 with activated cells is active when infused into selected anatomical sites and visceral cavities where tumor is present [44, 88]. It seems clear that targeting of monoclonal antibody and its conjugates may be improved by selective organ or region perfusion or infusion. Considerations for the design of early-phase trials in these spaces differ markedly from those using systemic administration.

Patient Selection Historically, patient selection for phase I drug trials has been broad, and patients bearing many tumor types have been entered into the trials. While this may be appropriate for drugs, and for selected lymphokines that act broadly on immunological responses or may act in a general antiproliferative manner across a broad spectrum of tumors, it is not appropriate for those biologicals that act more specifically. Thus, a monoclonal antibody that has been specifically designed to recognize a particular type of cancer can be appropriately tested only in patients with that type of cancer [45, 59]. In vitro determinations of specificity and activity may play a greater role in the selection of the patient populations for phase I biotherapy trials. The escalating-dose phase I trials, with the dose being escalated within individual patients, preselected on the basis of in vitro specificity and/or activity, appear to be most relevant and efficient for determining the distribution, biological effects, and toxicity of monoclonal antibodies. The same considerations may apply to certain lymphokines/cytokines, in which case selection as to activity may be on an individual patient basis. These trials may be more appropriately termed phase I/II trials. In fact, given the heterogeneity of cancer, both with respect to the specificity of recognition by antibody and with respect to activity by immunoconjugates and lymphokines/cytokines, in vitro determinations may play a major role in clinical trial design. Biological systems are diverse, and single patients may require specifically designed treatments when specificity and activity restrict the applicability of biotherapy. The design of early-phase [biotherapy] clinical trials needs to be radically changed [55, 58, 71]. There has been much speculation concerning the types of patients most appropriate for trials with biologicals. Many believe that these agents should be tested primarily in the adjuvant setting, where the tumor burden is quite small and the biological responses to be modified are still healthy [75]. Although this sort of strategy may be optimal, it is also prohibitively expensive

Development therapeutics and the design of clinical trials and very difficult to design. It would be virtually impossible to investigate a significant number of biologicals in phase II or III trials utilizing this type of clinical trial design. The number of patients required for trials in the adjuvant setting is enormous, in that the recurrence rate cannot be precisely predicted without a concurrent random control group. The lack of certainty for recurrence prompts ethical questions in designing phase II or III trials for these patients, since dose, route, schedule, OBRM and therapeutic efficacy have not been determined. It has now been well demonstrated that certain biologicals have activity in patients with bulky and resistant disease [4, 7, 13–16, 27, 57, 58, 67–68, 76, 89, 94, 108]. Even though biotherapy may be more effective, as chemotherapy and radiotherapy are, when the tumor burden is small, that does not mean that it is totally ineffective in patients with bulky disease. Given the large number of biologicals available to the clinic, there is a need to develop methods of rapid clinical testing in early-phase trials, and this will necessitate testing in patients with apparent disease [55, 57, 75, 108]. Indeed, the initial clinical trials with interferons, interleukins, monoclonal antibodies, and cellular therapies generally selected such patients with a good performance status and a reasonably functional immune system. These patients have shown evidence of biological response modification, and they have shown antitumor responses. Thus, such clinical trials can be used as indicators for biological activity of new agents [57, 67, 94].

Future Prospects Developmental therapeutics for biotherapies is well underway. From the inception of this field in 1980 to the present, a great number of new approaches have become available in biotherapy [42, 57, 71, 94, 108]. Unlike the field of drugs, where very large numbers of compounds are screened in a preclinical testing program and very few reach the clinic, a much higher percentage of the biologicals selected rationally and tested actually go on to clinical trials. This is due to selection based on the known physiologic activities of biologicals as opposed to random testing of chemicals in drug development. The strategies for these clinical trials need to be cost efficient, advantageous to the patient, ethical [71, 97], and scientifically interpretable to the clinician/scientist [64, 75]. Phase I strategies are most important in that they give the initial leads on which the design of early phase II and III trials are based. With proper selection clinical trial strategies, new biologicals can be more effectively screened and evaluated as potential anticancer agents [59, 71].

Robert K. Oldham

49

Biotherapy is the fourth modality of cancer treatment [57, 58, 84, 85, 86, 94]. These agents and this technology have far broader applications in medicine than cancer therapeutics. Given the large number of cloned biologicals and the virtually unlimited number of recombinant molecules that can be produced therefrom, along with unlimited variety of monoclonal antibodies that can now be produced, it is apparent that the coming years will produce new challenges for those involved in developmental therapeutics [33]. This tremendous expansion of agents and approaches available for cancer therapeutics will increase our opportunities and amplify the complexity in the preclinical evaluation of these agents and their translation to the clinic. The numbers of biologicals to be evaluated and their inherent selectivity call for new methods in the selection of the “most likely to succeed.” Additionally, the methods used historically by pharmaceutical manufacturers and the regulations established by governmental agencies for approving anticancer drugs are inhibitory to the rapid development of biotherapy [47, 55, 83]. Novel approaches will be necessary if these biologicals are to be effectively and rapidly translated to clinical trials [47, 55, 64, 95]. Alpha-interferon, IL-2 and monoclonal antibodies and their conjugates have anti-tumor effects, even in patients with bulky disease. This finding is likely to

emerge for other forms of biotherapy [42, 57, 59, 67, 71, 104, 108, 109]. This should give pause to those who believe the immunological dogma that biotherapy can only be active in minimal residual disease. Like chemotherapy and radiotherapy, biotherapy may be more active with lesser tumor burdens, but current data indicate that activity and hence selection of compounds for further study can be assessed in patients with advanced disease. The potential for the use of biotherapy is great. We are in a new era in cancer therapeutics. To develop biotherapy, we must begin to approach this new era in pharmaceutical research and to contemplate novel methods for the efficient and timely development of biotherapy rather than simply continuing in old paradigms (Fig. 1) [33, 55, 71, 82, 83]. Some would still say no new treatment can be judged without randomized, clinical trials. Such trials are valid in searching for small differences between a new treatment and an ethically acceptable control (standard treatment) [101]. Whether placebo controls are ever acceptable in cancer treatment is debatable; however, pilot studies without controls can be very useful even to the point of defining efficacy if the treatment effect is large and obvious. A recent and innovative “n of 1” trial also bears consideration in trials of biotherapy [35].

Preclinical Testing (in vitro/animal model) Phase I Clinical Trial Phase II Clinical Trial Phase III Clinical Trials

Unresponsive or poorly responsive neoplasm; Less than partial responses Partial responses to newer active single agents Increased partial responses and rare complete responses To some single and combined agents

Figure 1. Although radically different, chemotherapy and biotherapy share a systemic approach to cancer treatment. For those cancers that chemotherapy has been able to cure, reaching that goal required a sequence of steps in which new and increasingly more effective agents and combinations of agents had to be identified. Biotherapy can be expected to travel a similar path

Increased complete responses to more effective Combination therapy Increased survival in complete responders with or Without continued treatment during remission A portion of long-term survivors are cured

50

Development therapeutics and the design of clinical trials

We must not just continue to simply do what has been done historically [71]. Development therapeutics for biologicals certainly represents the kind of science Lewis Thomas described: It is hard to predict how science is going to turn out, and if it is really good science it is impossible to predict. This is in the nature of the enterprise. If the things to be found are actually new, they are by definition unknown in advance, and there is no way of telling in advance where a really new line of inquiry will lead. You cannot make choices in this matter, selecting things you think you’re going to like and shutting off the lines that make for discomfort. You either have science or you don’t, and if you have it you are obliged to accept the surprising and disturbing pieces of information, even the overwhelming and up heaving ones, along with the neat and promptly useful bits. It is like that.

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14. Dillman RO, Oldham RK, Tauer KW, et al. Continuous interleukin-2 and lymphokine activated killer cells for advanced cancer: an NBSG trial. J Clin Oncol 1991; 9:1233–1240. 15. Dillman RO, Church C, Oldham RK, et al. Inpatient continuous infusion Interleukin-2 in 788 cancer patients: the NBSG Experience. Cancer 1993; 71:2358–2370. 16. Dillman RO. The clinical experience with Interleukin-2 in cancer therapy. Cancer Biother 1994; 9(3):179–182. 17. Dillman RO, Dillman JB, Halpern SE, et al. Toxicities and side effects associated with intravenous infusions of murine monoclonal antibodies. J Biol Response Mod 1986; 5:73–84. 18. Dillman RO. Human antimouse and antiglobulin responses to monoclonal antibodies. Antibody, Immunoconj and Radiopharm 1990; 3:1–15. 19. Dillman RO Monoclonal antibodies for treating cancer. Ann Inten Med 1989; 111:593–603. 20. Dillman RO. Antibodies as cytotoxic therapy. J Clin Oncol 1994; 12:1497–1515. 21. Dillman RO. Magic bullets at last! Finally – approval of a monoclonal anbitody for the treatment of cancer!!! Cancer Biother Radiopharm 1997; 12:223–225. 22. Dillman RO. Radiolabeled anti-CD20 monoclonal antibodies for the treatment of B-cell lymphoma. J Clin Oncol 2002; 20: 3545–3557. 23. Ellenberg SS. Studies to compare treatment regimens: the randomized clinical trial and alternative strategies. J Am Med Assoc 1982; 246:2481–2482. 24. Fidler IJ, Berendt M, Oldham RK. Rationale for and design of a screening procedure for the assessment of biological response modifiers for cancer treatment. J Biol Response Modif 1982; 1:15–26. 25. Foon KA, Bernhard KA, Oldham RK. Monoclonal antibody therapy: assessment by animal tumor models. J Biol Response Modif 1982; 1:277–304. 26. Foon KA, Schroff RW, Bunn PA, et al. Effects of monoclonal antibody serotherapy in patients with chronic lymphocytic leukemia. Blood 1984; 64:1085–1093. 27. Foon KA, Sherwin SA, Abrams PG, et al. Treatment of advanced non-Hodgkin’s lymphoma with recombinant leukocyte alpha interferon. N Engl J Med 1984; 311:1148–1152. 28. Fost N. Consent as a barrier to research [editorial]. N Engl J Med 1979; 300:1272–1273. 29. Giardina SL, Schroff RW, Woodhouse CS, et al. Detection of two distinct malignant B-cell clones in a single patient using anti-idiotype monoclonal antibodies and immunoglobulin gene arrangement. Blood 1985; 66:1017–1021. 30. Ghielmini M, Schmitz SF, Cogliatti SB, et al. Prolonged treatment with rituximab in patients with follicular lymphoma significantly increases event-free survival and response duration compared with the standard weekly × 4 schedule. Blood 2004; 103: 4416–4423. 31. Goeddel D, Yelverton E, Ullrich A, et al. Human Leukocyte Interferon Nature (Lond) 1980; 287:411–416. 32. Guisti RM, Shastri KA, Cohen MH, et al. FDA drug approval summary: panitumumab (Vectibix). Oncologist 2007; 12:57–83. 33. Grillo-Lopez AJ. Regularity process for approval of biologicals for cancer therapy. Chapter in: principles of cancer biotherapy, 5th ed. Dordrecht, The Netherlands: Kluwer. 34. Gutterman JU, Rosenblum MG, Rios A, et al. Pharmacokinetic study of partially pure interferon in cancer patients. Cancer Res. 1984; 44:4164–4171. 35. Guyatt G, Sackett D, Taylor DW, et al. Determining optimal therapy – randomized trials in individual patients. N Engl J Med 1986; 314:889–892.

Robert K. Oldham 36. Hanna MG, Jr., Key ME, Oldham RK. Biology of cancer therapy: some new insights into adjuvant treatment of metastatic solid tumors. J Biol Response Modif 1983; 4:295–309. 37. Herberman RB, Thurman GB. Summary of approaches to the immunological monitoring of cancer patients treated with natural or recombinant interferons. J Biol Response Modif 1982; 2:548–562. 38. Horwitz RI, Feinstein AR. Improved observational method for studying therapeutic efficacy. JAMA 1981; 246:2455–2459. 39. Hurvitz SA, Timmerman JM. Current status of therapeutic vaccines for non-Hodgkin’s lymphoma. Curr Opin Oncol 2005; 17:432–440. 40. Hwang KM, Foon KA, Cheung PH, et al. Selective antitumor effect of a potent immunoconjugate composed of the A chain of abrin and a monoclonal antibody to a hepatoma-associated antigen. Cancer Res 1984; 44:4578–4586. 41. Kavvinavar FF, Hambleton J, Mass RD, et al. Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improved survival for patients with metastatic colorectal cancer. J Clin Oncol 2005; 23:3706–3712. 42. Kirkwood JM, Ernstoff MS. Interferons in the treatment of human cancer. J Clin Oncol 1984; 2:336–352. 43. Knost JA, Sherwin SA, Abrams PG, et al. The treatment of cancer patients with human lymphoblastoid interferon: a comparison of two routes of administration. Cancer Immunol Immunother 1983; 15:144–151. 44. Lembersky B, Baldisseri M, Seski J, et al. Phase IB study of intraperitoneal (IP) interleukin-2 (IL-2) for refractory ovarian cancer (OC). Proc Am Assoc Cancer Res 1990; 31:277. 45. Lewko WM, Ladd PA, Pridgen D, et al. Tumor acquisition propagation and preservation: culture of human colorectal cancer. Cancer 1989; 64:1600–1608. 46. Liao SK, Meranda C, Avner BP, et al. Immunohistochemical phenotyping of human solid tumors with monoclonal antibodies in devising biotherapeutic strategies. Cancer Immunol Immunother 1989; 28:77–86. 47. Madden BJ. A clinical trial for the FDA’s clinical trial process. Cancer Biother Radiopharm 2005; 20(6):569–578. 48. Maluish AE, Leavitt R, Sherwin SA, et al. Effects of recombinant alpha interferon on immune function in cancer patients. J Biol Response Modif 1983; 2:470–481. 49. Maluish AE, Ortaldo JR, Sherwin SA, et al. Changes in immune function in patients receiving natural leukocyte interferon. J Biol Response Modif 1983; 2:418–427. 50. Maluish AE, Ortaldo JR, Conlon JC, et al. Depression of natural killer cytotoxicity following in vivo administration of recombinant leukocyte interferon. J Immunol 1983; 131:503–507. 51. Miller RA, Maloney DG, Warnke R, et al. Treatment of B cell lymphoma with monoclonal anti-idiotype antibody. N Engl J Med 1982; 306:517–522. 52. Nowinski RC, Tam MR, Goldstein LC, et al. Monoclonal antibodies for diagnosis of infectious disease in humans. Science 1983; 219:637. 53. Oldham RK. Toxic effects of interferon. Science 1982; 219:902. 54. Oldham RK. Monoclonal antibodies in cancer therapy. J Clin Oncol 1983; 1:582–590. 55. Oldham RK. Biologicals: new horizons in pharmaceutical development. J Biol Response Modif 1983; 2:199–206. 56. Oldham RK. Guest editorial: biological response modifiers. J Natl Cancer Inst 1983; 70:790–796. 57. Oldham RK. Biologicals and biological response modifiers; fourth modality of cancer treatment. Cancer Treat Rep 1984; 68:221–232. 58. Oldham RK. Biologicals and biological response modifiers: new approaches to cancer treatment. Cancer Invest 1985; 3:53–70. 59. Oldham RK. Biologicals and biological response modifiers: the design of clinical trials. J Biol Response Modif 1985; 4:117–128.

51 60. Oldham RK. Biologicals and biological response modifiers: new strategies for clinical trials. In: Finter NB and Oldham RK, eds. Interferons, IV. Amsterdam: Elsevier, 1985; 235–249. 61. Oldham RK. Interferon: a model for future biologicals. In: Burke D, Cantell K, Gresser I, De Maeyer E, Landy M, Revel M, Vilcek J, eds. Inf. VI. New York: Academic Press, 1985; 127–143. 62. Oldham RK. Biologicals for cancer treatment: interferons. Hospital Practice 1985; 20:72–91. 63. Oldham RK, Foon KA, Morgan AC, et al. Monoclonal antibody therapy of malignant melanoma: in vivo localization in cutaneous metastasis after intravenous administration. J Clin Oncol 1984; 2:1235–1242. 64. Oldham RK. Therapeutic monoclonal antibodies: effects of tumor cell heterogeneity. In: Present status of nontoxic concepts in cancer therapy. Cancer symposium (Germany). Basel: Karger, 1986. 65. Oldham RK. Monoclonal antibody therapy. In: Chiao JW, ed. Biological response modifiers and cancer research. New York:Marcel Dekker, 1988; 40:3–16. 66. Oldham RK. Set my factors free. Mol Biother. 1990; 2(4):194–195. 67. Oldham RK. Cancer biotherapy: principles and practice. New York: Marcel Dekker, 1991. 68. Oldham RK, Dillman RO, Yannelli JR, et al. Continuous infusion interleukin-2 and tumor derived activated cells as treatment of advanced solid tumors. An NBSG trial. Mol Biother 1991; 3(2):68–73. 69. Oldham RK. Custom tailored drug immunoconjugates in cancer therapy. Mol Biother 1991; 3(3):148–162. 70. Oldham RK. The cure. Franklin, TN: Pulse Publications, 1991. 71. Oldham RK. BioEthics: opportunities, risks and ethics: the privatization of cancer research. Franklin, TN: Media America, 1992. 72. Oldham RK, Greco FA. Brief intensive chemotherapy and second look laparotomy in advanced ovarian carcinoma. In: William CJ, Whitehouse M, eds. Recent advances in clinical oncology. London: Churchill Livingstone, 1982; 165–185. 73. Oldham RK, Morgan AC, Woodhouse CS, et al. Monoclonal antibodies in the treatment of cancer: preliminary observations and future prospects. Med Oncol Tumor Pharmacother 1984; 1:51–62. 74. Oldham RK, Sherwin SA, Maluish A, et al. A phase I trial of immune interferon: a preliminary report. In: Goldstein AL, ed. Thymic hormones and lymphokines. New York: Plenum Press, 1984; 497–506. 75. Oldham RK, Smalley RV. Immunotherapy: the old and the new. J Biol Response Modif 1983; 2:1–37. 76. Oldham RK, Smalley RV. The role of interferon in the treatment of cancer. In: Zoon KC, Noguci PD, Lui TY, eds. Interferon: research, clinical application and regulatory consideration. New York: Elsevier, 1984; 191–206. 77. Oldham RK, Thurman GB, Talmadge JE, et al. Lymphokines, monoclonal antibodies and other biological response modifiers in the treatment of cancer. Cancer 1984; 54:2795–2810. 78. Oldham RK, Patient-funded cancer research. N Engl J Med 1987; 316:46–47. 79. Oldham RK. Drug development: who foots the bill? Bio/technology 1987; 5:648. 80. Oldham RK. Who pays for new drugs? Nature 1988; 332(28):795. 81. Oldham RK, Avent RA. Clinical research: who pays the bills? Oncology Issues 1989; 4(2):13–14. 82. Oldham RK. Clinical research in cancer: a time for consensus. Pharm Exec 1989; July. 83. Oldham RK. Regulatory hierarchies (editorial). Mol Biother 1988; 1(1):3–6. 84. Oldham RK. Biotherapy: the fourth modality of cancer treatment. Cancer: perspective for control symposium. J Cell Physiol Suppl 1986; 4:91–99.

52 85. Oldham RK. Biotherapy: the fourth modality of cancer treatment. In: Mak TW, Sun TT, eds. Cancer: perspective for control symposium. New York: Alan R. Liss, 1986; 91–99. 86. Oldham RK. The government-academic “industrial” complex. J Biol Response Modif 1986; 5:109–111. 87. Oldham RK. The cure for cancer. J Biol Response Modif 1985; 4:111–116. 88. Oldham RK, Bartal AH, Yannelli JR, et al. Intra-arterial and intracavitary administration of lymphokine activated killer cells in patients with advanced cancer: feasibility and laboratory results. Proc AACR (abstr) 1988; 29:396. 89. Oldham, RK, Lewko W, Good R, et al. Growth of tumor derived activated T cells for the treatment of cancer. Cancer Biother 1994; 9(3):211–224. 90. Oldham RK. Fundamentally different. Cancer Biother Radiopharm 1999; 14:413–15. 91. Oldham RK. Cracking the FDA code. Wall Street J Op/Ed. 2.2.2001. 92. Oldham RK. Informal opinions: editorial introduction. Cancer Biother Radiopharm 2002; 17:247. 93. Oldham RK. FDA trials cost lives. Wall Street J Op/Ed. 3.3.2002. 94. Oldham RK, Dillman RO. Monoclonal antibodies in cancer therapy: twenty-five years of progress. JCO. 2008; 26(11):1774–1777. 95. Oldham RK, Dillman RO. Gold standard or wrong standard? Cancer Biother Radiopharm 2004; 17(6):271–272. 96. Orr DW, Oldham RK, Lewis M, et al. Phase I trial of Mitomycin-C immunoconjugate cocktails in human malignancies. Mol Biother 1989; 1(4):229–240. 97. Relman AS. The ethics of randomized clinical trials: two perspectives [editorial]. N Engl J Med 1979; 300:1272. 98. Ricart AD, Tolcher AW. Technology insight: cytotoxic drug immunoconjugates for cancer therapy. Nat Clin Prac Oncol 2007; 4:1379–1389.

Development therapeutics and the design of clinical trials 99. Romond EH, Perez EA, Bryan J, et al. Trastuzumab plus adjuvant chemotherapy for operative HER2-positive breast cancer. N Engl J Med 2005; 353:1673–1684. 100. Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevcizumab for non-small-cell lung cancer. N Engl J Med 2006; 355:2542–2550. 101. Schafer A. The ethics of randomized clinical trials. N Engl J Med 1982; 307:719–724. 102. Sears HF, Herlyn D, Steplewski Z, Koprowski H. Effects of monoclonal antibody immunotherapy on patients with gastro-intestinal adenocarcinoma. J Biol Response Modif 1984; 3:138–150. 103. Sherwin SA, Knost JA, Fein S, et al. A multiple dose phase I trial of recombinant lymphocyte alpha interferon in cancer patients. JAMA 1982; 248:2461–2466. 104. Slamon DJ, Leyland-Jones B,Shak S, et al. Use of chemothe-rapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001; 344: 783–92. 105. Smalley RV, Oldham RK. Interferon as a biological response modifying agent in clinical trials. J Biol Response Modif 1983; 2:401–409. 106. Sylvester RJ, Pinedo J, De Pauw M, et al. Quality of institutional participation in multicenter clinical trials. N Engl J Med 1981; 305:852–855. 107. Weiss DG, Williford WO, Collins JF, Binham SF. Planning multicenter clinical trials; a biostatistician’s perspective. Controlled Clin Trials 1983; 4:53–64. 108. West WH, Tauer KW, Yannelli JR, et al. Constant infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. N Engl J Med 1987; 316(15):898–905. 109. Vitetta ES, Krokick KA, Miyama-Inaba M, et al. Immunotoxins: a new approach to cancer therapy. Science 1983; 219:644–649. 110. Zelen M. A new design for randomized clinical trials. N Engl J Med 1979; 300:1242–1246.

4

Recombinant proteins and genomics in cancer therapy KAPIL MEHTA, BULENT OZPOLAT, KISHORCHANDRA GOHIL, AND BHARAT B. AGGARWAL

Abbreviations ADA, adenosine deaminase; AML, acute myeloid leukemia; bFGF, basic fibroblast growth factor; CML, chronic mylogenous leukemia; CSF, colony-stimulating factor; EGF, epidermal growth factor; EPO, erythropoietin; FDA, Food and Drug Administration; HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; LIF, leukemia inhibitory factor; MAb, monclonal antibody; PDGF, platelet-derived growth factor; PE, Pseudomonas exotoxin; PEG, polyethylene glycol; TGF, transforming growth factor; TNF, tumor necrosis factor

Introduction Recently published sequence of the complete human genome represents a major milestone in the era of the modern molecular biology [318, 132]. The sequencing of approximately 3.2 billion nucleotides of the human genome that is estimated to contain about 20,000–25,000 protein-encoding genes signifies the first step down the long road. Gene identification does not necessarily translate into an understanding of gene function. Although mapping and cloning of several genes have linked them to heritable genetic disorders, the normal function of a majority of these genes remains unknown. Recombinant DNA technology has made it possible to generate large amounts of many biologically active proteins and to delineate their functions. The novelty of recombinant technology is the precision and efficiency with which scientists can manipulate single gene. The ability to isolate human genes and insert them into microorganisms, which then produce human proteins, thereby serving as biological factories, has revolutionized the field of biology. Interferons have special significance to recombinant DNA technology as paradigm modifiers of immune response. The interest in the therapeutic potential of interferon against cancer and viral diseases has served as catalyst to the emerging recombinant DNA industry. Despite its great promise as an antiviral agent, the clinical application of interferon had been rather slow, mainly

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

because of the lack of methods for producing adequate amounts of the pure protein. Interest in interferon beyond the field of virology began in the early 1960s, when workers began to recognize its growth-inhibitory and immune-activation properties. During the 1960s and early 1970s, reports of interferon’s antiviral and antitumor activity in laboratory animals and humans stirred up this interest and several groups decided to purify human interferon for clinical use. The first practical method of producing sufficient quantities of interferon was developed by Cantell et al. [31]. They were able to isolate 100–200 mg of interferon from 1,000 l of starting material that contained 2–5 kg of other contaminating proteins. The purified material had a specific activity of greater than 108 U/mg protein [69] and was sufficient to treat only a few patients [282], but the initial clinical results stimulated wider interest in expanding production of interferon for more extensive clinical trials. Enthusiasm intensified when interferonalpha was successfully cloned and the purified recombinant protein became available [225, 226]. The first trial to test dose levels and side effects of the purified bacterial product in human beings began in 1981 [227, 112]. The availability of pure recombinant protein led researchers to crystallize interferon, the first step toward analysis of the protein’s three-dimensional structure by X-ray crystallography. This permitted the production of individual molecular species of interferon free from other species and other proteins that were simultaneously induced in human cell cultures. By using this technique, our knowledge of the varied biological properties of interferons, previously determined with relatively crude preparations, has been confirmed and extended.

Isolation, Cloning and Expression of Genes A gene is a defined region of a chromosome comprising a specific sequence or part of a long polynucleotide. It codes for some specific function or characteristic

53

54

Recombinant proteins and genomics in cancer therapy GENE -A-G-A -T-C-T

a

b Intron

d

c

C-T-A-G- G-A-T-C- -

exon

Eukaryotic DNA Genome

Transcription a

b

c

d Primary RNA transcript

a

b

c

d Splicing

a

b

c

d

mRNA

Translation NH2

------

COOH

Protein

Protein processing, folding

Mature functional protein

Figure 1. The organization of a eukaryotic gene and processing of the RNA transcript

(phenotype) of a cell. The eukaryotic genome contains up to 109 nucleotides in 50–100,000 genes [40, 97]. To study the events in such a complex system it is necessary to be able to isolate and study a single gene in a purified form. Molecular cloning provides a method for isolating a single discrete segment of DNA from a population of genes and amplifying the DNA segment to produce enough pure material for chemical, genetic, and biological analysis. Cloning and expression of foreign genes has permitted access to such complex biological mechanisms as RNA splicing, oncogene dynamics and antibody diversity. It has also been the foundation for the new biotechnology industry. The organization of genes in higher organisms is more complex than in bacteria and viruses. The linear array of information in a complex gene contains one or more stretches of non-coding sequences, called introns. The remaining sequences, which encode information for proteins, are called exons. When these genes produce a protein, the DNA is transcribed into a large RNA molecule from which the introns are removed; this

mRNA molecule is then translated to produce the corresponding protein (Fig. 1). The organization of bacterial genes is simpler. They do not produce the enzymes necessary for RNA splicing, so a eukaryotic gene containing introns, if introduced into a bacterial cell as in recombinant techniques, will not be properly expressed. This problem can be circumvented by making a DNA copy (cDNA) from the appropriate mRNA by using the enzyme reverse transcriptase (Fig. 2). As an alternative, the DNA can be fragmented at specific target sequences with the help of restriction endonucleases [249]; the DNA fragment that contains the gene of interest is inserted into the purified DNA genome of a self-replicating genetic element, generally a virus or a plasmid. A DNA fragment containing a human gene, for example, when inserted into such a virus or plasmid, can be joined in a test tube to the chromosome of a bacterial cell. Starting with only one such recombinant DNA molecule which can infect only a single cell, the normal replication mechanism of the virus can produce more than trillion identical molecules in

Kapil Mehta et al.

55 Genomic DNA GENE

Restriction nuclease digestion ------

-----Transcription

mRNA Reverse Transcriptase

DNA Fragments

Alkali degradation Single-stranded DNA DNA Polymerase

Complementary DNA (cDNA)

pBR322

cDNA molecules are joined to vector DNA for propagation in bacteria

Selection of clone(s) carrying desired cDNA sequence, using a nucleic acid or antibody probe

Expression of cloned gene in host organisms such as bacteria, yeast or mammalian cells Figure 2. Gene cloning from mRNA or genomic DNA

less than a day, thereby amplifying the amount of the inserted human DNA fragment by the same factor, making it possible to infect millions of cells. The virus or plasmid used in this way is known as a cloning vector, and the DNA propagated by insertion into it is said to have been cloned. Cloning the gene or cDNA encoding a particular protein is only the first of many steps needed to produce a recombinant protein for medical and industrial use. Expression of foreign genes in a host organism requires vectors which contain specific control sequences governing transcription and efficient splicing of RNA. The most popular expression systems used for this purpose are the bacteria Escherichia coli and Bacillus subtilis, yeast, cultured insect cells and mammalian cells. The choice of expression system depends on the properties of the protein to be produced. Bacterial cells are most

common and convenient host organisms. They are simple to grow, have short generation times, provide large yields, and are most cost-effective. Particularly in the case of B. subtilis, the cells can be induced to secrete the product into culture medium, which facilitates the purification of the cloned protein. However, there are some disadvantages of using bacterial cells for gene expression (Table 1). Though most proteins are expressed in large amounts in bacteria, some of them fail to fold properly, leading to formation of insoluble ‘inclusion bodies.’ Protein extracted from these inclusion bodies is sometimes biologically inactive. Small proteins can be refolded into their native form, but larger ones containing several cysteine residues, in general, stay inactive, most likely because of the improper formation of disulfide bridges. Moreover, the expressed foreign protein is sometimes toxic to the bacteria, so that the culture

56

Recombinant proteins and genomics in cancer therapy

Table 1. Posttranslational processing of proteins in various expression systems Event

Bacteria

Proteolytic Cleavage Glycosylation Secretion Folding Phosphorylation Acylation Amidation % yield

+/−

Yeast

Insect cells

Mammalian cells

+

+

− +/− +/− −

+ + +/− +

+ + + +

+ + + +

− − 1–5%

+ − 1%

+ + 30%

+ + <1%

producing the protein cannot be grown to high cell density. This problem can be overcome by using an inducible promoter that is turned on to begin the transcription of the foreign gene only after the culture has been grown. Unlike eukaryotic cells, bacterial cells lack enzymes that catalyze posttranslational modifications of proteins such as phophorylation, acylation and glycosylation. These posttranslational modifications are sometimes essential for the normal functioning of a protein. Yeast cells may be preferable to bacteria for the production of some proteins biological by recombinant DNA procedures. Yeast is a simple eukaryote that resembles mammalian cells in many ways but, like bacterial cells, can be grown conveniently and economically. Yeast cells are capable of catalyzing many post-translational modifications that are found on mammalian proteins [126]. Also, they process the signal peptides needed for the secretion of protein and thus can be induced to secrete certain proteins into the growth medium for harvesting. Moreover, unlike bacteria, yeast cells do not have endotoxins. However, yeasts do produce active proteases that can degrade the foreign proteins, thereby reducing the yield of the final product. To overcome this problem, the construction of yeast strains with deleted protease genes is being attempted. Until recently, the expression of a biologically active protein from a complex eukaryotic gene in large amounts was a problem. The problems with prokaryotic expression systems have already been described. Even in yeast (eukaryotic) expression systems, the low biological activity of complex eukaryotic proteins remains a common problem. Mammalian expression systems provide all the post-translational modifications that may be necessary for full activity of eukaryotic proteins, but the yields from these systems are generally much lower than can be obtained from E. coli or yeast. Recently, protein yields as high as 10 mg/l have been obtained by

using mammalian expression systems [61], but this process is often lengthy and costly. Expression of foreign proteins in cultured insect cells by baculovirus vectors is an alternative to this problem. This relatively new system is becoming the system of choice for expressing mammalian and viral proteins. Baculovirus promoters are among the strongest known and can drive the expression of target genes at high levels (1–500 mg/l). The baculovirus expression system offers several other advantages over prokaryotic, yeast, and mammalian expression systems [178]. Some of the advantages include: high-level expression, correct folding, the ability to catalyze post-translational modifications like mammalian cells (Table 1), quick and easy growth in monolayers or suspension cultures without CO2, and safest – they do not infect humans, animals or plants. More than 400 different proteins have been expressed by using baculovirus vectors, and in most cases the protein produced is similar in structure, biological activity and immunological reactivity to the authentic protein [177]. Although the cost of culturing insect cells is currently more than that of culturing bacteria or yeast, it is still significantly less than that of culturing mammalian cells. Despite the significant advantages of producing human proteins in heterologous host cells, in some cases the best method for producing a mammalian protein is a mammalian cell system. Transient expression in mammalian cells is often used to check the function of a newly cloned gene and as a quick method to assess the function of engineered proteins. The extracellular domains of cell-surface receptors have been engineered for secretion from cells by introduction of a stop codon into the gene before the sequence of the transmembrane domain. These soluble receptors, as we will discuss later in this chapter, are valuable reagents for the study of ligand binding and for screening receptor agonists or antagonists that may eventually be used as therapeutics themselves. Although transient transfections yield enough protein for laboratory experiments, stably integrated amplified genes in mammalian cells are necessary for large-scale production of proteins [2]. With this goal in mind, great improvements have been made in recent years in identification of appropriate promoters, vectors, transformation protocols and host cell systems.

Recombinant Proteins as Cancer Therapeutics Biotherapy, as an alternative to the rigorous cytotoxic regimens used in the treatment of various malignant disorders, offers distinct and attractive advantages over

Kapil Mehta et al.

57

Antiproliferative Anti-angiogenic IFN TNF TGF Amphiregulin Oncostatin M Lymphotoxin FAS/APO ligand

Differentiation CSFs IFN-gamma TNF LIF EPO

Anti-VEGF antibody Endostatin Angiostatin

Approaches to the Biological Therapy of Cancer

Immunomodulatory

Cytotoxic Immunotoxins antibodies Fas/APO

IFNs TNF

Figure 3. Model for recombinant DNA-derived protein therapeutics for cancer

conventional chemotherapy. Biological therapy is based on the principle of stimulating the body’s own immune response and/or using biological substances against a disease. A perception of the malignant cell as one whose differentiation has been blocked due to the lack, deficiency or mutation of some key element led to the emergence of a biological therapy. The strategy of biological therapy contrasts with the immediate cell death induced by cytotoxic drugs, where there is no attempt to restore homeo-stasis. Biotherapy may offer the opportunity to use relatively nontoxic agents, the body’s own elements, to correct the underlying problem. It has long been recognized that the immune system plays a pivotal role in the patient’s response to disease. Therefore, recent cancer therapies have been directed at modulation of components of the immune system (Fig. 3). In particular, “immune messengers” or cytokines may play a vital role in regulating host antitumor defense mechanisms. Knowledge of cytokines and their functions is expanding rapidly [3, 6, 15, 122, 123, 126, 135, 179]. In the following section we will discuss the potential role of cytokines in the treatment of malignant disease.

on target cells and regulate the proliferation, differentiation and metabolism of either the same cell (autocrine) or another cell (paracrine). Cytokines are different from endocrine hormones in that they are produced by any number of different cells rather than by specialized glands. Different cytokines exhibit considerable overlap in their biological activities. Because of the advent of recombinant DNA technology, cytokines have become available in highly pure form and in sufficient quantities. This has accelerated the elucidation of in vitro and in vivo biological activities of these proteins and led to their rapid testing in the patients as therapeutic agents for the treatment of cancer. The cytokines that have been shown to have therapeutic potential in cancer include the interferons (IFNs), interleukins (ILs), colony- stimulating factors (CSFs), and tumor necrosis factors (TNF). Table 2 lists some recombinant cytokines that are currently being used for the treatment of malignant diseases.

Cytokines

Interferons are a family of regulatory glycoproteins produced by many cell types in response to viral infections and a variety of mitogenic and antigenic stimuli. Three major classes of IFNs have been described: IFN-α and IFN-β, which share components of the same receptor

In general, cytokines are low molecular weight (10– 50 kDa) proteins secreted by cells of the immune system that bind with great specificity and affinity to receptors

Interferons

58

Recombinant proteins and genomics in cancer therapy

Table 2. Cytokines Approved or in Development for Human usea Cytokine

Target disease

EPO

Countries approved

r.Protein biotherapeutics approved by FDA for human use IFN-α Chronic myelogenous leukemia USA, Europe, Japan Hairy cell leukemia USA, Europe, Japan AIDS-related Kaposi’s sarcoma USA Chronic non-A/non-B/C USA hepatitis Condylomata acuminata USA Lymphoma Essential thrombocythemia Melanoma Certain solid tumors Multiple myeloma Acute hepatitis B IFN-β Renal cell carcinoma Europe Advanced solid tumors Soft tissue carcinoma Adult T cell leukemia IFN-γ Chronic granulomatous USA disease (CGD) Advanced solid tumors Renal cell carcinoma Adult T cell leukemia Chronic myelogenous leukemia Lepromatous leprosy IL-2 Renal cell carcinoma Europe Metastatic melanoma Japan, Europe Advanced malignancies USA G-CSF Non-myeloid malignancies associated with chemotherapy-induced myelosuppression Myelodysplastic syndromes Severe chronic neutropenia (cyclic, idiopathic, congenital) Acute myelogenous leukemia Bone marrow transplantation GM-CSF Autologous bone marrow USA, Japan transplantation for nonHodgkin’s lymphomas, Hodgkin’ s disease, acute lymphocytic leukemia Allogeneic bone marrow transplantation for leukemias Myelodysplastic syndromes/ aplastic anemia Cancer chemotherapy associated myelosuppression Acute myelogenous leukemia AIDS, anti-AIDS drug treatment Associated myelosuppression

Anemia associated with chronic USA renal failure Malignancy, chemotherapy AIDS, AZT treatment rheumatoid arthritis anemia of prematurity Autologous blood donation prior to surgery Compensation of surgical blood loss Hepatitis B hepatomas surface antigen

r.Protein biotherapeutics currently under clinical trials IL-1αβ Malignant disorders Bone marrow transplantation Severe aplastic anemia Allotransplant patients IL-3 Advanced neoplasms Secondary hematopoietic failure Bone marrow recovery Breast cancer IL-4 Metastatic renal cell carcinoma Metastatic breast carcinoma Metastatic melanoma Disseminated cancer Advanced malignancies IL-7 Refractory solid tumors Metastatic melanoma Metastatic kidney cancer IL-11 Relapsed non-Hodgkin lymphoma Leukemia Melanoma IL-12 Lymphoma Advanced solid tumors (melanoma, neuroblastoma, breast cancer) IL-18 Ovarian carcinoma IL-21 Melanoma Ovarian carcinoma IL-29 Hepatitis C (associated with hepatocellular carcinoma) CSF-1 Endometrial carcinoma IFN-α Metastatic renal carcinoma, Metastatic melanoma Multiple myeloma Lymphoma TNF-α Advanced neoplasms Reduction of ovarian ascites M-CSF Solid tumors Breast cancer Fungal infections Acute myelogenous leukemia IL-1 AML, CML, sepsis, septic shock Receptor antagonist a

Disease in bold indicates approved use of cytokine.

Kapil Mehta et al.

59

and are referred to as type I; and IFN-γ, which uses a separate receptor system and is referred to as type II (Table 3). Additional IFNs have been discovered, but they are not well characterized [259]. Crude IFN was probably the first cytokine used in patients and was shown to delay recurrent growth of tumors in patients who had undergone surgery for osteogenic sarcoma [282, 283]. Pharmacological doses of partially purified IFN-α were reported to induce regression of tumors in significant numbers of patients with metastatic breast cancer, low-grade lymphoma, or multiple myeloma [110]. In 1981, IFN-α was successfully cloned by two groups, and the purified protein was immediately tested in the clinic. This was the first study of a recombinant cytokine in patients with cancer and, for the most part, the biological activity seen with the partially pure natural form was reproduced with the recombinant DNAderived form [111]. Treatment with IFN-α was also Table 3. Recombinant DNA-derived proteins relevant to treatment of cancer Cytokine

Receptor(s) used

Reference

Interferon-α (IFNα) Interferon-ß (IFN-β) Interferon-γ (IFN-γ) Transforming growth factor-β (TGF-β) Thymosin α1 Epidermal growth factor (EGF) Platelet derived growth factor (PDGF) Fibroblast growth factor (Basic) (bFGF) Fibroblast growth factor (Acidic) (aFGF) Transforming growth factor-a (TGF-α) Insulin like growth factor(IGF-I & II) Hepatocyte growth factor (HGF) Macrophage colonystimulating factor Granulocyte colonystimulating factor GM-colony stimulating factor Leukemia inhibitory factor (LIF) Stem cell factor (SCF) Erythropoietin (EPO) B cell growth factor Hepatitis B surface antigen

IFNα/β-R IFNα/β-R IFNγ-R TGF-β -R1 and -RII

[2, 225] [293] [103, 241, 261] [2, 56]

EGF-R

[329] [2, 101, 261]

PDGF-RI and RII

[116]

FGF-R

[98]

FGF-R

[98]

EGF-R

[57]

IGF-RI and IGF-RII

[54]

HGF-R

[199]

M-CSF-R

[2, 145]

G-CSF-R

[2, 120, 276]

GM-CSF-R

[2, 335]

LIF-R

[2, 99]

SCF-R EPO-R BCGF-R ?

[2, 331] [2, 78] [260] [65, 314]

shown to induce remissions in some patients with welldifferentiated B-cell tumors [282]. The most remarkable effects of recombinant IFN-α were observed in patients with hairy cell leukemia [237]. Treatment with partially purified or recombinant IFN-α suppressed peripheral blood cell production and rapidly increased platelet counts in these patients [112]. Their immune status improved and the number of leukemia cells in the bone marrow and blood declined. These patients stopped having opportunistic infections and required no further platelet and erythrocyte transfusions. These studies led to the approval of IFN-a for treatment of hairy cell leukemia in June 1986 by the United States Food and Drug Administration (FDA), an action adopted by regulatory agencies from 31 other countries. The mechanisms of IFN-α action in the compromised survival of the patients with hairy cell leukemia are not fully understood but may include differentiation, cell-cycle arrest and/or apoptosis. Other B-cell neoplasms also show variable degrees of sensitivity to IFN-α [110, 111, 282]. In patients with multiple myeloma or low-grade lymphoma, the cytokine often has demonstrated a positive clinical impact on survival when combined with chemotherapy [182, 272, 275]. Clinical results with IFN-α in patients with chronic myelogenous leukemia (CML) have been rather interesting. During the chronic phase of CML, IFN-α treatment causes hematologic remission [112]. Approximately 75% of the patients in the benign phase of the disease achieve complete normalization of blood counts. Moreover, IFN-α had the astonishing capacity to suppress selectively the cells bearing the Philadelphia chromosome, resulting in partial or complete restoration of the normal clone [290]. While showing great promise for leukemia, IFN-α therapy of solid tumors has been rather discouraging. Only 10–15% of patients with renal cell carcinoma or malignant melanoma undergo regression in response to IFN-α. However, the responses of carcinoid tumors to the cytokine were encouraging. A majority of patients showed improvement in symptoms, and a smaller fraction experienced tumor regression [210]. Both squamous and basal cell carcinomas of the skin show sensitivity to IFN-α, as a single agent or in combination with retinoids [170, 229]. Mycosis fungoides, a malignancy of the T helper cells, is also sensitive to IFN-α alone or with other modalities including retinoids [140, 247]. Kaposi’s sarcoma, an angioproliferative disease that commonly develops in individuals infected with human immunodeficiency virus (HIV), has responded well to IFN-α therapy: 40% of patients experienced significant regression of lesions [282]. This work led to the approval of

60 IFN-α in the U.S. and 21 other countries for the treatment of AIDS-related Kaposi’s sarcoma. After isolation of its gene and 10 years’ work identifying several of its biological activities, IFN-α was tested in human subjects for a wide variety of diseases including cancer. It was found to have impressive effects against chronic granulomatous disease, leading to its approval by the FDA for human use [298]. More recently, IFN-β was approved for treatment of ambulatory patients with relapsing/remitting multiple sclerosis [4]. Clinical toxic effects associated with administration of IFNs and other cytokines are summarized in Table 4. The IFNs, like most other cytokines, are produced by the body to act locally. When used as a systemic pharmaceutical, they can have substantial toxic effects [282].

Recombinant proteins and genomics in cancer therapy Table 5. Interleukins with relevance to cancer treatment Interleukin

Receptor

Reference

Interleukin-1-α (IL-1α)

IL-1R type I; IL-1R type II IL-1R type I; IL-1R type II IL-1R type I; IL-1R type II IL-2R . α, IL-2R . β, IL-2R .γ IL-3R . α, IL-3γ IL-4R, IL-2R . γ IL-5R . α, IL-5R . γ IL-6R, gp130 IL-7R (CDw127), IL-2R . γ IL-8R, Type I & Type II IL-9R, IL-2R R . γ; IL-10R1; IL-10R2 IL-11R, gp130 IL-12R IL-13R . α, IL-4R, IL-2R . γ IL-15Rα, IL-2R . β, IL-2R . γ CD4 IL-17R IL-18R

[59]

Interleukin-1-β (IL-1ß) Interleukin-1 receptor antagonist Interleukin-2 (IL-2) Interleukin-3 (IL-3) Interleukin-4 (IL-4) Interleukin-5 (IL-5) Interleukin-6 (IL-6) Interleukin-7 (IL-7) Interleukin-8 (IL-8)

Interleukins The interleukins are a family of cytokines that are essential to both cellular and humoral immune responses. To date, at least 34 interleukins have been identified and cloned (Table 5). Many of these molecules exhibit

Interleukin-9 (IL-9) Interleukin-10 (IL-10) Interleukin-11 (IL-11) Interleukin-12 (IL-12) Interleukin-13 (IL-13) Interleukin-15 (IL-15)

Table 4. Common toxic effects associated with administration of cytokines Fever Chills Nausea Vomiting Headache Anorexia Fatigue Myalgias Arthralgias Bone pain Flush Local erythema Inflammation at the site of injection Capillary leak syndrome Granulocytopenia Thrombocytopenia Anemia Hypotension Liquid accumulation in the lung, spleen, kidneys Reversible increase in body weight Reversible increase in the serum creatinine Oliguria Malaise Asthenia Rigors Diarrhea Hepatocytotoxicity Lethargy, depression Mental confusion EEG-abnormalities

Interleukin-16 (IL-16) Interleukin-17 (IL-17) Interleukin-18 (IL-18)/ IGIF Interleukin 19 (IL-19)a Interleukin-20 (IL-20) Interleukin-21 (IL-21) Interleukin-22 (IL-22) Interleukin-23 (IL-23) Interleukin-24 (IL-24) Interleukin-27 (IL-27) Interleukin-32 (IL-32)

IL-19R IL-20Ra. & IL-20Rβ IL-21R IL-22R IL-12Rα1, IL-12Rβ2 IL-20R1,IL-20R2, IL-22R, IL-22R2 IL27RA

[59] [17] [162] [131] [205] [288] [8] [46] [128, 193] [239, 240] [188] [64, 222] [304] [187, 347] [90] [49] [166] [200, 201] [86] [24] [217, 353] [149, 336–349] [213] [347, 354, 355] [349, 350] [351, 352]

a

IL-19 is a novel homologue of human IL-10.

antineoplastic activity. Currently, IL-2, IL-12, IL-15 and IL-18 are the most potent inducers of anti-tumor activity in preclinical and clinical studies. Recently, new interleukins have been characterized, including IL-21, IL-23, IL-24 and IL-27, which exert potent immunomodulatory and anti-tumor effects in vitro and in vivo settings, indicating that they may have considerable potential as future cancer therapeutics [348]. Interleukin 1 (IL-1): IL-1 was originally described as an “endogenous pyrogen” in 1940 because of its ability to cause fever when injected into animals. Two forms of

Kapil Mehta et al. IL-1 are now recognized: IL-1a is cell-associated and is involved in antigen presentation, whereas IL-1β, the predominant form, is readily secreted by macrophages. Though the two forms have limited amino acid homology, IL-1α and IL-1β bind to the same receptor and share several biological properties. Other cell types that produce IL-1 are endothelial cells, keratinocytes, neutrophils and B lymphocytes. Constitutive expression of IL-1 occurs in cells lining the external environment, i.e., skin and mucosal surfaces [58]. The cDNAs coding for both human IL-1s were reported in 1985 [39, 83]. Recombinant IL-1s induced fever, hepatic protein synthesis, production of prostaglandin E2, cartilage breakdown, bone resorption, and elevated ACTH and augmented the T lymphocyte response to antigens and mitogens [58]. Recombinant IL-1 exhibits cytostatic activity toward human melanoma tumor cells in vitro and direct cytotoxic effects against human melanoma cell line A375 [157]. Recently, recombinant IL-1 was shown to enhance the recovery of platelets in ovarian cancer patients following carboplatin therapy, suggesting a potential role for IL-1 in attenuating thrombocytopenia associated with chemotherapy [309]. Interleukin-2 (IL-2): IL-2 is a 15.5-kDa glycoprotein produced by peripheral blood lymphocytes and is a potent growth factor for activated T lymphocytes. It acts as a cofactor in development of cytotoxic T lymphocyte activity against tumors and has been shown to participate in tumoricidal activity by inducing the growth of natural killer (NK) cells and lymphokine-activated killer (LAK) cells [242]. Several cancers show sensitivity toward recombinant IL-2, both in animal models and in the patients. LAK cells are peripheral blood lymphocytes that can be generated in vitro by incubation with high doses of IL-2. They have the ability to kill tumor cells specifically while leaving normal cells unharmed. IL-2 has been used in combination with LAK cells to achieve more potent antitumor response [242–245]. In 1985, Rosenberg and associates published the results of their first study documenting tumor regression in patients with melanoma following administration of IL-2 and LAK cells [243]. An update of the results in 180 patients was published in 1991 [242]. Antitumor responses were seen in patients with advanced melanoma, renal cell cancer, colon cancer or non-Hodgkin’s lymphoma. Like LAK, tumor-infiltrating lymphocytes (TILs), the lymphoid cells that infiltrate solid tumors, can be grown in vitro in the presence of IL-2. These cells have unique lytic activity against autologous tumors. Treatment with TILs in combination with IL-2 was shown to mediate substantial tumor regression in some patients with advanced malignant melanoma [174]. Objective responses were

61 observed that lasted for 3–14 months in 29% patients with renal cell cancer and 23% of those with melanoma. Further potential of IL-2 in cancer therapy has been demonstrated by using recombinant IL-2 in combination with other cytokines [244, 306]. For example, recombinant IL-2 in combination with IFN-α elicited a potent antitumor response in several animal tumor models [175]. The most significant antitumor activity seen with IL-2 therapy has been in malignant melanoma and renal cell carcinoma. Other tumors treated with IL-2 include glioma, bladder carcinoma, ovarian carcinoma, neuroblastoma, lung carcinoma, head and neck carcinoma, breast carcinoma, lymphoma, colon carcinoma and mesothelioma [123]. As shown in Table 4, IL-2 administration in patients is associated with a wide range of toxic effects, the most common being fluid retention, anemia, thrombocytopenia and hypotension [175]. IL-2 is the first cytokine that has been employed so widely in clinical trials. However, because of its toxic effects, its clinical use has been limited. Interleukin-3 (IL-3): Recombinant IL-3 is a 15–17kDa polypeptide that is known to stimulate mast cells, neutrophils, macrophages and megakaryocytes. No direct antitumor activity has been observed for IL-3, but this cytokine has a role in increasing platelet and neutrophil counts in patients with advanced malignancy [87]. Phase I and II clinical trials with recombinant IL-3 have been carried out in patients with advanced malignancy. A dosedependent increase in platelet counts and substantial increases in the numbers of circulating neutrophils, eosinophils, monocytes and lymphocytes were observed in these patients [87]. Hematopoietic failure caused by prolonged chemotherapy, radiotherapy or infiltration of bone marrow by tumor cells could be restored by recombinant IL-3 treatment. The side effects of rIL-3 therapy in patients include fever, bone pain and headache [87]. Thus, recombinant IL-3 is a multilineage hematopoietic cytokine with promising effects on platelet and neutrophil counts. Interleukin-4 (IL-4): IL-4 is a T cell-derived glycoprotein of 20 kDa. The gene for human IL-4 has been cloned [16]. The antitumor functions of IL-4 include increased T cell, NK cell and monocyte proliferation. IL-4 has also been shown to enhance the generation of cytotoxic T lymphocytes and to participate in induction of LAK cell activity, and to synergize with I-2 in this activity [194]. Furthermore, IL-4 can stimulate the generation of TILs in human melanoma, increase the antigen-presenting ability of mouse and human monocytes and augment the expression of tumoricidal activity in murine macrophages. It appears to inhibit the release of TNF, IL-1 and IL-6 by human monocytes [296].

62 Interleukin-4 has been shown to exert potent antitumor activity against several transplantable tumors in a murine model. Using IL-4-transfected tumor cells, the potential of transfecting lymphokine genes into tumor cells as a method of cancer therapy has been demonstrated [93]. Because of its antitumor effects in vitro and in murine models, IL-4 may be useful in inhibiting the growth of solid tumors and cell lymphomas.

Other Interleukins The family of interleukins has continued to grow and new members have been included (Table 5). IL-18 is the most recently cloned member of this family [200], which is synthesized as a 24 kDa proform that is processed into an 18 kDa mature form. The mature form of IL-18 induces IFN-gamma secretion and plays an important role in antitumor immunity [219]. IL-18transgenic mice show increased CD8+ CD44 high T cells and macrophages, while B cells are decreased in these mice [129]. Similarly, IgE, IgG1, IL-4 and IFN-γ levels are substantially increased in the sera of IL-18transgenic mice. In contrast, the IL-18 knock out mice exhibit impaired clearance of neurovirulent influenza A virus-infected neurons from the brain [190]. Another relatively newer member of the interleukin family is IL-15 that acts as a strong cofactor for Th1 T cell development and as a growth factor for T lymphocytes and TILs [112]. Like IL-2, it binds to the beta and gamma chains of the IL-2 receptor to exert its action [100]. However, specificity for IL-15 versus IL-2 resides in the unique private α-chain receptors that complete the IL-15Rαβ.g and IL-2αβγ heterotrimeric high-affinity receptor complexes and thereby allow differential responsiveness depending on the ligand and high affinity receptor expressed [323]. A recent review discusses the role of IL-15 in human diseases and its potential clinical implications as a therapeutic target [70]. Other important members of the interleukin family include IL-5, IL-6, IL-7, IL-12, IL-21 and IL-24. IL-5 is an 18-kDa product of T lymphocytes that has been cloned and shown to be a lineage-specific eosinophil growth and differentiation factor [30, 250]. Murine IL-5 induces antibody-dependent killing of tumor cells by blood eosinophils and enhances phagocytosis by eosinophils. Interleukin-6, initially described as β2-interferon, is a 19–28-kDa protein produced by a variety of cells, including mononuclear phagocytes, fibroblasts, keratinocytes and endothelial cells. The experimental data support a potential clinical role for IL-6. Its hematopoietic activity and thrombopietic activity, in particular, may make this cytokine a useful agent for inducing the

Recombinant proteins and genomics in cancer therapy recovery of bone marrow in patients with myelosuppression that usually follows aggressive chemotherapy regimens. The results of phase I clinical studies of IL-6 have recently been reported [325]. IL-7 is a 22 to 25-kDa glycoprotein that was originally characterized on the basis of its ability to promote the growth of precursor B lymphocytes [96]. Recombinant IL-7 induces the proliferation of both thymocytes and mature T cells and is known to activate macrophages for tumor cell killing. In human monocytes, IL-7 induces the expression of IL-8, IL-6, IL-1a, IL-1.b and TNF-α. IL-12, a product of B cells and mononuclear phagocytes, has multiple effects on both T cells and NK cells. It induces IFN-γ production in T and NK cells and sustains the cell-mediated immune response [304]. Interleukin-21 (IL-21) is a cytokine with structural and sequence homology to IL-2 and IL-15 that has antitumor activity alone in experimental tumor models and a tolerable safety profile in phase I trials in patients with metastatic melanoma and renal cell carcinoma [353]. Several monoclonal antibodies targeted at tumor-associated antigens also have improved antitumor activities in mice when used in combination with IL-21, suggesting that the use of IL-21 in adjuvant settings induces a strong T cell-mediated immune responses [353]. Interleukin-24 (IL-24) is a cytokine belonging to the IL-10 family of cytokines that signals through two heterodimeric receptors (IL-20R1/IL-20R2 and IL-22R1/ IL-20R2). IL-24, a recently identified cancer gene therapeutic, is melanoma differentiation associated gene-7/ IL-24 (mda-7/IL-24) that has the unique ability of inducing apoptosis in a variety of cancer cells with no toxicity to normal cells or tissues. It exerts immunomodulatory, anti-angiogenic, and radiosensitizing effects when applied as adenovirus expressing mda-7/IL-24 (Ad.mda-7) in human xenograft animal models and has been successfully used in a Phase I clinical trial in patients with advanced cancers [347, 354].

Colony-Stimulating Factors The colony-stimulating factors (CSF) are a family of glycoproteins that have the ability to induce proliferation and differentiation of progenitor hematopoietic cells and have effects on the functional status of their mature progeny. Multi-colony stimulating factor (IL-3), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), stem cell factor (SCF), and leukemia inhibitory factor (LIF) are some of the clinically important members of this family (Table 3). All the

Kapil Mehta et al. known CSFs have been produced by recombinant DNA methods and tested in human subjects (Table 2). The potential for using hematopoietic growth factors in the treatment of disease is enormous. Their ability to control the production of blood cells has been realized, and the results of clinical trials to date suggest that the side effects of these growth factors are relatively minor [329]. Three recombinant hematopoietic growth factors, G-CSF (filgrastim), GM-CSF (Sargramostim), and EPO (epoetin alfa), are now commercially available for clinical use in the United States. Extensive clinical and preclinical data on recombinant human G-CSF and GM-CSF indicate that both these cytokines are effective in accelerating neutrophil recovery and shortening the duration of neutropenia following chemotherapy with or without bone marrow transplantation [48, 77, 185, 193, 206]. Administration of recombinant human G-CSF as an adjunct to cyclophosphamide, doxorubicin and etoposide chemotherapy for small cell lung carcinoma significantly reduced duration and severity of neutropenia and associated clinical sequelae [303]. Similarly, patients with transitional cell carcinoma of the bladder treated with methotrexate, vinblastin, doxorubicin and cisplatin experienced up to fourfold increases in neutrophil count on administration of G-CSF with few or no toxic effects [84]. GM-CSF may also exert antitumor effect by inducing tumoricidal activation of macrophages. Administration of GM-CSF has been shown to decrease the tumor burden in a murine Lewis lung carcinoma model [121]. Clinical trials of M-CSF have been performed in an attempt to ameliorate leukopenia. A phase I trial of M-CSF in patients with metastatic melanoma showed an increase in the number and function of circulating monocytes [18]. In a non-randomized, controlled study (32 patients with urinary tract malignancies) and a randomized controlled study (98 patients with gynecological malignancies), M-CSF administration reduced the period of post-chemotherapy leukopenia [192, 207]. Among the cytokines whose role can be predicted from in vitro studies, EPO is perhaps the best example. EPO is produced mainly by the kidneys and is responsible for regulating the production of erythrocytes. EPO acts on erythroid precursors in the bone marrow, spleen and fetal liver and stimulates the colony formation of the burst-forming unit-erythroid. When infused in mice, EPO markedly increases both peripheral blood erythrocytes levels and the number of erythroid progenitor cells present in bone marrow. These results led to the clinical trials with a human recombinant EPO, the findings suggested that EPO can reverse anemia in patients with end-stage renal cell disease. EPO produced dose-

63 dependent increases in hematocrit and hemoglobin levels, and in most cases eliminated the need for regular blood transfusions [67]. The major side effect reported is increased blood pressure. EPO also increases the ability of patients undergoing elective surgery to donate autologous blood [94]. Double-blind placebo-controlled studies with recombinant EPO suggested that it is an effective treatment for predialysis patients [168]. Stem cell factor (SCF) has recently been used in the clinic as a single agent following chemotherapy. SCF by itself appears to have limited efficacy and significant toxicity-mainly due to mast cell stimulation at higher doses. However, Tong et al. [300] showed that patients receiving CSF have an increase in primitive progenitor cells suggesting that SCF might be highly effective in combination with later acting hemopoietins. From these data it is clear that recombinant CSFs are effective in correcting hematopoietic disorders of various etiologies. Whether these mediators improve morbidity and mortality in patients will be decided by further clinical results. However, combinations of cytokines, for example, those with relatively restricted biological activity (EPO, G-CSF, M-CSF etc.) and those that have a broad range of action (SCF, IL-3, GM-CSF, IL-6 etc.), are likely to show more promising effects on hematopoiesis than any single cytokine alone [114].

Tumor Necrosis Factors Tumor necrosis factor (TNF) is a proinflammatory cytokine that is produced primarily by mononuclear phagocytes in response to endotoxin. In recent years, several new members belonging to the TNF superfamily of molecules has been described and the list has continued to grow (Table 6). There are two most studied forms of TNF; TNF-α is a cytotoxic factor with a molecular weight of 17-kDa, and TNF-β, also known as ‘lymphotoxin,’ has a molecular weight of 25-kDa and is released from stimulated lymphocytes [102, 203, 204, 223]. Both forms have been produced by recombinant DNA technology and appear to have antiproliferative, cytostatic and cytolytic effects against human tumor cells in vitro as well as in vivo when injected into nude mice [212]. TNF-α exerts synergistic effects with different cytokines, such as IFNs, IL-1, IL-2 etc., and can induce secretion of series of mediators, including IL-1, IL-6, prostaglandins etc. It has been implicated in both the generation and the cytotoxicity of LAK cells and cytotoxic T lymphocytes [37]. Based on these observations, a large number of phase I and II studies were initiated to investigate the antitumor properties of TNF-α. Unfortunately, in clinical settings the efficacy of TNF-α has been very limited,

64

Recombinant proteins and genomics in cancer therapy

Table 6. Members of the TNF family and their receptors Cytokine

Other names

Receptor

TNF-α LT-α3 LT-β1α2 LIGHT

TNFR1 (CD120a); TNFR2(CD120b) (TNF-β) LTβR (HVEML)

TNFR1,TNFR2

OX40L 4-1BBL

CD134L CD137L

CD27L CD30L CD40L FasL TRAIL

CD70) CD153 CD154, TRAP, gp39) Apo-1L, CD95L Apo-2L

RANK

TRANCE, OPGL

TWEAK APRIL

Apo-3L TALL-2

BAFF TALL-1, zTNF4) GITRL EDA-A1 EDA-A2 ? VEGI ? 75NTR ?

AITRL

HVEM (TR2, ATAR, LIGHTR), LtbR, DcR3 OX40 (CD134) 4-1BB, TNFRSF9, CD137, ILA CD27 CD30 CD40 Fas (Apo-1, CD95); DcR3 TRAIL-R1 (DR4, Apo-2) TRAIL-R2 (DR5, KILLER) TRAIL-R3 (DcR1, TRID) TRAIL-R4 (DcR2, TRUNDD) OPG (OCIF) RANK (TRANCE-R) OPG (OCIF) Fn14 (TWEAK-R), DR3 BCMA TACI (BlyS, THANK, TACI MCMA GITR EDAR XEDAR TAJ (TROY); TRAIN ? DR6 NGFR (p75, NTR) RELT

APRIL, A proliferation-inducing ligand; BCMA, B cell maturation antigen; BAFF, B cell activating factor; BlyS, B lymphocyte stimulator; THANK, TNF homologue that activates apoptosis, NF-kB and JNK; TACI, Transmembrane activator and CAML-interactor; TWEAK, a weak homologue of TNF; HVEM, herpes virus enetery mediator; LIGHT, homologous to lymphotoxin, shows inducible expression and competes with HSV glycoprotein D for HVEM; TRANCE, TNF-related activation-induced cytokine; RELT, receptor expressed in lymphoid tissues. For details, see references [5, 52, 109, 262, 269, 324, 339, 340].

and its use is associated with serious toxic effects [125]. Of 127 eligible patients enrolled in nine different phase II protocols between 1988 and 1990 for the treatment of diverse malignancies, including breast, colon, gastric, pancreatic, endometrial, and bladder cancers, multiple myeloma and sarcomas, only one patient responded (response rate, 0.8%), whereas 13% experienced grade four or fatal toxic effects. Despite the initial disappointing results with TNF-α as an antitumor agent, investigators have continued working on new and improved approaches

for its use. In a recent study, Lienard and co-workers [167] used an intra-arterial route to administer of high doses of TNF-α in conjunction with melphalan, hyperthermia and IFN-γ. Of the 23 patients treated (19 with melanoma and four with sarcoma), all responded, 21 with complete remission and two with partial remission. Eleven of these patients were previously unresponsive to melphalan alone and one had failed to respond to cisplatin therapy. The toxic effects observed (neutropenia, hypotension, thrombocytopenia and kidney failure) were reversible. The overall rate of survival was 70% and of disease-free survival over 12 months, 76%. These results suggested that further understanding of the mechanisms of TNF-α’s antitumor action could help improve its clinical efficacy. For example, decreasing the agent’s systemic toxicity without reducing its anticancer effects could lead to substantial therapeutic advantages. TNF-α mediates its effects by interacting with two different surface receptors, p55 and p75 [296]. Studies have suggested that TNF-α’s interaction with p75 may be responsible for its systemic toxicity [118, 296]. Thus, mutant TNF molecules that interact with p55 but not p75 could induce antitumor effects with reduced systemic toxicity, permitting higher doses of TNF-α. Indeed, such mutant human TNF molecules that specifically bind to p55 have already been described and shown to exert cytotoxic effects against transformed cells in vitro [214]. In addition, concomitant use of drugs that are able to decrease TNF-α systemic toxicity could permit use of higher, more effective doses of this cytokine in cancer therapy. Combination regimens of TNF-α with other cytokines, concomitant use of TNF toxicity inhibitors and use of mutant TNF molecules may provide better clinical outcomes. Moreover, regional therapy with TNF-α requires further exploration in view of the fact that such regimens have already produced some very promising results [167].

Soluble Cytokine Receptors Certain membrane receptors are enzymatically cleaved from the cell-surface and released into the extracellular medium in the form of soluble fragments. Soluble receptors corresponding to the ligand-binding domains of many polypeptide hormones and cytokine receptors have been described (Table 7). The function of soluble receptors is not yet known [73, 115]. However, it is likely that this process represents an important mechanism for regulation of surface expression of such receptors and may determine the effects of cytokines and growth hormones on the target cells. For example, the cell growth, differentiation and immunomodulatory effects

Kapil Mehta et al.

65

Table 7. List of soluble receptors identified for various cytokines Receptor Proteolytic cleavage: IL-1R IL-2R IL-4R (murine) IL-6R IL-6R (gp 130) TNFR IFN-α R IFN-γ R NGFR M-CSFR Hergulin R EGFR EPOR PDGFR Alternative splicing: IL-4R IL-5R (murine) IL-7R LIFR GM-CSFR G-CSFR EGFR c-erbB3

Reference [115, 267, 268, 279] [274] [74] [195, 198] [195, 198, 202] [12, 13, 88, 139] [208] [209] [60] [62] [161] [108] [21, 196] [299] [191] [178] [95] [159] [251] [79] [228] [143]

of cytokines are exerted in response to their binding to specific cell-surface receptors. The presence of soluble receptors in the biological milieu may thus promote direct binding of the ligand to the soluble receptor, neutralizing and preventing its action. The in vivo relevance of soluble cytokine receptors is well illustrated by several viruses. Vaccinia and cowpox viruses encode a protein that displays homology with soluble IL-1 receptor and is able to bind IL-1β [279]. Furthermore, proteins that bind to TNF have been identified in the open reading frame of pox viral strains [274]. Herpes and myxoma viruses encode proteins that can effectively bind IL-8 and IFN-γ ligands, respectively [7, 307]. Such soluble receptors assist virus infection by suppressing host defense mechanisms. From the studies on induction of antiviral soluble LDL receptors by IFNs, it seems that host organisms make use of a similar mechanism for the opposite role of controlling viral infection. During the release of cell-surface receptors, it is usually the extracellular domain of the receptor that is shed; thus, soluble receptors act as inhibitors of cytokines. The soluble receptors may originate via two separate mechanisms, one involving alternate splicing in which a receptor gene lacks a transmembrane domain. As an alternative, the receptors can be shed from the cell-surface as a result

of activation of specific proteolytic enzyme or enzymes. The identity of enzymes involved in proteolytic cleavage of the receptors is not known; however, it is a highly regulated process and appears to be controlled by phosphatases and kinases [4]. The treatment of cells with ligand can also lead to down modulation of the receptors and their subsequent shedding. The significance of soluble cytokine receptors as a therapeutic modality for treatment of cancer will be determined by further research and evaluation. Since soluble receptors can provide highly specific biological inhibitors for cytokines and growth factors, and because the majority of transformed cells require cytokines for their growth and survival, soluble receptors may have therapeutic potential as antagonists to cytokine action. For example, hematopoietic growth factors are known to maintain the viability of hematopoietic cells through the prevention of apoptosis [332]. Several investigators have reported that autocrine production of hematopoietic growth factors such as IL-1β [82, 104] or GM-CSF [344] supports the growth and survival of acute myeloid leukemia (AML) cells in vitro. In contrast, their soluble receptors and receptor antagonist could inhibit the growth of leukemic cells including AML [343], chronic myelocytic leukemia (CML) [68], and juvenile CML [257]. Receptor proteins for most of the cytokines have been cloned and expressed [2]. However, the information available on their therapeutic potential in cancer is very limited. Like the soluble and membrane-bound forms of cytokine receptors, the cytokine ligands also exist in these two forms. For example, the cytokines IL-1, TNF, FGF, TGFα, TGF-β and SCF have been reported to exist in both the soluble and membrane-bound forms. This process, commonly initiated by cell stimulation, may regulate the surface expression of such cytokines and play an important role in determination of cytokine activity.

Genomics and Proteomics in Cancer Therapeutic Advances in the analytical tools and recombinant DNA technology have improved our understanding of the signaling pathways encoded in cytokine, growth factors and hormones [158, 85]. In most cases, the focus of such investigation is often a single molecular target. Compilation and integration of this voluminous literature demonstrates a high level of complexity in cell’s repertoire for the detection and processing of molecular signals. Despite all this information, the relative contribution of each and more importantly, the cross-talk among different pathways that influence

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Recombinant proteins and genomics in cancer therapy

the process of differentiation, proliferation or apoptosis remains unclear. Recent advances in the techniques for quantitative and comprehensive analysis of all the mRNAs (transcriptome, ∼40,000 unique molecules [53, 317]) or all the proteins (proteome, >40,000 unique proteins) of the cell, offer a promise for defining the cellular functions in terms of causative molecular changes. In the following sections we define some of the basic concepts of these recent techniques and their early contribution in our understanding of cancer cells. These early data suggest considerable molecular heterogeneity in an apparently homogeneous group of tumors classified by conventional tools, such as immuno- and histochemistry [92, 9]. Interpretation of such results offers possible explanation for the observed differences in the outcome of a specific anticancer therapy. In addition, the spectrum of molecular targets for a therapeutic agent can also be defined by the application of global gene expression analysis [181]. Such treatment specific mRNA profiles offer the opportunity to predict diverse effects of anticancer drugs and contribute towards selection and design of tumor specific therapies. The Fig. 4 represents some essential molecular players that are involved in gene expression pathways and are recruited during changes in cell proliferation, differentiation and apoptosis initiated by extracellular signals. mRNAs and proteins are obligatory intermediates in the Cytokines Micronutrients

Growth factors

activated transcription factors

Inactive transcription factors

Translocation to nucleus

? histone acetylases histonedeacetylases

Euchromatin (active)

heterochromatin (inactive)

RNA Splicing protein

mRNAs

cDNA arrays Oligonucleotide arrays SAGE Differential display Subtraction hybridization

Proteins

Proteomic analysis (2D separation-Mass Spec)

Figure 4. Molecular players involved in gene expression pathways

signal transduction pathways [277]. It is now abundantly clear that single transcription factors such as NF-κB which is translocated to the nucleus from cytoplasm in response to binding of a cytokine to its receptor, activates a large number of genes [19, 255]. Similarly intranuclear p53 [234, 345] and MYC [41] proteins also affect the expression of large numbers of genes that are important determinants of cell proliferation and cell death. Such large-scale gene profiling studies begin to address the relative importance of individual genes in the determination of cell’s destiny.

Oligonucleotide Arrays for Quantitative Analysis of >30,000 Unique mRNAs GeneChips commercialized by Affymetrix offer a reliable tool for the evaluation of distinct mRNAs present in extracts of total RNA or mRNA by selection with oligo dT. The GeneChips are unique with respect to at least two notable features: the first that they have oligonucleotide probes prepared by a combination of photolithographic technologies and combinatorial chemistry [169]. The second unique feature of GeneChips is the distribution of the probes on the glass slide. There are 16–20 pairs of antisense oligonucleotides arrayed on a glass slide for each of ∼30,000 unique mRNAs. Each pair of oligonucleotides contains a set of perfectly matching antisense sequence of 23 nucleotides and a set of identical sequence of nucleotides with one “mismatch” base at the 13th nucleotide. Hence each mRNA is probed 16–20 times for specific binding and an equal number of times for non-specific binding. A comprehensive statistical analysis of 16–20 pairs of data for each target then evaluates the specific mRNA to be either present or absent and gives a numerical value for absolute hybridization. A major disadvantage of the technique is its inability to detect low abundance mRNA. For example, in authors’ experience, the GeneChips were unable to detect mRNAs for nitric oxide synthase genes that could be detected by RT-PCR. The application of oligonucleotide arrays to human cancers have initiated a more detailed compilation of molecular changes that are cancer specific [285–328]. Such a data-base will prove useful in diagnosis and treatment of cancers.

cDNA Arrays are Dotted Arrays of PCRAmplified Products of Cloned Genes cDNA arrays of large numbers of genes were invented and pioneered in laboratories of Patrick Brown and David Botstein [28, 256]. Unlike oligonucleotide arrays,

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cDNA arrays are constructed by robotic dotting of PCR amplified fragments of cDNA on to glass slides or nylon membranes. The cDNA arrays of human genes have been used to define disease specific gene profiles. Such analyses have contributed towards a better understanding of cancer and normal cells [9, 10]. The techniques for constructing cDNA arrays are more readily available to laboratories and tissue or cell-specific arrays can be constructed in a specialized and dedicated core facility [254]. The application of cDNA arrays to various cancers cells ex vivo [254] and in vivo [92] have resulted in a better classification of cancers and will lead to improved paradigms of anti-cancer drug development in the near future.

methods for structural determination, and to improve methods for predicting protein structure. While efforts to improve the crystallographic and NMR techniques have continued to receive a major share of intellectual effort, structural prediction studies have received new impetus from the development of new models and methods [273]. The molecular modeling serves several functions: (1) it provides the tool to visualize protein structure in three dimensions; (2) it permits protein-to-protein comparisons; (3) predicts the structure of proteins; and (4) facilitates the computer-aided drug design. Such integrated information that can define the transcriptome and the proteome will help identify novel molecular targets for therapeutic interventions of malignant growth.

Proteomic Analysis and Cancer

Antibodies and Conjugates

One major aim of scientists is to understand mutations that can cause cancer. To accomplish this aim, a thorough understanding of the workhorse of all biological systems, the proteins, is required. Currently, a major effort is being made to understand protein–protein interactions. There is an equal emphasis on understanding the structural features of proteins and the structural basis of protein–protein, protein–ligand, and protein–drug interactions. However, initially lack of techniques that enable comprehensive and quantitative labeling and separation technology for proteins, have been limiting factors these efforts [1]. The separation of cellular proteins by two dimensional, denaturing gel electophoresis combined with determination of peptide sequence to identify proteins has been used to define changes in proteins in response to oxidative stress in yeast [91]. More recent developments of analytical techniques that use novel methods of labeling and separating large numbers of proteins [1] are beginning to define the pathways of flow of molecular information from the genome to the proteome [130]. Various stable isotope labeling techniques have recently emerged to improve the efficiency and accuracy of protein quantitation by mass spectrometry. We have developed a mass-tagging strategy to incorporate stable isotope tagged amino acids into cellular proteins in a residuespecific manner during cell growth [356]. The major emphasis for new drug development in the modern era is based on thorough understanding of the relationship between the drug and its target protein, whether it is an enzyme, receptor, structural, or transport protein. Detailed information regarding the physical, chemical, biochemical, genetic, and physiological characteristics of that relationship must be well understood. Hence, a major efforts is being devoted to enhance the pace and resolution of classical crystallographic and NMR

Antibodies are highly selective proteins that can bind to a single target among millions of irrelevant sites. Because of this specificity, the antibodies have been used extensively to target drugs, prodrugs, toxins and other agents to particular sites in the body. It is this use of antibodies as targeting devices that led to the concept of “magic bullets,” a treatment that could effectively seek and selectively destroy tumor cells wherever they resided. The major problem in the therapeutic use of antibodies was their production in large quantities, but the development of ‘monoclonal antibody’ technology changed the situation dramatically [113, 186, 333]. Monoclonal antibodies (MAb) are already widely used for the diagnosis and treatment of cancer and for imaging of tumors for radiotherapy. Despite the rapid progress being made in application of MAbs as therapeutic agents, their use has been limited because of their immunogenicity problem. MAbs are usually mouse proteins; when injected into patients they are eventually recognized as foreign proteins and cleared from the circulation. To overcome this problem, researchers set out to engineer fully “humanized MAbs” that will be indistinguishable from natural proteins [32, 334]. Humanizing MAbs is a technology that uses recombinant DNA techniques to improve or change the function of these antibodies [233]. The first fully humanized MAb recognizes an antigen on the surface of human lymphocytes and is being evaluated as an immunosuppresant and for treatment of lymphoid tumors. Another potentially useful humanized MAb recognizes a growth factor receptor in large numbers on the surface of several breast tumor cells. This MAb successfully inhibited tumor cell growth in culture and is currently being evaluated in patients [322]. In the following section, we will briefly discuss the potential use of Mab-based immunotherapies

68 that have been used for the treatment of malignant diseases. Detailed aspects of this approach will be discussed somewhere else in this book.

Monoclonal Antibodies as Agonists Antibodies directed against cell-surface molecules on many types of tumor cells can act as ligands, resulting in powerful antitumor effects mediated by signal transduction [320]. For example, MAb 4D5 against erbB-2, when added to breast or ovarian carcinoma cells that overexpress erbB-2, induces a strong antiproliferative effect [164]. The erbB-2 protein product is a member of the EGF receptor family and is shown to act as a signaling receptor for a recently identified ligand, heregulin, in regulation of growth and differentiation of breast cancer and other cell types [47]. Herceptin is a humanized monoclonal antibody directed against Her-2/neu protein that is abnormally abundant in 25–30% breast cancer patients [141]. In view of the encouraging results observed in Her-2/neu positive breast cancer, Herceptin (trastuzumab) was recently approved by the FDA for treatment of metastatic breast cancer [165]. Data from pivotal trials suggested that trastuzumab is active when added to chemotherapy in patients with advanced metastatic breast cancer [271]. In particular, the combination significantly prolonged the median time to disease progression, increased overall response rate, increased the duration of response, and improved median survival time by approximately 25% compared with chemotherapy alone [271, 22]. As a single agent, trastuzumab induces durable objective response in women with HER-2 positive metastatic breast cancer and is rapidly becoming a standard of care for these patients [23]. Similarly, the ligation of CD95 (APO-1/Fas) protein with an anti-Fas MAb resulted in apoptosis (programmed cell death) of malignant cells. Using MAbs, the human Fas and APO-1 proteins were identified as cell-surface proteins of 200- and 48-kDa molecular mass, respectively, in two different laboratories. Both induced apoptosis in a variety of cell types upon binding. Subsequent isolation of cDNAs encoding the two proteins revealed that they were identical despite a difference in apparent molecular weight [133, 211]. Apoptosis triggered by the anti-CD95/Fas/APO-1 Mab has been successfully used for the treatment of mice bearing human hematopoietic tumors [302, 151]. The clinical use of anti-Fas/ APO-1 therapy for cancer treatment will be based on further studies. Besides negative signaling, Mabs have other potential uses in tumor therapy. Some MAbs can block interactions between tumor cells and neighboring cells,

Recombinant proteins and genomics in cancer therapy stroma, or matrix that are necessary for tumor growth or metastases. For example, injection of anti-CD44 MAb or its F(ab’)2 fragment 1 week after inoculation of human melanoma cells in mice with severe combined immunodeficiency (SCID) prevented metastases but not the development of primary tumor [78]. The antibody had no effect on growth of tumor cells in vitro. MAbs against growth factors or their receptors can also exert significant antitumor effects. For example, antibodies against IL-6 and IL-6 receptor were effective in the treatment of human myeloma in SCID mice [287] and produced transient responses in patients bearing IL-6dependent tumors [146]. MAbs against the IL-2 receptor have been used to treat adult T-cell leukemia with some partial or complete remissions [322]. Thus MAbs selected against tumor surface antigens to exert either potent growth-inhibitory effects or host-tumor interactions should lead to new strategies for selecting the antitumor activity of Mabs.

Monoclonal Antibodies-Conjugated Drugs The clinical progress with conjugates of Mabs and cytotoxic drugs has been rather slow. An important factor that has limited the use of this approach for treatment of cancer is the relatively low potency of standard chemotherapeutic agents. The potency of these compounds is further reduced by their conjugation with MAbs [148]. However, a recent report that such a conjugate, BR96doxorubicin (BR96-Dox), is highly effective in curing xenografted human carcinoma-bearing mice [301], has rejuvenated great interest in Mab-drug conjugates. BR96 is a chimeric monoclonal antibody that contains a framework region of human immunoglobulin and the binding region of a murine antibody. The antibody binds to an antigen that is expressed on the surface of many human carcinomas. Treatment of tumor-bearing athymic mice with BR96-Dox induced complete regressions and cures of xenografted human lung, breast, and colon carcinomas and cured 70% of mice with extensive metastases from a human lung carcinoma [301]. Clinical trials with BR96-Dox were recently initiated to determine its safety in patients. Similar results with Mab-vinblastine conjugates were reported years ago, but evaluation of this Mab-drug conjugate was discontinued because of unacceptable gastrointestinal toxic effects [14, 230]. Recently, several groups have concentrated their research on more potent immunotoxins conjugated with agents such as calicheamicins, maytansines and trichothecenes. Calicheamicin is a family of antibiotics that produce double-stranded DNA breaks; when conjugated with Mab CT-M-01, which recognizes PEM antigen and

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is located on the surface of human cancerous epithelial cells, and injected into breast carcinoma-xenografted mice, it significantly inhibited tumor growth and produced long-term tumor free survivors [124]. In a more recent study, 40 patients with relapsed or refractory CD33+ AML were treated with humanized anti-CD33 antibody linked to the calicheamicin [266]. In eight of the 40 patients, leukemia was eliminated from the blood and marrow whereas, in 3 additional patients the blood counts returned to normal. Similarly, patmaytansinoids, which are 100 to 1,000-fold more potent than doxorubicin and vinblastine, when conjugated to Mab A7, which recognizes an antigen expressed on human colon cancer cell lines, showed high antigen-specific in vitro cytotoxicity against cancer cells and low systemic toxicity in mice [34]. Similar specificity and potency have been observed in Mab-trichothecenes conjugates (protein synthesis inhibitors) has been observed in terms of their tumor cell-killing ability [189]. More recently, Mabs have been used to deliver enzyme inhibitors to tumor cells. Thus, conjugation of Geninstein (an inhibitor of Src protooncogene family protein tyrosine kinases) to MAb B43, which recognizes the B cellspecific receptor CD19, selectively bound to B-cell precursor leukemia cells, inhibited CD19-associated tyrosine kinases and triggered rapid apoptotic cell death [307]. Treatment of B-cell precursor leukemia-xenografted SCID mice with less than one tenth the maximum tolerated dose of B43-Geninstein resulted in more than 99.999% killing of human leukemia cells, which led to 100% long-term even-free survival from an otherwise

invariably fatal leukemia of these mice [307]. It remains to be seen whether antibody-drug conjugates will be effective anti-cancer agents in clinical settings as well.

Immunotoxins Immunotoxins are chimeric molecules in which antibodies or the ligand that interacts with the cell-surface molecules are coupled to toxins or their subunits (Fig. 5). The antibody or growth factor binds with high selectivity to the target cells. The toxins are derived from plants or bacteria. DNA sequences encoding the bacterial toxins; Pseudomonas exotoxin (PE) and diphtheria toxin and the plant toxins; ricin and gelonin have been cloned and expressed in E. coli, [2, 221]. The topic of immunotoxins is discussed in detail elsewhere in this book, therefore, we will concentrate only on the therapeutic potential of recombinant immunotoxins in this section. Initially, the toxins produced in bacteria were chemically linked to antibodies to make immunotoxins. In recent years, significant progress has been made in engineering recombinant immunotoxins by fusing the cell-binding ligand genes to modified toxin genes [221]. For example, a truncated form of Pseudominas exotoxin (PE40) has been produced by deleting the first 252 amino acids; this toxin has extremely low toxicity because of its inability to bind to cellular receptors [136]. However, when chemically conjugated to an antibody [76, 156] or the recombinant chimeric toxin generated by fusing the PE40 gene to DNA fragments encoding growth factors, antibody-binding sites, or other target-recognition elements, Pseudomonas

Immunotoxin (IT) Receptor

Toxin (Ricin, Pseudomonas exotoxin, gelonin etc.)

Ligand

Cell surface MAb or antigen or cytokine A chain Cytokine hormone Receptor Killing Domain

TUMOR CELL

Figure 5. Schematic representation of immunotoxin

B chain Translocation Native enhancing domain binding domain

NORMAL CELL

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Recombinant proteins and genomics in cancer therapy

exotoxin becomes highly specific and potent in killing target cells [220, 221]. The gene encoding this chimeric toxin is expressed in E. coli. Table 8 lists some of the recombinant immunotoxins that have been produced by fusing the PE40 gene to cDNAs encoding different targeting molecules. Anti-Tac(Fv)-PE40 is one such recombinant immunotoxin that was generated by fusing the truncated form of the Pseudomonas exotoxin (PE40) gene with the variable region of an antibody against the IL-2 receptor [36, 155]. This immunotoxin is highly toxic to cells from patients with adult T-cell leukemia and induced regression of IL-2 receptor-bearing carcinoma tumors in athymic mice [66, 153, 154]. TGFα-PE40 recombinant chimera toxin targets PE40 to cells with EGF [66, 258]. Although many normal cells contain EGF receptors, tumor cells often have extremely large number of receptors because of amplification and overexpression of the EGF receptor gene [238]. When administered systemically, TG&Fbdot;α-PE40 caused regression of subcutaneous epidermoid carcinoma and prostate carcinoma tumors in mice [221]. Interleukin-2-PE40 is a recombinant chimeric protein designed to deliver the toxin to cells with IL-2 receptors [172]. Normal resting lymphocytes do not express IL-2 receptors, but when they are activated with an antigen or IL-2, the receptors are induced. IL-2-PE40 has been shown to be highly toxic to activated mouse and rat T cells and had some therapeutic effect against mouse lymphoma [150]. Similarly, IL-6-PE40 chimeric toxin killed many human myeloma and hepatoma cell lines

Table 8. Recombinant toxins derived from Pseudomonas exotoxin Immunotoxin PE40 Anti-Tac (Fv)-PE40 TGF-α-PE40

IL-2-PE-40 IL-6-PE40 B3(dsFv)-PE38KDEL e23(Fv)PE40

Target

Reference

Human IL-2 receptor (leukemia) EGF receptor, epidermoid carcinomas adenocarcinomas, glioblastomas smooth muscle cells IL-2 receptor (leukemia) IL-6 receptor myelomas, hepatomas, prostate Many carcinomas erbB2 lung, breast, ovary and stomach adenocarcinomas

[36, 153–155] [66, 113, 258, 252]

[150, 172] [264, 265] [25, 26] [20]

that express IL-6 receptors at high numbers and also several other carcinomas [150, 263, 264]. The first clinical trial of a genetically engineered immunotoxin (B3LysPE38) was initiated in April 1993 in breast and colon carcinoma patients. Some responses were evident, but toxic effects appeared to be greater in human patients than seen in mice and primates [270]. In contrast to Pseudomonas exotoxin-immunotoxins, the immunotoxins composed of ricin or its A subunit and MAbs have generally been constructed using chemical cross-linking reagents [89, 173]. Recombinant ricinbased chimera molecules have been difficult to produce, because the A chain of the plant toxins must be attached to the cell recognition domain by a disulfide bond, and disulfide-linked subunits are difficult to produce in bacteria [238]. Many reviews have already described the activities and properties of immunotoxins made with ricin and other plant toxins [89, 142, 249, 308, 315, 320]. Ricin-containing immunotoxins have been used to eliminate selected populations of lymphocytes. Vitetta, Uhr and associates have produced ricin conjugates of antibodies to B cell-specific antigens and shown such conjugates to cause complete regression of B-cell lymphomas in mice [80]. Significant antitumor activity of ricin A chain immunoconjugates has been observed against solid or ascites tumors in animal models [75, 105]. Because of encouraging results in preclinical studies, several ricin-containing immunoconjugates have been developed and approved for human trials, and two kinds of human trials have been conducted. The first involves the ex vivo treatment of harvested bone marrow to eliminate contaminating tumor cells prior to re-infusion in patients undergoing autologous bone marrow transplantation. The second kind of trial involves the parenteral administration of immunotoxins [231, 308, 315, 320]. Some patients with B-cell lymphoma responded to immunotoxin therapy [89]. Currently, an anti-CD22 dgA immunotoxin is being evaluated in phase II clinical studies in lymphoma patients. The side effects observed with administration of immunotoxins are different from those of conventional chemotherapy; immunotoxins do not exert cytotoxic effects against normal rapidly dividing cells. Immunotoxins such as the bacterial toxins, Pseudomonas exotoxin and diphtheria toxin, induce hepatotoxicity, whereas the ricinbased immunotoxins cause reversible vascular leak and myalgias [320]. Several groups of researchers are currently working on second- and third-generation immunotoxins to eliminate the immunogenicity and side effects of the first generation immunotoxins. Continued refinement in design of these pharmaceuticals may eventually prove useful in the treatment of cancer.

Kapil Mehta et al.

Monoclonal Antibodies and Radioimmunotherapy The use of MAbs to deliver radioisotopes for treatment of certain cancers has yielded some encouraging results [106, 137, 138]. Some of the most promising of these results radioimmunoconjugates in bone marrow transplantation. In the treatment of leukemia, radioiodine-MAb conjugates ablate marrow safely, delivering up to fourfold more radiation to the marrow than to other normal tissues. Responses have been less frequent in solid tumors treated with radioimmunoconjugates in clinical trials [183, 184]. Objective tumor regressions, however, were seen in four patients with non-bulky disease who were treated with rhenium186-labeled Mab NR-LU-10 [134]. Because of their size and high molecular weight, diffusion of MAbs within bulky tumors can be a problem, as illustrated by the results of a clinical trial that used radiolabeled anti-CD20 and anti-CD21 MAbs to treat patients with B-cell lymphomas [236, 253]. Among the most promising approaches to overcoming this problem is the use of genetically engineered, low molecular weight F(ab’)2, Fab and singlechain Fv fragments that may diffuse better at tumor sites. Early results of clinical trials employing humanized MAbs appear encouraging. In addition to reduced immunogenicity, F(ab’)2, Fab and Fvs, with their improved ability to penetrate tumors, may prove useful carriers for radiotherapy. Newer regimens combining radioantibody-based therapy with other treatment modalities may prove even more effective.

Chimeric Proteins One interesting aspect of recombinant DNA technology is the potential for producing of new proteins with novel properties. For example, the hybrid proteins formed by fusion of two or more genes (chimera) offer several advantages in terms of their stability, affinity, efficacy and pharmacology over the individual component proteins. A chimera protein formed by fusion of the IFN-γ and LT genes was shown to have better antiproliferative activity than IFN-γ or LT alone [71]. Similarly, PIXY321, a genetically engineered hybrid of the GM-CSF and IL-3 proteins exhibits greater colony-stimulating effects in vitro than the combination of GM-CSF and IL-3 [50]. In preclinical studies, PIXY321 has been shown to accelerate both neutrophil and platelet recovery in rhesus monkeys subjected to sublethal irradiation [330]. Because of the preclinical observations, PIXY321 was tested in patients for its ability to ameliorate disease- or treatmentrelated bone marrow suppression. The clinical results were encouraging and suggested that the hybrid protein

71 elicits the biological effects of both its component cytokines [29, 311, 312]. Thus, PIXY321 became the first recombinant fusion of two hematopoietins to enter the clinic. Early clinical experience has shown great potential in the prevention and treatment of hematopoietic suppression. The other recombinant fusion proteins have been made, including chimeric toxins constructed by fusion of genes encoding human cytokines and Pseudomonas exotoxin [35, 55, 284]. For example, IL-4-PE, a chimera of human IL-4 and Pseudomonas exotoxin proteins, is highly potent against many cancer cells [55], suggesting that it might be useful in the therapy of many cancers. Generation of homologues and analogues of natural biotherapeutic proteins is another potentially important application of recombinant DNA technology. The technology involves alteration or deletion of key nucleotide sequences in the gene that will result in the modification of only a few amino acid sequences in the resultant protein, compared with the natural protein. Synthesis of novel human TNFα, IFNα and IF&Nbdot;γ homologues has already been reported [11, 214]. All three of these homologues are distinct from their parent protein. Prolonged activity of insulin has been achieved by various substitutions that increase the isoelectric point. The prolonged activity seems to occur because the novel homologues precipitate when they encounter the neutral pH of the body [180]. These examples indicate an interesting way by which protein modifications can be exploited for therapeutic potential. Furthermore, the ability afforded by newer techniques in recombinant DNA technology such as “phage display” to correlate protein structure and function in a systematic way makes it possible to design novel drugs [38, 51, 176]. Such a technique could be used to design or even select a small peptide that binds to the receptor with the same affinity as the larger protein. And then, using computer modeling to display the molecular contacts between ligand and receptor, small nonprotein molecules that make the same contacts could be designed and synthesized. The end product would be a small organic molecule that could be produced more cheaply than the recombinant protein, yet would retain the full biological activity of the protein hormone. What’s more, such molecules could be administered orally, eliminating the major disadvantage of most recombinant protein therapeutics, which must be delivered directly into the blood stream by injection.

Cancer Vaccines The issue of genetically engineered vaccines for cancer treatment will be discussed in detail in another part of

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Recombinant proteins and genomics in cancer therapy

this book. We will, therefore, focus only on the novel aspects of recombinant vaccines that may have some potential as cancer therapeutics. The function of a vaccine is to give the immune system a boost, thus helping it recognize and destroy the ‘non-self’ antigens on the surface of cancer cells. Though the idea of inciting the immune system to fight cancer has been around for a long time, recent developments in biotechnology and better understanding of the immune-system network have caused an explosion of research and development in the field of cancer vaccines. Table 9 lists some cancer vaccines that are currently undergoing clinical trial. Prior to the advent of recombinant DNA technology, two types of vaccines were used, inactivated vaccines (chemically killed derivatives of actual infectious agents) and attenuated vaccines (actual infectious organisms altered so that they do not multiply). However, these types of vaccines are potentially dangerous, as they can carry over the infectious contaminations. For example, a small number of children each year contract polio from their live polio vaccinations. Thus, one of the most promising applications of recombinant DNA technology is the production of subunit vaccines, consisting solely of the surface protein to which the immune system responds and thus eliminating the risk of infection [27]. The arrival of biotechnology in the late 1970s enabled targeting of Table 9. Cancer vaccines under clinical trials Immunogen

Target Cancer

Reference

Recombinant vaccinia encoding for CEA Gene therapy using patient’s own cells

Colorectal, lung, breast

[152]

Renal cell, carcinoma, melanoma prostate, colorectal Melanoma

[152, 287]

Melanoma Lymphoma, Cervical prostate, Prostate, Melanoma, Renal, Colorectal Melanoma, Cervical

[163] [163, 232, 346]

Recombinant poxvirus encoding for MAGE antigen Heat shock protein Naked DNA

Synthetic peptides Synthetic antigens

Anti-idiotypic antibodies Inactivated tumor cells with the cytokine IL-2 Recombinant antigens Gene transfer

[315]

[72, 224, 342] [44, 163]

Ovarian, Breast, Melanoma, Colorectal Melanonoma, Colorectal, [278, 295] gastric, ovarian Colon [163] Colorectal, lung prostate Melanoma

[63, 119, 278] [163]

specific cell-surface antigens, and monoclonal antibody technology permitted identification of tumor-associated antigens, their characterization and tissue distribution. The polymerase chain reaction (PCR) brought the technology of cloning and expressing gene products. These technologies jointly led to the development of the subunit vaccines of the 1990s. The human melanoma antigen MAGE-1 was the first tumor-specific antigen identified [315]. Poxvirus containing the MAGE-1 gene is currently being evaluated as a candidate vaccine for treatment of melanoma and breast cancers in humans [163]. Similarly, the recombinant vaccinia virus containing carcinoembryonic antigen (CEA) is being evaluated for treatment of certain cancers. CEA is expressed on the surface of virtually all colorectal cancers, 70% of lung cancers, and 50% of breast cancers. Clinical results if these phase I trials of this vaccine in late-stage cancer patients were promising [152]. A recombinant fusion of a tumor-derived idiotype and GM-CSF, yielded a strongly immunogenic protein that was capable of inducing idiotype-specific antibodies and protected the recipient animals from challenge with an otherwise lethal dose of B-cell lymphoma [295]. These results can be applied not only to B-cell lymphoma but perhaps can be generalized to other classes of tumor antigens as well. The discrete peptide fragments from certain tumorspecific oncoproteins (such as mutant p53 and the protein products of the ras and HER-2/neu genes) are rapidly progressing as potential vaccine candidates for cancer treatment [72, 342]. This strategy is based on the principle that intracellular proteins are degraded and presented back on the cell surface as small peptides in the groove of class I major histocompatibility complex (MHC) antigens. A nine amino acid peptide from the HER-2/neu oncoprotein was shown to be recognized by both breast and ovarian cytotoxic T lymphocytes, and this small peptide was able to induce a tumor-specific immune response [224]. It is now possible to isolate and custom synthesize tumor-specific immunogenic oncopeptides by using the patients’ own mutation to prepare an autologous peptide vaccine that is selectively targeted to tumor cells containing the mutant gene product [342]. The ability of human oncopeptide vaccines to generate a peptidespecific CD8+ cytotoxic T-lymphocyte response in animal models has formed the rationale for clinical trials of autologous peptide immunizations in patients with diverse epithelial malignancies (breast, lung, gastrointestinal) that are commonly accompanied by p53 and ras mutations [142]. It is likely that similar considerations will apply to other recently identified oncoproteins such as the product of the BRCA-1 gene in breast cancer.

Kapil Mehta et al. Recently, there have been several attempts to generate tumor cell vaccines engineered to secrete various cytokines [42, 43, 127, 235]. The strategy seeks to alter the local immunologic environment of the tumor cell so as to enhance either the antigen presentation of tumorspecific antigens to the immune system or the activation of tumor-specific lymphocytes. Many cytokine genes have been introduced into tumor cells with varying effects on both tumorigenicity and immunogenicity. Some of these cytokines, when produced by tumor, induce a local inflammatory response that results in elimination of the injected tumor. The local inflammatory response is, in general, dependent on leukocytes other than classical T cells. Many cytokine genes have been introduced into tumor cells, including IL-1α, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, GM-CSF, IFNα, IFNγ and TNFα [42, 215]. Preclinical data from various animal models suggest that tumor cells engineered to produce cytokines indeed provide a novel approach for tumor therapy [42, 107, 216]. Clinical trials are currently in progress to assess the therapeutic efficacy of cytokinetransduced tumors as vaccines for the treatment of established solid tumors. A remarkably straightforward and potentially useful approach in the field of cancer vaccines was recently developed. It involves the direct in vivo delivery of MHC-associated tumor antigens to provoke a tumordirected immune response. This approach for treatment of diverse malignant diseases is currently under clinical investigation (Table 9). In essence, this approach is based on the ability of some viral and human ‘naked DNA’ genes to transfect certain cells without the need for elaborate genetic engineering maneuvers, sometimes using modified DNA/liposome complexes to deliver genes by direct injection at the tumor site or by systemic administration [232, 346]. For example, injection of naked DNA or mRNA transcripts encoding human CEA in mice has been shown to elicit strong cytotoxic T-lymphocytes and antibody responses against this antigen [44, 45]. The development of this simple and direct approach for in vivo gene therapy-based immunotherapy is an extremely important avenue for continued clinical and preclinical investigations. Most tumor vaccines must be employed subsequent to the development of cancer. However, in some instances, for example, in those geographic areas in which human papilloma virus infection is highly endemic and thus rates of cervical carcinoma are high, it may be useful to vaccinate children prophylactically. In China, where endemic hepatitis virus is causally related to hepatic cancer, broad immunization to prevent hepatitis and subsequent cirrhosis and hepatic cancer is underway.

73 Ultimately, as we discussed earlier in this chapter, development of multiple strategies that could be applied in synergy are most likely to yield beneficial results in cancer treatment and control. It remains to be seen what place the immunotherapy will have in this armamentarium. However, it is obvious that availability of recombinant DNA technology has made it possible to design vaccines on a molecular basis.

Problems Unique to Recombinant Biotherapeutics The development of protein therapeutics by using recombinant DNA technology has presented many new and interesting challenges to pharmacologists and drug delivery scientists. Many of these biotherapeutics have multiple biological effects [6]; thus, an important priority in their development is the evaluation of their potency, pharmacological profile and toxic effects. One strategy to understand the potency and toxicity of anticancer agents is the use of appropriate animal models. However, many animal models that have been developed for testing conventional low molecular-weight drugs may not be useful for testing rDNA-derived therapeutic proteins. Species specificity of protein therapeutics further narrows down the choice for appropriate animal models. Certain in vitro biological properties of the interferons for example, did not translate to their efficacy in intact animals or in patients [280]. The lack of information on preclinical pharmacological behavior also limits a general analysis of the toxic effects of therapeutic proteins. For example, agents such as IFÑγ, IFN-α and IL-2 are produced in E. coli and are nonglycosylated. EPO, in contrast, is produced in Chinese hamster ovary cells and is glycosylated. Rats, dogs, hamsters and monkeys were used to study the toxicity of these agents. Comparison of results on gross morphology, histology, blood chemistry and hematology obtained from these studies demonstrated no general responses that might be attributed to the use of biotherapeutic proteins. The effects observed were primarily related to the known or anticipated biological effects of the agent and, as expected, occurred only in the species in which the agents were known to be biologically active. Considering these results, it is apparent that toxicologic findings in animals essentially reflect the pharmacological effects of biotherapeutic agents. Therefore, the use of biotherapeutic agents in species in which they are active should be most informative. However, observations of the lack of toxic effects in other species may be important in addressing some concerns about nonspecific

74 toxic effects. Cross-species activity may be seen for some if not all biological effects. For example, human IFN-α has pyrogenic activity in rabbits. Clearly, the pharmacological effects observed with materials that lack pronounced species specificity are likely to be more dependable, especially if different species have manifested the same toxicity profile. Similarly, human TNF was found to be less toxic in mice than in human subjects because one of the receptor subunits with which TNF interacts is species-specific and the other is not. In contrast, the pharmacological effects of agents like EPO are highly cell type and species specific. There have been relatively few preclinical studies of the immunogenicity of recombinant therapeutic proteins. The useful information has come from the clinical studies. The generation of antibodies to proteins administered over long periods may result in formation of soluble immune complexes. These immune complexes can induce vascular and tissue injury, particularly glomerulonephritis [281]. As an alternative, immune complexes can elicit the release of inflammatory mediators from cells. However, to date there have been no examples of antibody responses to any recombinant therapeutic protein that have been shown to clearly cause clinical pathologic effects. Another potential problem is incorporation of the wrong amino acids when a high level of expression of recombinant proteins is enforced. Such errors are generally difficult to pinpoint, since current analytical methods for amino acid composition and sequence are not really amenable to detecting variations below 10% of the major constituents. It is conceivable that such altered sequences may resemble a toxic peptide or a protein with different biological functions. Also, the altered sequences in the protein may render them immunogenic and may provoke enhanced immunogenic responses.

Delivery of Therapeutic Proteins The potential use of therapeutic proteins in medicine is severely limited because of their poor activity when administered orally. Proteins are rapidly degraded by proteolytic enzymes in the gastrointestinal tract and have been primarily administered by injection. Moreover, proteins are generally characterized by short biological half-lives in the circulatory system, so that the repeated injections are generally needed. Even after intramuscular or subcutaneous administration, their bioavailability is often low because of their small size and the widespread distribution of proteolytic enzymes. In addition, most proteins pass through biological barriers rather poorly because of low diffusion and a low partition coefficient.

Recombinant proteins and genomics in cancer therapy These considerations have led to the development of different strategies to prolong the bioavailability of therapeutic proteins. Conjugation of certain proteins to synthetic polymers can circumvent the problems of rapid clearance from the circulation, immunogenicity and instability. The general requirements of any polymer used for this purpose are that it be water-soluble, biocompatible, nonimmunogenic and devoid of biological activity. Zoladex® and Nafarelin®, the decapeptide agonists of luteinizing hormone releasing hormone (LHRH), have been formulated in slow-releasing polymer base and used effectively in clinical studies [81, 117]. Attempts have also been made to stabilize the proteins against degradation at the site of injection as well as in the circulation, but these studies are still preliminary in nature [160]. Covalent conjugation of certain proteins with watersoluble polyethylene glycol (PEG) enhances their solubility and permits the design of stable formulations suitable for clinical use. This is particularly important for recombinant proteins produced in E. coli that are usually recovered as insoluble refractile bodies, and unlike many of their native counterparts, are not glycosylated. For example, conjugation of both IL-2 and IFN-β with PEG increased their solubility, and aqueous solutions were stable for long periods of time [144]. Moreover, a recently published study revealed that PEGlated IFN-α is better tolerated and may be more effective in treating CML patients [291]. Similarly, PEGylation of TNF-α alters its pharmacokinetics and reduces its in vivo toxicity [144]. G-CSF conjugated to PEG has a four times slower clearance rate in rats than unmodified G-CSF. In addition, PEGylated G-CSF administration exerted a sustained biological effect on peripheral blood neutrophils [294]. PEGylated adenosine deaminase (ADA), an enzyme unrelated to cancer, is now approved for use as replacement therapy for severe combined immunodeficiency diseases that are due to inherited ADA deficiency. Patients who received PEG-ADA did not develop neutralizing antibodies to ADA activity [33]. The key factor with any drug delivery system is to achieve adequate concentrations of the drug at the desired sites while avoiding significant concentrations at sites that mediate toxic effects. Novel delivery systems such as liposomes may prove to be useful in achieving this. The ability of IFN-γ to stimulate the tumoricidal activity of monocytes was increased 1,000-fold by its encapsulation in liposomes [147]. Encapsulation of TNF-α in liposomes ameliorated the systemic toxicity of this cytokine in dogs [171]. Anti-HER2 immunoliposomes, consisting of long circulating liposomes linked to anti-HER2 MAb fragments proved highly efficient for intracellular delivery

Kapil Mehta et al. of the drugs and showed superior anti-tumor activity in animal models [218]. Delivery can also be modified by a combination of the biotherapeutic protein with an antibody [61]. For example, the in vivo clearance of human IFN-α in rats is threefold slower when it is combined with a specific MAb [246]. However, it remains to be seen whether such delivery systems will confer any advantages to biotherapeutic proteins in vivo in terms of local delivery, reduced toxicity or altered pharmacokinetics.

Conclusions It is clear that recombinant DNA technology has produced a revolution in the field of cancer therapeutics. The dream of biological therapy, thought to have a great potential for cancer, can now be realized. The ability to manufacture cancer drugs by using genetically engineered organisms has given rise to a novel biotechnology industry within the last decade that has earned as much as $15 billion. The revolution has been not only in the industrial sector; but also in the academic sector. It has enabled scientists to discover new molecules, redesign molecules for lower toxicity and more efficacy, and investigate the pathology of disease at the molecular level. This technology, however, has given rise to new set problems in the area of drug manufacturing and delivery. One of the major problems in the manufacturing area is ensuring that the recombinant biological product is identical or very similar to the natural counterpart. Another problem is the rapid degradation of proteins in the circulation and their short circulating half-lives, which restrict oral delivery of biotherapeutic proteins and may require continuous injection. It is now possible to design from the crystal structures of the ligand and the receptor small molecules that can mimic these large proteins and thus may help to overcome delivery problems. Some of these problems might be circumvented by directly injecting the gene for a given protein: although this approach seems attractive, it also suffers from the delivery and organ/cell-specificity problems. Treatment with antisense DNA and RNA to inhibit the expression of oncogenes in tumor cells and the new technologies based on viral vectors for the delivery of vaccines and genes may find widespread application in the near future. Many of these approaches work effectively in the test tube, and the main challenge now is to translate these laboratory techniques into commercially viable processes to produce active, effective biotherapies with acceptable toxicity.

75 Acknowledgements The research in authors’ laboratories was supported in part by a grants from Food and Drug Administration, Clayton Foundation, and Department of Defense.

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5

Current concepts in immunology ROBERT K. OLDHAM

The main function of the immune system is to protect the host from certain death due to numerous potential pathogens present in the environment. The development and maintenance of immunity is dependent on a complex and highly sophisticated defense organization functionally divided into the innate and adaptive immune systems. Innate immunity provides the first line of defense against most pathogens. Phagocytes, including neutrophils and monocytes/macrophages, and natural killer cells are the most important cell types participating in this form of immunity. In addition, several soluble factors, including the complement cascade, lysozyme, and acute-phase reactants, contribute to and reinforce the physical barriers that prevent most infectious organisms from penetrating the body. These factors also promote the activation of the inflammatory reaction that contains the injury caused by an invasive infectious agent. These cells and factors are suitable to accomplish this function because their activity does not depend on a prior encounter with the antigens in the microbial agent or tumor cells, and they lack the fine specificity of the cells and humoral factors of the adaptive immune system. Specificity and memory are two cardinal features of the adaptive immune system. The main cellular components are the T and B lymphocytes. Each clone of these cell populations has an extraordinary and unique antigen specificity via the expression of genetically programmed, antigen-specific receptors on the cell surface. The adaptive immune system has the ability to differentiate between foreign and self-molecules and in this way, most destructive reactions against the host’s own tissues are prevented. When the immune system loses its capacity to differentiate self from nonself, autoimmunity develops. The innate and adaptive systems communicate with each other directly by cell–cell interactions and through soluble mediators termed cytokines. For example, macrophages are important not only in innate immunity but also in specific immune responses, since they can present antigen and activate T and B cells. In turn, T cells regulate macrophages, B cells, and natural killer cell activity through the synthesis and release of cytokines. Some of the cytokines also behave as messengers that

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

mediate the communication between cells of the immune system and other organs of the body, such as the central nervous and endocrine systems. This chapter presents a summary of the structure and function of the immune system with emphasis in those areas relevant to the defense against tumors and to cancer biotherapy. The following sections will briefly describe the characteristics and functions of the major cells and mechanisms that control immune responses and the clinical methodologies for assessing the general state of immunocompetence.

Current Concepts of Immunity All the major cells of the immune system, that is, monocytes/macrophages, B and T lymphocytes, natural killer cells, polymorphonuclear cells, etc., originate in the bone marrow from pluripotent stem cells [58]. Although the exact mechanisms and mediators involved are not completely understood, it is now accepted that stem cells, under the influence of growth and differentiation factors such as erythropoietin, colony-stimulating factors, and interleukins, differentiate into two main progenitors: stem cells that have the potential to originate erythrocytes, eosinophils, platelets, granulocytes and monocytes/macrophages (GEMM-CFC); and stem cells that are the precursors for mature lymphoid cells (L-CFU) [37, 78, 109] (Fig. 1). We now know many of the factors that induce proliferation of the committed stem cells. Most of these cytokines have been purified, sequenced, and are currently produced by recombinant techniques [37, 78, 109, 134]. The availability of these recombinant cytokines has contributed significantly to defining and characterizing their biological activities (Fig. 1). Four separate colony-stimulating factors (CSFs) have been recognized. They are interleukin 3 (IL-3; multiCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte-colon-stimulating factor (M-CSF) and granulocyte-stimulating factor (G-CSF) [37, 109]. These factors, together with interleukin 1 (IL-1), interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 6 (IL-6), are essential for the proliferation and differentiation

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Bone Marrow Pluripotent Stem Cell

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Figure 1. Origin of the cells of the immune system. All the cells of the immune system are derived from the multipotential stem cell in the bone marrow. Proliferation, maturation, and differentiation of the different cell lineages are driven by a multitude of cytokines. GEMM-CFC, granulocyte/erythroid/megakaryocyte/monocyte colony-forming cell: L-CFU, lymphocyte colony-forming unit; CFU-GM, granulocyte/monocyte colony-forming unit; B, B lymphocyte; Th/i, T helper inducer; Ts, T suppressor; Tc, T cytotoxic; Mo, macrophage; LGL, large granular lymphocyte; Pc, plasma cell; Ab, antibody; PMN, polymorphonuclear cell; EP, erythropoietin; BFU-E, burst-forming unit – erythroid; RBC, red blood cell; GM-CSF, granulocyte/monocyte colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; M-CSF, monocyte colony-stimulating factor; IL-1, interleukin 1; IL-2, interleukin 2; IL-3, interleukin 3; IL-4, interleukin 4; IL-6, interleukin 6; IFN-γ, interferon-γ

of committed stem cells, such as GEMM-CFC, GM-CFC, and L-CFU. For example, IL-3 and GM-CSF are required to induce GEMM-CFC to differentiate into granulocyte/ monocyte-colony forming cells (GM-CFC), while M-CSF, GM-CSF, and IL-3 are necessary for GM-CFC to differentiate into precursors of the monocytic lineage (Fig. 1) [37, 78, 109]. Some of these cytokines act on targeted cell lineages; for example, IL-5 regulates eosinophilic maturation and B-cell activity while erythropoietin (EP) has its predominant effects on committed erythroid cells (BFU-E) (Fig. 1). Macrophages, activated T lymphocytes, fibroblasts, and endothelial cells are the major sources of GM-CSF, G-CSF, M-CSF, IL-1, IL-3, IL-5, and IL-6 [16, 37, 44, 51, 78, 90, 109, 134].

Some of these cytokines (i.e., C-CSF, GM-CSF) are already approved for the treatment of patients with chronic severe neutropenia associated with cancer chemotherapy, AIDS, bone marrow transplantation, myelodysplasia, and aplastic anemia [27, 37]. Platelet growth activation factors have been approved and others, along with receptor agonists, are in clinical testing. Lymphocyte-precursors originating from the L-CFU migrate to the thymus where they differentiate into mature T lymphocytes. This cell population is comprised of several subpopulation, including T helper/ inducer (Th/I) lymphocytes, which modulate the activity of virtually all other cell types of the immune system (Fig. 1), T-cytotoxic (Tc) lymphocytes, which are the

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effectors of the cytotoxic responses against tumors, foreign tissues, virus-infected cells, etc. and T-suppressor or regulatory (Ts or Treg) lymphocytes, which are involved in the down-regulation of the immune response. In addition to T lymphocytes, the bone marrow lymphocyte-precursor cells also give rise to natural killer cells (NK), which are involved in the defense against tumors, and B lymphocytes (B), which after maturing in the bursa of Fabricius (in birds) or bursa equivalent (bone marrow in humans), migrate to peripheral tissues, where in response to antigens, they differentiate into antibody-producing plasma cells (Fig. 1). Mature lymphoid cells circulate in blood, from where they populate peripheral lymphoid tissues, such as the paracortical areas of the lymph nodes and the periarteriolar sheaths of the spleen, the skin, and the mucosal linings of the digestive, respiratory, and genitourinary tracts. The migration of circulating B and T lympho-

cytes into specific lymphoid tissues appears to be governed by complementary adhesion molecules present on lymphocytes and on endothelial cells of high endothelial venules (HEV) and postcapillary venules (homing receptors, specialized adhesion molecules) [115, 129]. The expression of some specialized adhesion molecules by endothelial cells is induced by cytokines produced at the inflammatory sites. For example, the intracellular adhesion molecule 1 (ICAM-1), which serves as a receptor for the leukocyte-function-associated antigen-1 (LFA-1) molecule present on leukocytes [5, 23, 110], is induced by IL-1 in endothelial cells. The rapid increase in these surface proteins facilitates the adhesion of leukocytes to the endothelium, thus accelerating the initial stages of the inflammatory reaction (Fig. 2). In addition to traffic to and from secondary lymphoid organs, a small number of lymphocytes travel through most non-lymphoid tissues of the body. This traffic Macrophage IL-6 TGF-β

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Fever Sleep Acute Phase Reactants Insulin Lipoprotein Lipase Steroid Synthesis Shock Neutrophilia GM – CSF Bone Marrow Stimulation Appetite Hypotension

Figure 2. Cytokine participation in inflammatory and immune responses. Early monouclear cell recruitment is induced by platelet-derived transforming growth factor β (TGF-β). Activated macrophages produce additional TGF-β and other cytokines, including interleukin 1 (IL-1), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6), that have local and systemic effects as well as effects on the immune response. PDGF, platelet-derived growth factor; PMN, polymorphonuclear cell; Plt, platelets

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pattern is designed to optimize the interaction of foreign antigens with the appropriate receptor specificities in T and B cells and to assure the fast development of a specific immune response. This process is also facilitated by migration of antigen presenting cells (APC) from the peripheral tissues to local lymph nodes [115]. Following cognitive interaction (i.e., involving antigen and class II histocompatibility complex molecules) between APC and T cells and T-B cell cooperation, lymphocyte proliferation and maturation take place, with the generation and subsequent recirculation of antigen specific effector and memory cells (Fig. 3). During these events, lymphoid cells secrete a number of cytokines, such as transforming growth factor β (TGF-β), IL-1, IL-6, tumor necrosis factor α (TNF-α), and platelet-derived growth factor (PDGF), that affect the inflammatory and healing

GM–CSF M–CSF TGF–β

Tr

T lymphocytes T cells, which comprise 70–80% of all circulating lymphocytes, are central to the development of normal immune responses. They play a critical role in cellular immune reactions, including delayed-type hypersensitivity (DTH), resistance against certain bacteria, viruses and tumor, and as effector cells mediating the rejection of organ transplants [2, 54]. T cells also have important regulatory function, due to their ability to produce cytokines that act on other T cells, B cells and

B

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processes by modulating the activity of endothelial cells and fibroblasts, thus linking the inflammatory and the immune response (Fig. 2) [124].

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IL–1; IL–2; CSF IFN–γ ; TNF–β Tumor cell killing

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Figure 3. Simplified scheme of the lymphokine cascade and its role in the modulation of immune response. Various cytokines with unique and overlapping biological activities are produced during the immune response. These cytokines act in concert to regulate the activation, proliferation and maturation of cells participating in the specific and nonspecific arms of the immune system. Mo, macrophage; Tr, T activated lymphocyte; Tind, T inducer; Tm, T memory; Th, T helper; B, B cell; Ts, T suppressor; Tc, T cytotoxic; Pc, plasma cell; LGL, large granular lymphocyte; IL-1, interleukin 1; IL-2, interleukin 2; IL-3, interleukin 3; IL-4, interleukin 4; IL-5, interleukin 5; IL-6, interleukin 6; CSF, colony-stimulating factor; TNF-β, tumor necrosis factor β/lymphotoxin; IFN-γ, interferon γ TGF-β, transforming growth factor β; GM-CSF, granulocyte/monocyte colony-stimulating factor; M-CSF, monocyte colony-stimulating factor; Ag, antigen; Ab, antibody; II, class II histocompatibility complex molecules

Robert K. Oldham macrophages, and by directly interacting with other lymphocytes [18, 67, 74, 112, 113, 116]. In general terms, the functions of T cells can be divided into regulatory and effector categories. The positive and negative regulatory functions are mediated by different lymphocyte subsets. The positive signals are associated with the helper/inducer subpopulation of T cells (Th/I) and the negative with the suppressor T-cell subpopulations (Ts) [2, 19, 95, 105, 125]. The effector category includes the cytotoxic (killer) T cells (Tc) reactive against virally infected cells and foreign histocompatibility antigens, and the sensitized Th(TDTH) cells, mediating delayed hypersensitivity reactions [24, 91]. T cell subpopulations can be identified by immunofluorescence or flow cytometry with the use of specific monoclonal antibodies (mAb), which react with specific surface markers. An International Nomenclature Subcommittee has examined a large number of mAb directed to leukocyte antigens and clustered them into groups of antibodies with the same reactivities (clusters of differentiation or CD), and a number was assigned to each group [46]. Helper/inducer T lymphocytes bear the CD4 marker, while cytotoxic/suppressor T cells are recognized by the presence of the CD8 marker [41, 46, 95, 96]. Other mAb, which recognize the T3 portion of the T-cell antigen receptor complex (CD3), and others that recognize the CD2, react with virtually all T cells and are widely used for the determination of total T-cell numbers [41, 46, 95, 96]. In human peripheral blood, there are twice as many CD4 as CD8, a proportion that is altered in many diseases including immunodeficiencies and cancer. More recently, the phenotypes of the two main subsets of CD-4 lymphocytes have been defined as helper-inducer (CDw29+) and suppressor-inducer (Treg) (CD25 + FOXp3 +, CD127neg) Th/I subpopulations, respectively [60, 107]. The T-cell antigen receptor (TCR) has been isolated and characterized [1, 12]. It is a disulfide-linked heterodimer composed of an α and β polypeptide chain, each containing a constant and a variable (antigen binding) region somewhat similar to the structure of immunoglobulins (Fig. 4). In a small number of T cells present in peripheral blood (0.5–10% T cells), thymus, epidermis, and gut epithelium, a different type of T-cell receptor consisting of two distinct polypeptide chains (γ and δ) has been recognized [92]. The majority of these cells do not express CD4 or CD8 on the cell surface and their functions are not completely understood, but they are believed to represent a mature functional lineage of lymphocytes that can be activated by triggering through the γ/δ receptor.

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CD4 or CD8 Th/I or

Processed Antigen T cell receptor CD3 Tc/s

Figure 4. A current model for T-cell antigen recognition. Antigen-presenting cells (APC) incorporate, process, and express the modified antigen in conjunction with MHC molecules for presentation to T cells. The interaction of T helper (Th/i) cells with APC is class II restricted; that is, it occurs when the antigen is presented to the Th/i cell together with class II molecules that interact with the T-cell receptor and CD4 molecules, respectively. Class I restriction occurs between APC expressing class I and the T-cell receptor in CD8+ (Tc/s) cells. In both cases the delivery of the transduction signal for cell activation appears mediated by the CD3 molecular complex

Both α/β and γ/δ receptors are non-covalently associated with the CD3 molecular complex, which is composed of at least four polypeptide chains, γ,δ,ε,and ζ one of which (the γ chain) has a long intracytoplasmic portion with several phosphorylation sites [12]. It is believed that the CD3 complex is involved in mediating signal transduction during the interaction of antigen with the specific binding site in the α/β receptor (Fig. 4) [12]. Antigen recognition by T cells requires that the antigen be presented to the T cells in association with the appropriate major histocompatibility complex (MHC) molecules [32] (Fig. 4) and is amplified by co-stimulatory molecules T-helper cells recognize antigen in the context of class II MHC molecules expressed by antigen-presenting cells and B cells (class II restriction), while T-suppressor and most cytotoxic T cells recognize antigen in association with class I molecules (class I restriction). Recent work has demonstrated that CD4 molecules present on T cells bind with low affinity to certain invariable regions in the class II molecules. These findings support the theory that a complex involving the TCR α/β-CD3 and CD4 or CD8 molecules is formed on the surface of the T cells during antigen recognition (Fig. 4). In this situation, the specificity is determined by the variable region of the α/β receptor, the reaction is stabilized by binding

90 of CD4 and CD8 to class II or Class I molecules, respectively, and the activation signal is transduced by the CD3 complex [64, 106].

T-helper Cells T-helper cells were first described as the T lymphocyte subpopulation that “helps” B lymphocytes mount an optimal antibody response and “induces” the generation of cytotoxic T cells. These T cells recognize processed antigen presented by macrophages or other APC in the context of Class II (DR) products (Fig. 4). The induction of lymphoid cell proliferation upon stimulation by mitogens or antigens is characterized by two distinct phases: competence and progression. The activating signals (competence signals) cause the resting T cells to move into the early G1 phase of the cell cycle. During this “competence stage,” binding of antigens to the TCR/CD3 (TCR complex) induces the generation of intracellular signals, such as increases in intracellular free calcium, membrane depolarization, generation of diacylglycerol (DG) and phosphatidyl inositol (PI) turnover, activation of protein kinase C (PKC), and changes in the levels of cAMP, cGMP, protein phosphorylation, and express of c-fos, c-myc, and other proto-oncogenes [13, 93, 94, 108, 111, 126]. These events (some of which are triggered by IL-1) lead to the expression of specific genes [including interleukin 2 and IL-2 receptor (IL-2R)] critical for the progression phase [6, 126]. During the progression stage, binding of IL-2 to its high-affinity receptor is a critical event leading to passage of competent T cells from early G1 through the other phases of the cell cycle, culminating in cell proliferation [6, 7, 29]. Other changes noted during activation are increased numbers of interleukin 1 receptors (IL-IR), de novo expression of Class II molecules, and acquisition of transferrin receptors (TR) to ensure incorporation of iron, since this element is essential for cell division [75]. The rate of T-cell proliferation is tightly regulated by the transient expression of IL-2R and the limited production of IL-2. The high-affinity IL-2R (HA-IL-2R), a heterodimer consisting of an α chain (p55) associated with a β chain (p70), is rapidly internalized in the presence of the ligand and is widely recognized as the IL-2R species that mediates the biological responsiveness to IL-2 [20, 53, 97, 98, 118, 122]. Small numbers of α-chain (MW 55 kD) molecules are normally expressed by resting CD4 + T cells. Normally, IL-2R expression lasts for about 1 week, and IL-2 production for 2 or 3 days.

Current concepts in immunology Antigen-activated CD4 T-cells release a number of cytokines including IL-2, gamma-interferon (IFN-γ), colony-stimulating factors (CSFs), B-cell growth factors (IL-4, IL-5, IL-6), etc. [38, 40, 49, 73, 79, 87, 88, 117, 132] (Fig. 3). These cytokines, in turn, induce activation, proliferation and differentiation of other antigenspecific T and B cells resulting in a specific immune response and the production of memory T cells (Fig. 3). There is some evidence that lymphokine release from TH cells can lead to tumor regression (see Chapter 8). Although human counterparts are not fully characterized, there are two well-defined subpopulations of murine Th/I lymphocytes, termed TH1 and TH2 [69]. These subpopulations are characterized by the secretion of different sets of cytokines, leading to different functional properties [69]. Although some cytokines such as GM-CSF, TNF-α, and IL-3 are produced by both cell types, TH1 but not TH2 clones produce IL-2, γ-interferon, and lymphotoxin (TNF-β) [69]. In contrast, TH2 but not TH1 cells synthesize IL-4, IL-5 and IL-6. TH2 cells induce growth and immunoglobulin (Ig) secretion of B cells in response to specific antigens and polyclonal activators. These functions require not only IL-4 and IL-5 synthesis but also TH2-B cell-cell interactions. In addition, IL-4 is essential for IgE production, and IL-5 appears to play a role in B-cell hyperactivation observed in mice prone to develop a severe lupus-like autoimmune syndrome. TH1 cells appear to be involved in proliferation (but no Ig synthesis) of B cells, induction of T-cell activity, and generation of cells participating in the late phase of delayed hypersensitivity reactions. Immune responses characterized by predominant activation of TH1 cells may result in strong induction of macrophage-mediated cytotoxic reactions induced by IFN-α and TNF-β and increased expression of Fc receptors for IgG2a in macrophages. Thus, these responses would result in effective killing of target cells with intracellular viral or parasite infections and strong DTH reactions. In contrast, activation of TH2 cells lelads to immune responses characterized by high levels of antibody production. Normal immune responses probably involve the participation of both cell types [69].

T-suppressor Cells T-suppressor (regulatory) cells suppress (down-regulate) T cell activity, antibody production by B cells, DTH, contact sensitivity, cytolytic T-cell function, proliferation of T cells, and immune responses against tumors [2, 19, 105]. The Ts cells are activated during normal

Robert K. Oldham responses to a variety of antigens, thereby providing a safety mechanism that continuously controls the magnitude of the immune response. Although T-suppressor cells are antigen-specific, they are also able to bind antigen in the absence of accessory cells or specific products of the MHC. T-suppressor cells appear to be selectively activated when the antigen is presented in certain routes, that is, intravenously or orally, or when administered in very low doses (low zone tolerance). T-cell induced suppression might be mediated directly by Ts cell or by antigenspecific and non-antigen-specific soluble factors released by them (TSF). Ts binding consists of an antigen-binding portion and an I-J molecule (28 kD) that binds to the acceptor cell through the antigen molecule and an I-J binding site [68]. Regulatory T cells inhibit tumor immunity in some experimental systems [105], and recent clinical trials [20] utilizing antibodies to Treg cells have demonstrated that these biological drugs have clear therapeutic activity (see Chapter 6).

T-cytotoxic Cells T-cytotoxic cells were originally described as the effector cells of specific cell-mediated cytotoxicity against allografts, virus-infected cells, bacteria, tumor cells, etc. The Tc lymphocytes also interact with the antigen (most commonly a foreign cell, tumor cells, or virus-infected cells) through the T-cell receptor [54, 71]. The interaction of specific cytotoxic T cells with the target structure results in lysis of the latter. “Killing” of a target cell, including tumor cells, consists of a number of mechanisms that can be divided in three phases [35, 36, 131]. Initially, binding between target and effector cells must take place. The majority of cytotoxic T cells are CD8+ and therefore recognize the antigen in the context of MHC 1 molecules (class I restriction [32]). In addition, they can also recognize foreign class I antigens alone, indicating that they can be effective in the destruction of allogeneic transplanted tissues. A small percentage (10%) of cytotoxic T cells bearing the CD4+ phenotype recognize antigens in association with class II molecules and might play a role in lysis of virus-infected cells [36]. The second phase (“programming for lysis”) involves the reorganization of cytoplasmic organelles, including polarization of the microtubule organizing center and tubulin and actin. This leads to an increased area of contact between the target and effector cells, leading to an increase in the efficiency of the cytolytic process. This is followed by reorientation of the cytoplasmic granules toward the binding region, fusion to the membrane, and release of lytic molecules (perforins) contained in those

91 granules. The presence of calcium is the limiting factor in this phase, since polymerization of the perforin molecules on the membrane of the target cell with channel formation does not occur in the presence of calcium chelators [42, 66, 71]. The granules also contain other factors known to mediate cytotoxic and cytostatic effects on tumor cells, such as esterases and proteoglycans [89]. The third phase includes the delivery of the lethal hit and death of the target cell. Following interaction with the cytotoxic T cell, disturbances in the ion concentrations and DNA fragmentation and fluid extrusion occur, culminating in target-cell disintegration. Interestingly, cytolytic cells are resistant to the cytolytic mechanisms that they generate, and in this way each effector Tc may kill more than one target cell. Studies of Tc cells infiltrating human tumors has helped define therapeutically active T cells leading to tumor regression. These cells have proven exceedingly useful in defining relevant human tumor associated antigens for vaccine formulation.

B lymphocytes B cells are responsible for humoral immunity, and, like T cells, are present in peripheral blood where they represent about 10–20% of the circulating lymphocyte pool. After activation, B cells mature into specific antibody-secreting cells or plasma cells [48, 128, 133]. Mature B cells express surface immunoglobulins (Ig) with identical specificity to the antibodies (Ab) they secrete [128]. The majority of peripheral blood B cells express both IgM and IgD, while most B lymphocytes present in body tissues express IgG, IgA, and IgE. Most B cells also express Class II molecules in the cell membrane. These molecules participate during the physical cell–cell interaction that takes place during T-B cell cooperation. In addition, receptors for the Fc portion of IgG, the third component of complement (C3b), IL-2 and a large number of other markers (CD19, CD20, CD21, CD22, etc.) have been found on the surface of mature B lymphocytes [48, 128, 133]. The process of activation and maturation of B lymphocytes into plasma cells involves the interaction of antigens with the specific immunoglobulins on the surface of B lymphocytes, which triggers cell activation and proliferation. The cooperation between B cells and Th/I, macrophages, and other accessory cells, a process requiring identity of Class II products of the MHC among these cells, is necessary to induce optimal B lymphocyte responses. However, binding of antigen to B cells is necessary but not sufficient to initiate antibody

92 production. Nonspecific maturation and differentiation factors (i.e., cytokines) mainly produced by Th/I cells are also involved in the activation and progression of mature B cells into antibody-forming cells [49, 128]. Among these factors are IL-1 (produced by macrophages and other accessory cells), IL-2, IL-4, IL-5, IL-6, IFN-γ and other factors secreted by activated T lymphocytes [17, 22, 40, 44, 49, 52, 78, 79, 88, 90, 128]. Several of these cytokines, including IL-1 and IL-4 are also produced by B cells [22, 49]. Most of the circulating B lymphocytes are in the resting state (G0 phase of the cell cycle), but they become activated after interaction with the specific antigen or antigen-presenting cells in the presence of IL-4 and IL-1, progressing to the G1 phase of the cell cycle. Activated B cells undergo cell division in the presence of T cells and secreted cytokines. Differentiation of proliferating B cells into plasma cells is mediated by IFN-γ, IL-4, IL-5 and IL-6 [40, 78, 79]. Alternatively, proliferating cells may return to the resting state and remain as memory B lymphocytes (Fig. 3). During the maturation process, they not only increase their rate of Ig synthesis and actively secrete Ig, but they also switch the class of heavy chains that carry the variable region of Ig involved in antigen recognition [49, 128]. It has been demonstrated that a single clone of proliferating B cells may switch at any division. Not all the proliferating clones mature into secreting plasma cells. Some return to the resting state and remain as long-lived memory cells [128].

Monocytes and Macrophages Monocytes are large cells (15–30 μm in diameter) that comprise about 10–25% of peripheral blood mononuclear cells (PBMC). They originate in the bone marrow from monoblasts, circulate in peripheral blood as monocytes, and then enter various tissues where they are termed resident tissue macrophages (Fig. 1). The large degree of heterogeneity that exists within the macrophage population is believed to represent different stages of the maturational process and reflect environmental conditions at the tissue level, rather than distinct macrophage subpopulations [57]. Monocytes/macrophages can be identified by a number of methods, including morphology, ingestion of particles (such as latex), histochemical staining of cytoplasmic enzymes (such as nonspecific esterase), and by flow cytometry with a number of mAb that recognize markers present on their membrane (i.e., CD40, CD54, CD80 and 86, etc.) [46]. Monocytes/macrophages play a critical role in the defense against bacterial and other infections by ingest-

Current concepts in immunology ing and killing the attacking microorganisms [57, 123]. They have also been shown to be very effective in destroying neoplastic cells and removing dead or injured cells (scavenger function). In addition, monocyte/macrophages are critical for the development of normal immune responses since they process and present antigen (dendritic cells) to T lymphocytes and secrete cytokines that play a major role in the initiation of specific immune responses, such as IL-1 and IL-6 [10, 11, 17, 22, 44, 51, 57, 90, 104, 119] (Fig. 3). Monocytes/macrophages have been shown to secrete more than a hundred different molecules that mediate their functions. These products include (a) enzymes with bactericidal capacities, such as lysozyme and lysosomal hydrolases, neutral proteases (e.g., collagenase and plasminogen activator); (b) arachidonic acid metabolites (e.g., prostacyclin, prostaglandin E2, and leukotrienes), which have profound effects in the regulation of the immune response; (c) reactive oxygen metabolites (e.g., superoxide, O2 radical, and H2O2), which are important mediators in macrophage-mediated cytotoxicity; (d) complement components; (e) coagulation factors; and (f) cytokines, which in turn exert a multiplicity of regulatory actions including playing a critical role in antigen presentation to T cells [10, 11, 17, 22, 44, 51, 57, 90, 104, 119, 123] (Fig. 3). Antigen-presenting cells, which include dendritic cells, Langerhans cells, veiled cells, interdigitating cells, and others in addition to macrophages, are characterized by their ability to process and present antigen to T cells in conjunction with MHC class molecules, as well as cytokine production, resulting in T-cell activation. Macrophages incorporate antigens nonspecifically by phagocytosis or by binding of immune complexes to the Fc receptors. In contrast, activated B lymphocytes bind antigen via the specific antigen receptor, and dendritic cells are likely to process antigen directly in the cell membranes. These short peptides (8–24 amino acids) are then linked to the antigen cleft in the class II molecules and returned to the cell membrane for interaction with the Th/s cells [32] (Fig. 4).

Natural Killer Cells Natural killer cells were first discovered by Oldham and co-workers [80, 81]. Later studies defined their ability to bind and lyse sensitive tumor and virus-infected normal cells without the need of previous sensitization [31, 59, 84, 121]. These cells, which constitute approximately 15% of peripheral blood lymphocytes, are a relatively homogeneous cell type identified as large granular lymphocytes

Robert K. Oldham (LGL). LGL are nonphagocytic, express receptors for the Fc portion of IgG (FcR-positive), and have a high cytoplasm to nucleus ratio, indented nucleus, and a few discrete azurophilic granules in the cytoplasm [84, 121]. Several monoclonal antibodies have helped define the surface markers and the phenotype of natural killer cells. Some human NK cells express the following T-cell markers: CD2, CD8; and after activation they express T10 (a marker present in thymocytes and activated T cells) [46, 59, 84, 121]. NK cell function is regulated by stimulatory and inhibitory mechanisms. The generation and function of NK cells are regulated mainly by IFN-α, IFN-γ, IL-2 and IL-4 [59]. IL-2 and IFNs increase the lytic activity of mature NK cells, promote the recruitment and activation of nonlytic cells, and induce proliferation of precursors and mature cells either in vivo or in vitro [121]. In addition to their nonspecific cytotoxic activity against tumor cells, NK cells may exert a regulatory role in specific immune responses mediated by T and B cells because of their ability to produce a variety of cytokines including IFN-α and γ, IL-2, IL-1, CSF, and TNF-α [59, 84, 121]. Natural killer cells play an important role in the resistance to growth and metastasis of malignant tumors [81]. Results obtained using several in vivo experimental models suggest that NK cells are of paramount importance during the early stages of tumor development. For example, NK-sensitive clones of tumor cell lines take longer to develop into palpable tumors when injected in the footpads of syngeneic hosts than NK-insensitive clones with similar doubling times [4, 39, 47]. Furthermore, selective depletion of natural killer cells in experimental animals has been correlated with increased frequency of spontaneous tumor metastasis.

Lymphokine-activated Killer Cells The lymphokine-activated killer phenomenon (LAK) was originally defined as the ability of PBMC incubated for several days in vitro in the presence of IL-2 to lyse freshly isolated tumor cells and NK-resistant targets, such as the HL-60 cell line [33, 34, 76, 100, 102, 130]. The LAK phenomenon, which is not MHC restricted [85], can be mediated by cells expressing markers present in both T and NK cell populations. For example, the presence of cell populations with LAK activity expressing CD16 (Leull, an NK marker) and/or CD56 (Leu19, a marker present in both Tc and NK cells) has been reported in short-term cultures. Most of the LAK activity

93 in long-term cultures stimulated with IL-2 and anti-CD3 was observed in the CD3+ CD16−; CD3− CD16+; CD3+ CD4− CD8−; CD16− and CD3− CD56+ cell populations. On the contrary, the CD3+ CD4+ or CD3+ CD8+ populations present in these cultures exhibited a significantly lower level of LAK activity [77]. Interleukin-2 mediated induction of LAK cells can be enhanced by the addition of IL-1α or IL-1β, probably by rendering LAK precursors more susceptible to the activity of IL-2 [14, 21]. Interestingly, recent evidence indicates that the generation of LAK cells is dependent upon the expression of the p70/75 (intermediate affinity) IL-2 binding protein in the cell membrane. The expression of p70 occurs in the absence of the p55 (low affinity, Tac) IL-2 binding protein, which is critical for the expression of high-affinity IL-2R in activated T lymphocytes [86]. In addition to their remarkable tumoricidal activity, LAK cells are able to secrete IL-1α, IL-1β, IFN-γ, TNF-α, and TNF-β (lymphotoxin) [56]. Experiments in murine models have clearly established the in vivo antitumor activity of the cells mediating LAK activity (i.e., LAK cells) when administered in conjunction with high doses of IL-2 [55, 56, 70, 103]. Based on these observations, a number of clinical trials in advanced cancer patients were done and these have demonstrated clear, but limited, antitumor activity [16, 101, 127].

Clinical Assessment of Immune Competence In Vivo Delayed Type Hypersensitivity (DTH) DTH is a test of cell-mediated immunity based on the response against a test antigen after it is injected intradermally, or applied topically to the skin (Table 1). Reddening and induration of the test site occur in 8–12 h, reaches a peak at 48–72 h, and thereafter slowly subsides. The DTH lesion is characterized by the accumulation of mononuclear cells in the subcutaneous and deep and superficial dermis [8]. It should be stressed that DTH reactions are complex immunological phenomena requiring the participation of effector T lymphocytes as well as monocytes/macrophages as accessory cells. Thus, a deficit of T cells or monocyte/macrophages, or the presence of certain serum inhibitory factors that impair T cell or monocyte/macrophage activity, or active suppressor cells, leads to impaired DTH reactivity. Antigens used in DTH testing can be divided into two classes: recall antigens and neoantigens (an antigen to

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Current concepts in immunology

Table 1. Selected immunologic tests Parameter T Cells Total and subpopulations Responses to antigens Cytokine production T-receptor gene rearrangement Cytotoxicity B Cells Total number Surface Ig Ig gene rearrangements Serum immunoglobulins Serum Ig classes Reticuloendothelial system: Monocytes, macrophages Cytokine production Phagocytosis Chemotaxis Tumor cell killing Activation

Test Flow cytometry Cell proliferation elicited by PHA, Con A, MLR Delayed-type hypersensitivity Bioassays/RIA/ELISA Southern blot analysis Release of radiolabeled compounds from target cells Flow cytometry Flow cytometry Southern blot analysis Serum electrophoresis Nephelometry, RID

Bioassays, RIA, ELISA Cytotoxicity in vitro Production of oxygen radicals

which the subject has never been previously exposed). One of the most commonly used neoantigens is 2,4-dinitrochlorobenzene (DNCB), which has been used extensively in cancer patients. Among the recall antigens, tuberculin (PPD), mumps, tricophytin, and candida have been the most commonly used [8]. In general, several recall antigens are studied simultaneously to ensure that a patient will have been exposed to at least one or more of them. The interpretation of skin tests can vary widely between studies, since antigen concentration, reader variability, patient’s prior exposure to the antigen, boosting effects of repeated antigen administration, time course of the reaction, and definition of a positive reaction are all variables that can influence the final assessment of a positive or negative response. Of the available agents, only DNCB has been a consistently useful skin test agent. However, DNCB is an awkward reagent to employ, as repeated testing will clearly yield anamnestic responses. Thus, only the original test on an individual can provide clear information on the responsiveness to a neoantigen.

Humoral Immunity The testing of the levels of total and different classes of Ig, as well as the primary or secondary antibody responses to a variety of antigens in the sera of cancer

patients, has been used as an indication of the existence of alterations of immunoregulatory processes or of defects in the production of Ig. Quantitation of the levels of Ig is usually measured by the single radial immunodiffusion method, ELISA, or by nephelometry [62, 63]. Among the antigens used to elicit primary and secondary antibody responses are brucella endotoxin, salmonella extract, hemocyanin, etc. Antigens have been divided into T-cell dependent or T-cell-independent depending upon whether or not Th/I cells are required for maximal antibody production by B cells.

Reticuloendothelial System The in vivo phagocytic cell function can be assessed by measuring the rate of clearance from the bloodstream after intravenous injection of a variety of materials including colloidal gold, bacterial proteases, lipid emulsions, or aggregated human albumin labeled with radioactive iodine [72, 114]. Another technique that has been used to study reticuloendothelial function is the Rebuck skin window. In this procedure, a microabrasion of the skin surface is made, followed by application of a cover slip to the raw area, and assessment of the accumulation of macrophages onto the coverslip [45].

In Vitro Immune Cell Quantitation The accuracy in the quantitation of the levels of the different cells and cell subsets involved in the immune response has improved dramatically with the advent of the monoclonal antibody technology. It is possible to accurately estimate the numbers of monocytes/macrophages, Th/I, Tc, and Ts (regulatory) lymphocytes, LGL, B lymphocytes, granulocytes, etc. with the use of flow cytometry using mAb that recognize specific markers on the surfaces of individual cell types [46, 99].

Lymphoproliferative Responses This has been one of the most widely applied tests for the determination of lymphocyte function. It is based on the property of lymphocytes to undergo blast transformation and proliferate in response to mitogenic or antigenic stimulation [61]. The most commonly used mitogens include phytohemagglutinin (PHA) and concanavalin A (Con A), which primarily stimulate T cells, and pokeweed mitogen (PWM), which stimulates both T and B cells. Whereas mitogens nonspecifically activate broad subpopulations of

Robert K. Oldham cells, antigens activate specifically sensitized antigenreactive clones. The assay can be performed with whole blood but most laboratories usually first isolate PBMC (from whole blood, lymph nodes, or tumor specimens) by density-gradient fractionation on Ficoll-Hypaque followed by incubation with the appropriate mitogen at various concentrations. Seventy-two hours later cultures are pulsed with radiolabeled [3H]thymidine, which is incorporated into the cellular DNA. Cells are then harvested and the radioactivity quantitated in a liquid scintillation counter and expressed as counts per minute (cpm). A similar technique has been extensively employed to measure proliferative responses to antigenic stimulation. In these studies, mitogen is replaced by the specific antigen under study and longer incubation periods are usually required [61]. Antigen responses are usually lower in magnitude that those observed with mitogens, since they represent the activation of specific T-cell clones, rather than most lymphocytes as is the case with mitogens. Another proliferative response assay is the mixed leukocyte reaction (MLR). This test is based in the ability of T cells to proliferate in response to alloantigenic stimulation (that is cells expressing different MHC antigens). This is the in vitro correlate of in vivo graft rejection, and is usually performed by incubating PBMC obtained from the patient (responder cells) with irradiated PBMC obtained form one or more histoincompatible individuals (stimulator cells) for 6 days. Response is measured by [3H]thymidine incorporation as described above [61].

Cytotoxicity Cytotoxic reactions mediated by antigen-specific T cells, NK cells, antibody-dependent killer cells, lymphokine-activated killer cells (LAK), and activated macrophages are measured most commonly in microcytotoxicity assays [9, 30]. In general, target cells are labeled with a radioactive element able to bind to some intracytoplasmic structure, which is released to the media when the cell dies. Fixed numbers of labeled target cells are incubated in microtiter plates with different numbers of effector cells. The assays last for from 4 to 48 h, after which the supernatants are collected and the radioactivity counted. The extent of radioactivity present in the supernatants is in direct relationship with the amount of killing. The results are expressed as percent cytotoxicity [9, 31].

95 been developed for their quantitation in the clinical setting [28]. For example, the measurement of IL-2 is based on (a) the fact that T lymphocytes produce IL-2 in response to mitogenic and antigenic stimulation and (b) the ability of IL-2 to induce lymphocyte proliferation and maintain in vitro T cell lines (called IL-2 dependent cell lines). The first step is the generation of cell culture supernatants containing IL-2 by incubating PBMC with mitogens (usually PHA or Con A) for 24–48 h. The IL-2 content in these supernatants is then determined by their ability to support the in vitro growth of an IL-2 dependent T-cell line, as assessed by [3H]thymidine incorporation after 24–48 h of culture [30]. Currently, the concentrations of a variety of cytokines, including IL-1, IL-2, IL-4, IL-6, GM-CSF, TNF-α, IFN-γ, etc., are measured using commercially available immunoassays [radioimmunoassay (RIA) and/or ELISA], which employ specific monoclonal antibodies (see Chapter 8).

Immunoglobulin Production Immunoglobulin production can be assessed in vitro by exposing a suitable effector cell population (e.g., PBMC) to either a polyclonal B-cell mitogen (e.g., PWM) or specific antigen and following a period of incubation, detecting the immunoglobulin produced. Secreted Ig can be detected either in the culture supernatants by using ELISA or radial immunodiffusion (RID) techniques.

Phagocytic Cell Function Both mononuclear (e.g., monocytes/macrophages) and polymorphonuclear (PMN; e.g., granulocytes) leukocytes exhibit phagocytic activity. Well-established techniques exist for the assessment of monocyte phagocytosis, hemotaxis (motility), and response to lymphokines (e.g., migration inhibition factors, MIF) [25, 26, 65, 70, 108]. Established techniques also exist for the assessment of monocyte/macrophage cytotoxicity [50]. Various tests are also available to measure PMN functions, including tests of phagocytic cell activity, bactericidal capacity, and the ability to take up and reduce nitroblue tetrazolium dye (NBT) [3].

Immunoregulatory Cell Functions

Lymphokine Production

These assays are technically difficult to perform and are not usually part of the routine assessment of immune competence. Three broad classes of immunoregulatory cell assays have been utilized:

As the roles of some of the soluble mediators in the immune response have become clearer, assays have

1. Coculture assays. In these assays effector cells (e.g., B cells, T cells) are exposed to a polyclonal activator

96 (e.g., PWM, MLR) in the absence or presence of test cells. The test-cell population might induce either enhancement (e.g., increases in antibody production or enhanced MLR responses) or reduction (suppression) of the response of the effector cells [125]. 2. Mitogen-induced suppressor T-cell activity. In this system suppressor cells are generated from resting T cells by exposing them to a polyclonal T-cell lectin such as Con A. Con A induced suppressor cells are then added in coculture to an effector-cell assay as described above. 3. Adherent suppressor cells. The presence of suppressor monocytes can be assessed by either a positive or negative effect. In the former situation, removal of adherent cells (e.g., monocytes) leads to augmentation of effector cell function (e.g., lymphoproliferative responses to mitogens), while in the latter case, readdition of adherent cells leads to suppression of the response [120].

Quality Control of in vitro Assays In practice, it has been difficult to standardize lymphoproliferative or cytolytic assays to insure the universal validity and reproducibility of results. A number of conditions affect the comparability of in vitro assays as performed in different laboratories such as assay incubation time, presence or absence of physiological buffers and/ or serum supplementation, choice of methodology to prepare the responder cell population (i.e., purified lymphocytes or unfractionated mononuclear cells), and the purity and/or concentrations of mitogen/antigen, or radioactive label. In addition, various in vivo confounding variables, such as the influence of concurrently administered drugs, or seasonal and/or diurnal effects, provide further sources of assay variability. A number of procedures have been employed in an attempt to minimize interassay variability such as the use of cryopreserved reference lymphocyte preparations [43, 82] or indices based on results from a panel of reference lymphocytes [15]. Nevertheless, because of the inherent variability of in vitro assays, only limited successes have been achieved in clinical trials to date. Indeed, neither appropriate guidelines for interpreting the results of clinical trials employing immunorestorative agents nor identified statistically significant end points upon which to select appropriate sample sizes for such clinical trials currently exist [83]. Acknowledgment Dr. Susana A. Serrate-Sztein (National Institutes of Health, Bethesda, Maryland) and Dr. Marcelo B. Sztein (University of Maryland School of Medicine,

Current concepts in immunology Baltimore, Maryland) contributed much of this chapter in the second edition, which was revised by the current author in the third, fourth, and fifth editions. I would especially like to acknowledge the assistance of Dr. Richard S. Schulof, who co-authored this chapter in the second edition, but died in an accident during the preparation of the third edition.

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6

Therapeutic approaches to cancer-associated immune suppression ROBERT K. OLDHAM

It is now well established that many cancer patients exhibit in vivo and in vitro evidence of immune suppression, which often correlates with tumor-cell burden, stage of disease, and prognosis. Except in transplantation associated cancers, cancer-associated immune suppression appears to be a direct result of the presence of disease, or follows treatment for it, rather than being an antecedent or predisposing condition. However, the precise role that nonspecific and/or specific antitumor immunity plays in the control of human cancer remains controversial. Indeed, there is some evidence that suggests that the development of certain immune responses may lead to augmented tumor cell growth rather than tumor regression [178, 287, 406, 407, 582]. The clinical relevance of the relative state of general immunocompetence in determining whether or not a patient can be cured of cancer also remains controversial. A case in point is the dissociation between immunodeficiency and curability of patients with Hodgkin’s disease. Despite the well-recognized immune suppression associated with this malignancy, and the immunosuppressive therapies used to treat patients (e.g., lymphoid irradiation and steroid-containing combination chemotherapy), it is one of the most curable of all cancers. Such a discrepancy suggests that unique biological properties of malignant cells, rather than the general immune competence of the patient, are the more critical factors in determining the curability of cancer. Even though the precise relationship between the general state of immune competence and cancer curability has not been established, investigators have administered a variety of agents to cancer patients including biologicals, vitamins, hormones, and drugs, hoping that the reversal or prevention of general immune suppression might translate into prolonged disease-free remissions and improved patient survival. This chapter will review the multifactorial basis of cancer-associated immune suppression and the therapeutic strategies that have been utilized. The sections on immune suppression will focus exclusively on the general assessment of immunity, and not specific antitumor immunity. The sections on therapy of immune suppression will be limited to those biological response modifiers (BRMs),

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

both chemical and biological, that were administered either to restore depressed immunity to normal, or to prevent the deterioration of immune competence due to surgery, radiation therapy, or chemotherapy. BRMs that have been employed as adjuvants along with tumor cell or tumor antigen vaccines to boost specific anti-tumor immune responses, or whose primary mechanisms of action are by activating effector cells directly, such as interferons and/or interferon inducers, muramyl dipeptide and cogeners, or interleukin-2 and other interleukins (e.g., tumor necrosis factor), will not be covered, nor will more “traditional” whole organisms from the older literature [e.d., Bacillus Calmette-Guerin (BCG), Corynebacterium parvum, or mixed bacterial vaccine].

Immunosuppression and Cancer It is clear that preexisting immunodeficiency plays a permissive role in the development of certain cancers, such as malignant lymphoma or Kaposi’s sarcoma [393, 448]. Patients with primary (e.g., Wiscott-Aldrich syndrome, at ataxia-telangiectasia) or secondary (e.g., acquired immune deficiency syndrome, AIDS) immunodeficiency syndromes in which defects in cell-mediated immunity predominate and patients receiving immunosuppressive drugs following organ transplantation all exhibit an increased incidence of Burkitt’s and non-Burkitt’s lymphoma, Kaposi’s sarcoma, and a variety of other tumors not otherwise commonly seen. In several instances, following the cessation of immunosuppressive therapies, cancer regressions have been noted, suggesting that when immunocompetence is restored, control of tumor growth may occur. Patients with primary (e.g., Bruton’s-type agammaglobulinemia) and secondary (e.g., chronic lymphocytic leukemia) immunodeficiency syndromes in which defects in humoral immunity predominate also exhibit an increased incidence of malignancies including skin cancer, primary brain neoplasms, sarcomas, carcinomas, and leukemias [167, 240, 394, 436]. Since the malignancies associated with underlying immunodeficiency states are not those common in the

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general population, it would appear that most adult malignancies do not reflect underlying immunodeficiency. It is more likely that a combination of genetic factors, chronic immune system stimulation (possibly as a result of recurrent infections), the presence of infectious carcinogenic agents (e.g., viruses), chronic chemical carcinogenesis, and other undefined factors leads to the high incidence of certain cancers in patients with primary or secondary immunodeficiency states. There is evidence to indicate that certain carcinogens – for example, asbestos – can suppress immune functions such as NK activity [430]. However, for the majority of common cancers, the overwhelming evidence suggests that immunodeficiency arises secondarily as a consequence of cancer and the therapies used to treat it; that is, cancer itself is an immunosuppressive disease. Cancerassociated immunodeficiency is further influenced by other factors, including age and genetic background, as well as environmental factors, such as nutritional status, stress, and infections. For example, nutritional status is frequently impaired in patients with head and neck cancer [87], which accounts for many of the immunodeficiencies reported in these patient populations such as decreased T cell numbers. Thus, it is the balance of many different endogenous and exogenous factors that ultimately contributes to the overall immune deficiency state of cancer patients.

times impossible) to directly compare the results of in vitro and in vivo immunologic assays from different studies. Nevertheless, a number of general conclusions have been reached concerning cancer-associated immune suppression. No single explanation, or generally agreedupon concept, has emerged to explain the immunodeficiency. Rather, there is a complex set of interactions involving a number of different mechanisms. This multifactorial basis of immunodeficiency is outlined conceptually in simplified form in Fig. 1. The scheme is equally applicable to T−, B−, NK−, or phagocytic effector-cell immune mechanisms. Five major factors each play a role in determining the immune responsiveness of cancer patients: (a) the proportions and absolute numbers of circulating, tissue-derived, or intratumoral effector cells; (b) the intrinsic functional capabilities of the effector cells on a per-cell basis; (c) the influence of immunoregulatory helper and suppressor cells; (d) the influence of local systemic-circulating and immunomodulatory soluble factors; and (e) the influence of systemic treatment. In Fig. 1, it is shown schematically how the relative contributions of these five factors modulate immune responsiveness of cancer patients as assessed with in vitro and in vivo assays.

Multifactorial Basis of Immunodeficiency in Cancer Patients

Any basic immune response reflects both the number (or relative proportion) of effector cells present and the intrinsic functional capability of the effector cells. Thus, impaired immunity can result purely from a deficiency in absolute numbers (or proportions) of effector cells

Effector Cell Numbers and Function

Because of subtle variations in methodologies employed by different investigators, it is often difficult (and some-

Helper Cells

Tumor-derived Factors

Ontogenesis

Precurser (Stem) Cells

Local Immunomodulatory Factors

EFFECTOR CELLS

Immune Boosters (adjuvants)

Lymphokines

EFFECTOR CELLS FUNCTION

In Vivo Assays

IMMUNITY

In Vivo Assays

Suppressor Cells

Serum Inhibitory Factors

Figure 1. Multifunctional basis of immunodeficiency in cancer patients

Cancer Therapy (i.e. RT, Chemo)

Robert K. Oldham that otherwise exhibit normal function on a per-cell basis, from effector cells that exhibit intrinsic functional defects despite normal cell numbers, or from a combination of both decreased cell numbers and impaired function of the individual cells. The primary immunodeficiency syndromes, such as the DiGeorge syndrome, ataxia-telangiectasia, and severe combined immunodeficiency syndrome (SCID), are examples of immune deficiency resulting from defective ontogenesis of the immune system. However, since the development of the immune system occurs during fetal and neonatal life, defects in ontogeny are not a consideration in the immune deficiency of cancer patients. Cancer patients do, however, often exhibit lymphocytopenia and decreased T-cell numbers [75, 101, 320, 352, 376, 384, 416, 481, 547], which contribute to their overall state of immunodeficiency. In contrast to primary immunodeficiency states, the effector cell abnormalities detected in cancer patients arise secondarily, probably as a result of the suppressive effects of cancer-derived factors on effector cell production and/or survival. When PBMC is used as the source of effector cells in a lymphocyte or monocyte function assays, it cannot be established whether a depressed functional immune response results from a decreased proportion of effector cells within the PBMC mixture, from intrinsic functional defects of the effector cells themselves, from the presence of excessive suppressor cell activity, or from a combination of abnormalities in cell proportions, functions, and immunoregulation. There is considerable evidence indicating that cancer patients exhibit depressions in mitogen and antigen-induced proliferative assays [75, 94, 97, 101, 102, 141, 171, 174, 197, 239, 284, 310, 320, 333, 352, 376, 408, 420, 429, 481, 547, 574] and in cytolytic activity [29, 287, 409, 496, 542] using PBMC as effector cells. In some cancers, for example, head and neck cancer [399] and Hodgkin’s disease [466], it has been possible using purified T cells to document intrinsic functional T-cell defects. Studies in cancer patients have shown an impaired ability of T cells to produce the lymphokine IL-2 [190, 321, 353] following activation by mitogens; but that spontaneous IL-2 production (reflecting possible stimulation due to circulating tumor antigens) is increased [235]. However, in Hodgkin’s disease, although IL-2 production and/or IL-2R expression of PBMC has been reported to be low [48, 359, 490, 580], the abnormality in T-cell lymphoproliferation does not appear to be related to defects in the IL-2 system [48]. Peripheral blood monocytes and polymorphonuclear leukocytes isolated from patients with a variety of cancers also exhibit depressed functional activity [31, 73, 103,

103 136, 172, 203, 487]. NK-cell activity is also frequently depressed in cancer patients [158, 409, 542]. At the local level, NK activity may be more impaired than other lymphocyte functions such as LPRs [17]. Thus, many studies now suggest that intrinsic functional defects of effector cells contribute, at least in part, to the immunodeficiency of cancer.

Immunoregulatory Cells The intrinsic functional capabilities of T, B, NK, and phagocytic cells are modulated by interactions with a variety of immunoregulatory cells. The T- and B-cell functions can be influenced either positively or negatively, depending upon the relative balance between Tind and Tc/s cells, as well as by number and function of T-regulatory cells (Treg)[178, 540, 582]. Once again, both absolute numbers (or proportions) and immunoregulatory function per cell are considerations in determining the overall influence of immunoregulatory cells on effector cell function. Another important immunoregulatory cell is the monocyte/macrophage. Most normal immune responses require the presence of monocytes/macrophages as accessory cells for optimal processing of antigen and appropriate activation of effector lymphocytes. In many disease states, including cancer, monocytes/macrophages exhibit suppressor-cell activity rather than helper-cell activity [540]. It is likely that such “suppressor” cells may develop in cancer patients to release factors capable of suppressing tumor growth as their primary action and thus that the suppression of immune reactivity is an “innocent bystander” effect. The influence of monocytes/macrophages is dependent upon cell numbers (or proportions) and their state of activation [540]. There is considerable evidence for immune suppression mediated by activated monocytes/macrophages in cancer patients [5, 28, 30, 50, 80, 193, 194, 195, 250, 257, 330, 461, 523, 532, 540]. For example, monocyte-mediated suppression of lymphocyte-proliferative functions have been demonstrated in Hodgkin’s disease [154, 175, 189, 336, 454, 535] as well as in patients with lung cancer [5, 93, 250], breast cancer [194, 253], malignant melanoma [363], colorectal cancer [28, 194, 524], head and neck cancer [30, 50, 550], bladder cancer [195], and a variety of other malignancies [86, 540]. Monocyte-mediated suppression of cytolytic effector-cell activity has also been demonstrated in cancer patients [14, 18, 123, 151, 228, 229, 540], including NK-cell activity [246], and autologous T-cell-mediated anti-tumor cytotoxic responses [151, 461]. In comparative studies, it has been demonstrated that such suppressor-cell activity is of

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greater magnitude at the local level (i.e., intratumoral, effecting TIL cells and in draining lymph nodes) than in the systemic circulation [14, 18, 123, 228, 229]. Of interest is the finding that monocyte/macrophages activated to kill cancer cells can suppress T-cell cytotoxicity toward the very same cells [532]. The contribution of suppressor lymphocytes to the generation of cancer-associated immunodeficiency is less certain and still somewhat controversial. Several studies that identified helper and suppressor T-cells on the basis of the presence of surface Fc receptors for IgM (Tμ cells) or IgG (Tγ cells), respectively, have suggested that the Tind/Tc/s ratio is depressed in cancer patients [204, 267, 531]. However, more recent studies using monoclonal antibodies to detect surface antigens indicate that the relative proportions of Tind and Tc/s cells are preserved in most untreated cancer patients, unless they have extremely widespread disease [130, 200, 266, 310, 462]. Few reports have focused on functional aspects of suppressor lymphocyte activity in cancer patients. Definitive conclusions are lacking, although such activity has been reported in patients with Hodgkin’s disease [217, 480] and various solid tumors [151, 236, 237, 283, 537]. Inducible suppressor T-cell activity has generally [104, 183, 236, 464] but not always [521] been found to be depressed in cancer patients. Over the last 10 years, studies of immunoregulation have focused increasingly on a subset of T-cells called T-regulatory cells (Treg). Tregs have been well defined as a distinct population of CD4 T-lymphocytes, which constitutively express CD-25 and are further characterized by the molecules CTLA-4, FOXP-3, and a TNF receptor. Treg cells also produce immunosuppressive cytokines, such as IL-10 and TGF beta [307, 444, 467]. Treg cells with the phenotype CD-25+, FOXP-3+, CD-127− have been found in tumor specimens and are suspected as cells which suppress the activation of CD-4− and CD-8+ tumor-infiltrating lymphocytes active in tumor destruction. The presence of these cells correlate with poor clinical outcome in cancer patients [218, 450, 467]. Clinical studies are now underway, using antibodies to CTLA-4, to attempt to lessen the immunosuppressive effects of Treg and, thereby, enhance the effectiveness of the CD-4 and CD-8 T-cells and immune effectors [573].

Immunomodulatory Factors It is now well established that, in normal immune reactions, the influence of immunoregulatory cells is mediated at least in part by the local release of cytokines such as IL-1 by monocytes, and IL-2, 6 and 10, as well as γ-interferon by helper T cells (see Chapter 8). In a vari-

ety of disease states including cancer, there may be a deficiency in the production of cytokines by cells that normally produce them [159, 190, 206, 279, 353, 360, 548, 572, 579]. The release in cancer patients of a variety of immunomodulatory factors produced by monocytes/macrophages and/or the tumor cells themselves has been described. Immunosuppressive factors, for example, the E series of prostaglandins, are released in excessive quantity from both monocytes/macrophages [5, 28, 30, 50, 80, 154, 189, 195, 363, 386, 453, 523] and tumor cells [72, 139, 140, 221, 261, 358, 523], which could suppress cytolytic activities [358] and proliferative functions [123] of TIL cells as well as of circulating lymphocytes. However, prostaglandins alone do not mediate the complete suppressor cell activity of monocytes [28, 30, 154, 453, 523]. Monocyte-derived toxic oxygen metabolites, for example, hydrogen peroxide [336], and other as yet undefined mediators also appear to play a role. Increased monocyte production of prostaglandins has been shown to directly impair lymphocyte proliferative and cytolytic functions [139, 140] as well as the phagocytic function of monocytes themselves [150]. A variety of other factors have been identified that are shed by tumor cells and modulate both local as well as systemic immune responses. These include tumor-associated glycoproteins [536] and lipids [196]. For example, various melanoma-associated gangliosides have been shown to both up-regulate and downregulate lymphocyte responses to IL-2 [230]. Thus, it has been suggested that the unique tumor-antigen-associated phenotype of each individual tumor (based on the proportion of various immune-suppressing and augmenting factors released by it) determines whether the individual tumor will exhibit immune stimulation or suppression. In this regard, it has been demonstrated that whereas primary melanomas stimulate autologous lymphocyte responses, metastatic melanomas suppress immune responsiveness [510]. It is also apparent that a number of different substances with immunosuppressive properties can be detected in the blood of patients with cancer [469]. Many investigators have shown that serum from cancer patients is immunosuppressive in that it suppresses mitogen and antigen responsiveness of lymphocytes from normal donors [11, 55, 98, 164, 182, 188, 226, 416, 421, 475, 482, 502]. Cancer serum inhibitory factors include acute-phase reactants, such as α1-acid glycoprotein [39, 509], α-globulins [234], C-reactive protein [383], and immune complexes [35, 259]. A serum factor in young cancer patients has been reported to inhibit serum thymic-hormone bioactivity [121]. Circulating immune

Robert K. Oldham complexes have been shown to produce immunosuppressive effects by a variety of mechanisms including (a) blocking of B-cell differentiation and antibody production [435, 516]; (b) stimulating the production of anti-idiotype antibody which then interferes with the immune response to the original antigen [435, 516]; (c) inducing suppressor T-cells [105]; (d) reducing IL-2 levels [418]; and (e) blocking Fc receptors on effector cells [168]. Theoretically, the quantitative removal of tumor antigens, antitumor antibodies, and/or immune complexes could lead to a specific or nonspecific stimulation of the immune system, leading to an increase in general immune competence as well as in specific antitumor immune responsiveness. This has formed the basis for therapeutic trials with extracorporeal treatment of cancer with immobilized staphylococcal protein A [335, 362] and for attempts at plasma ultrafiltration. The mechanism of antitumor activity of such approaches has still not been defined. However, plasmapheresis has been associated with increased LPRs and antitumor immune responses [468, 470], probably due to removal of immunosuppressive serum factors. In conclusion, it is clear that the state of immunocompetence of an individual cancer patient is dependent upon a number of complex interactions among effector cells, immunoregulatory cells, and local and systemic immunomodulatory factors. A simplified explanation for the immunosuppression of cancer is that products released from the malignant cells themselves lead to (a) activation of suppressor cells; (b) impaired effector cell production and survival; and (c) direct inhibition of effector cell function. In assessing the state of immune competence with in vitro and in vivo assays, the underlying basis of immunodeficiency may or may not be identified.

Immunosuppression and Tumor Cell Burden In newly diagnosed, untreated cancer patients, the degree of immunodeficiency generally parallels the extent of disease [564]. The most reasonable explanation for such an association is that the release of tumor-derived immunosuppressive factors relates directly to the tumor cell burden. Immune parameters of which impairment correlates with extent of disease include DTH to recall antigens, blood effector cell levels (e.g., T-lymphocyte levels), lymphocyte functions, including proliferative responses to mitogens and antigens, cytotoxic activity, phagocytic cell activity, serum immunoglobulin levels, and primary antibody responses to a variety of immunizing antigens. Whereas quite early cancer is associated with only subtle

105 abnormalities of immune competence, more advanced disease, particularly after treatment, leads to abnormalities in all measurable immune parameters [131].

Solid Tumors Delayed-Type Hypersensitivity (DTH) Many reports have confirmed that as the extent of disease increases, the incidence of positive DTHS reactions decreases [457]. For example, in one large study of 234 patients with various types of cancer, patients without metastases exhibited positive reactions to DNCB (83%) more often than those with regional metastases (67%) or with distant metastases (41%) [401]. Similar findings using both DNCB as well as recall antigens have been noted in a wide variety of solid tumor-bearing patients, including those with breast cancer [2, 34, 36, 75, 76, 146, 296, 496], gastrointestinal cancer [76], head and neck cancer [495], lung cancer [8, 227, 389, 350, 561], renal cancer [86, 355], gynecologic cancer [561], urologic cancer [39, 85, 456], malignant melanoma [145, 408], sarcomas [145], and primary brain cancers [84, 317]. The incidence of positive DTH responses varies according to type of cancer. For example, patients with localized head and neck cancer had a much lower reactivity (42%) than patients with sarcomas (73%) and lung cancer (80%) [76]. This result has been explained, at least in part, by the alcohol intake and general state of malnutrition associated with head and neck cancer patients [54, 311]. In another study, it was found that the correlation between DNCB reactivity and clinical status was more pronounced in patients with squamous-cell carcinomas of the head and neck than in those with sarcomas or melanomas [562]. These findings suggest that although DNCB reactivity generally correlates with extent of disease, the relationships are modified depending upon the particular type of cancer. It has been easy to demonstrate the marked degree of impairment in DTH reactions found in patients with metastatic cancer; however, it has been much more difficult to discern relative differences in reactivity of patients with similar stages of disease whose tumors differ in size, regional lymph-node involvement, or local invasiveness. Therefore, DTH responses are insensitive immunological tests [101].

Lymphoproliferative Responses (LPR) An exhaustive literature has accumulated in which various in vitro lymphoproliferative assays were employed to assess the state of immune competence of cancer

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patients. In general, PBMC were the responder cells, and T-cell mitogens (e.g., PHA, Con A) were used to induce blastogenic transformation. Many reports have documented an inverse correlation between PHA response and stage of disease for patients with a variety of solid tumors. For example, among 179 patients with gastric carcinoma, 87% with stage I-II, 49% with stage II, and only 24% with stage IV disease could exhibit morphological evidence for blastogenic transformation of PBMC following exposure to PHA [376]. A study of 154 patients with carcinoma of the lung revealed that lymphoproliferative responses were significantly decreased in patients with stage III disease, but not in those with stages I and II [547]; other studies have reported similar results [102, 141, 197, 420]. Depressed LPR to alloantigens in mixed leukocyte culture (MLR) were observed in 46% of patients with small (TINOMO) stage I lung cancers [97]. Depressed mitogenic responsiveness has been correlated with advancing stage of disease in breast cancer [49, 239, 320, 333, 464] and, in one study, lymphocyte responses to PHA were impaired in earlier stages of disease than was DTH reactivity [496]. Similar inverse correlations between stage of disease and LPRs have been noted in colorectal cancer [171], malignant melanoma [174, 284, 481], and head and neck cancer [101]. Although many reports have documented that LPR generally decrease with advancing stage in solid tumor patients, this finding has not been universal. For example, lack of correlation between blastogenic responses and disease stage has been reported in patients with breast cancer [71, 310, 429], malignant melanoma [94, 174, 408], and colorectal cancer [352]. These reported inconsistencies have probably resulted from differences in techniques and quality control procedures for assays with a great deal of inherent variability. Nevertheless, the same general conclusion holds for in vitro LPR and DTH skin testing, namely, that there tends to be an inverse correlation between immune reactivity and tumor burden and/or stage of disease.

Immune Cell Quantitation There are many monoclonal antibodies available to quantitate blood, organ, and intratumoral levels of various immune effector cells; however, most of the early observations concerning lymphocyte quantitation and cancer were made using more primitive assay methods. The quantitation of lymphocytes in the peripheral blood of cancer patients has been studied as a measure of immunocompetence since the early 1920s, when lymphocytopenia was found to be common in patients with

advanced malignancies [578]. Other studies found an inverse correlation between total lymphocyte count and stage of disease in breast [416] and lung cancer [547]. The observation that human T lymphocytes could be easily identified by a binding reaction to sheep red blood cells to form E rosettes (E-RFC), coupled with the emerging recognition of the importance of T cells in immune responsiveness, led to numerous studies on cancer patients. Using E-rosette techniques, the percentage of circulating T cells has been determined for patients with a wide variety of solid tumors and correlated with clinical stage of disease [320, 481, 547, 567]. Other studies have not found such a correlation [75, 352, 376]. In general, however, as with the in vitro assays of T-cell functions, peripheral blood T-cell numbers tend to decrease in association with more advanced stages of cancer. Although assessments of B cells, monocytes, and Tind and Tc/s ratios have been performed less frequently in cancer patients, in general, B-cell numbers have paralleled T-cell numbers [266, 310, 352, 440], whereas absolute monocyte counts tend to increase with advancing disease [310]. T-cell subset abnormalities have been found in some patients with malignant melanoma [266], head and neck cancer [130, 204], and lung cancer [13, 527, 565]. T-cell subset abnormalities were more pronounced in lung lavage cells than in PBMC from patients with lung cancer [160], suggesting that such perturbations occur first at a local level before systemic abnormalities become detectable. In general, abnormalities of the Tind and T c/s ratios are found in patients with advanced or progressive disease. The ratio between Treg and CO8+ cytotoxic T cells correlates with immunosuppression and clinical outcome [218, 450].

Cytolytic Functions The measurement of nonspecific lymphocyte-mediated cytotoxicity has been employed with increasing frequency as part of the general assessment of immune competence in patients with solid cancers. In general, cytotoxic activity (most often NK activity) was depressed in cancer patients, particularly those with metastatic disease, but clear correlations have not been identified between impaired function and clinical stage of disease [262, 307, 505, 542]. Studies in patients with small-cell lung cancer and stage I and II malignant melanoma have found an inverse correlation between NK activity and amount of clinically detected tumor [158]. Similarly, NK-cell activity was more likely to be depressed in stage II breast cancer than in stage I disease. A large study of 247 cancer patients showed that

Robert K. Oldham circulating NK cell numbers, assessed by monoclonal antibody methods, were significantly reduced in patients with colon, lung, and breast cancer, but not in those with melanoma or sarcomas [29]. Thus, a depression of NK cell numbers could explain the depressed NK function reported in some, but not all, cancer patients [3]. The relationship between NK activity and other measures of immunocompetence has been explored. A lack of association was found between DTH reactivity to PPD and NK activity [409]. A comparison between TIL activity and PBMC revealed that TIL expressed diminished NK activity compared with PBMC [225]. In one study, although PBMC exhibited depressed NK activity, proliferative responses to PHA and in MLR were maintained [409]. This suggested that cytolytic and proliferative effector-cell mechanisms represent distinct functional entities.

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Phagocytic Cell Function Other impairments of immune responsiveness in solidtumor patients have been documented. For example, the inflammatory response of patients with advanced cancer is associated with a reduced capacity to mobilize monocytes [31, 137, 172, 546]. Reticuloendothelial function in patients with breast and colorectal cancer is depressed [138]. Depressed monocyte chemotaxis has been correlated with disease stage for a variety of tumor types [73, 203, 487]. Detailed evaluations of monocyte function in solid-tumor patients reveal abnormalities in patients with malignant melanoma, breast cancer, colorectal cancer, and head and neck cancer, but no consistent correlations have been identified between tumor type, monocyte defect, and clinical stage of disease.

Correlations Among Immune Cell Numbers and Function Antibody Formation A variety of humoral immune abnormalities have been found to occur in association with solid tumors. A large study of 984 patients with nonhematopoietic cancers found no general trends, but did find increases in serum IgG and IgA in males with skin and lung tumors, increases in IgM in males with sarcomas and females with melanoma, decreases in IgM in patients with ovarian cancer, and increases in IgA in patients with oral, gastrointestinal, and uterine cancers [238]. Increased IgA has also been demonstrated in head and neck cancer [319, 552] and prostate cancer [6], but to date, no correlations have been observed between serum immunoglobulin level and clinical stage of disease [125]. The serologic response to several different B-cell immunogens has also been studied in solid-tumor patients. Patients with nonlymphomatous malignancies were found to exhibit decreased specific antibody responses to Salmonella extract [297]. Patients with stage III squamous-cell lung cancer exhibited deficits in IgG and IgA production following immunization with Helix pomatia hemocyanin, a T-cell-dependent antigen [248]. A significant impairment in the ability of certain patients with solid tumors to produce both IgM and IgG antibody in response to primary challenge with monomeric S. Adelaide flagellin has been reported [297]. Both complete and incomplete primary antibody responses to heat-killed Brucella were reduced in patients with breast and lung cancer [544]. However, precise kinetic data for humoral antibody production in most patients with solid tumors are lacking, as are correlations between humoral immunity and extent of disease [143].

A number of studies have attempted to find correlations between in vitro and in vivo abnormalities of immune cell numbers and function. For example, both in vitro PHA responses and in vivo DNCB reactivity were normal in patients with early bladder cancer, but significantly impaired in patients with advanced disease. Similar correlations have been seen in patients with lung cancer [424] and breast cancer. In contrast, in a study of 48 patients with lung cancer, LPRs were more often suppressed than were skin test responses [8], and no correlations between DNCB reactivity and in vitro immune functions were found in patients with head and neck cancer. It has also not been possible to identify any consistent association between alterations in effector-cell numbers and function [103, 251].

Hematopoietic Malignancies Reed’s report in 1902 that patients with Hodgkin’s disease (HD) failed to react to tuberculin skin tests even if they were known to have had active tuberculosis [410] led to a vast number of immunologic studies for this malignancy. Many of the immune abnormalities first reported – for example, alterations of absolute lymphocyte counts, impairments of T-cell LPR, and the presence of suppressor monocytes/macrophages – were subsequently also found in patients with a variety of other cancers. Even though the primary immune defects described in untreated patients with HD involve T-cell immunity, other defects have also been documented [58, 264, 433] including impaired in vitro B-cell production of antibodies, phagocytic cell function, and NK-cell activity [161].

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The clinical relevance of the T-cell immunodeficiency in patients with HD has been recognized for many years [419]. Patients with HD have an increased susceptibility to infections associated with defective T-cell immunity, including Pneumocystis carinii pneumonia and viral infections such as herpes simplex, herpes zoster, and cytomegalovirus [22, 433]. In most of these studies, however, it has been difficult to assess the role of the treatment for HD (radiation therapy, chemotherapy) in exaggerating the T-cell immune deficiencies, since many of the infectious complications occurred only during or after the completion of therapy. A number of studies have now attempted to correlate defects in T-cell functions with depression in T-cell numbers in untreated patients with HD [71, 224, 460]. In general, no such relationships could be identified, although lymphocytopenia patients were more likely to exhibit impaired LPR. There is an easily identifiable correlation between defects in immune responsiveness and clinical stage of disease, in that most immune abnormalities are more readily apparent in patients with advanced stages of disease than in those with localized disease [460]. Immune deficiencies have also been shown to correlate with lymphoma subtype and extent of disease in the non-Hodgkin’s lymphomas. These include impaired in vitro LPR to mitogens [12, 312] and impaired in vivo DTH responses [12]. In general, patients with highgrade lymphomas exhibit more profound abnormalities than those with more favorable histologies [258]. It has been very difficult to relate the extent of immunodeficiency to tumor burden in patients with cancers involving the bone marrow (e.g., leukemia, multiple myeloma) since, with the exception of multiple myeloma, staging systems based on tumor cell burdens do not exist. However, a wide variety of in vitro as well as in vivo abnormalities have been documented in patients with lymphoid and nonlymphoid leukemias [199, 209]. Furthermore, a spectrum of intrinsic functional abnormalities has been identified in B cells and also purified T-cell populations of patients with chronic lymphocytic leukemia and multiple myeloma [236, 247]. Both of these diseases are often associated with profound depressions of normal serum immunoglobulin levels and impaired ability to mount primary humoral immune responses.

Prognostic Implications of Immunosuppression Since immunodeficiency generally correlates with the stage and extent of disease, and since stage and extent of

disease are the most important prognostic indicators for any particular cancer, it is logical that the general state of immunocompetence and prognosis should be closely related. The potential importance for correlating immunocompetence and prognosis at the time of diagnosis or at the onset of therapy is obvious [155]. Assessment of immune status could, in theory, help to select patients with a poor prognosis who might require more aggressive therapy than usual, or adjuvant therapy even if all disease appeared to be surgically excised. In a now classic study, it was observed that patients who could be sensitized to DNCB and freed of disease by surgery had a good prognosis, whereas those who were apparently freed of disease by surgery but could not react to DNCB had a poor prognosis and relapsed within 6 months to 1 year [562]. The DNCB responses of patients in the poor prognosis group were identical to those of patients who were found to be inoperable at surgery. The two groups of patients were otherwise comparable with regard to type of tumor, extent of disease, and all other usual clinical prognostic factors. This report formed the basis for a number of subsequent studies attempting to correlate immunodeficiency with prognosis for previously untreated patients with solid tumors and hematopoietic malignancies.

Solid Tumors Since 1970, there have been numerous attempts to correlate impaired DTH skin reactions with prognosis. Associations between impaired DNCB reactivity and disease recurrence have been observed in lung cancer [299], head and neck cancer [318, 441], gastrointestinal cancer [77], and breast cancer [146]. A number of reports have suggested that patients with or without metastases but without reactivity to DNCB have a poor prognosis [74, 85, 147, 265, 298, 318, 402]. Lack of reactivity to common skin test recall antigens has also been correlated with poor prognosis in breast [160, 349], lung [15, 244, 256, 299], gastrointestinal [382], urologic [334], and head and neck cancer [494], although this has not been a universal finding [54, 296, 350, 361, 397, 406, 452]. Several studies have subcharacterized patients with identical clinical stages of disease on the basis of impaired skin test reactivity to define further the relationship between anergy and prognosis. Such correlations have been found, for example, in patients with stage I [19] or stage III and IV [295] malignant melanoma, and in limited-disease small-cell lung cancer [256]. However, other studies of accurately staged patients with breast cancer, although DNCB-negative patients had a worse overall survival, when survival distributions of DNCB-positive and –negative patients

Robert K. Oldham with either primary operable or advanced breast cancer were compared separately, significant differences were not seen. Thus, studies to date have yielded conflicting conclusions concerning the correlation between impaired in vivo immunity and prognosis. Associations between poor prognosis and impaired in vitro LPR have also been reported [97, 392]. Other studies have not produced such correlations [94, 174, 429, 446]. Studies correlating absolute lymphocyte counts or absolute T-cell levels with prognosis have provided conflicting results [376, 479]. Furthermore, the use of multiparameter immunological assessments has not generally improved the ability to assess prognosis for previously untreated solid tumor patients [128, 174, 305, 320, 352]. However, some investigators have developed predictive indices based on multiple immunological parameters. For example, in one study, an integrated score of immunocompetence based on various in vivo and in vitro assays showed that the recurrence of breast cancer was significantly higher in suboptimal (61%) as opposed to optimal (28%) responders [3]. Similarly, an immunological staging system based on absolute lymphocyte counts and serum immunoglobulin level was found capable of predicting the outcome off stage III head and neck cancer patients in 86% of cases [268]. However, in a report using logistic regression methodology, it was demonstrated that only the level of complement binding activity, which may reflect levels of circulating immune complexes, correlated with the likelihood of responding to induction chemotherapy [452]. No associations could be determined between response to chemotherapy and abnormalities of a variety of other immune parameters, including lymphoproliferative responses, NK activity, lymphocyte subset numbers and percentages, and serum immunoglobulin levels.

Hematopoietic Malignancies Among the hematopoietic malignancies, most attention has focused on correlating defects in T-cell immunity with prognosis in HD [488, 577]. More recent reports suggest that there is a correlation between extent of immune dysfunction and prognosis [57, 153, 533]. Relationships between immunocompetence and prognosis have also been reported for a variety of other hematopoietic tumors. For example, in acute leukemia, patients with showed DTH skin reactivity and, to a lesser degree, in vitro blastogenic responses to mitogens exhibited the best overall survival, outranking such prognostic variables as age, type of leukemia, and absolute blast cell count [177, 303, 304]. In chronic lymphocytic leukemia,

109 both cell-mediated and humoral immune functions have been found to correlate with prognosis [59, 120]. While DTH was only moderately decreased, diminution in antibody responses and PHA responses correlated with the duration of disease, status of therapy, degree of lymphocytosis, and immunoglobulin levels [120]. A correlation has been reported between depressed absolute circulating NK-cell levels and poor prognosis in patients with largecell lymphoma [38]. Patients with high-grade lymphomas who have high levels of serum IL-2 receptor have been shown to have more advanced disease [410] and a worse prognosis [543]. However, in this instance, the IL-2 receptor is synthesized by the tumor cells so that serum levels parallel tumor cell bulk. Nevertheless, the predictive value of soluble IL-2 receptor was superior to that of other markers that reflected tumor cell bulk such as lactic dehydrogenase level (LDH) or clinical stage [410].

Perioperative Immunosuppression Although it is perhaps not universally recognized, the operative procedure and its associated general anesthesia result in a variety of transient immunological defects that can persist for several weeks after surgery [559]. During operative procedures under general anesthesia for a variety of benign and malignant conditions, patients exhibit inhibition of skin reactivity to DNCB [511] and DTH recall antigens [527], suppression of circulating T-cell levels [273, 528, 529], diminished LPR to PHA and other mitogens [260, 437, 483], and depressed NK activity [456, 513]. Patients with cancer appear more likely to experience these periods of postsurgical immunosuppression than do patients who undergo surgery for benign conditions. Among the conditions associated with the greatest degree of postoperative immunosuppression are intraabdominal and intrathoracic procedures, blood transfusions, and longer operating times [439, 471].

Perioperative Blood Transfusion Several studies have suggested that perioperative blood transfusion, possibly by inducing a greater degree of immunosuppression, results in an adverse effect on prognosis for postoperative patients with colorectal cancer [222], breast cancer [514], non-small-cell lung cancer [512], prostate cancer [205] and soft-tissue sarcoma [434]. However, three recent studies in breast cancer [541], colorectal cancer [550], and lung cancer [271] have not confirmed initial reports. Thus, it has not been conclusively proven that perioperative blood transfusions worsen the prognosis of patients with cancer. It is possible that the requirement for transfusion is a marker for other risk

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factors such as advanced stage of disease, need for more extensive operation, or greater blood loss during surgery, thus accounting for the worse prognosis following surgery. The precise mechanisms for postoperative immune suppression have not been fully defined. Among the considerations are the immunosuppressive effects of surgical stress [124], or of the anesthetic agents themselves [511], a relative decrease in circulating T-cell levels compared with other cell types [180, 181], and/or the generation of suppressor cells [360]. In nonsurgical patients, chronic blood transfusion is associated with depressed Tind/Tc/s ratios and impaired NK activity. Theoretically, the associations between perioperative blood transfusion and earlier cancer recurrence, and between prolongation of renal allograft survival and transfusion, may be attributed to a transfusion-induced immune suppression resulting from a graft-versus-host reaction mediated by transfused T-lymphocytes. Although the precise mechanisms to explain perioperative immune suppression have not yet been defined, this well-documented abnormality has nevertheless formed the basis for a number of therapeutic trials in which various putative immunorestorative agents have been administered as surgical adjuvants.

Radiation Therapy-induced Immunosuppression The immunological effects of external-beam radiation therapy have been delineated in detail. Radiation therapy to a variety of portals, including mediastinal [146, 273, 306, 371, 498], pelvic [65, 306, 375, 413, 498], head and neck [306, 371, 483, 494, 551], lymphoid [165, 185, 432], and breast [33, 191, 306, 413, 522, 556] portals, results in similar acute and chronic changes characterized by a generalized lymphocytopenia involving primarily a depletion of circulating T cells as well as marked depressions in various T-cell functions such as in vitro LPRs to mitogens and antigens and in vivo DTHS reactions [265, 484]. The depression of T-cell numbers and function occurs progressively with radiotherapy. The magnitude of immune depression reflects both the dose of radiation received and the volume of tissue, blood, lymph, or bone marrow included within the radiation portals. This acute immune suppression is short-lived, and substantial recovery is apparent within 3 weeks of cessation of therapy. However, most patients show a modest chronic depression in both numbers and functions of T cells, which may last for years following the completion of radiotherapy [252]. A number of other observations have been made concerning the mechanism of functional immune impairment

following radiation therapy. Mediastinal irradiation for treatment of localized lung cancer has been associated with a decrease in proliferative responses of purified peripheral blood T cells, suggesting that radiation directly impairs their functional capabilities [462]. Several studies have assessed the effects of radiotherapy on T-cell subset proportions. Although there was a drop in the absolute numbers of both helper and cytotoxic/ suppressor T-cells following mediastinal irradiation, the drop in cytotoxic/suppressor T-cells was greater, so that treatment resulted in a significant increase in the Tind/Tc/s ratio [462]. These observations are consistent with in vitro functional data indicating that suppressor T-cells are more radiation-sensitive than helper T cells [184, 398]. In contrast, radiotherapy for patients with breast cancer [452, 397], head and neck cancer [249], and Hodgkin’s disease [404, 432] has been associated with a relative increase in the proportion of T cells bearing surface antigens and/or Fc receptors (IgG) of cytotoxic/ suppressor cells, leading to a decrease in the Tind/Tc/s ratio. Indeed, in postradiotherapy patients with breast cancer [397] and Hodgkin’s disease [185], helper T-cell levels remained low for years after irradiation. Several studies have assessed the influence of therapeutic irradiation on suppressor cell function. They indicate that radiation therapy can activate suppressor monocytes [63, 68, 313] as well as increase the sensitivity of lymphocytes to the suppressive effects of prostaglandins [313]. External-beam radiotherapy, such as pelvic [375, 413, 498, 499] or chest wall [64, 513, 498, 499, 522, 556] radiotherapy, has also been associated with a decrease in B-cell and NK-cell numbers and functions in many, but not all, reports. Mediastinal irradiation more severely depressed NK activity than treatment to nonmediastinal sites [332], possibly reflecting the relatively large volume of blood and/or lymph node volume included within the mediastinal radiation portal, leading to damage of NK cells, or their more radiation-sensitive precursor cells. However, within 3–4 months following the completion of radiotherapy, NK-cell activity returned to pretreatment levels, which then persisted for at least 2 years [67]. There was a partial recovery in B-cell function, which remained below pretreatment levels for 12–18 months after completion of therapy [438, 500]. Reports concerning the effects of radiotherapy on absolute blood monocyte levels have been conflicting [522, 549]. Other immunological parameters have not been significantly affected. For example, radiotherapy does not produce changes in serum immunoglobulin levels or neutrophil function. However, the ability of monocytes to differentiate into macrophages was impaired in breast cancer

Robert K. Oldham patients treated by radiotherapy [515], and absolute monocyte levels increased after treatment [522], whereas pelvic radiotherapy produced a decrease in both monocyte numbers and functions in patients with colorectal cancer [549]. In the earlier literature it was not possible to correlate the extent of radiotherapy-induced immunosuppression with relapse or survival [133, 462, 555]. In general, however, patients who exhibited skin reactivity to DNCB prior to radiotherapy had a better prognosis than those who did not [79]. In two more recent studies of patients who received primary radiation therapy for breast cancer [499, 555] and/or cervical cancer [499], those who exhibited the greatest postradiotherapy depressions of LPRs to mitogens or antigens had the shortest survival [555]. Several studies on postradiotherapy patients with lung cancer have serially assessed the immune status of those who responded transiently to treatment but relapsed at a later date [133]. Lung cancer patients who responded clinically to radiotherapy showed some improvement of absolute T-cell levels in the months following cessation of treatment, whereas those who progressed did not show such partial recovery [133]. In addition, for postradiotherapy patients with locally advanced non-small-cell lung cancer, there was a gradual and progressive decrease in T-cell and helper T-cell percentages, and in the Tind/Tc/s ratio that preceeded relapse [462].

Chemotherapy-induced Immunosuppression It has been much more difficult to assess the effects of cancer chemotherapy than of radiation therapy on immune responsiveness. In part, this is due to the fact that a wide variety of different cancer chemotherapy drugs are available for clinical use, many of which can be administered by a variety of routes and doses, either alone or in combination with other drugs. The majority of anticancer drugs produce a dose-dependent pancytopenia, which itself is immunosuppressive because of the associated granulocytopenia. In addition, most, if not all, cancer chemotherapy drugs exhibit immunosuppressive properties as assessed with conventional in vivo and in vitro assays. This includes the alkylating agents, antimetabolites, and antitumor antibiotics. Cancer chemotherapy may lead to profound depressions of cellular as well as humoral immunity [202, 207, 213]. Newly acquired DTH and primary humoral immune responses appear to be more sensitive to drug-induced immunosuppression than are secondary responses. Less often, the administration of selective drugs, for example, cyclophosphamide (CTX), at low doses can lead to a

111 temporary enhancement of immune responsiveness due to preferential inhibitory effects on suppressor cells. Traditionally, it has been felt that intermittent chemotherapy schedules of administration using single or multiple drugs tend to be associated with relatively little effect on DTH responses and with transient depressions of lymphocyte numbers, in vitro LPR, antibody responses, and inflammatory responses, with at least some recovery several weeks after discontinuation of treatment [210, 417]. In contrast, continuous therapy, if given in adequate doses, has been shown to lead to a progressive decline in all phases of immune reactivity [166, 207, 210, 331, 417], which is reversible after the cessation of administration of drug(s). In several more recent studies, however, it has been demonstrated that intermittent combination chemotherapy regimens lead to cumulative depressions of lymphocyte numbers and functions over months to years [315, 501], which may or may not be reversible. These changes include depressions of absolute T- and B-cell levels, depression in the Tind/Tc/s ratio, and depressions of LPR to mitogens and antigens. Another phenomenon that has been described is the recovery or transient, rebound overshoot of immune responsiveness after a single cycle of chemotherapy [81, 108, 201]. Rebound is found in patients who are responding clinically to treatment. Patients whose immune functions remain suppressed either have not responded clinically to treatment or are at high risk for relapse compared with those who exhibit recovery to levels above those pretherapy. It has been suggested that the rebound overshoot in immune reactivity following chemotherapy may be due to a reduction in monocyte suppressor cell function [81]. The effect can result from a transient, chemotherapy-induced decrease in relative numbers of monocytes/macrophages, from a reduction in suppressor cell function on a per-cell basis, or from a combination of both. Recovery of suppressor monocyte/ macrophage function is responsible for the eventual decline in immune function following drug-induced rebound overshoot.

Antimetabolites All of the antimetabolites commonly used for treating cancer patients, including thiopurines such as 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), antifolates such as MTX, fluorinated pyrimidine analogs such as 5-fluorouracil (5-FU), cytosine arabinoside (Ara-C), and DTIC (5-[3-3-dimethyl-1-triazeno]-imidazole-4-carboxamide) are immunosuppressive in humans. 6-MP has been one of the most extensively studied agents [207, 213, 300]. Azathioprine, a nitroimidazole derivative of

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6-MP, is a potent immunosuppressive drug, and has found more use in clinical transplantation than as a cancer chemotherapy drug [343]. 6-MP, 6-TG, MTX, and 5-FU have all been shown to preferentially depress primary antibody responses and the development of new DTH reactivity [213, 343, 345, 346]. However, 5-FU treatment restored DTH to recall antigens such as PPD, mumps, and trichophytin [62, 345], but caused a transient decrease of in vitro LPRs to PHA and PPD [370]. Intravenous 5-FU produced a rapid (within 1–2 days) decrease in absolute T- and B-lymphocyte levels, which returned to baseline over the ensuing 1–3 weeks [152]. Ara-C has been shown to partially suppress primary and secondary antibody responses [117]. Although both MTX and Ara-C abolished established DTHS to recall antigens, Ara-C was much more potent in inhibiting DNCB reactivity [346]. In vitro MTX did not affect the phagocytic or cytolytic activities of human neutrophils [255]. DTIC has been considered only weakly immunosuppressive in humans, based on studies in patients with malignant melanoma in which only a minority of treated patients exhibited depressions in antibody production to typhoid vaccine or in DTHS reactions to primary sensitization [89]. More recent studies with DTIC have indicated that treatment is associated with a cumulative decrease of T-cell numbers, but no change in B-cell numbers [52]. A normalization of Tind cell numbers occurred after cessation of treatment.

Alkylating Agents The alkylating agents comprise a variety of drugs including cyclophosphamide (CTX), nitrogen mustard (HN2), chlorambucil (CLB), thiotepa (TT), and busulfan (BS). These compounds have found widespread clinical use, particularly for the treatment of leukemias and lymphomas. Their antiproliferative effects also extend to normal host hematopoietic precursor cells of all lineages, including lymphoid precursors. In animals, these agents are potent immunosuppressants of antibody formation [447]. Recent clinical interest has focused on the novelty of CTX as an immunomodulatory agent [37, 144, 202, 207, 213, 216, 325, 380]. Administration of oral daily CTX maintenance therapy to patients with lymphomas did not result in interference with the development of normal humoral and cell-mediated immune response to keyhole limpet hemocyanin. Other studies have revealed that prolonged oral therapy with CTX can produce lymphopenia and suppress in vitro LPRs to PHA. CTX administered intravenously at conventional doses for 7 days inhibited the production of primary antibody responses, but did not significantly interfere with DTHS. Low intravenous doses of CTX (100–600 mg/m2) pref-

erentially decreased circulating B-cell numbers. Doses from 200 to 600 mg/m2 selectively depleted cytotoxic/ suppressor T cells, leading to a transient increase in the Tind/Tc/s ratio, whereas higher doses affected all T-cell subsets equally. Intravenous administration of conventional doses of CTX for 5 days has been shown to depress established DTH by 7–10 days after the cessation of therapy, at a time when the peripheral white blood cell count was at its nadir. Perhaps the most intriguing aspect of CTX-induced immune changes has been the elucidation of the selective immunopotentiating effects of CTX [144, 325]. For example, in a study of 22 patients with metastatic cancer, CTX pretreatment significantly augmented the development of DNCB reactivity as well as DTHS to new antigens [44], even though absolute lymphocyte counts fell within 1–2 days and did not recover for 21 days [46]. The T-cell, B-cell, and T-cell subset numbers were all affected equally. Lymphoproliferative responses to mitogens and alloantigens also fell significantly within a day, but recovered to pretreatment levels by day 3; some cases exhibited rebound overshoot by day 7. Inducible T-cell suppressor cell activity was also diminished within 1 day after CTX administration; however, in contrast to LPRs, suppressorcell activity remained significantly impaired on day 3 and only partially recovered by day 7. Thus, between 3 and 7 days after intravenous administration of CTX, there appears to be a preferential impairment of nonspecific T-cell-mediated suppressor cell activity, which could account for the augmented DTHS noted in CTX-treated patients. Most recently, a single intravenous low dose (300 mg/m2) of CTX was shown to inhibit the generation of inducible suppressor cell activity in cancer patients for up to 19 days without affecting lymphoproliferative responses to T-cell mitogens or the Tind/Tc/s ratio [45]. This CTX dose (300 mg/m2) 3 days prior to immunotherapy has been employed clinically in a variety of circumstances in an attempt to abrogate suppressor cell activity, for example, as an adjunct to active specific immunotherapy with a melanoma tumor cell vaccine [43], and in combination with low-dose intravenous IL-2 [344]. Although the mechanism by which CTX augments the immune response has not yet been elucidated, the leading hypothesis, at present, is an abrogation of suppressor-cell function, with blockage of the subset of helper T-cells that is the inducer of suppressor cells [144].

Antitumor Antibiotics Surprisingly, clinical information concerning the immunosuppressive effects of this class of anticancer drugs that includes such widely employed drugs as adriamy-

Robert K. Oldham cin (ADR), daunomycin (DNR), mitomycin (MTC), and bleomycin (BLEO) is limited. However, in animal models, these agents affect a variety of immune cells, in particular, macrophages and immunoregulatory cells [144, 340], suggesting that they should exert a spectrum of immunomodulatory effects in humans. On the other hand, ADR has been shown to augment both monocyteand lymphocyte-mediated cytotoxicity of human PBMC [20, 21, 282]. Following a single intravenous dose (25 mg/m2), PBL from cancer patients showed an increase in lymphocyte cytotoxicity as well as an increase in Tc/s levels [20]. In cancer patients, intravenously administered ADR has been shown to produce a rapid but reversible lymphocytopenia of both T and B cells [152]. In vitro, ADR has been shown to impair phagocytic function of human polymorphonuclear leukocytes [536], whereas similar effects were not seen with a variety of other drugs, including MT, VCR, 5-FU, and cisplatin. In addition, ADR has also been shown to inhibit granulocyte degranulation and release [95]. Some evidence has been provided that low doses of ADR can also augment both T- and B-cell mediated immune responses [144]. Thus, it now appears as if ADR, like CTX, can exhibit a variety of immunomodulatory activities.

Vinca Alkaloids and Podophyllotoxins The vinca alkaloids, vincristine (VCR) and vinblastine (VLB), possess a unique mechanism of antitumor activity involving drug-induced microtubular dysfunction leading to mitotic arrest. VCR has been shown to depress granulocyte aggregation, lysozyme release, and chemotaxis [95, 492]. However, there remains a substantial lack of studies concerning the immunomodulatory effects of these agents in humans. They are often administered in conjunction with steroids as treatment for hematopoietic cancers, making it impossible to define their selective immunosuppressive effects. Not unexpectedly, they inhibit human LPR in vitro, and they should exert similar effects in vivo. Both VCR and VLB have not been particularly immunosuppressive in humans [208]. Vindesine, a new semisynthetic vinca alkaloid, was devoid of immunosuppressive activity in preliminary clinical trials [425]. Podophyllotoxins such as VP-16 have mild immunosuppressive effect, similar to the vincas.

Other Drugs Other frequently employed anticancer drugs, such as cisplatin, nitrosoureas (BCNU, CCNU) and taxol also

113 exhibit varying degrees of immunomodulatory activity when incubated in vitro with human PBMC [492], but clinical data are sparse. Single intravenous doses of cisplatin have been associated with a rapid depression of LPR, with recovery in 1–2 days, whereas a single dose of CCNU leads to depressed PHA responsiveness for up to 6 weeks, in the absence of changes in T- and B-cell proportions. Long-term therapy with CCNU has been associated with cumulative depressions of absolute levels of both and T cells [52]. The reduction of T-lymphocyte levels was due mainly to a depletion of Tind cells. Taxanes have moderate immunosuppressive effects.

Hormones Hormonal agents such as tamoxifen (TAM) and medroxyprogesterone acetate (MPA) are used widely in the treatment of breast cancer. Both estrogen and progesterone receptors have been identified in human lymphocytes [117, 126]. There is considerable experimental and clinical evidence suggesting that hormones can modulate immune mechanisms [114, 371, 381]. Longterm treatment of patients with TAM has not produced significant immunosuppression [254, 451], whereas MPA depressed LRP as well as the Tind Tc/s ratio of treated patients (451).

Combination Chemotherapy When multiple cancer chemotherapy drugs are administered together, it is impossible to predict what effects their interactions will have on their individual immunosuppressive properties. Nevertheless, at the present time, most cancer chemotherapy regimens include a combination of drugs. A number of recent studies have begun to define the immunological consequences of combination chemotherapy as currently used for the treatment of patients with advanced disease, as well as for patients treated in the adjuvant setting. A comparison of intravenous doses of single drugs, namely ADR and 5-FU, with combinations such as COBAM (CTX, VCR, BLEO, ADR, MTX) or DOMF (DTIC, VCR, methyl-CCNU, 5-FU × 5) revealed that both the single agent and multiagent regimens produced a rapid drop (within several days) in the percentage and absolute numbers of circulating B and T cells, which was more pronounced for the combined drug regimens [152]. Such decreases were transient, and either a partial or complete recovery (with or without rebound) to baseline levels was then observed over the 1 or 2 weeks after drug administration [152]. Similar acute effects of multiagent chemotherapy regimens have been noted with respect to

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functional immune parameters such as in vitro LPRs [87], B-cell activation [232], reticuloendothelial cell function [151], and cytotoxic cell (NK) activity [83, 442]. In general, there has not been a good correlation between the depressions in immune cell numbers and functions. Cyclic combination chemotherapy can result in cumulative immunosuppressive sequelae [315, 396, 501]. Three similar studies have all involved patients receiving cyclic adjuvant chemotherapy for breast cancer who did not exhibit evidence of immune suppression before treatment. Serial immune assessments revealed that cyclic chemotherapy was associated with a marked initial (within 1 month) decrease in T- and B-lymphocyte numbers, and then gradual progressive further decreases [501], the same being observed in Tind Tc/s ratios [396]. There was also a rapid initial decrease in functional immune parameters, such as in vitro LPRs to antigens, with stabilization or some improvement over the ensuing year of treatment. In general, the functional immunological parameters tended to normalize during or, more often, within several months of stopping chemotherapy, whereas the abnormalities of immune cell numbers were still apparent 1 year after cessation of treatment [315]. Thus, it appears that prolonged administration of cyclic combination chemotherapy results in cumulative impairments of immune cell numbers and functions, some of which are long-lasting [439, 501]. In none of these studies were investigators able to predicate relapses in individual patients based on immune parameters. Recent attempts to manipulate the immune system have involved the combination of total body irradiation with aggressive, but non-ablative, chemotherapy. Such therapeutic studies are being done to deplete regulatory cells that negatively influence T-cell function followed by the reinfusion of T-lymphocytes derived from the patient’s own tumor, so-called tumor-infiltrating lymphocytes (TIL). These studies, which will be described in more detail elsewhere in this book, are producing clinical responses in 50% or more of patients with advanced melanoma [573]. Such aggressive approaches indicate that the immune system can be manipulated with chemotherapy and radiation therapy, but the future will probably in the area of more selective immune depletion with monocloncal antibody of the targets agents imparting less general toxicity.

Immune Status of Patients in Clinical Remission A number of studies have evaluated the immune status of patients in remission following the successful treatment of their cancer. In general, there tends to be

improvement and/or normalization of immune cell numbers and function for patients who achieve clinical remission following chemotherapy or radiation therapy. Nevertheless, some immunologic impairment persist for years after completion of successful treatment. Thus, in many cases, it may be extremely difficult to dissociate the chronic immunologic consequences of therapy from the presence of persistent immune defects while in clinical remission.

Solid Tumors Patients surgically cured of their cancers generally have a restoration of immune cell numbers and function after the perioperative periods [260, 437, 483]. Although patients in clinical remission following potentially curative radiation therapy may have some normalization of immunity, it does not usually become apparent until years after the completion of treatment [375]. Finally, as discussed previously, it has also become apparent that adjuvant chemotherapy for breast cancer results in cumulative depressions of both lymphocyte numbers and functions [315, 396, 501].

Hematopoietic Malignancies Much important information concerning the long-term effects of chemotherapy and radiation therapy in cured patients has resulted from studies of children who received maintenance chemotherapy for acute lymphoblastic leukemia and of adult patients with HD. In acute lymphoblastic leukemia patients who remained in clinical remission for 1–3 years after completion of chemotherapy, immune competence was found to recover. Those patients destined to relapse showed a subsequent deterioration in immune status, which was detectable some months before clinical relapse [178, 210, 211, 212, 214, 472]. The immune status of patients with HD in long-term clinical remission after treatment with chemotherapy or radiation therapy has also been evaluated in detail. At the completion of radiotherapy, DNCB reactivity was lost in almost all patients who were initially sensitive. However, many patients regained their DTHS responses during the first year after discontinuation of chemotherapy [7, 427] or radiotherapy [165, 428]. NK-cell activity also improved following successful therapy for HD. [161]. Several studies have shown that, following the completion of successful MOPP chemotherapy, patients in remission from HD exhibited gradual improvements of in vitro T-cell functions [7, 427]. In contrast, other studies have revealed a persistent depression of LPRs at 1–10 years after the completion

Robert K. Oldham of treatment, with no evidence for recovery [56, 57, 100, 154, 189, 428], along with a preferential chronic depletion of Tind cells [29, 185, 404, 427, 432]. In some of these studies, patients had received radiation therapy, which was felt possibly to contribute to the prolonged immune deficiency [165, 185, 404, 432]. Several authors have argued, however, that patients with HD in prolonged clinical remission continue to manifest immune abnormalities as a reflection of their underlying disease [56, 57, 100, 154, 291]. For example, persistent defects in T-cell functions were more severe in irradiated patients with HD than in similarly treated patients with testicular cancer [47, 432]. Although some improvements were noted on serial immune assessments following discontinuation of treatment, there have been no correlations between the status of immunity during clinical remission and likelihood of relapse [427, 428]. It has been suggested that an increased sensitivity of T-cells to the inhibitory effects of suppressor cells may account for the persistent depressions of T-cell functions in cured HD patients [535].

Treatment of Cancer-associated Immunodeficiency The term immunotherapy was introduced to clinical oncology 2 decades ago following the independent observations that Bacillus Calmette-Guerin (BCG) administration could prolong the survival of patients with acute lymphoblastic leukemia [326] and induce regressions of injected as well as noninjected malignant melanoma lesions [357]. Since these reports, a wide variety of chemicals and biologicals have been administered to immunosuppressed cancer patients in the hope of reconstituting or boosting host immune mechanisms (Table 1). A broader term, biotherapy, is now being used since there are many biological substances that stimulate cells outside of those from the immune system. These stimulations with colony stimulating factors, growth and maturation factors may have secondary effects on cancer and immune function.

Immunorestorative Agents Chemicals Levamisole, an orally active synthetic phenylimidazole, 2,3,5,6-tetrahydro-6-phenyl-imidazol[2, 1-6]thiazole, is the levo isomer of tetramisole, a potent, broad-spectrum antihelminthic agent introduced in 1966 [517]. The demonstration in mice that tetramisole could augment

115 Table 1. Biological response modifiers with immunorestorative properties Chemicals Azimexone Cimetidine Copovithane Coumarin DTC Ibuprofen Indomethacin Interferon inducers Isoprinosine Levamisole NPT 15392 Oxyphenbutazone Piroxicam Ranitidine

Biologicals Bestatin CSFs FK-565 IMreg-1 ImuVert Interleukin 1-–32 Interferon gamma Lentinan OK-432 Retinoids T-Cell reconstituting factor (SR 270258) Thymic factors Transfer factor Tuftsin

the protective effect of a Brucella vaccine [423] led to widespread clinical investigation of the immunomodulatory activity of both tetramisole and levamisole. In various animal models, and also following in vitro incubation with effector cells from human donors, levamisole has been found to increase both T-cell numbers and functions (e.d., LPR), if initially depressed [424, 574], as well as phagocytic and chemotactic activities of polymorphonuclear leukocytes and monocytes [111]. Its immunorestorative mechanism of action is currently unknown [220]. It has been shown to induce thymic factor-like activity, which has been attributed to the presence of a sulfur atom in its structure [186]. Levamisole, which contains an imidazole ring, may function like imidazole in affecting enzymes that control cyclic nucleotide levels in lymphoid precursors of lymphocytes. Both imidazole and levamisole, which themselves are not mitogenic, elevate cyclic GMP levels in lymphocytes in vitro and enhance their proliferative responses to mitogens or foreign antigens [111]. Only a limited number of dose- and schedule-seeking trials for cancer patients were performed with levamisole. In general, it has been administered intermittently, using two or three daily doses every 1–2 weeks [9]. The drug is well absorbed, and a single oral dose of 150 mg produces a peak plasma level in 2 h (0.49 + 0.05 μg/ml), which is the concentration required for in vitro activity. The plasma half-life of levamisole is 4 h. It is widely distributed and can be detected in all tissues and fluids, with the highest levels in liver and kidneys. It is excreted primarily in the urine, most of it by 24 h, although much of the excreted product has already undergone extensive metabolic changes.

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A number of side effects of levamisole have been reported [411, 414, 415, 431, 503, 568]. In studies on 3,900 patients with a variety of diseases (including rheumatic and inflammatory diseases and cancer), reactions included idiosyncratic or allergic ones, such as a rash or febrile influenza-like illness, sensorineural reactions such as alterations of taste and smell, and gastrointestinal symptoms. Rashes and fever resulted in the cessation of levamisole treatment in 7% and 1.5% of cases, respectively, but were quickly reversible. The major serious side effects have been agranulocytosis and/or neutropenia and, less commonly, thrombocytopenia, which have been observed in between 0.2% and 2% of all treated patients. Agranulocytosis could not be related to dose or schedule of administration, and was always spontaneously reversible following discontinuation of treatment [9]. The incidence of side effects in various clinical trials has varied from insignificant to major, requiring interruption of therapy in up to 21% of cases [385]. Levamisole was approved for the adjuvant treatment of stage III colon cancer as an immunomodulator to be used with adjuvant chemotherapy. It enjoyed some use for several years in this setting, but is no longer used because of improved regimens for the adjuvant treatment of stage III colon cancer. Isoprinosine (IPS), a synthetic antiviral agent, is a complex of inosine and the p-acetamidobenzoate (PacBA) salt of N,N-dimethylamino-1-propanol (DIP) is a 1:3 molar ratio [111, 186]. In early clinical trials with rhinovirus-infected humans, IPS increased the titers of circulating antiviral antibody, suggesting it had B-cell immunomodulatory activity [489]. Subsequent in vivo studies in animal models and in vitro studies with human PBMC have indicated that IPS enhances T-cell functions such as LPR to mitogens and alloantigens [111, 186, 356]. Helper T cells appear to be the main target for the drug in humans. IPS has also been shown to induce the appearance of T-cell surface markers in mouse prothymocytes, similar to that of thymic factors, as well as increase the proportions of various T-cell subpopulations following incubation with human PBMC. In vitro, IPS at a concentration of 100 μg/ml restored LPRs, NK activity, and monocyte chemotaxis of PBMC isolated from cancer patients [526]. Because the immunomodulatory activity of IPS is similar to that of a variety of thymic factors, it has been classified as a thymomimetic drug. Although IPS has been investigated clinically in several different viral diseases (including human immunodeficiency virus [HIV] infections), only a limited number of studies have been performed with cancer patients [111,

517]. Extensive tolerance and safety studies have been conducted in which IPS was administered orally for periods of 1 week to 2 years at doses of 1–8 g/day. Minimal side effects have been noted, including transient rises in serum and urine uric acid levels and, occasionally, transient nausea associated with higher daily dosages. Following oral or intravenous administration to rhesus monkeys, the inosine moiety of IPS is rapidly metabolized, with a half-life of less than 4 h. Azimexone (BM 12.531) is an orally active aziridine dye, 2-[2-cyanaziridinyl-(1)-2-[2-carbamoylaziridinyl(1)]-propane, which has been found to increase the number of cytotoxic autoreactive cells. It exhibits antitumor activity in animal models [53, 111, 186] and exerts a variety of immunomodulatory effects on T cells, monocytes, phagocytic cells, and NK cells. Azimexone has exhibited immunorestorative activity in various animal models of infectious diseases. In vitro studies using PBL from advanced cancer patients revealed that azimexone at various concentrations (0.2–10 μg/ml) enhanced the LPR to PHA with a maximal effect at 0.2 μg/ml. Other studies have indicated that azimexone also enhances the percentage of activated T lymphocytes in vitro. In a limited number of clinical studies, the only significant side effect observed with intravenous administration has been a dose-dependent self-limiting hemolysis [358]. Oral absorption of azimexone is almost complete, and the serum half-life is 6 h.

Histamine Receptor Antagonists Cimetidine is a histamine type II receptor antagonist widely used for the treatment of gastrointestinal ulcers. A growing body of evidence has suggested that suppressor T cells that possess histamine receptors (H2 type) may play an important immunoregulatory role in normal immunologic responses [88, 149, 377]. The rationale for administering H2 blockers to cancer patients is based on the observation that cimetidine could abrogate in vitro histamine-induced suppressor T-cell activity using human PBL. Results of in vitro preclinical testing with cimetidine have been summarized [329]. It has been shown to augment LPRs and IL-2 production of PBMC from cancer patients to mitogens and alloantigens [148, 289, 513]. In vitro cimetidine also enhances NK-cell activity of PBMC from cancer patients, probably through its inhibitory effects on suppressor cells [156]. No effects have been seen on transformation of peripheral blood monocytes to macrophages [169]. Cimetidine was employed in a phase I/II study [323] and in combination with coumarin [322, 518, 519]. Varying degrees of antitumor activity have been noted

Robert K. Oldham in patients with malignant melanoma [518, 519] and renal cancer [322] treated with the combination of cimetidine plus coumarin. The mechanism of antitumor activity has not been established but the doses used are approximately 2x that for treatment of ulcers. Histamine has been shown to suppress human LPR to mitogens, and cimetidine might block the inhibition. More recently, histamine was used in clinical trials as a drug to assist IL-2 induced T cell function with some evidence of useful clinical effects in melanoma metastases to the liver. The clinical effects were marginal.

Nonsteroidal Anti-inflammatory and Antipyretic Agents (NSAID) Indomethacin and ibuprofen are prototype inhibitors of prostaglandin synthesis. A large number of in vitro studies using PBMC from patients with solid tumors and HD have suggested that (a) the depressed LPRs of cancer patients result, at least in part, from the immunosuppressive effects of prostaglandins released by activated monocytes/macrophages, and (b) indomethacin is capable of abrogating the suppressor cell influence. In addition, it has been shown in vitro that indomethacin also acts directly on T cells of patients with malignant melanoma to augment mitogen responsiveness [520]. It has been postulated that this direct immunomodulatory action results from specific pharmacologic effects such as alteration of intracellular cyclic AMP levels. Finally, a correlation has been observed between tumor spread and content of prostaglandin E2 of the tumor [42]. These observations have formed the basis for administering prostaglandin inhibitors to cancer patients [96, 405, 563].

NPT 15392 Studies with IPS suggested that the inosine moiety might be responsible for its immunomodulatory activity on LPRs. Subsequently, a number of purines with inosine-like structures were synthesized, one of which is NPT 15392 (9-erythro-2-hydroxy-3-nonylhypoxanthine) [157, 186, 578]. In animals, NPT 15392 has exhibited effects on T-cell and monocyte/macrophage functions and T-cell differentiation similar to those of the parent compound [157]. Toxicological studies have shown that it is nontoxic at oral doses up to 35 mg/day. This drug has been tested in clinical trials in cancer patients [575]. DTC, sodium diethyldithiocarbamate (Immunothiol), was developed as a result of preliminary studies with levamisole in an attempt to synthesize a chemically defined sulfur-containing compound that would be la more potent immunorestorative agent than the parent

117 compound [186, 422]. DTC is a chelating agent in use for the treatment of heavy-metal poisoning. In animal models, it exhibits a variety of immune-augmenting effects on T-cell dependent immune responses as well as on the induction of T-cell differentiation. No significant toxicity has been noted following long-term administration. Clinical trials in cancer patients and in patients with HIV disease have not been promising. Coumarin (1,2 benzopyrone) has been reported to exhibit immunomodulatory activity [49, 581]. Unlike warfarin, coumarin is devoid of anticoagulant activity. In cancer patients, coumarin administration was reported to enhance LPRs to PHA but did not effect T-cell numbers [49]. In vitro coumarin has augmented HLA DR expression and NK activity [581]. It has been administered in combination with cimetidine to patients with malignant melanoma [518, 519] and renal cancer [322] with varying degrees of antitumor activity reported. The mechanism of antitumor activity of the combination has not been established. Copovithane (BAYi7433) is a copolymer of 1,3-bis (methyl amino carboxy)-2 methylene propane carbamate, has exhibited antitumor activity in a variety of pre-clinical models [458]. A phase I trial in advanced cancer patients using weekly intravenous dosing revealed minimal fatigue, and occasional nausea and proteinuria, as the only side effects, some antitumor effects, and some improvements in Tind/Tc/s and in vitro toxicity responses and LPRs [233].

Biologicals Thymic Factors A functioning thymus gland is an essential requirement for the normal development and maintenance of cell-mediated immunity [73]. The thymus is responsible for the normal maturation of all the various subclasses of T-lymphocytes, including various effector cells, as well as immunoregulatory cells. The thymus exerts its influence during the ontogenesis of the immune system of releasing, in situ from its epithelial stroma, a variety of differentiating factors that induce the maturation of resident pre-T stem cells (thymocytes) into mature T cells, which ultimately circulate in the peripheral blood and lymph. It is now well established that the thymus gland is an endocrine organ and that at least several of its locally released differentiating factors are also secreted into the bloodstream. Thymic hormone-like bioactivity has been demonstrated in the blood of animals and humans. This activity decreases following thymectomy, as well as with age, in parallel to the physiological agedependent involution of the thymus. The presence of the

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thymus is important well into adulthood in order to maintain immune T-cell mechanisms. A number of factors with thymic hormone-like activity have been prepared from thymus tissue and blood, and these are in varying stages of characterization [173, 372, 463, 478, 485]. The best studied are thymosin fraction 5 (TF5), thymosin α1 (Tα1), prothymosin α (Pro Tα), thymostimulin (TP-1), thymulin (FTS-Zn), thymopoietin (TP), thymic humoral factor (THF), and thymic factor x (TFX). These agents all exhibit a broad spectrum of immunorestorative effects on T-cell numbers and functions in animal models and humans. In some bioassays, many of the well-characterized thymic preparations have identical, or even opposite, effects. An increasing number of thymic factors have been employed therapeutically in treating a variety of diseases – predominantly cancer and HIV disease [134]. The well-characterized thymic preparations are listed in Table 2 [173, 192, 463]. Among the thymic factors that have been administered to cancer patients are TF5, Tα1, TP-1, THF, and TFX. Both TF5 and TP-1 are partially purified extracts of calf thymus glands. They include a mixture of different biologically active as well as inactive polypeptides. The purification procedures for TF5 and TP-1 are similar but not identical. TF5 consists of ten major – and at least 30 minor – polypeptides on analytical isoelectric gel focusing with molecular weights ranging from 1,000 to 15,000. The first biologically active polypeptide isolated from TF5 is Tα1. Biologically active Tα1 has a molecular mass of 3,108 daltons. It has been synthesized by classical chemical,

Table 2. Chemical properties of thymic hormones Name

Chemical properties

Thymosin fraction 5 (TF5) Heat-stable, acidic Peptides MW 1,000–15,000 Thymosin α1 (Tα1) Peptide of 28 residues, MW 3108 Prothymosin α (Proα) 113 Amino acids, MW 13,500 Acidid peptide, MW 2000, pl 3.5 Thymosin α9 Thymosin α11 (T α11) Peptide of 35 residues, 28 residues identical To Tα1 Thymosin β3 (T β3) Peptide of 49 residues, MW 5700, 43 residues Identical to Tβ4 Thymosin β4 (Tβ4) Peptide of 43 residues, MW 4963 Thymic factor X (TFX) Mixture of peptides; active compound is a peptide, MW 4200 Thymostimulin (TS/TP-1) Mixture of peptides Thymulin (FTS-Zn) Nonapeptide, MW 857, Thymic humoral factor Peptide MW 3200 (THF) Thymopoietin (TP) Peptide of 49 residues, MW 5562

solid-phase, and recombinant DNA techniques, but only the chemically synthesized material has been employed in clinical trials. Thymopoietin, THF, and TFX are purified thymic peptides with molecular weights ranging from 3,220 to 5,562. TP-5 is a biologically active synthetic pentapeptide that represents amino acids 32–36 of thymopoietin. In contrast to all other thymic preparations, thymulin (FTS-Zn) has usually been isolated from pig blood rather than thymus tissue. Although thymulin, Tα1, and thymopoietin are all detectable in the blood, only thymulin levels drop significantly following thymectomy, and are restored by thymic grafts. None of the well-characterized thymic peptides exhibit any significant homology with the other characterized peptides. However, a 50% homology has recently been identified between a 35 amino acid region of Tα1 and that of the p17 core protein of the AIDS retrovirus (HIV) [449]. Thymic factors, which have been administered to more than 1,000 cancer patients, have shown minimal toxicity, but some have been approved for general clinical use. The purified preparations that have been administered by intramuscular or subcutaneous injection have not produced any significant side effects [173, 463]. Partially purified bovine preparations, such as TF5, have produced rare (less than 1%) allergic reactions and erythema and/or pain at the sites of injection in about one third of treated patients. However, potentially immunizing treatment schedules, such as daily injections for 1 week followed by 3 weeks of rest, and reinstitution of treatment, have been associated with a high (10%) incidence of anaphylactoid-like reactions. As a single agent, TF5 has exhibited minimal antitumor activity in patients with renal cancer in one study [465] and none in another [135]. More recently, thymic factors have been used less due to the availability of specific lymphokines with more powerful activities.

T-cell Reconstituting Factor This is a highly purified protein fraction isolated from human serum that has been shown to exhibit immunomodulatory effects on T-cell numbers and functions [364]. In preliminary phase I/II trials, no unexpected toxicities have been observed following SQ administration. TsIF is a thymic isolate, distinct from other thymic factors and cytokines, that induces immature bone marrow cells to differentiate into competent suppressor T cells. It has a molecular mass of 75,000 daltons as determined by gel filtration and high-performance liquid chromatography. Recent studies in mice indicated that TsIF administration suppresses the development of autoimmune disease in lupus-rheumatoid arthritis-prone animals [341].

Robert K. Oldham

Transfer Factor In 1955 it was demonstrated in humans that the transfer of DTHS to streptoccal M substance and tuberculin could be accomplished by administration of a suspension of leukocytes disrupted by either distilled water or repeated freeze-thaw cycles [294]. The substance (or substances) responsible for this transfer was resistant to RNAase and DNAase and was termed transfer factor. This initial work was extended using dialyzable human leukocyte extracts from patients sensitized to various antigens [25], including skin allograft antigens [493]. The crude dialyzable preparation has subsequently been shown to enhance LPRs in vitro [24, 142]. The further chemical characterization of transfer factor has been hampered by the lack of a unique biological assay to monitor final purification. Preliminary fractionation studies of human leukocyte extracts capable of transferring DTHS responses have indicated that transfer factor is probably a low-molecularweight material (approximately 1,000) with the electrophoretic mobility of slow γ-globulin but with no reactivity to anti-immunoglobulin antisera. Its possible composition, that is, a short polypeptide chain joined with a threeor four-base segment of RNA, has been difficult to verify. Unlike other nonspecific immunorestorative BRMs, transfer factor appears to exert antigen-specific immunerestorative effects. Despite the lack of readily reproducible in vitro or in vivo assays, the effects of transfer factor have been studied following subcutaneous administration to patients with a variety of immunodeficiency diseases, including cancer [354].

Interleukins Many of the BRMs currently being synthesized by recombinant DNA techniques are products of lymphocytes (lymphokines), monocytes (monokines), or other cells (cytokines). Various interferons and interleukins have been purified to homogeneity produced by genetic engineering to make large quantities available for clinical trials. Various other cytokines are undergoing active clinical investigation [187]. The characteristics and results of clinical trials with these materials are discussed in Chapter 8.

Retinoids Considerable interest has recently been focused on the influence of vitamin A (retinol) and its natural and synthetic derivatives (Aretinoids) on the growth and differentiation of neoplastic cells. Although retinoids have been administered to cancer patients primarily as chemopreventive agents [74, 308], accumulated evidence also indicates that they have beneficial effects on the host immune system. For example, in animals they

119 have been shown to restore PHA responsiveness, to induce augmentation of cytotoxic and helper T-cell numbers and functions, and to inhibit prostaglandin synthesis by host tumor-activated macrophages. However, the role that altered immune mechanisms play in the chemopreventative action of retinoids, such as 13-cis-retinoic acid, remains unclear (see Chapter 16). FK-565 [heptanoyl-8-D-Glu-(L)meso-α1-A2pm(L) AlaOH] is a heptanoyl tripeptide analogue of FK-156, a biologically active acylpeptide isolated from fermentation products of Streptomyces. Preclinical studies in mice have shown that FK-565 augments NK-cell activity and macrophage functions as well as T-cell functions both in vitro and in vivo [507]. However, in animals, mitogeninduced IL-2 production is decreased [4]. Clinical trials in cancer patients have not been promising. Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoylL-leucine] is a low-molecular-weight immunomodifier found in supernatants of Streptomyces olivoreticuli [327]. It is a competitive inhibitor for the enzymes aminopeptidase B and leucine aminopeptidase. These enzymes are associated with the outer membranes of most mammalian cells, including lymphocytes. In animal models, bestatin augmented both humoral as well as cell-mediated immune responses [60, 69], particularly in immunosuppressed mice. Macrophage activation, but not NK activity, was also observed both in vitro and in vivo [506]. It has also been shown to enhance T-cell numbers and cytotoxic functions following in vitro incubation with human lymphocytes, but it has not significantly influenced LPR [69]. It has been shown to augment phagocytic cell function in vitro. Bestatin has now been chemically synthesized and is in clinical trials. The drug has been well tolerated in cancer patients at daily doses of 30 mg for up to 2 years [60, 69, 369, 576], but has shown little clinical activity. Tuftsin is a naturally occurring tetrapeptide (Thr-LysPro-Arg), found normally in human and animal plasma, and represents residues 289–292 of the heavy chain of γ-globulin [121, 157, 365, 395]. It is released enzymatically from a protein carrier (Leukokinin) and is capable of activating various functions (particularly phagocytosis) of polymorphonuclear leukocytes and monocytes/ macrophages, T-cell cytolytic activity, and IL-2 production at physiologic concentrations [125, 327]. It has also been shown to enhance antibody production to thymicdependent as well as thymic-independent antigens in animals and Ia-suppression. It has also been able to reduce tumor necrosis factor in animals [571]. Lentinan is a purified polysaccharide obtained from the extracts of the edible mushroom Lentinus edodes

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[66, 110, 243, 373]. Chemically, lentinan is a β-(1,3)glucan with some β-(1,6)-glucoside side chains. It has a molecular weight of about 500,000. Evidence has been presented that lentinan is a T-cell adjuvant, but a variety of immunomodulatory activities have been observed [109, 327]. In animal models, it has been shown to augment antibody production only in the presence of T cells. It has also been shown to augment a variety of cytotoxic effector-cell mechanisms following administration to animals and to trigger production of various kinds of serum factors including IL-1, CSF, and IL-3 [109]. In vitro, it activates monocytes/macrophages and NK-cell activity of PBMC from cancer patients but does not enhance LPR or LAK-like cytotoxicity [263]. OK-432 is a heat-killed substrain of Streptococcus pyogenes that has been studied extensively in Japan. OK-432 predominantly acts by augmenting NK-cell activity [378, 539, 545], as well as augmenting macrophage [269] and T-cell [223] cytotoxicity. It has also been shown to induce various cytokines such as interferon, IL-1, and IL-2 [242, 368, 443]. A variety of Japanese phase I, II, and III studies have been performed in cancer patients in which it has been administered intradermally [106, 115, 339, 545], intramuscularly or intralesionally [539]. The major toxicities have been fevers and local inflammatory reactions at injection sites. ImuVert is prepared from the bacterium Serratia marcesseus. Its primary components are vescicles derived from the bacterial membrane and ribosomes [554]. It augments natural killer-cell activity and has antitumor cell activity in animals. It has demonstrated poor activity in clinical trials in patients with malignant gliomas and other cancers. Imreg-1 is a natural, leukocyte-derived immunomodulator containing two low-molecular-weight peptides: a dipeptide (tyrosine-glycine) and a tripeptide (tyrosineglycine-glycine). In vitro Imreg-1 enhances the production of various cytokines, including IL-2 [176]. Clinical trials to date have been limited to patients with HIV disease and have not shown much promise.

Treatment of Cancer-associated Immunosuppression: Phase I and II Studies Most of the agents described in the preceding section have already been employed therapeutically in patients with cancer. Of the chemical BRMs, levamisole has been by far the most exhaustively studied, whereas among the biologicals, the interleukins have received the most attention. No organized approach has been employed for the clinical evaluation of the immunorestorative BRMs. In general, a small phase I or phase

II study to assess the tolerability and immunomodulatory effects of the agent in advanced cancer patients has been followed by phase III trials with random experimental designs in which survival was the end point and immunological monitoring was minimal, or even omitted. Thus, it has been difficult to make any firm conclusions concerning the immunorestorative properties of these agents in cancer patients. A number of preliminary small-scale phase I and II studies have indicated that levamisole [111, 302, 349, 366, 558], isoprinosine [111, 356, 403], azimexone [53, 388], bestatin [69, 288, 328, 369, 560, 576], OK-432 [530, 545], retinoids [337], NPT 15392 [575], DTC [422], coumarin [49], various thymic factors [173, 463], lentinan [245], cimetidine [289, 329, 426, 488], and transfer factor [493] could improve T-cell, NK-cell, or B-cell numbers and/or functions in advanced cancer patients with pretreatment abnormalities. In general, however, the effects have not been striking. When administered to patients without immunodeficiencies, however, therapy was often followed by a deterioration of immune competence. In several reports, no immunomodulatory effects of levamisole were observed on T-cell numbers or functions [200, 558]. A study of patients with colorectal cancer suggests that the major in vivo effect of levamisole is augmentation of monocyte chemotaxis [179]. Of the thymic factors, THF and TFX have been employed predominantly in patients with infectious complications, and reports have been mostly anecdotal. Cimetidine has increased an in vivo local graft-versus-host reaction of advanced cancer patients [508]. However, in other studies, it had no effects on immune cell numbers or functions, or on DTH [316]. Similarly indomethacin has been of only limited utility in restoring immunity in advanced cancer patients [219]. The administration of cimetidine or ranitidine to advanced cancer patients has been associated with improvements in performance status [92]. In one study, treatment with the combination of coumarin plus cimetidine increased the percentage of monocytes and of DR+ monocytes of treated patients [323].

Chemicals Hodgkin’s Disease HD was chosen because of the well-documented abnormalities of T-cell immunity that are present prior to treatment and that persist in patients during clinical remission. Levamisole has been shown in vitro to enhance T-cell proportions [50, 451] among PBMC of HD patients. Treatment of patients with HD in remission for less than 2 years after the completion of radiotherapy resulted in

Robert K. Oldham improvement in T-cell percentages and functions [301]. DTH reactions and PHA responses also increased [50]. Continuation of treatment beyond 3 months led to a slight but consistent decline of these parameters.

Solid Tumors A pilot study was performed with levamisole in surgically resected patients with malignant melanoma and squamous-cell carcinoma of the head and neck [57]. Patients treated with levamisole (150 mg orally twice a week) for 6 months exhibited improvements in absolute circulating T-cell levels, and only one of eight melanoma patients treated with levamisole relapsed. Based on these results, large-scale phase III were designed, the results of which have been reported as negative in one and marginally positive in the others [412, 491]. Based on phase III data in resected colon cancer, levamisole was approved for use with 5-fluoruracil as adjuvant treatment of resected stage III colon cancer. However, more active regimens have recently been available, making levamisole little used in the current treatment of colon cancer.

Biologicals

121 negative prior to treatment. In a more recent report from the same group, 19 patients in remission for at least 6 months were randomized to receive TP-1 at 50 mg IM daily, every other day for 35 days, or no treatment [304]. Patients who received TP-1 were then maintained on TP-1 twice weekly. Before treatment, patients exhibited depressed percentages and absolute numbers of circulating T cells and Tind cells. Following 5 weeks of daily TP-1, the proportions and numbers of all T-cell subpopulations increased significantly, while alternateday treatment was not as effective. Maintenance TP-1 therapy did not produce any further improvements in T-cell or B-cell numbers or proportions. PBMC from some patients also exhibited improvements in mitogeninduced lymphokine production (e.g., IL-2, IFN-γ), but the improvements were not statistically significant overall. No significant changes were observed in a variety of serum markers including neopterin and β2-microglublin. It was concluded that TP-1 has the potential to expand the T-cell pool, and, in particular, Tind cells, in patients with HD in remission, but that intensive (daily) induction therapy is required. In this small sample, no conclusions could be established and large-scale clinical trials have not been done.

Hodgkin’s Disease

Malignant Melanoma

In one study, 19 consecutive untreated patients with HD received TP-1 at a dose of 1 mg/kg daily for 7 days, and immunological monitoring was performed prior to treatment and again on day 8 [324]. A majority of untreated patients exhibited depressed T-cell percentages as well as depressed DTHS and LPRs to PHA. The mean T-cell percentage increased significantly, from 47% to 55.7% (normal is 58.9%) following treatment with TP-1, whereas the mean PHA response improved but did not totally normalize. Similarly, DTH responses to recall antigens were positive in 53% of patients before therapy, and in 95% after therapy. The patients most likely to respond to TP-1 were the most lymphopenic, for whom an in vitro enhancing effect of TP-1 was observed on T-cell percentages and LPRs of PBMC prior to therapy. In a similar study, 15 patients with HD (in remission and off therapy for at least 1 year) were treated with 50 mg TP-1 by daily intramuscular injections for 60 consecutive days [303]. For patients with initially depressed T-cell numbers, including depressed helper (Tind) cell numbers, reconstitution to normal occurred within 30 days after the onset of therapy, but returned to pretreatment levels after the discontinuation of therapy. There was also a significant increase in NK activity, but in vivo DNCB reactivity did not become positive in any patient who had been

TP-1 has been studied in a novel random-design trial involving 32 patients with localized stage I malignant melanoma who were rendered disease-free following surgery [51]. Subjects were selected from a cohort of 211 postsurgical patients who were monitored at 3-month intervals in the first 2 years after surgery. In prior work, it was demonstrated that patients who developed low circulating T-cell levels were at high risk for relapse. Patients who either exhibited presurgical depressions of total T-cell numbers or on serial monitoring developed a depression of absolute circulating T cells to less than 1,000/mm3 were considered eligible for random assignment to one of three treatment arms: TP-1 alone (25 mg IM once a week, eight patients); chemotherapy alone (DTIC, 200 mg/m2 intravenously for 5 days, repeated monthly, eight patients); or no further therapy (16 patients). In the eight patients who received TP-1, T-cell numbers began increasing within 3 days after the first injection, and weekly immunological monitoring revealed that levels returned to normal and were maintained at that level. Only inconsistent effects on T-cell functions were seen in the group that received TP-1, and no changes were observed in any parameter in patients who were untreated or who received chemotherapy. Overall survival was better for the TP-1 group, but the improvement was not statistically significant.

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Chronic Lymphocytic Leukemia (CLL) Patients with Cll have also been treated with TP-1 [290]. TP-1 significantly increased Tind-cell proportions, leading to an improvement in the Tind/Tc/s ratio accompanied by an increase in LPRs to PHA and helper-cell function. In this study, the in vitro incubation of purified T-cells with TP-1 did not produce modifications of subset proportions or of immune functions. In contrast, in vitro incubation of T-cells with Tα1 from patients with stablephase CLL led to an increase in T-cell proportions and an improvement in T-cell functions [41].

Phase II/III Surgical Adjuvant Studies Chemicals Lung Cancer The vast majority of large-scale phase III surgical adjuvant studies have been performed with levamisole. Two randomized, double-blind trials in patients with nonsmall-cell lung cancer have been reported [10, 16]. In the first trial, a fixed dose of levamisole was administered preoperatively for 3 days, and then for 3 days every 2 weeks [10]. Treatment was continued for 2 years or until relapse. Among 211 patients, although there was no difference in overall survival, levamisole treatment was associated with a reduction in distant metastases and in cancer deaths. When analyzed on the basis of adequacy of drug dosage, it was found that the greatest benefit of levamisole was seen with patients who received a daily dose of 2.1 mg/kg or more. The second study was designed as a confirmatory trial, and a weight-related dose of levamisole was employed, with a schedule identical to that in the original study [16]. Nevertheless, among the 217 evaluable patients, overall survival was decreased in those who received levamisole, because of non-cancer-related deaths in the perioperative period.

Gastrointestinal Cancer In the early 1980s, results of several small-scale trials were reported that suggested that levamisole could prevent perioperative immune suppression. A British report in 1979 first indicated that the administration of levamisole on 3 postoperative days could accelerate the recovery of antitumor immunity (in an LMI assay) but not LPRs to PHA [570]. In a Japanese trial of 50 patients with colon cancer, 15 were treated with levamisole at a dose of 150 mg daily for 2 consecutive days, every other week, beginning 3 days before surgery. Treatment with levamisole appeared to prevent the postoperative depression of LPRs to PHA, as compared with a control group of

patients, but did not affect DNCB reactivity [477]. A Belgian colorectal trial used levamisole (150 mg for 3 days every 2 weeks) in a matched group of patients operated on by the same surgeon. In both the overall group and the 40 patients with Dukes B2 and C cancers, survival was prolonged with levamisole [538]. Improved survival of resected patients was reported in a broad trial of 177 patients with various gastrointestinal cancers treated with levamisole [347]. There was also improvement in DTHS reactions and in vivo LPRs. Similar results from the Japanese literature have been reported for patients with advanced gastric cancer treated with levamisole [348]. An interesting recent report of a randomized trial involving 181 resected patients with gastric cancer showed an increase in median survival for patients who received cimetidine [524].

Phase III Levamisole Trials in Colon Cancer Within the past decade, results of a series of phase III trial results have been reported, in which levamisole was administered alone, or in combination with adjuvant chemotherapy in patients with resected colorectal cancer [23, 32, 113, 244, 292, 293, 351, 474, 569]. A large-scale trial that was designed to compare treatment with levamisole to surgery only reported by the European Organization for Research and Treatment of Cancer (EORTC) Gastrointestinal Tract Cancer Cooperative Group [23]. This double-blind placebocontrolled trial involved 297 patients with Dukes C colon cancer. Levamisole dosage was based on body weight and ranged from 100 to 250 mg/day for 2 consecutive days each week for a period of 1 year. Treatment was started as soon as possible after surgery, usually within 2 weeks. With a median follow-up time of 3 years, no benefit was seen in disease-free survival for patients who received levamisole. Although the proportion of patients alive at 5 years was 51% in the levamisole group versus 39% in the placebo group, this difference was not statistically significant. Other large-scale trials convincingly demonstrated that postoperative adjuvant therapy with levamisole plus 5-FU impacts significantly on survival of patients with colon cancer [244, 292, 293, 351]. Thus, levamisole plus 5-FU was considered standard adjuvant treatment for resected Dukes C colon cancer until folinic acid plus 5-FU proved as effective, less expensive and easier to administer. Levamisole is currently not used in the treatment of colon cancer or any other human malignancy.

Gynecologic and Urologic Cancer Positive surgical adjuvant studies have been reported with levamisole in postoperative patients with cervical

Robert K. Oldham cancer [447] and bladder cancer [486] but is no longer being used.

Malignant Melanoma Only one study has focused on assessing in detail the immunorestorative effects of levamisole in patients with locally advanced malignant melanoma following surgical resection [253]. Levamisole was administered at 150 mg/twice weekly. No improvements were noted in T-cell numbers, or in LPRs to mitogens or antigens. A large-scale phase III trial with levamisole involved 203 postsurgical patients with malignant melanoma. No improvement in either relapse rates or overall survival was noted in patients who received levamisole [491]. In stage I patients, there was a trend in favor of levamisole regarding time-to-recurrence and survival. More recently, a large study involving 548 patients with completely resected malignant melanoma having a poor prognosis (Clark levels 3, 4, or 5, greater than 0.75 mm, satellite lesions, in-transit metastases, or regional lymph node metastases) revealed that patients treated postoperatively with levamisole (2.5 mg/kg orally on 2 consecutive days weekly for 3 years) survived significantly longer than those who received either no postoperative therapy or weekly BCG, or a combination of BCG alternating with levamisole [412]. Median follow-up time for this trial was 5.1 years. In a randomized trial involving 156 stage I Clark level 3, 4, and 5 patients, there was a trend for a delay in appearance of distant metastases in patients receiving levamisole (30 months versus 9 months for patients receiving placebo), but overall survival was not significantly improved [309].

Biologicals Lung Cancer Several studies have focused on the effects of transfer factor administered as an adjuvant to surgery. A tumor antigen-specific preparation, prepared from household contact family members with positive reactivity to lung cancer antigen, was administered to 28 resected patients with non-small-cell lung cancer twice by subcutaneous injection. Treatment was associated with a significant improvement in a variety of specific as well as nonspecific immune parameters, including DTH reactions and LPR to PHA [163]. In a randomized study of 63 postoperative patients with non-small-cell lung cancer (some of whom received mediastinal radiotherapy), those who received transfer factor from normal donors beginning 1 month after surgery and continuing every 3 months showed a significant improvement in survival at 2 years compared with nontreated patients [276].

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Head and Neck Cancer The impact of preoperative perilesional therapy with OK-432 was studied in 13 patients [539]. Treatment was associated with less pronounced decreases in NK activity of PBMC, and higher NK activity in draining lymph nodes, than in patients treated by surgery alone.

Malignant Melanoma TP-1, transfer factor [90], isoprinosine [403], and TF5 [463] have been administered to postoperative patients with malignant melanoma. Each of these trials involved random treatment assignments and varying degrees of immune monitoring. No adjuvant effects could be demonstrated in these small studies. The trial with TF5 was complicated by the fact that only 45 patients were allocated to treatment with either a low dose (4 mg/m2) or high dose (40 mg/m2) administered subcutaneously, concurrently with BCG, with or without DTIC chemotherapy. A large-scale, randomized, double-blind phase III trial that involved 168 resected patients with highrisk stage I and stage II malignant melanoma has been reported [342]. Therapy (normal donor transfer factor or placebo) was initiated within 90 days of resection and continued for 2 years. With a median follow-up period of 25 months, patients who received placebo exhibited a trend for improved disease-free survival and overall survival. Thus, this large-scale trial has indicated that normal donor transfer factor is not an effective postsurgical adjuvant therapy for malignant melanoma.

Treatment of Radiotherapy-induced Immunosuppression Although a number of clinical trials using immunorestorative BRMs in irradiated patients have been reported, little information is available concerning whether or not treatment can prevent and/or reverse radiotherapy-induced immunosuppression.

Chemicals Levamisole Of the putative chemical immunorestorative agents, levamisole has been most extensively evaluated as an adjunct to radiation therapy. Since the larger trials have focused almost exclusively on survival as an end point, very little information has been obtained concerning its immunorestorative properties. Several of the smaller trials have included serial immune monitoring. In a study involving 57 patients with colorectal cancer treated with surgery and radiation therapy, levamisole at a dose of 150 mg/day for

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2 consecutive days every other week, beginning concurrently with postoperative radiation therapy, enhanced LPRs to PHA as compared with an untreated control group [293]; no effects were observed on DNCB reactivities. In a randomized double-blind study involving 71 postoperative patients with stage II breast cancer, 38% of those who received levamisole (2.5 mg/kg/day on 2 consecutive days, twice weekly, beginning with the initiation of radiation therapy) exhibited improvement in DTHS to at least three recall antigens, as compared with only 19% of those given a placebo [277]. Similar results were noted in an earlier trial using DNCB reactivity [473]. Thus, such studies suggested that levamisole may improve some aspects of T-cell functions in irradiated patients. A number of large-scale cooperative group trials have now demonstrated that levamisole administration does not improve the survival of patients treated with radiotherapy. Four separate negative trials have been reported involving patients with non-small-cell lung cancer (NSCLC). The Southeastern Cancer Study Group reported that levamisole was without significant clinical benefit in a large randomized, placebo-controlled trial of 251 patients undergoing radiotherapy for inoperable non-small-cell lung cancer [285]. In this study, levamisole was administered at a dose of 2.4 mg/kg twice weekly beginning at the initiation of radiotherapy. The median survival of patients treated with levamisole was shorter than that of those who received placebo. Negative results have also been reported in a SWOG similar trial [566]. The Radiation Therapy Oncology Group (RTOG) reported results of two separate randomized, placebocontrolled trials, one involving 74 patients with resected NSCLC and positive lymph nodes [215], and a second involving 285 patients with unresectable tumors [395]. Levamisole (2.5 mg/kg on days 1 and 2, weekly) or placebo was initiated at the beginning of radiation therapy and continued for up to 2 years. Accrual to the first trial was terminated prematurely when survival data became available from the second study indicating a worse survival (9 months versus 12 months). Thus, it appears to be conclusively established that levamisole combined with radiation therapy has no benefit in the treatment of NSCLC [400]. In the randomized trial involving 71 stage II breast cancer patients treated with radiotherapy postoperatively, those who received levamisole exhibited a slight prolongation of disease-free survival [277]. Among postmenopausal patients, levamisole significantly increased both disease-free and overall survival, and the levamisole group showed fewer distant metastases as the first sign of recurrence. In another trial involving 150 patients ran-

domized to postoperative radiotherapy, chemotherapy, or both with or without levamisole, patients receiving radiotherapy plus levamisole exhibited improved diseasefree and overall survival [278, 280]. These results are in contrast to two other similar studies. In a randomized but not double-blind study, levamisole treatment was associated with an increased recurrence rate [127]. In a trial involving 198 patients with resectable axillary nodepositive disease, levamisole, when begun following completion of postoperative radiotherapy, had no effect on either disease-free or overall survival [525]. Nearly a third of patients treated with levamisole had to terminate therapy prematurely because of toxicity (primarily leukopenia, skin rash, nausea, fever, and mucosal infection). It has been argued that differences in patient characteristics led to these discordant results. In head and neck cancer, three reports from the same Italian group have indicated that radiotherapy led to a decrease in both T-cell numbers and functions (LPRs), and that treatment with levamisole accelerated the restoration of T-cell counts, compared to patients who received placebo [26, 27, 382]. With a median follow-up of 30 months, there was some improvement in diseasefree interval for patients who received levamisole. Another similar trial failed to demonstrate an improvement in survival for patients with head and neck cancer treated with levamisole [374].

Isoprinosine In a double-blind, placebo-controlled trial designed to evaluate the immunorestorative effects of isoprinosine following radiotherapy, one half of 106 irradiated patients with breast, head and neck, or uterine cancers were randomly assigned to receive isoprinosine or placebo in discontinuous courses for 5 months. After 3 months of treatment, 64% of the isoprinosine-treated patients exhibited evidence of improvement of DTHS and in vitro functional tests, whereas only 23% of placebo-treated patients did [356]. No correlations have yet emerged between immune reconstitution and clinical status.

Prostaglandin Antagonists The nonsteroidal anti-inflammatory drug oxyphenbutazone was found to improve survival of patients with stage III cervical cancer when administered during radiation therapy [563]. This trial involved 160 patients. Thirty of 73 patients with stage III disease were treated with 100 mg oxyphenbutazone three times daily. Treatment was associated with an improved survival, but no studies were performed to assess the drug’s effect on immune responsiveness.

Robert K. Oldham

Biologicals Thymic Factors Several clinical trials with TF5 and Tα1 have indicated that thymic factors may accelerate the reconstitution of T-cell functions following radiation therapy to head and neck or mediastinal portals. The first clinical trial involved 75 patients with localized but unresectable head and neck cancer [553]. Patients were randomly assigned to receive TF5 (60 mg/m2) subcutaneously in a loading dose (daily for 10 days, then twice weekly) schedule or no further therapy. Treatment with TF5 began concurrently with the initiation of radiotherapy and continued for 1 year or until relapse. TF5 administration did not prevent, nor could it restore, the marked T-cell lymphocytopenia that followed radiotherapy. Patients treated with TF5 exhibited a prolongation of disease-free survival, but of only borderline statistical significance. Similar findings have been reported with Tα1 in postradiotherapy patients with non-small-cell lung cancer [459]. This study was a double-blind, randomdesign trial involving 42 patients with localized, unresectable disease who had just completed a course of radiotherapy to the primary tumor and mediastinum. Patients who received mediastinal radiation were chosen specifically as the study population, based on an in vitro study, indicating that TF5 could increase T-cell proportions of PBMC from patients who had received mediastinal radiation to other portals [272]. Patients whose disease regressed or remained stable at the completion of radiotherapy were randomized to receive Tα1 (900 μg/m2 subcutaneously) either by a loading dose (daily for 14 days, then twice a week) or on a twice-weekly schedule; treatment began within a week after completion of radiotherapy. All patients exhibited a marked depletion in absolute circulating T-cell levels and in T-cell LPRs at the completion of radiation therapy. Serial immunological monitoring revealed that the patients who received Tα1 had a complete normalization of T-cell function in MLR, which became apparent following 7 weeks of treatment. However, only patients treated on the twice-weekly schedule maintained normal Tind/Tc/s ratios throughout the study period. The Tα1 administration, however, did not influence absolute circulating T-cell numbers or T-cell subset numbers, and all treated patients remained lymphocytopenia over the 15-week follow-up period. These results were interpreted to indicate that more intensive schedules of Tα1 administration are optimal for inducing restoration of T-cell functions, whereas less intensive schedules are optimal for maintaining immune balance of T-cell subsets.

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Transfer Factor In a randomized, double-blind trial involving 100 patients with nasopharyngeal carcinoma, transfer factor (derived from young adults with a proven history of infectious mononucleosis and from normal blood donors with high anti-Epstein-Barr virus capsid antigen antibody levels) was administered for 18 months in conjunction with radiotherapy [170]. This trial was based on the association of Epstein-Barr virus with nasopharyngeal cancers. No significant effects of transfer factor were noted on disease-free and overall survival. Immune competence studies were not performed. A study was reported involving 111 patients with NSCLC who had surgery followed by radiotherapy [93]. Twenty-six patients received transfer factor (normal donor) bimonthly beginning after RT was completed. Increased DTHS and a lower relapse rate were observed in patients who received transfer factor, but the small sample size of this trial precludes any definitive interpretations. In a small randomized, placebo-controlled trial of 47 patients receiving radiotherapy with or without chemotherapy for Hodgkin’s disease, DTHS skin responses were markedly enhanced in 22 patients who received transfer factor (prepared from normal donor buffy coat cells), but no improvements were noted in a variety of other immunologic parameters, nor in the prevention of infection, including varicella/zoster [198].

T-cell Reconstituting Factor A pilot clinical trial has been performed in which this factor was administered by SQ injection (2 mg/m2 TIW) for 1 month to 11 patients who had just completed radiotherapy for a variety of locally advanced solid tumors. A variety of immune parameters were followed serially and compared to those of five patients randomized not to receive treatment. Treatment produced a generalized lymphocytosis involving both Tind and Tc/s cell numbers, but no effects were noted on DTHS or LPRs at the dose and schedule employed [112].

Retinoids Therapeutic and immune restorative effects of vitamin A have been evaluated in a randomized trial of 42 patients undergoing radiation therapy for inoperable cervical carcinoma [338]. Vitamin A palmitate was administered orally at a daily dose of 1.5 × 106 IU on days 1–5, 8–12, 16–20, and 23–27, and radiotherapy began on day 22 for 8 weeks. Vitamin A was well tolerated, although most of the 21 treated patients developed scaling of the skin. Serial immunological assessments were performed on some of the patients. No effects were found on DTHS reactivity, in that only about 50% of

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patients reacted in both the treatment and control groups. Vitamin A treatment increased in vitro LPRs (albeit to a low degree) from pretreatment values, whereas the control patients showed no change or decrease in LPRs during radiation treatment. Thus, it was concluded that vitamin A administered concurrently with radiation could prevent the radiation-induced depression of at least one T-cell function.

Bestatin Several studies have explored the in vivo effects of bestatin in irradiated cancer patients [61, 66, 70, 373]. In a prospective randomized trial, the clinical efficacy of bestatin was evaluated in 151 evaluable patients who had completed a course of local radiotherapy for bladder cancer. Patients were randomly assigned to receive 10 mg bestatin orally, three times daily, for at least 1 year, or no further treatment. The recovery of LPRs to PHA and PPD proceeded at an accelerated rate for 9 months in the bestatin-treated patients [70]. Patients treated with bestatin exhibited a greater percentage of circulating T cells after 1 month of treatment, but levels declined to control values within 3 months. Similar transient changes were noted in NK-like activity. It was concluded that the schedule or dose of bestatin was not optimal, and that either higher doses or intermittent schedules require evaluation. An update of the trial has shown that the bestatin-treated patients exhibited an improvement in disease-free survival compared to the radiotherapy-only group, but no improvement in overall survival [65]. The disease-free survival benefit was more apparent in patients with earlier stages of disease.

OK-432 A large multicenter trial in Japan randomized 382 patients with cervical cancer, stratified by presence or absence of surgery and clinical stage, to radiotherapy with or without intradermal OK-432 starting concurrently at 2-day intervals with the initiation of radiation treatment [106], and continuing biweekly for 2 years. Patients who received OK-432 exhibited a decrease in 3-year recurrence rate, a more rapid restoration in DTHS to PHA, a-polysaccharide antigen, and peripheral blood lymphocyte counts than the control patients.

Combined-modality Studies with Chemotherapy Three major problems in combined-modality studies (Table 3) have made it difficult to draw conclusions concerning the influence of putative immunorestorative agents on chemotherapy-induced immunosuppression:

(a) the changing and varied combination chemotherapy regimens available for clinical use; (b) the lack of welldefined doses and schedules of administration for the immunorestorative agents; and (c) the almost universal omission of detailed serial immunologic assessments of patients receiving treatment. For the most part, these studies have emphasized conventional chemotherapy parameters as their end point, that is, tumor regression rates and overall patient survival, on the assumption that if improvements were noted in patients who received an immunorestorative agent, they would result from undefined immunomodulatory effects on the immune response.

Advanced Disease Three broad approaches are feasible for the treatment of advanced cancer patients with combined chemotherapyimmunorestorative therapy. The putative immunorestorative agent could be administered concurrently with chemotherapy, following the completion of chemotherapy when clinical remission has been achieved, or during maintenance chemotherapy if it is continued. All three approaches have been applied, and most studies have employed levamisole and/or thymic factors.

Chemicals Levamisole This compound has been evaluated in several largescale clinical trials as an adjunct to conventional chemotherapy, with mixed results. In general, levamisole has been administered as either 150–200 mg or 2.5 mg/ kg on 2 consecutive days each week between chemotherapy courses. Serial immunological studies have usually not been performed. In 82 patients with metastatic colorectal cancer, survival of those receiving 5-FU and levamisole was significantly greater than those receiving 5-FU alone [78]. Improvements in response rates and/or survival have also been seen in patients receiving levamisole in addition to combination chemotherapy for advanced breast cancer [278, 281, 497]. In one study, an improvement in DNCB reactivity was noted with levamisole treatment; however, negative reports have also appeared concerning breast cancer [99, 387], colorectal cancer [91], nonsmall-cell lung cancer [107, 129], and malignant melanoma [122]. A report in 669 patients with non-small-cell lung cancer indicated that patients who received combination chemotherapy along with levamisole plus warfarin plus tranexamic acid survived longer than those receiving chemotherapy alone [473].

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Table 3. Summary of combined modality studies designed to treat chemotherapy-induced immunosuppression Cancer types

Outcome

References

Colon

+ − + − + − − + + − − + + +

[78] [91] [278, 281, 497] [99, 387] [475] [107, 129] [122] [445] [390, 391] [441] [119] [111, 286] [82] [295]

+ − + − + − − + + +

[478] [51] [314] [132, 312] [118] [455, 476] [40] [557] [231, 504] [379]

Gastric Ovarian

+ − + − + −

[113, 292, 293] [244, 351] [279, 280] [270] [367] [274]

Breast Non-small-cell lung Non-small-cell lung

+ + +

[241] [162] [557]

Advanced disease Chemicals Levamisole

Breast Non-small-cell lung

Isoprinosine Azimexone Piroxicam Cimetidine Biologicals Thymostimulin

Thymosin Fr. 5 OK-432 Lentinan Bestatin Adjuvant treatment Chemicals Levamisole

Melanoma Multiple myeloma Acute lymphoblastic leukemia Acute myeloblastic leukemia Colon Breast Non-small-cell lung Ovarian Gastrointestinal Melanoma Small-cell lung Non-small-cell lung Small-cell lung Non-small-cell lung Non-small-cell lung Gastric Acute nonlymphoblastic leukemia Colon Breast

Biologicals Thymostimulin Transfer factor OK-432

Levamisole has also been evaluated as an adjunct to maintenance chemotherapy for a variety of hematologic malignancies. Improvements in survival from the start of maintenance chemotherapy have been noted in patients receiving levamisole in cases of multiple myeloma [445] and acute lymphoblastic leukemia [390, 391]. Thus, levamisole did appear to have potential as an adjunct to maintenance chemotherapy for patients with hematopoietic cancers; however, no effects were observed when levamisole was administered concurrently with intensive induction chemotherapy for ANLL [534]. Isoprinosine has been administered concurrently with intravenous 5-FU at various doses without any observable antitumor effects [119].

Azimexone has been administered to ten patients with breast cancer after remission was induced by chemotherapy (CTX, MTX, 5-FU, VCR, prednisone) to assess whether it could ameliorate the immunosuppressive effects of treatment. Detailed weekly serial immunological assessments, performed while patients were receiving 100-mg weekly intravenous injections, revealed that chemotherapyinduced immunosuppression was markedly reversed during azimexone administration. Significant increases in peripheral blood lymphocyte counts and in vitro LPR were noted without change in T- or B-cell percentages [111, 286].

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Prostaglandin Inhibitors It has been demonstrated that the concurrent administration of nonsteroidal anti-inflammatory drugs can prevent some aspects of chemotherapy-induced immune suppression [82].

Cimetidine A study of the immunomodulatory effects of cimetidine was performed in patients with advanced ovarian cancer who received concurrent chemotherapy (cisplatin, ADR, CTX) [275]. Treatment with chemotherapy produced a decrease in CD4 cell counts and in IL-2 production of PBL, which was significantly improved in patients who received concurrent cimetidine.

Biologicals Thymic Factors Two studies performed in the late 1970s with TP-1 and TF5 suggested both a rationale and role for the use of thymic factors in conjunction with combination chemotherapy [478]. Although overall response rates were not altered by TF5, its administration at a high dose (60 mg/ m2) led to a significant improvement in overall median survival [116]. Improved survival was limited to patients who had exhibited pretreatment depressions of total T-cell levels below 775/mm3 and serum HS glycoprotein levels below 60.5 mg/dl. Although serial immunological assessments were not performed, it was concluded that, whereas TF5 had no detectable direct antitumor effects, its administration to immunosuppressed patients may have improved survival by ameliorating the immune defects due to the presence of tumor, and exacerbated by the chemotherapy. These two reports culminated in the performance of a number of confirmatory trials, as well as a variety of other studies in which TF5 and TP-1 were administered concurrently with combination chemotherapy for patients with solid tumors, generally small-cell or NSCLC [40, 51, 132, 312, 314, 455, 476]. In only one of these studies did the administration of thymic factors improve the overall response rate to the chemotherapy and survival [314]. Although this trial involved very small patient numbers, the favorable effects of TP-1 on both myelotoxicity and survival indicate that further studies should be performed adding TP-1 to conventional chemotherapy. In a randomized trial involving 91 of patients with small-cell lung cancer [455, 476], TF5 (60 mg/m2 subcutaneously, twice weekly) had no effect on survival when combined with chemotherapy, compared to chemotherapy alone (CTX, ADR, VCR alternating with

VP-16, cisplatin). An analysis based on pretreatment immune function, total white blood cell count, and absolute lymphocyte count revealed no difference in survival distributions. These results could not confirm the prior reported study [118]. However, this latter trial differed from the original study in that it used different chemotherapy regimens and included prophylactic chest and whole-brain radiotherapy for patients who responded to chemotherapy. Thus, no firm conclusions can be reached regarding the role of TF5 as an adjunct to chemotherapy for small-cell lung cancer. Several different trials in patients with advanced non-small-cell lung cancer treated with combination chemotherapy with or without TF5 [41] or TP-1 [132, 312] and in metastatic melanoma patients treated with TP-1 [51] failed to find improvements in survival for patients treated with thymic factors. In one of the lung cancer trials, only immunosuppressed patients were considered eligible for study; however, no significant immunorestorative effects were noted in the TP-1-treated patients except transient mild improvement in absolute T-cell levels. In vitro LPR to mitogens remained suppressed following treatment, and no improvements in DTHS were observed. Thus, follow-up combined modality studies with TF5 and TP-1 have not provided evidence that thymic factors could either prevent chemotherapy-induced immunosuppression or improve survival.

Lentinan In small studies, administration of lentinan in combination with tegafur [504] or 5-FU and mitomycin [231] to patients with advanced gastric cancer has been reported to improve patient survival.

Bestatin In a randomized controlled trial of 101 patients with ANLL, patients over 50 years of age who received bestatin along with induction chemotherapy exhibited a better remission duration and survival than the chemotherapy-only group [379].

Adjuvant Chemotherapy Adjuvant chemotherapy has become an accepted treatment modality for patients with breast and colon cancer. Evidence is also accumulating that postsurgical adjuvant chemotherapy for other solid tumors may also improve patient survival. The acute and chronic immunosuppressive effects of contemporary adjuvant chemotherapy regimens have recently been evaluated [315, 396, 501].

Robert K. Oldham

Chemicals Levamisole In a randomized trial of 135 postoperative breast cancer patients with positive axillary nodes, patients received L-phenylalanine mustard with levamisole (150 mg for 3 days every 2 weeks) or placebo for up to 2 years [270]. No significant effects of levamisole were noted, although trends for improved survival were seen in postmenopausal patients and in those with four or more positive lymph nodes. In other studies involving patients with stage III breast cancer treated by either radiotherapy or adjuvant chemotherapy (ADR, CTX, VCR), there was a high incidence of toxicity (primarily transient agranulocytosis), requiring discontinuation of levamisole. Follow-up is continuing on this trial. In a study comparing postoperative radiation therapy and adjuvant chemotherapy (vincristine, adriamycin, and cyclophosphamide), levamisole administered concurrently appeared to improve both disease-free, as well as overall, survival for patients who received chemotherapy alone or combined radiation therapy plus chemotherapy [279]. Improvements were also noted in various immune parameters in the levamisole treatment group [305]. Although this trial involved 150 patients, the multiple randomizations made it difficult to analyze. In other recent studies, levamisole was shown to improve the survival of resected patients with gastric cancer treated with MIT and tegafur [367], but there was a deleterious effect of levamisole in a trial of 140 patients with ovarian cancer who received adjuvant chemotherapy following maximal surgical reduction of tumor [274].

Biologicals Transfer Factor The administration of transfer factor, prepared from leukocytes from household contacts, has been reported to improve survival for stage I and II patients with resected non-small-cell lung cancer who were treated further with a variety of adjuvant combination chemotherapy regimens [162].

OK-432 In a trial of 311 patients, OK-432 was shown to improve survival of patients with resected stage I, II and III nonsmall-cell lung cancer when combined with three-drug combination chemotherapy, compared to chemotherapy [557].

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Thymic Factors In a randomized trial 51 patients received adjuvant CMF chemotherapy with or without TP-1 (50 mg/m2 IM daily × 2 weeks, then twice weekly for a minimum of 3 months) following radical mastectomy for breast cancer [241]. Although details of the patients’ clinical characteristics were not reported, patients who received TP-1 exhibited a significant decrease in the incidence of infections (mostly cystitis, conjunctivitis, and mucositis), and an increase in Tind/Tc/s compared to the control (no treatment) group. There was also a lower incidence of myelotoxicity in the TP-1 treated patients. These results have not been confirmed with a large-scale trial.

Current Status of Therapeutic Alterations for Cancer-associated Immune Suppression The results with levamisole in combination with 5-FU as adjuvant therapy for Dukes C colon cancer have provided strong evidence that drugs with immunorestorative properties can play a role in the treatment of human cancer. What has been learned about the reversibility or prevention of cancer-associated immunosuppression? All of the agents discussed in this chapter, at least in small phase I and II studies, appear to have the capability of improving immune cell numbers and/or functions in immunosuppressed patients. The large-scale phase III surgical adjuvant trials for the most part have provided only limited information concerning whether or not perioperative immunosuppression could be prevented. Part of this relates to the fact that it is extremely difficult – and probably impossible – to perform adequate, quality control assays of immunity in multiple different institutions participating in the same large-scale trial. The mechanism by which levamisole improved survival was not addressed, and it is not clear whether the drug exerted its effects as a result of immunomodulation or by other, as yet undefined, mechanisms. Several smaller studies in patients with GI cancers suggested that certain aspects of T-cell immunity (i.e., LPR) could be maintained during the perioperative period by treatment with levamisole. Since these studies, FA/5-FU and later FOLFOX regimens have become the regimen of choice in adjuvant therapy of colon cancer. Studies with levamisole, TF5, Tα1, bestatin, and vitamin A have suggested that various immunorestorative agents could ameliorate radiotherapy-induced depression of T-cell numbers or functions, or accelerate the

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reconstitution of immunity following radiotherapy. In no case, however, did treatment totally normalize both T-cell numbers and functions, and so efficacy can be considered partial at best. In several clinical trials, levamisole has not improved survival in irradiated patients with non-small-cell lung cancer. Analysis of the trial in postradiotherapy patients with non-small-cell lung cancer has suggested that Tα1 can improve overall patient survival when used as an adjunct to radiotherapy. However, the mechanism by which Tα1 improved survival is not known. In addition to accelerating the reconstitution of T-cell dependent immunity, it is possible that thymic factors can protect bone marrow stem cells from the myelotoxic effects of radiotherapy or chemotherapy [241]. Such a mechanism has been proposed to explain the lower incidence of myelotoxicity in breast cancer patients who received TP-1 along with adjuvant CMF chemotherapy [241]. Because radiation therapy results in a uniform and marked depression of immune cell numbers and functions, patients who have received this treatment are ideal candidates for studies of the immunorestorative effects of BRMs. However, it must be recognized that if putative immunorestorative agents are administered concurrently with radiation, their effects on immunity might be negated to some degree. An agent could exert a beneficial effect during radiation treatment only if it provided a direct protective action on immune cells from the deleterious effects of radiation. Although a number of large-scale clinical trials have been performed with patients receiving concurrent chemotherapy, there are only very limited data concerning the prevention of chemotherapy-induced immunosuppression. Several studies have suggested that levamisole may have a role as a therapeutic adjunct for patients with hematopoietic malignancies who are receiving maintenance chemotherapy, yet the mechanism by which levamisole exerts its effects remains unknown. Although none of the immunorestorative agents have proven effective when administered concurrently with intensive combination chemotherapy for patients with advanced metastatic cancers, their potential role in patients treated with adjuvant chemotherapy remains to be explored. The success or failure of future studies with BRMs will be determined primarily, if not exclusively, by the selection of suitable phase II and III clinical models for study and by the avoidance of confounding variables or inadequate sample sizes, which could lead to uninterpretable results. It is likely that a two-phased approach will be necessary to evaluate BRMs with immunorestorative properties: focusing initially on phase I/II,

single-institution pilot studies to establish the pharmacokinetics and immunomodulatory potential of the agent, and then in properly stratified, large-scale phase III randomized trials performed by a cooperative cancer group to establish clinical efficacy using the optimal immunomodulatory dose and schedule and an appropriate patient population. Acknowledgment Dr. Richard Schulof co-authored this chapter in the second edition, but died in an automobile accident during the preparation of the third edition.

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7

Cancer vaccines KENNETH A. FOON AND MALEK M. SAFA

Introduction Immune approaches to the therapy of cancer have substantially evolved over recent years, from treating patients with nonspecific immune stimulants to a focus on the use of tumor-associated antigens (TAAs) either by passive immune therapy with antibodies targeted directly to tumor cells or by active immune therapy via vaccinations with tumor cells, tumor cell lysates, peptides, carbohydrates, gene constructs encoding proteins, or anti-idiotype antibodies that mimic TAAs.

Approaches to Cancer Vaccines Specific active-immunotherapy differs from nonspecific immune-based therapies such a BCG in that the goal is not general but rather specific activation of the immune system to eliminate tumor cells and not affect surrounding normal tissue. Theoretically, specific immunotherapy through vaccines activates a unique lymphocyte (B-and/ or T-cell) response, which has an immediate antitumor effect as well as memory response against future tumor challenge. The first and most obvious type of vaccines is autologous or allogeneic tumor cell preparations (Table 1). Alternatively, membrane preparations from tumor cells have been used. In either instance these vaccines have been combined with a variety of cytokines. Genemodified tumor cells expressing antigens designed to increase immunogenicity or gene modified to secrete cytokines have been a valuable tool for vaccination. In addition, increase in our knowledge of TAA biology has led to the use of purified TAAs, DNA-encoding protein antigens, and/or protein-derived peptides. All of these approaches are being tested in the clinic. Mechanistically, the ultimate aim of a vaccine is to activate a component of the immune system such as antibodies or lymphocytes against TAAs presented by the tumor (Table 2). Antibodies must recognize antigens in the native protein state at the cell surface. Once bound, these molecules can mediate ADCC or complement-mediated cytotoxicity. T lymphocytes, on the other hand, recognize proteins as fragments or peptides of varying size, presented in the context of MHC anti-

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

gens on the surface of the cells being recognized (Fig. 1) [1–3]. The proteins from which the peptides are derived maybe cell surface or cytoplasmic proteins [4, 5]. MHC antigens are highly polymorphic, and different alleles have distinct peptide-binding capabilities. The sequencing of peptides derived from MHC molecules has led to the discovery of allele-specific motifs that correspond to anchor residues that fit into specific pockets on MHC class I or II molecules [6, 7]. There are two T lymphocyte, helper and cytotoxic, which recognize antigens through a specific TCR composed of both α and β subunits arranged in close conjunction to the CD3 molecule, which is responsible for signaling. CD4 helper T cells secrete cytokines and lymphokines that enhance immunoglobulin production as well as activate CD8 CTLs. CD4 helper T cells are activated by binding via their TCR to class II molecules, which contain 14–25 amino acid (mer) peptides in their antigen-binding cleft [8–10]. Extracellular proteins are endocytosed and degraded (exogenous processing into 14–25 mer peptides in endocytic compartments (acidified endosomes) ) and bind to newly synthesized MHC class II molecules. The MHC peptide complex is transported to the cell membrane, where it can be recognized by specific CD4 helper T cells. In most cased the MHC class II antigen-containing peptide is presented to the CD4 helper T cells by a specialized cell called an antigen presenting cell (APC). More specifically, a variety of cells are capable of processing and presenting exogenous antigen including B cells, monocytes, macrophages, and the bone marrow derived derndritic cells (DC). DCs are the most efficient APCs and express high levels of MHC class I and II molecules, costimulatory molecules such as CD80 and CD86, and specific markers such as CD83. After antigen uptake, DCs migrate peripherally to lymph nodes, where antigen presentation to CD4 helper T cells takes place [11, 12]. There are two types of CD4 helper T cell capable of generating either an antibody-or a cell-mediated immune response. This is based on the type of signaling they receive. Th1 CD4 helper T cells stimulate cell-mediated immunity by activating CTLs thought the release of lymphocytokines such as IL-2. Th2 CD4 helper T cells mediate an antibody response through the release of lymphocytokines such IL-4 and IL-10. In some instances

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Cancer vaccines Table 1. Tumor vaccines Autologous tumor vaccines Allogeneic whole cell vaccines Dendritic cell vaccines Viral oncolysates Polyvalent shed antigen vaccines Peptide vaccines Anti-idiotype vaccines Genetically modified vaccines Recombinant viral vaccines DNA vaccines

Table 2. Characteristics of different vaccines Response characteristics Vaccine Autologous cells Allogeneic cells Shed antigens Carbohydrate Peptide Anti-idiotype antibody Dendritic cell DNA

Multiple antigens

Single antigen

Antibody response

T cell response

+ + + − − −

+ − − + + +

+ + + + + +

+ + + − + +

NA −

NA +

+ +

+ +

the generation of one type of response may serve to inhibit the generation of the other (ref. 13, i.e., IL-10 secretion by Th2 helper T cells inhibits the generation of CTLs). CD8-positive CTLs are activated in most cases by peptides derived from intracellular proteins that are cleaved to 9–10 mer peptides in the cytosol of tumor cells or APCs by proteosomes. The peptides are then transported via specialized transporter molecules called TAP proteins to the endoplasmic reticulum, where they become associated with newly synthesized MHC class I molecules [14]. The complex is then transported via the Golgi apparatus to the cell surface membrane, where the complex is recognized by CD8 cytotoxic T cells via a specific TCR. Any endogenously processed protein can be presented to the immune system in this way. Several reports suggest a subset of APCs can present exogenously processed proteins on MHC class I molecules to CTLs [15–19].

Pitfalls in Developing Cancer Vaccines Tumor cells have developed a variety of mechanisms to escape immune surveillance. DC’s are actively inhibited in the tumor milieu. Both immature and defective DCs

+, present; −, absent; NA, not applicable Vaccine (cells, lysates, protein, peptide, cDNA)

14 25mer peptide

Antigen Presenting Cell (APC)

MHC Class II

Exogenous protein from lysed tumor

CD4 T -cell

TCRS CD

IL - 2

TC CD R S

MHC Class

9 10 mer peptide

CD8 T -cell

R TC S CD

Tumor cell undergoing lysis

IL - 4 IL - 5 IL - 10

Tumor cell

B -cell

Figure 1. T cell activation: T cells recognize antigens as fragments of proteins (peptides) presented with major histocompatibility complex (MHC) molecules on the surface of cells. The antigen-presenting cell processes exogenous protein from the vaccine or from the lysed tumor cell into a peptide, and presents the 14/25-mer peptide to CD4 helper T cells on a class II molecule. There are also data to suggest that exogenous proteins can be processed into 9/10-mer peptides that may be presented on MHC class I molecules to CD8 cytotoxic T cells. Activated Th1 CD4 helper T cells secrete Th1 cytokines such as IL-2 that up-regulate CD8 cytotoxic T cells. Activated Th2 CD4 hepler T cells secrete Th2 cytokines such as IL-4, IL-5, and IL-10 that activate B cells

Kenneth A. Foon and Malek M. Safa are described in a variety of tumors. In addition, DC’s that present tumor antigens, may fail to reach the T cells in lymph nodes that generate active immune responses against tumors. The immune response may be skewed toward a Th2 response or T cells may be anergic. Immune regulatory cells may contribute to immune tolerance. CD4 positive T cells (T-reg) with a high affinity receptor for CD25 that co-express the intracellular marker Foxp3 also play an important role in immune tolerance [20, 21]. Mutation or down regulation of immunodominant tumor antigens, MHC molecules, or molecules involved in the antigen processing machinery may also in part explain the escape of tumor cells from immune recognition [22–24]. Down regulation or mutation of pro-apoptotic molecules and expression of anti-apoptotic molecules may also render tumor cells resistant to apoptosis. Tumor cells may acquire mechanisms that may actively contribute to immune tolerance. For instance, Fas ligand (FasL) – expressing tumors can deliver an apoptotic signal to activated T cells and natural killer (NK) cells expressing Fas receptor. The tumor microenvironment may also contain soluble factors that inhibit T cell function. Factors such as TGF-beta, prostaglandins, IL-10, and catabolizing enzymes are produced by tumor cells themselves or by stromal cells that may lead to T cell hyporesponsiveness. Countering these various tumor escape mechanisms is a key component to successful vaccine therapy.

Mechanisms to Improve the Immune Response Potent adjuvants improve the effectiveness of vaccines by accelerating the generation of immune responses and sustaining responses for extended periods of time. Commonly used adjuvants such as alum or Freund’s are effective in elevating antibody titers but do not elicit significant Th1 responses or activate cytolytic T lymphocytes (CTL’s). The current focus is on the generation of adjuvants that are designed to specifically elicit cellular immune responses. Toll-like receptors (TLRs) are known to be involved in the initiation of immune responses. CpG stimulates TLR9 and has been used with vaccines to augment immune responses [25]. TLR8 may be involved in the activity of T-reg cells. Strategies aimed at TLR8 are proposed to neutralize T- reg cells [26]. One approach to decrease the role of T-reg cells is to use immunotoxin directed against the high affinity IL-2 receptor [27, 28]. Monoclonal antibodies specific for the negative regulatory signals mediating the CTL

149 antigen 4 (CTLA4) on T-reg cells has also been tested to enhance the anti-tumor immunity to vaccines [29]. Cyclophosphamide has been used for many decades to boost immune response [30–32], as have other chemotherapy agents [33, 34].

Cancer Prevention Vaccines A major success story in cancer vaccinology is cancer prevention targeting the human papillomavirus (HPV) to prevent cervical cancer. Cervical cancer is the second most common cancer in women responsible for over 250,000 deaths annually worldwide. Seventy percent of cervical cancers are caused by the two most common oncogenic HPV types, HPV-16 and HPV-18, while another 10% are caused by HPV-45 and HPV-31 [35]. The Food and Drug Administration has approved the cancer prevention vaccine HPV-16/18/6/11 (Gardisil, Merck & Co. Inc, Whitehouse Station, NJ). This vaccine uses recombinant DNA technology to develop subunit vaccines which include only the epitopes from the pathogen recognized by the immune system. Copies of the L1 viral capsid protein, the same protein which antibodies are generated against in the natural immune response to HPV, spontaneously self-assemble into noninfectious virus-like particles which are used as the antigen in the prophylactic vaccine. The vaccine is formulated with ASO4 (aluminum hydroxide and monophosphoryl lipid A) adjuvant [36, 37]. Only half the women with HPV infection develop protective immunity because the natural infection by HPV evades detection of the immune system. Clinical studies demonstrated seropositivity in women who receive the HPV 16/18/6/11 vaccine was 100% for HPV-16 and HPV-18 1 month after vaccination and remained at 100% 4.5 years after vaccination [38–40]. Efficacy against cervical intraepithelial neoplasia associated with HPV-16 was 100%. Of course, many women are already infected with HPV and will develop cervical intraepithelial neoplasia and remain at risk for developing cervical carcinoma. A number of HPV based cervical vaccines are in development that are designed to eliminate HPV induced disease after infection.

Therapeutic Cancer Vaccines There has not yet been a therapeutic cancer vaccine approved by the US FDA, however, there are a number of success stories and near success stories that should lead to approval in the near future. Melacine is composed of lyophilized melanoma lysates from two melanoma cells

150 lines and the adjuvant Detox. In a phase 3 trial of 604 patients with resected stage 3 melanoma, patients were administered Melacine and low dose interferon alpha2b versus high dose interferon alpha-2b [41]. Patient’s were stratified by sex and number of nodes and randomly assigned to receive either 2 years of treatment with Melacine and low dose interferon alpha-2b or high dose interferon alpha-2b alone for 1 year. The median overall survival exceeded 84 months on the melancine low dose interferon alpha-2b (arm 1) versus 83 months in the high dose interferon alpha-2b (arm 2) (p = 0.56). Five year overall survival was 61% in arm 1versus 57% in arm 2 and estimated 5 year relapse-free survival was 50% in arm 1 versus 48% in arm 2 with median relapsefree survival times of 58 months in arm 1 and 50 months in arm 2. Overall survival and relapse-free survival were clearly indistinguishable in the two arms. The incidence of neuropsychiatric severe adverse experiences were similar, although they were more severe in the high dose interferon alpha-2b arm. The primary aim of this study was that Melacine plus low dose interferon alpha-2b would prolong overall survival compared with high dose interferon alpha-2b. Unfortunately, this primary aim was not met, and their results failed to demonstrate rejection of the null hypothesis, with nearly identical survival curves. Melacine was approved in Canada based on quality of life improvements. DC vaccines are an attractive approach to vaccine therapy although they are labor intensive requiring unique autologous DC preparations from individual patients (Table 3). Sipuleucel-T and DCVax-Prostate are both vaccines based on DC’s engineered to present T-cell antigens associated with prostate cancer [42, 43]. Sipuleucel-T consists of autologous DC and a fusion prostatic acid of prostates and phosphatase and granulocyte macrophage colony stimulating factor (GM-CSF). A randomized phase 3 placebo controlled trial in patient with metastatic asymptomatic androgen-independent prostate cancer was completed. Eighty-two patients were randomized in a 2:1 ratio [44]. The primary endpoint of this study which was progression-free survival was not reached (p = 0.052). However, overall survival was significantly different for vaccine (26 months) versus placebo (21 months) with a hazard ratio of 1.7 and p = 0.01. A second study showed a trend toward increased survival (19 months versus 16 months) but did not reach significance. A phase 3 clinical trial using survival as an end point is ongoing. GVAX consists of two irradiated allogeneic prostate cancer cell lines engineered to secrete GM-CSF [45, 46]. Two phase 2 trials have been completed in patients with asymptomatic metastatic androgen-independent

Cancer vaccines Table 3. Selected therapeutic vaccines in phase 3 development Vaccine Antigen-loaded dendritic cells Sipuleucal-T DCVax-prostate Monified tumor cells OncoVax-CL GVAX Idiotype vaccines Favid BiovaxID MyVax Viral vector INGN-201 TroVax Peptides TV-1001 (telomerase) BLP-25 (MUC1) Carbohydrate GMK

Company

Indication

Dendreon Corp Northwest Biotheraputics Inc

Prostate Prostate cancer

Intracel Corp Cell Genesys Inc

Colon cancer Prostate cancer

Favrille Inc

Follicular lyphoma Follicular lyphoma Follicular lymphoma

Biovest International Inc Genitope Corp Introgen Therapeutics Inc Oxford BioMedica

Head and neck cancer Renal cell cancer

GemVax AS

Pancreatic cancer Non-small cell lung cancer

Merck KGA Progenics Pharmaceuticals Inc

Melanoma

prostate cancer. In the first study, patients were treated at two dose levels with median survival at the low-dose of 24 months and at the high dose of 35 months. A second study used five vaccine doses. The median survival at the low and middle doses was 23 months and 20 months respectively and was not yet reached at the high dose (>29 months). Phase 3 trials are ongoing. There are a number of non-cell based patient specific vaccines that are currently in phase 3 trials. Favid, BiovaxID and MyVax are idiotype vaccines that use KLH as a carrier. All three are patient specific for patient’s with B-cell lymphoma. Anti-idiotype immune responses have been shown to correlate with better clinical outcomes in follicular lymphoma patients who received idiotype vaccines [47, 48]. MyVax is also being studied in phase 2 trials for mantle cell lymphoma, diffuse large B-cell lymphoma and chronic lymphocytic leukemia. INGN-201 is a recombinant adenovirus-p53-based vaccine for head and neck cancer. The adenovirus theoretically functions to deliver the p53 protein in large quantities to the tumor cells. While this is classified as a gene therapy, there is evidence that suggests an immune response is elicited by p53 which is overexpressed in

Kenneth A. Foon and Malek M. Safa

151 endocytosis of anti-Id by APC

processed to 9/10 mer peptides

MHC class II

MHC class I+ peptide CD8

IL-2r

processed to 15/25 mer peptides

MHC class I

MHC class II+ peptide TCR

APC TcR CD4

CD8 T cell clonal proliferation

Activated CD8 T cell

Th1 cytokines (IL-2, IFN-γ)

CD4 T cell

Th2 CD4 Mφ cytokines NK T (IL-4, IL-10) direct ADCC lysis TcR

MHC class II

CD8

Granzymes Preforin INF-γ TNF-β

Anti-antiId (Ab11)

MHC class I+ peptide

Tumor cell

TCR MHC class II+ peptide

processed to 15/25 mer peptides

B cell recognition of anti-Ιd and endocytosis of anti-Ιd

B Cell

mediates ADCC and direct anti-metastatic effect

Figure 2. Putative immune pathways for anti-id vaccines

the tumor. Therefore, p53 may be considered a tumorassociated antigen and may aid in the elimination of tumor cells [49]. Interestingly, an adenovirus-p53 gene therapy vector (Gendicine) has been approved in China for head and neck cancer. TroVax is a vaccine in which the tumor associated antigen 5T4 is expressed in a modified vaccinia Ankara (MVA) vector, which induces strong immune response similar to the live virus. It is expected that the recombinant 5T4 antigen will be included in the antiviral immune response. This vaccine is in phase 3 trials for renal cell cancer. TV-1001 is a telomerase peptide based vaccine that is in phase 3 trials for pancreatic cancer. Telomerase is over expressed in many cancers and is theoretically a good target antigen. GMK is a ganglioside conjugate vaccine in which ganglioside GM2 is coupled to KLH and formulated with QS-21 adjuvant [50]. The goal of this vaccine is to induce an antibody response rather than a T-cell response

[51]. This vaccine is in phase 3 trials for melanoma. BLP-25 is a liposome-encapsulated synthetic peptide that corresponds to the variable number 10 tandem repeat region of the mucin (MUC)-1 molecule. MUC-1 is overexpressed and underglycosylated on tumor cells. The MUC-1 target is highly represented on most epithelial tumors. There is currently a phase 3 trial in non-small cell lung cancer using BLB-25.

Conclusion The platform for therapeutic vaccines is broad including a variety of antigens, both non specific antigens represented by whole cell based vaccine approaches and recombinant antigens as represented by the protein and virus based approaches. DC’s are an extremely appealing vaccine approach, however, they are limited by the

152 difficulties associated with patients specific cell therapies. No specific approach to vaccine therapy has stood out as clearly superior. Strategies to enhance the immune response will be the next most important step in therapeutic cancer vaccines. Monoclonal antibodies inhibiting T-regs, the use of a variety of cytokines and TLR stimulation are among the strategies that will be employed.

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8

Cytokines WALTER M. LEWKO AND ROBERT K. OLDHAM

Cytokines are regulatory proteins, produced and secreted by various cells, which control immune response, hematopoiesis, inflammation, wound repair and tissue morphogesis. Cytokines may be secreted or membrane bound. Secreted cytokines may act locally as autocrine or paracrine factors or over some distance as would a hormone. Membrane bound cytokines act by cell–cell contact, communicating information from one cell to another, often bidirectionally. There are cell surface receptors for each cytokine that bind the cytokine specifically. Receptor subunits may be shared between different cytokines. Binding the cytokine brings about signaling and a series of cell activating events. For many cytokine receptors (not all), this involves increased phosphorylation of certain tryrosine residues on key cellular proteins. Kinases are the enzymes that carry out phosphorylation. Receptors may themselves be kinases, which activate upon binding. More typically, the activated receptor may recruit cytosolic kinases, for example, mitogen activated protein kinase (p38 MAPK)1 and Janus kinases (Jak1, Trk and Jak 3) [97, 533, 556, 1009, 1699]. As the kinases bind the receptor complex, their enzymatic activities increase and an array of cellular proteins is phoshorylated, in certain cases including the receptor itself. These modifications bring about changes, increases or decreases, in each protein’s activity. Among these proteins are the STAT proteins that control gene expression. When phosphorylated, STAT proteins translocate from the cytoplasm to the nucleus and bind specific enhancer segments allowing the expression of the genes needed to bring about a cytokine’s response. In this chapter, several of the major cytokines will be discussed. Regulation of cellular immunity will be emphasized for its role in the elimination of tumor and virus infected cells. Inflammation is discussed for its important role in not only in immune response but also cancer progression. Unfortunately, it is not possible to cover all of the interesting cytokine research in detail. We have tried to summarize past results and highlight certain interesting trends. The interferons, colony stimulating factors and certain growth factors are covered in other chapters in this

1

Abbreviations are listed at the end of the chapter.

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

book. Table 1 summarizes cell sources and effects of cytokines discussed.

Cytokine Receptors: Many Belong to Receptor Families Cytokines usually induce their effects by binding to target cell receptors, which generate intracellular signals. Often the result is gene activation. The receptors for the various cytokines tend to be grouped into families reflecting common genetic origin, structure, signaling and cell response. Some of the major receptor groups include the IL-1 family, cytokine/hematopoietin receptor family, the IL-6 family, the IL-17 family and the tumor necrosis factor family. For example, the receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-15, G-CSF, GM-CSF and Prolactin are all members of the hematopoietin receptor family. Receptors for these cytokines have been cloned and analyzed. Amino acid sequence reveals segments that are similar between members of the same family [1472]. The IL-6 family members share the gp130 signaling subunit [1943]. Receptors are generally composed of two or more subunits. In a basic model, one subunit binds the ligand; the other subunit generates the signal; together in a complex, the subunits bind the cytokine with higher affinity than they do separated. Different receptors may share subunits that carry out common functions. For example, several receptors (IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21) share a common gamma subunit (γc) that is involved in signaling. The importance of this gamma chain’s function is shown in various forms of combined immunodeficiency disease that appear due to mutations [259, 1097, 1699]. Since this receptor subunit is shared by so many cytokines, these genetic defects have far reaching and debilitating results in the immune response system. Cytokine activity is of necessity transient. Uncontrolled activity may result in inflammatory diseases, allergy or autoimmunity. There are several levels at which regulation may occur, from cytokine secretion to receptor binding to intracellular signaling to target cell responsiveness. For example, there are cellular proteins that regulate signaling [492, 1413, 1882, 2256]. Among them are the 155

156

Cytokines

Table 1. Summary of cytokine sources and effects related to the growth and treatment of cancera Cytokine

Cell source

Cells influenced

Effectb

IL-1

Mono/Macro B cells T cells NK DC Some tumor cells Fibroblasts Keratinocytes Endothelial Glial cells Eosinophils Neurons Epithelial Synovial

IL-2

T cells (active)

IL-3

T cells (active) Thymic epithelium Mast cells Keratinocytes Neurons Eosinophils

IL-4

Th2 Mast cells Basophils NK

Macrophages B cells T cells Th2 Mast cells LAK NK Tumor cells Fibroblasts Chondrocytes Keratinocytes Endothelial Vascular smooth muscle Eosinophils Basophils Liver Synovial Glial cells Neurons Pancreatic Islet Muscle DC Stem cells Hypothalamus Some tumors Melanoma B16 NK LAK T cells PBL B cells Macrophages DC Keratinocyte Eosinophils Tumor cells Stem cells Megakaryocyte RBC precursors Mast cells Natural cytotoxic Granulocytes Macrophages NK T cells Osteoclasts Hematopoietic tumor B cells T cells Th0 Mono/Macro

Activation, TNF, IL-6, PGE2, NO (w/IFNγ) Growth/Ig (w/IL-4, IL-6), chemotaxis Growth (w/IL-2), migration, IL-2, IL-2R, IFNγ Growth, IL-4, IL-5, IL-6 IL-3, IL-4, IL-5, IL-6, IL-9 (Th2-like cytokines) Induction (w/IL-2) IFNγ (w/1L-12), cytotoxicity (w/IFNγ) Growth/inhibition/metastasis/no effect Growth, PGE2, collagen ↓ Growth, ↓ collagen, ↑ protease Growth PGE2, VCAM, ICAM, angiogenesis Growth, IFNβ Degranulation Degranulation, histamine release Metabolism, protein production PGE2, collagenase, phospholipase A2 Growth Survival Insulin Negative protein balance (w/insulin, IL-1, TNF) Maturation (w/GM-CSF) Viability/survival (w/IL-3, G-, M-, GM-CSF) Corticotrophin releasing factor ↓ Growth ↑ Metastasis (↑ endothelial VCAM) Activation/cytotoxicity Activation/cytotoxicity Growth/cytotoxicity/tolerance/AICD Growth/CTL generation Growth/differentiation (w/IL-6, IL-12) Activation, TNFα, β, NO Proliferation Proliferation Eosinophilia Growth/inhibition/no effect ↓ ICAM/↓ MHCI Growth Platelet production RBC production Survival/growth (w/IL-4) Growth/TNF Growth/survival/differ Growth/differ/activation Activation (w/IL-2) Activation/growth (w/IL-2) ↑ or ↓ Growth stimulation/inhibition/no effect Growth, Ig secretion (IgG, IgE), MHCII Growth stimulation/inhibition/no effect Differentiation to Th2, inhibition formation Th1 Growth/activation/AP/HLA DR ↓ Inflammatory cytokines Growth/VCAM Growth Growth (IL2-primed) Growth/IL5 secretion (w/IL-2, IL-12) Growth/ICAM (w/IL-3) Activation (IL-2 primed) Growth/activation (w/IL-2, IL-7, GM-CSF)

Eosinophils DC γδ T cells NK T cells

Endothelial Fibroblasts PBL NK Mast cells LAK DC

(continued)

Walter M. Lewko and Robert K. Oldham

157

Table 1. (continued) Cytokine

Cell source

IL-5

Th2 Eosinophils Mast cells NK

IL-6

Fibroblasts Macrophages Epithelial Endothelial Eosinophils Neutrophils Mast cells B cells Keratinocytes Th2/CD8 T Osteoblasts Synoviocytes

IL-7

IL-8

IL-9

IL-10

Megakaryocytes Langerhans cells Astrocytes DC Some tumors Stroma, marrow Stroma, thymus Keratinocyte B cells Intestinal epithelium DC Endothelial Some carcinomas Leukemia/lymphoma Macrophages Endothelial Neutrophils Eosinophils Mast cells Epithelial cells Fibroblasts Keratinocyte Some melanomas Some carcinomas Th2 Mast cells

Th2, mice Th1 Th2, humans Tr1 nTreg B cells

Cells influenced

Effectb

Hematopoietic cells Certain tumors Eosinophils B cells Mast cells T cells LAK NK B cells T cells Megakaryocytes NK Hepatocytes Intestine cells Fibroblasts Osteoclasts Endothelial Neurons Melanoma Leukemia/lymphoma↓ growth Breast cancer Cervical cancer DC

Growth (+/−) ↓ Growth, ↓ COX-2, ↓ PGE2 Growth/differ/survival/activity/chemotaxis Growth/survival/Ig (IGA, IgM) Growth (w/other factors) Growth/differentiation (w/IL-2), IL-2R Activation (w/IL-2) Activation (w/IL-2), IL-2R Differentiation/Ig (w/IL-2)/survival Growth/activation (w/IL-2) Growth/platelets Activation/cytotoxicity Acute phase proteins, fibrinogen Acute phase proteins Growth/collagen Growth/activation Growth Regeneration ↓ Growth ↓ Growth

Pre-B cells Pre-T cells Mono/macro NK LAK T cells (w/IL-2) DC TIL Pre-eosinophils Melanoma Some leukemia/lymp Neutrophils T cells Macrophages Endothelial Eosinophils NK Cancer

Growth/differ (w/SCF/FLT3L) Growth/differ/survival (w/SCF/FLT3L) Growth/activation/antitumor activity Activation Activation Growth/different/survival/memory/cytotoxicity Growth/antigen presentation Activation/proliferation Growth ICAM Growth Chemotaxis, superoxide, degranulation, hydrolase Chemotaxis Chemotaxis Chemotaxis/angiogenesis Chemotaxis Chemotaxis Autocrine growth/metastasis/angiogenesis

T cells (active) Fetal thymocytes Mast cells Pre erythroid B cells Lymphoma T cells NK Mono/macro Mast cells B cells

Growth (w/IL2), IL-22 Growth (w/IL2) Survival, growth (w/IL-3, IL-4), IL-22 Growth (w/erythropoietin) IgE Growth/survival ↓ Growth/IFNγ/cytotoxicity/chemotaxis. ↓ Cytotoxicity/IFNγ ↓ Activivity/IL12/AP/collagenase; ↑ TIMP Growth (w/IL-3, IL-4) Growth/differ/survival/MHCII (w/IL-2)

↓ Growth ↓ Growth Antigen presentation, especially self antigen

(continued)

158

Cytokines

Table 1. (continued) Cytokine

Cell source

Cells influenced

Effectb

Activated B cells Fibroblasts DC

Apoptosis ↓ Collagen/fibrosis ↓ Antigen presentation

IL-11

Monocytes Eosinophils Mast cells Keratinocyte Some tumors Stroma, marrow Fibroblasts Epithelial Chondrocytes Synovial Smooth muscle

Progenitor cells Megakaryocytes B cells Macrophages Th2 Fibroblasts Liver Synovial cells Osteoclasts NK CD8 T cells Th1 Th2 PBL TIL Macrophages B cell DC Mono/macro B cells Neutrophils NK Endothelial DC Th17 Epithelial Synovial Macrophages Renal ca B cells B cell lymphoma

Growth/colony formation Thrombopoiesis (w/IL-3) Growth ↓ IL-12 ↓ IL-4, IL-5, IL-13 ↓ Differ to adipocytes Acute phase proteins TIMP Growth/differentiation Growth/cytotoxicity/IFN (w/IL-1) Growth/cytotoxicity/IFN (w/IL-2) IFNγ, IL-2, growth ↓ IL-4, IL-10, growth LAK induction/CTL growth cytotoxicity Growth/cytotoxicity IFNγ Growth/differentiation (w/IL-2) Activation/antigen presentation, IFNγ ↓ Cytokines/NO/PGE2/CD14/ADCC ↑MHCII/CD11b c/IL-1RA/CD23 Growth/differ/Ig/IgE switching/CD23 ↑ IL1Ra, ↓ CD14 IFNγ, cytotoxicity VCAM/MCP-1/angiogenesis Growth/maturation,↓ IL-23, IL-1, IL-6. Suppression ↓ NO ↓ IL-1β, TNF ↓ HIV production ↓ Growth Growth/differentiation/memory Autocrine growth

T cells γδ T cells B cells NK LAK TIL Mast cells Keratinocyte CD4 T cells Mono/macro DC Eosinophils HIV NK

Growth/cytotoxicity/memory Growth/activation Growth/IgG, IgA, IgM Growth/cytotoxicity/chemotaxis Induction/cytotoxicity Growth Growth/response Growth; psoriasis Migration/growth w/IL-2, IL-15 Migration/activation/antigen presentation Migration/antigen presentation/tolerance (w/TPO) Migration ↓ Replication/↓entry Cytokines/migration

Macrophages Fibroblasts

Inflammatory cytokines, IL-10, IL-12. Inflammatory cytokines, GM-CSF

IL-12

B cells Macrophages DC Neutrophils Microglial Keratinocytes

IL-13

Th2 cells Mast cells Basophils Eosinophils DC Some B lines Tumor cells

IL-14

IL-15

IL-16

IL-17

CD8 T cells Th1, Th2 NKT cells Follicular DC B lymphoma Epithelial Stroma, marrow PBMC Fibroblasts Keratinocyte

CD8 T Macrophages CD4 T cells B cells Fibroblasts Eosinophils Mast cells DC (immature) Epithelial Brain Th17 Neutrophils

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Walter M. Lewko and Robert K. Oldham

159

Table 1. (continued) Cytokine

IL-17B IL-17C IL-17F

IL-18

IL-19 IL-20

Cell source

Cells influenced

Effectb

CD8+ T cells

Epithelial Endothelial Keratinocyte DC Chondrocytes Granulocytes Synoviocytes CH ovary cells Monocytes Osteoclasts Monocytes Neutrophils

Inflammatory cytokines Inflammatory cytokines Inflammatory cytokines, ICAM Maturation/cytokine production Inflammatory cytokines/MMP/NO Production (w/GM-CSF) IL-6, LIF Invasiveness Osteoclast differentiation Activation IL-1β, TNFα Migration (indirect effect)

Monocytes

IL-1β, TNFα.

Endothelial Bronchial epithelium

↓ Angiogenesis. ↑ IL-2, TGFβ, MCP-1 ICAM, IL-6, IL-8 (neutrophil recruitment)

Th1

(w/IL-2, IL-12) IFNγ, growth/differ/IL-2, FAS/apoptosis/escape, IL-13 IFNγ (w/IL-12), IgG (w/IL-2, IL-12) IFNγ, growth, anti tumor activity FAS/apoptosis/escape, IL-13 IFNγ (w/IL-2) IFNγ (w/IL-2) IL-4 Degranulation, cytokines, CD11b (complement R) Migration/regulation of angiogenesis Activation, IL-4 Activation, IL-6, TNF, ROS IL-10, IL-19 Proliferation, psoriatic character Proliferation bFGF, VEGF, IL-8, angiogenesis Inflammatory cytokines IL-6, IL-8 Proliferation, activity Proliferation, activity Inhibition Class switching, ↑IgG, ↓ IgE Regulation, ↓maturation, ↓activation Growth stimulation, inhibition, or no effect Acute phase proteins ↓ IL-4 Inflammatory cytokines Inflammatory cytokines, migration, β-Defensin Survival, acute phase proteins Proliferation IFNγ, Il-17, IL-6, IL-1, TNF, ICOS IL-12, IFNγ IFNγ Activation (IL-17) Proinflammatory, IL-6, TNF Differentiation, ↓Proliferation Proliferation ↓Growth, ↓migration, ↓angiogenesis TNF, IL-6 ↓Growth, ↑apoptosis, ↓angiogenesis, ↑radiosens ↓ Angiogenesis

Pancreas Intestine Stomach Prostate Fetal kidney CD4 T cells Monocytes Basophils Mast cells Macrophages DC Kupffer cells

B cells NK

Keratinocyte Airway epithelium Adrenal cortex

Macrophages DC NK T cells Neutrophils Endothelial Th2 Monocytes PBMC Keratinocyte Hemato progenitors Endothelium Synovial fibroblasts NK T cells, Th17 Treg B cells DC Hematopoietic tumor Liver Th2 Epithelium Keratinocytes Hepatocytes CD4+ T memory Th DC Macrophages Th17 PBL Melanocytes Keratinocytes Vasc sm muscle Monocytes Tumors

Monocytes Keratinocytes PBMC Keratinocytes Monocytes Glial cells

IL-21

Th2 Th17 Follicular T cells

IL-22

Th17 Mast cells Th1 Th2 NK DC Macrophages Keratinocytes

IL-23

IL-24

Th2 Monocytes (activ) Melanocytes PBMC (activ) Some tumor cells

Endothelium

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160

Cytokines

Table 1. (continued) Cytokine

Cell source

Cells influenced

Effectb

IL-25

Th2 Eosinophils Mast cells Microglial cells Basophils

Eosinophils Th2 Th17 Cartilage Fibroblasts (Lung) Epithelial cells

Growth, infiltration, ICAM, L-selectin, IL-6, IL-8, MCP-1, MIP-1α Activation (IL-4, IL-5, IL-13) Regulation Catabolism Inflammatory cytokines ICAM-1, IL-8, IL-10

T cells NK Th1 Th17 Granulocytes Macrophages Endothelium Most cells DC Tumor cells Th2 Myeloid progenitors Monocytes Epithelial cells Skin epithelium Lung epithelium Sensory neurons Monocytes/DC T cells

Proliferation, IL-12R, IFNγ IFNγ Differentiation, proliferation, activity ↓ Differentiation response to TFGβ/IL-6 ↓ ROS response to endotoxin ↓ ROS response to endotoxin ↓ Angiogenesis Antiviral, oligoadenylate synthetase, MHCI/II Maturation, Treg induction ↓Growth, ↑apoptosis, ↑ Immune response ↓ Activation (↓ IL-4, IL-5, IL-13) Survival Inflammatory cytokines Inflammatory cytokines Dermatitis EGF, VEGF, inflammatory cytokines Itching sensation, development TNFα, IL-1β, IL-6, IL-8, PGE2 Apoptosis, TNF

Th2 Mast cells Endothelium Fibroblasts, heart Cardiomyocytes nTreg CD4+CD25− Th17 cells CD8 T/CD4 Th1 CD4 Th2 Monocytes/macro B cells (active) NK/NKT DC T cells B cells B cells T cells γδT cells NK

Activation (IL-4. IL-5, IL-13) IL-8, IL-4, IL-5, IL-13, survival Nuclear protein/transcription repressor ↓ Collagen ↓ Hypertrophy Proliferation, IL-10 Suppression Suppression, ↓ IL-17 Proliferation/cytokines/↓ AICD/↓ apoptosis Suppression/anergy Activation/survival Proliferation/survival IFNγ/IL-2 IL-12/1l-6 ↓ Activation (by reverse signal) Differentiation (+/−), memory/apoptosis/AICD Activation/ab production (by reverse signal) Stimulate or inhibit, memory/apoptosis/AICD Activation/cytotoxicity (reverse signal) Activation/cytotoxicity (w/IL-2)

B cells Neutrophils T cells Thymocytes NK Mast cells

Memory/↓ class switching, Ig prod (by rev signal) Oxidative burst/IL-8 (by reverse signal) Proliferation/apoptosis/AICD, IL-6 (by rev signal) Apoptosis/negative selection Apoptosis/AICD Chemokines (rev signal)

IL-26 IL-27

T cells NK DC Monocytes (LPS) Macrophages

IL-28A, B, IL-29 IL-31

IL-32

IL-33

Monocytes Macrophages DC Th2

NK cells T cells Epithelium Monocytes Synoviocyte (RA) Endothelium Fibroblasts

IL-35

nTreg

4-1BBL

T Cells DC Monocytes/macro B cells Stromal cells Some tumor cells

CD27L

B cells T cells NK DC B cell malignancy Renal cancer cells Brain tumor Macrophages B cells T cells Megakaryocytes Neutrophils Eosinophils

CD30L

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Walter M. Lewko and Robert K. Oldham

161

Table 1. (continued) Cytokine

CD40L

FASL

FLT3L

Interferon γ

Cell source

Cells influenced

Effectb

pre-erythrocytes Some leukemias Some lymphomas CD4 CD8 Mast cells Basophils Fibroblasts Platelets

Eosinophils Lymphomas

Apoptosis Apoptosis/no effect/growth

B cells DC Macrophages T cells NK Fibroblasts

Proliferation/class switch/Ig/survival Activation/B7.1/B7.2/ICAM/IL-12, survival Activation/proinflammatory cytokines Costimulation/activation/growth/longevity. Activation/cytotoxicity Activation/proinflammatory cytokines/ICAM/ VCAM ICAM/VCAM/Angiogenesis factors IL-4 (by reverse signal) Activation/cytotoxicity (by reverse signal) RANTES/Inflammation Apoptosis/AP Apoptosis/AICD/Immune privilege Apoptosis/AICD/Immune privilege Apoptosis; chemotherapy response Damage (VLS) Apoptosis Growth/survival Growth/survival Growth/maturation (w/SCF, GM-CSF, IL-4, TNFα) Growth (w/IL-7, SCF) Growth of progenitors; response to IL-2 Differentiation Survival (w/NGF) Growth regulation Growth Growth (especially neural crest origin) MHCI/II, FcR, P1 Kinase, 2 -5 oligoadenylate synthetase Activation, H2O2 Cytotoxicity Altered Ig isotype secreted Inhibition of IL-4-induced functions Differentiation/cytotoxicity (w/IL-2) Growth/cytokine production ↓ Growth/cytokine production ↓ Differentiation ↓ Growth/angiogenesis ↓ Growth; increased differentiation ↑ MHC expression; ↓ NK sensitivity Differentiation Colitis; progression to colon cancer Development Growth/activation Implantation/trophoblast differentiation Survival/↓ maturation ↓ Growth↑ differentiation, ACTH secretion Differentiation/↓ growth Differentiation/↓ growth Growth (breast, colon, renal, prostate) ↓ Differentiation Proliferation, IL-2/IFNγ/IL-4/TNFα Costimulation for AP to T cells T cell help Proliferation Apoptosis

T cells B cells NK cells Some tumors DC (Immature) Hematopoietic Nonhematopoietic

Th1 CD8 T cells NK

Endothelium CD4 CD8 Platelets Some tumors T cells B cells Some tumors Endothelium Some tumors Hematopoietic SC Progenitor cells DC B cells NK/LAK Osteoclasts Neurons Neuron SC Some leukemias Some tumors Cells in general Macrophages NK B cells CD8 T cells Th1 Th2 Th17 Endothelium Tumor cells

LIF

Fibroblasts Endothelium Macrophages Epithelium, thymus Synoviocytes

LIGHT

T cells Granulocytes Monocytes DC (immature)

T cells Colon Mammary Osteoclasts Endometrium Neurons Corticotropes Myeloid leukemia Glioma Some carcinomas Embryonic SC T cells DC NK Hepatocytes HVEM+ LTβR+

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162

Cytokines

Table 1. (continued) Cytokine

Cell source

Lymphotoxin α (TNFβ)

Th1 CD8 T cells B cells (early) NK Astrocytes T cells B cells NK cells DC (Immature) LN cells Spleen cells

Lymphotoxin β

Novel Neurotrophin Oncostatin M

T cells Mono Neutrophils Certain tumors

Osteopontin

T cells Mono/Macro Osteoblasts Endothelium Some epithelial Brain Certain tumors

OX40L

DC B cell Endothelium T cells

RANKL

T cells B cells DC Stroma, BM

SCF

Stroma, BM Fibroblasts Liver Spleen Certain tumors

TNFα

Mono/macro T cells (active) NK Endothelial Mast cells Neutrophils Keratinocytes Adipocytes Pancreatic β Osteoblasts Astrocytes Neurons Adrenal cells

Cells influenced

Effectb

Some tumors B/T/DC etc. Endothelial NK T cells Osteoblasts Osteoclasts Lymphoid cells T cells B cells

Apoptosis Lymphoid organogenesis VCAM/ICAM Growth/development Growth/activation Growth/activation Lymphoid organogenesis Survival Survival

B cells Macrophages Neurons LN cells Fibroblasts Osteoclasts Vascular sm muscle Synoviocytes Some gliomas Sarcoma (Kaposi) Th DC Osteoclasts Osteoblasts Mono/macro Endothelial Epithelium, kidney Melanocytes CD4 T cells DC B cell Endothelial T cells DC T, B, DC etc. Preosteoclasts Mono B cells Stem cells Progenitor cells Mast cells γδ T cells Tumors

Growth/IgM/IgE/IgG Growth Survival/development T cell development/migration Growth/wound repair/collagen/fibrosis Growth/activation Growth/wound repair Inflammation regulation (+/−) Differentiation/↓ growth Growth Activation (favors Th1) Activation (favors Th1) Binding/recruit/remodel/↓Hydroxyapatite deposit Binding/remodeling/↓Hydroxyapatite deposit Chemotaxis/IL-1, TNF, IL8/↓ NO Survival/angiogenesis/↓ NO ↓ NO Survival Costimulation/IL-2/IL-4/IL-5/longevity/memory Maturation/TNF/IL-12/IL-1β/IL-6 (by back signal) Growth/differ (by back signal) Inflammation (by back signal) Leukemia (w/HTLV) Activation/AP/survival Lymphoid organogenesis Maturation; bone development Osteoclast differ (w/GM-CSF, TGFβ) B development; osteoclast development Growth Growth Growth/differentiation/survival/fibrosis Growth Growth (breast, lung, testicular, utererine, cervical, ovarian, melanoma) Growth/development (wIL-1)/IL-2/IFNγ Activation/Ig production NO/cytotoxicity NO/cytotox/IL-12/IL-10/apoptosis Growth/maturation (w/GM-CSF) Degranulation/complement R/ adhesion/ADCC/apoptasis Cytotoxicity Cytotoxicity Growth VCAM, damage, vascular leak syndrome Mitosis Activation

T cells B cells Endothelial Macrophages DC Neutrophils TIL LAK γδT cells Endothelial Osteoblasts Osteoclasts

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Walter M. Lewko and Robert K. Oldham

163

Table 1. (continued) Cytokine

TRAIL

Tweak

a b

Cell source

Cells influenced

Effectb

DC (Immature) Epithelial cells CD4 T cells NK cells Monocytes DC (Immature) Many cell types

Synovial Fibroblasts Tumor cells DC Autoimmune T cells Mast cells Fibroblasts Synoviocytes Bronch epithelium Macrophages Mesangial cells Astrocytes Keratinocytes Liver Endothelium Some tumors

Enzyme secretion Growth Apoptosis; apoptotic bodies for AP AP/T cell activation Apoptosis Apoptosis IL-8, IL-6, RANTES, IP-10 Inflammatory cytokines Inflammatory cytokines IL-6, MCP-1, IL-8, MMP-9 MCP-1, RANTES, IP-10, VCAM IL-8, IL-6, ICAM RANTES Proliferation Proliferation, ICAM, E-selectin, IL-8, angiogenesis Proliferation, survival

This is a partial listing that emphasizes cancer related activities. Activity increased unless otherwise indicated.

suppressors of cytokine signaling (SOCS) which are also referred to as cytokine-inducible suppressor proteins [332, 1882]. They act by binding Jak/STAT components, interfering with their binding to the receptor and their function [333, 492, 1882]. They are tightly regulated and required for normal lymphoid development [1882]. Various cytokines induce SOCS to regulate their own activities (a form of feedback inhibition) [1857] and the activities of other cytokines [428]. For example, SOCS may be increased by immune suppressor cytokines such as IL-10 [256]. Cytokines may act synergistically. For example, IL-18 and IL-12 are synergistic in the way they induce IFN-γ secretion. Synergism may be explained in a number of ways, but frequently, it is due to the upregulation of one cytokine’s receptor or its signaling pathway by the other cytokine [521, 965, 1724, 2214, 2253].

Toll-Like Receptors and the Response to LPS There is a family of receptors, molecularly related to cytokine receptors, called the pattern recognition receptors [520, 1089]. They are better known as the Toll-like receptors (TLR) for their genetic similarity to an important class of Drosophila morphogenesis genes. In addition to their role in fruit fly embryogenesis, Toll also has an antifungal immune function in the adult flies [1089]. These receptors have the important role of sensing the

presence of microbial invaders and initiating immune response. They do this by specifically binding molecules (danger signals) shed from microbes, collectively called pathogen-associated molecular patterns (PAMPs) [1282, 1654]. Bacterial lipopolysaccharide (LPS) (endotoxin) is a well-studied PAMP. LPS is a major cell surface component of Gram-negative bacteria. It binds TLR on macrophages, endothelial cells and dendritic cells where it is a potent activator and stimulator of cytokine production [1983, 2081]. Activation may be very vigorous. LPS induces toxic shock syndrome [1629]. Knockout gene studies in mice have shown that TLR4 recognizes LPS whereas TLR2 is essential for response to several gram positive PAMPs [1586, 1956]. There are at least six human Toll-like receptors. They are integral membrane proteins. The intracellular region is homologous with the signaling domain of the IL-1 receptor family [1303]. TLR signaling activates several cells that are important in immune response [516, 2081]. Immature dendritic cells contain these receptors. Binding LPS induces DC activation, maturation and secretion of cytokines such as IL-12, allowing antigen presentation to occur. Toll receptors are an important bridge between the innate and specific immune response systems.

The Helper T Cell System Mosmann and coworkers proposed a paradigm for the differentiation of CD4+ helper T (Th) cells, cytokines secreted and their roles in regulating of the development

164 of cellular versus humoral immunity [1375, 1376, 1659, 1709, 1937]. This paradigm has been expanded to include additional cells and newly discovered cytokines. The following is a very brief summary of an involved system. A model for the differentiation of Th1 and Th2 helper T cells is shown in Fig. 1. Thp cells are naive precursors to helper cells; IL-2 is the major cytokine they produce. Antigen presentation stimulates their conversion to Th0, the intermediate precursor cells. Th0 cells secrete IL-2, IL-4 and IFN-γ [1314, 1376]. Depending on environmental conditions, Th0 cells differentiate into Th1, Th2, Th17 and regulatory T cells. This process is commonly referred to as polarization; the cells become polarized to secrete specific cytokines and perform certain functions. Th1 cells favor the development of cellular immunity. Intracellular microbes (such as viruses) are eliminated by this path. Cancer cells are also killed by cellular immune response. Th1 responses are important in delayed type hypersensitivity and in certain autoimmune diseases. Th1 cells secrete several cytokines, among them INF-γ, IL-2 and TNF-β, which are collectively referred to as Th1 cytokines. In particular, IFN-γ is considered a key marker for Th1 response. IL-12 and IL-2 favor the Th1 path. IL-4, a product of Th2 cells, generally inhibits the Th1 path. Th2 cells favor the development of humoral immunity. Th2 response also stimulates eosinophil recruitment and macrophage function. This path eliminates extracellular microbes and large parasites. Th2 responses are important in infectious diseases and in allergy. Th2 cells secrete IL-4, IL-5, IL-6, IL-9 and IL-13. IL-4 is the hallmark Th2 cytokine. Mouse Th2 cells secrete IL-10. (In humans, both Th1 and Th2 cells produce IL-10). IL-4 stimulates the Th2 path whereas IFN-γ, a major Th1 cytokine, inhibits it [2, 1746]. In addition to cytokines, there are further influences that fine tune Th1 versus

Figure 1. Differentiation of Th1 and Th2 helper T cells. Antigen presentation (AP) stimulates the conversion of naive precursor Thp cells to immediate precursor Th0 cells. Th1 cells favor the development of cellular immunity and response to intracellular microbes. Th2 cells foster humoral immunity and response to extracellular microbes and parasites. The major positive and negative stimulators of each path are indicated. Cytokines within the boxes are produced by the indicated cells. IL-10 is a Th2 cytokine for mouse cells; in humans, both Th1 and Th2 cells produce IL-10

Cytokines Th2 path selection. (a) Antigen dose; generally, low antigen dose favors the Th1 path, while higher antigen dose favors Th2 cells [798]. (b) Route of antigen administration [1993]. (c) Type of antigen presenting cell [1775]. (d) Type of costimulation: during antigen presentation, B7.2 favors Th2 [565]. Th17 cells induce inflammation. This path responds to extracellular microbes. Th17 cells have important roles in autoimmunity. Th17 cells secrete IL-17A, IL-17F, IL-21, IL-22, IL-6 and TNF-α. TGF-β and IL-6 induce differentiation of Th17 cells; IL-23 maintains and stimulates cytokine production. IL-2 and IL-13 inhibit Th17 cells [55, 869]. Regulatory T cells (Treg) are defined functionally by their suppressive effects on immune response, inflammation and autoimmunity. Tregs are the subject of intense research; they have important roles in the cause and prevention of diseases including cancer. Many types of cells show regulatory character. Here we will mention three major types. Th3 cells secrete the immunosuppressive cytokine TGF-β. Tr1 cells produce immunosuppressive IL-10. Natural Treg cells have the phenotype CD4+ CD25+ Foxp3+. IL-2 and TGF-β induce nTreg cells. The nTreg mechanism of suppression involves cell contact (cell surface receptors) and possibly the secretion of immunosuppressive IL-10 and TGF-β [1714, 2002]. Polarization is not limited to Th cells. Based on the cytokines secreted, NK cells [285], cytotoxic CD8 T cells [251, 1708], macrophages [1313] and dendritic cells [1206, 1596, 1647] may develop type 1, type 2 and regulatory cell profiles. Imbalance in type cells (exaggerated polarization/cytokine secretion) appears to drive development of diseases including autoimmunity (exaggerated Th1 or Th17) [6, 290], allergy (exaggerated Th2) [923, 1101], and cancer (Treg) [1590, 2056].

IL-12 + IL-4 -

Thp

AP

IL-13 IL-17

Th0 IFNγγ -

IL-2

IFNγ IL-2 TNF

Th1

IL-2 IL-4 IFNγ IL-13

IL-4 + OX40 + B7.2 +

Th2

IL-4 IL-5 IL-6 IL-9 IL-10 IL-13

Walter M. Lewko and Robert K. Oldham

Antigen Presentation; Dendritic Cells T-cells and B-cells do not typically respond to antigens directly, rather a family of cells referred to as professional antigen presenting (AP) cells make the presence of an antigen known to these effectors in a complex and highly regulated process referred to as antigen presentation [1159, 1888]. Dendritic cells (DC), macrophages, B cells, Langerhans’ cells, and eosinophils are antigen presenting cells. These cells have the necessary ability to migrate (between tissues and lymphnodes), they contain the appropriate antigen processing machinery (antigen is broken down to small peptides in the process) and surface binding proteins (including MHC to display antigen peptides) to help insure that an effective and safe immune response occurs. AP cells also have the necessary costimulatory molecules such as CD80 (B7.1) and CD86 (B7.2). Among the AP cells, dendritic cells are particularly good at inducing specific T cells with anticancer and antiviral activity. Different subpopulations of AP cells may favor different types of immune response (e.g. Th1 vs. Th2) [1595, 1596]. Cytokines control the proliferation and development of AP cells. AP cells are also a source of cytokines which influence innate as well acquired immunity. Abnormalities in antigen presentation may result in autoimmune disease or, in the opposite extreme, a failure to mount any immune response at all.

Tumor Necrosis Factor Family of Cytokines Tumor necrosis factor (TNF) was originally discovered as a component of blood, produced by host cells in response to infection and bacterial products such as LPS [150, 151, 152, 250, 1471]. A number of related factors turned up [6, 150, 657, 685]. Now it is known that TNF (now called TNF-α) is the founding member of a family of at least 19 cytokines and related virus proteins. The members of the TNF family are involved in cell growth, differentiation, inflammation, wound repair, costimulation of immune response and the regulation of cell death. There is homology in amino acid sequence among most TNF family members, though it is not particularly high [685]. Nerve growth factor (NGF) is not homologous but its receptor is related to the TNF receptor. TNF-β and NGF are soluble cytokines. TNF-α and TWEAK are mostly soluble, partly membrane bound. The remaining TNF family members are membrane-

165 bound proteins [685]. The active, receptor binding form of most TNF family members is a trimer. These trimers induce receptor clustering and signal transduction [75]. Membrane-bound TNF family members act by direct contact between the cytokine bearing cells and receptor bearing target cells. Certain membrane-bound cytokines, upon engaging receptor, may signal back (reverse signaling) inducing effects in the parental cell; where reverse signaling occurs, both cells are, at the same time, targets and effectors. TNF family members bind receptors that are related molecularly. They are transmembrane glycoproteins. Some TNF family receptors have soluble/shed forms that are the result of proteolytic cleavage or alternative mRNA splicing. The amino acid identity between the human receptors is in the range of 25–35%. The extracellular regions characteristically have three to six copies of cysteine-rich pseudo repeats, each containing six cysteines in a segment of about 40 amino acids. The cytoplasmic region of certain receptors contains a conserved “death domain,” involved in apoptosis (e.g. TNFRI, Fas, DR3, DR4, DR5). There are intracellular receptor associated proteins such as TRAF (TNFRassociated factors), TRADD (TNFR1-associated death domain protein), and RIP (receptor interacting protein) which bind and communicate between the TNF receptor family members to regulate apoptosis and other cytokine responses [2137]. Members of the TNFR family share these intracellular signaling proteins to activate similar transduction pathways. Certain members of the TNF receptor family are found in many types of cells (TNFRI, TNFRII, Fas, Fn14) whereas others are restricted to hematopoietic cells (CD27, CD30, CD40, HVEM, OX40, 4-1BB) or specific tissues (nerve growth factor receptor) [75, 2137].

Interleukin-1 Inflammation, Immune Regulation, Hematopoiesis, and Wound Repair IL-1 was originally called lymphocyte activating factor for its effects on mitogen-treated thymus cells [617]. It has also been called leukocyte pyrogen and endogenous pyrogen, for its fever inducing effects [436, 1401]. Monocytes, macrophages, keratinocytes, T cells, B cells, NK cells, eosinophils, dendritic cells, fibroblasts, epithelial cells, endothelial cells, neurons, glial cells and astrocytes produce IL-1 [551, 1170, 1735, 1972, 2140]. IL-1 is involved in inflammation, hematopoiesis, immune regulation, wound healing and metabolic regulation.

166 It is responsible for, or at least involved in, a number of inflammatory diseases. IL-1 also appears to influence cancer growth and metastasis. The IL-1 gene family has three main members: IL-1α, IL-1β and IL-1 receptor antagonist (IL-1Ra). IL-1α and IL-1β are synthesized by separate, distantly related genes [1225, 1500]. The proteins have 26% homology. Both cytokines are synthesized as 31,000 kDa pro-IL-1 molecules. They lack the usual signal peptides characteristic of most secreted proteins. Pro-IL-1α and the processed 17,000 mw product are both active cytokines. Newly synthesized IL-1α accumulates within the cytosol. Not much IL-1α is secreted. Intracellular IL-1 may serve an autocrine function in endothelial cells, keratinocytes and fibroblasts. Pro-IL-1α is found on the cell surface, associated with lectin; bound IL-1α has cytokine activity [201]. Pro-IL-1β is inactive. It is processed to a 17,000 mw form, which is functional and the major secreted form of IL-1. Processing is carried out by the protease, IL-1β converting enzyme (ICE, caspase 1) [68, 160, 272, 299, 1992]. ICE is involved in the processing of other cytokines (IL-16 and IL-18), but not IL-1α, which is cleaved by another protease. mRNA synthesis and posttranslational processing control the production of IL-1β. Bacterial products (LPS), TNF-α, IL-1 itself, and the immunostimulatory drug OK-432 stimulate IL-1β secretion by blood monocytes and other cells [782, 971, 435, 437]. Extracellular ATP triggers activation of ICE and a burst of IL-1β secretion [529, 528, 1557]. ATP is a signal that is released by activated lymphocytes and from damaged cells [782, 1925]. IL-1Ra is an inhibitor of IL-1. It competes with IL-1 for its receptor [454, 482, 658, 723]. Secretion of Il-1Ra generally follows IL-1 production. In patients and in cultures of human PBMC, IL-1Ra secretion is stimulated by IFN-α, IL-4 and to a lesser extent by IFN-γ [1997]. Patients treated with IL-1 had increased serum IL-1Ra and with time the antagonist remained measurable after IL-1 was no longer detected [1007]. There are two receptors for IL-1, IL-1RI that is responsible for activity and IL-1RII that is a regulatory decoy receptor. They are distinct membrane bound proteins, produced by separate genes [310, 1829]. The active signaling complex contains several components. IL-1 binds the IL-1RI. IL-1 receptor accessory protein (IL-1RAcP) binds and enhances affinity for IL-1; it is required for signal transduction [663, 1008, 2144]. MyD88 is an adapter protein that joins the complex [2145]. IL-1R associated kinase (IL-1RAK) then binds; the kinase is phosphorylated and activated [354].

Cytokines Then TNFR-associated factor-6 (TRAF-6) binds. Now this multicomponent receptor complex is functional and it induces the activation of transcription factors, in particular NF-κB, which translocates to the nucleus, binds DNA response elements and induces the production of mRNA’s responsible for IL-1 effects [243, 354, 1008]. The second receptor, IL-1RII binds IL-1 but cannot signal due to the lack of a binding site for MyD88. There is a soluble form of RII, which is found in tissue fluids; it is increased during inflammation [628, 1593, 1938]. Soluble receptor II may be generated by proteolytic cleavage [1506] or by alternative mRNA splicing [1136]. The cellular form of RII binds IL-1α, IL-1β and IL-1Ra; soluble RII binds IL-1α and IL-1β but not IL-1Ra [65]. RII appears to function as a competitive decoy for IL-1 [334, 1446, 1621] and IL-1RAcP [1056] available to bind IL-1RI. The biological properties of IL-1 are far reaching and complex; it is often referred to as a two edge sword. On one hand IL-1 functions in hematopoiesis, the regulation of metabolism, wound healing and the control of infection. On the other hand, IL-1 appears to be involved in the debilitating effects of several inflammatory diseases and septicemia [846, 1482]. IL-1 has a remarkable effect on hematopoiesis. In early progenitor cells, IL-1 is survival factor; it protects stem cells from environmental insults, cytotoxic chemicals, radiation and the like. IL-1 also primes progenitor cells for outgrowth, by placing them in the G0-G1 stage. IL-1 by itself does not induce outgrowth [191, 530, 1442, 1501, 1762]. IL-1 increases IL-3, G-CSF, M-CSF and GM-CSF; together with these cytokines, IL-1 synergistically stimulates the expansion of specific cell lineages [100, 1329, 1349, 2306, 2309]. Patient treatment with moderate doses of IL-1 caused elevated neutrophil and platelet levels [1842]. Interestingly, prolonged treatment and higher doses caused an apparent decrease in neutrophils, platelets and erythrocytes. There are two possible explanations for this decrease: First, IL-1 induces TNF and TNF has a depressive effect on hematopoiesis [594, 845, 898]. Second, IL-1 induces adhesion molecules on capillary endothelial cells. Studies in culture showed that endothelial cells with elevated adhesion molecules bind a number of different types of cells; these cells may be sequestered in the capillary bed resulting in an apparent decrease in cell numbers [406, 1380, 1681]. IL-1 stimulates dendritic cells. It acts together with GM-CSF to promote maturation [744]. Dendritic cells have an important function as antigen presenting cells in the development of lymphocytes. T cells and B cells synthesize IL-1 and have the IL-1 receptor [1163, 1504].

Walter M. Lewko and Robert K. Oldham IL-1 stimulated T cells, induced IL-2 production, IL-2 receptors and cytokine production; IL-2R induction appeared to be a key step [940, 1039, 1827]. Immature T cells appeared to require IL-1 in order to synthesize IL-2; IL-6 acts synergistically with IL-1 in T-cell maturation and the production of IL-2 [1686]. In more mature T cells, IL-1 favored Th2 cells. It stimulates the proliferation of Th2 cells and the production of IL-4, IL-5 and IL-6. IL-1 had no apparent effect on Th1 cells. IL-1 also acts as a chemoattractant for T cells and it stimulates production of IL-8 [822, 1039, 1335]. In B cells, IL-1 acts together with B cell growth and differentiation factors IL-4, IL-6 and IL-2 to stimulate growth and the production of immunoglobulin. IL-1 increases the levels of glucocorticoids. Glucocorticoids stimulate B cell IL-1 receptors. Glucocorticoids and IL-1 together stimulate antibody production [14]. In natural killer cells, IL-1 and IL-12 together stimulated the secretion of IFN-γ in response to infection [824]. The activities of IL-1, TNF and IL-6 are interrelated. IL-1 and TNF have similar biological properties and their combined effects are usually synergistic [1216, 2093]. IL-1 induces TNF production [1077, 2173] and TNF induces IL-1 production [197, 435]. The receptors for IL-1 and TNF share a common accessory protein and signaling path. As part of a regulatory loop, IL-1 tends to depress TNF receptors [788, 2093]. In mice IL-1 induced IL-6 production. Blocking IL-1 by IL-1Ra depressed IL-6 levels and inflammation [616, 1090]. TNF stimulates both IL-1β and IL-6 [550]. IL-6 regulates IL-1 activity by increasing IL-1Ra levels [1749] and by decreasing IL-1 formation [1742]. IL-1 stimulated mast cell production of IL-3, IL-4, IL-5, IL-6, IL-9 and TNF. Thus, IL-1 may enhance Th2related events including humoral response and allergic inflammation. IL-1 may also influence the natural response of mast cells to parasites, viral infection [820] and cancer. The nervous system has receptors for IL-1 and brain cells are capable of IL-1 production [196, 1072]. IL-1β is secreted by microglial cells in response to brain trauma. IL-1 induces production of nerve growth factor [1873] and ciliary neurotrophic factor [742], which are involved in nerve cell survival and wound repair. In this way IL-1 released during brain inflammation may have a beneficial effect on CNS regeneration [742]. IL-1 treated patients often suffer from sleepiness, fever and lack of interest in eating. IL-1 activity in the nervous system appears to be involved. IL-1 injections in animals induced slow wave sleep and depressed rapid eye movement [1815]. IL-1 induces fever. There are several other cytokines capable of doing so including TNF, IL-6

167 and IFN-α. IL-1 increases IL-6 levels; blocking IL-1 activity lowered IL-6 and depressed fever [327, 616, 2277]. IL-1 also induces anorexia in animals. The mechanism appears to involve increased brain cyclooxygenase and prostaglandin production [733]. IL-1 has several effects on metabolism. It interacts closely with a number of hormones, in particular the glucocorticoids and insulin. IL-1 influences the brainpituitary-adrenal axis. IL-1, injected i.v., increased the release of corticotropin releasing hormone and ACTH as well as endorphins, vasopressin and somatostatin. Glucocorticoid levels increased in response to injected IL-1 and during stress and trauma [1639, 1812]. Glucocorticoids influence metabolism throughout the body; hydrocortisone feeds back on the production of IL-1 [144, 148, 410, 1080]. In this way glucocorticoids appear to regulate inflammatory response. IL-1 promotes bone resorption and cartilage degradation [424]. It induces the release of collagenase, phospholipase A and prostaglandin E2 from synovial cells [154, 1326, 2304]. IL-1 altered the production of several liver proteins; fibrinogen, clotting factors, metallothionein and complement were increased. Albumin, transferrin and lipoprotein lipase were decreased. Negative nitrogen balance in muscle protein is associated with inflammatory diseases. IL-1, IL-6, TNF and insulin are involved. Pancreatic β islet cells have IL-1 receptors [706]. Both IL-1 and IL-6 stimulate production of insulin [2182]. IL-1 and IL-6 together with TNF have insulin-like effects on metabolism. In rats treated with endotoxin, for example, IL-1 blockade using IL-1Ra spared muscle protein [2272]. IL-1 stimulates the growth of several different types of cells including keratinocytes, smooth muscle cells, glial cells, mesangial cells and fibroblasts [996, 1126]. In this way, IL-1 has a role in wound repair and angiogenesis. In fibroblasts, IL-1 increased proliferation [1744] and the uptake of glucose [156]. IL-1 also stimulates collagen production by fibroblasts and epithelial cells. Tissue fibrosis is associated with inflammatory disease; it appears to be related to paracrine and autocrine IL-1 secretion. In cultured fibrotic kidney cells, IL-1 blockade using IL-1Ra depressed fibroblast proliferation [1150]. There are interesting relationships between IL-1 and endotoxin. IL-1 is one of several cytokines (IL-2, IL-15, IFN-γ and TNF-α), which stimulate Toll-like receptor (TLR) levels in macrophages [1247]. TLRs bind microbial products and stimulate innate immunity and inflammatory responses to pathogens. Endotoxin and IL-1 have similar biological properties including the induction of IL-1 and TNF levels. Interestingly, TLR and the

168 IL-1R have homologous intracellular signaling domains. Activation of these receptors brings about similar proinflammatory responses [164]. IL-1 is involved in several inflammatory diseases. In the intestine, IL-1 and TNF are produced by epithelial cells and appear to prevent microbial invasion. In inflammatory bowel disease, these cytokines were increased. Somatostatin inhibited basal IL-1β production and prevented induction by TNF and bacteria. These results suggested that somatostatin regulates IL-1 and may have a role in prevention of bowel inflammation [314]. In patients with rheumatoid arthritis, synovial tissues produce IL-1 [538] and the levels of IL-1 are elevated in plasma. IL-1 is a chemotactic factor for neutrophils [1732]; IL-1 induces nitric oxide synthetase and the production of nitric oxide, which mediates many inflammatory processes [504]. IL-1 also induces cyclooxygenase II and the production of prostaglandins, which have inflammatory effects [1217, 1360]. IL-1 directly stimulated levels of the proteolytic enzymes involved in joint destruction, collagenase, tissue plasminogen activator, and stromelysin [424, 1403]. In chondrocytes, chronic IL-1 inhibited proliferation and the production of collagen and proteoglycan [1156, 1885, 1968]. IL-1 and tumor necrosis factor act mutually to stimulate joint inflammation [197, 2173]. Clinical and animal studies have shown that therapies blocking TNF-α activity were beneficial. In mice with collagen-induced arthritis, anti-IL-1 antibodies and antibodies to the IL-1 receptor [607, 901, 2055, 2173] reduced the severity of the disease. Blocking both IL-1 and TNF-α had an additive effect [2173]. IL-1Ra and IL-1 RII are being studied clinically to see if they are of benefit in treating inflammatory diseases. In patients with rheumatoid arthritis, rhIL-1Ra reduced the progression of joint erosion [199]. Unfortunately, rhIL1Ra did not significantly increase survival in patients with severe sepsis [1499]. In patients with arthritis, exercise has a beneficial effect on joints. A biochemical rationale was provided in an interestingly cell culture model for physical therapy. Cyclic tensile strain on cultured chondrocytes decreased the effects of IL-1β by interfering with IL-1 receptor signaling. Markers for inflammation were decreased and the extracellular matrix was restored [2217]. IFN is used to treat viral hepatitis. However, 60% of patients do not respond well. IL-1 may be a reason for this lack of response. IL-1β appears to depress the antiviral activity of type I interferon [1994]. When mice were injected with IL-1β, liver cell antiviral response was depressed. IL-1 appeared to interfere with IFN signaling at the level of STAT 1 phosphorylation. It is

Cytokines possible that IL-1 blockade may be a way to intensify IFN response in the treatment of viral disease [1994] and possibly cancer. IL-1 is of interest in the treatment of cancer [1838]. In culture, IL-1 has a direct inhibitory effect on the growth of several types of human cancer cell lines [378, 959, 1498]. In animal studies, intratumoral treatment with IL-1 induced regression of injected tumors but not distant metastases [1418]. Fibrosarcoma cells expressing cell surface IL-1α, implanted in mice showed decreased growth and induced protective immunity [1858]. While preclinical studies showed promise, little or no benefit has been observed using IL-1 in patients with melanoma [1879] or renal cancer [1625]. The combination of IL-1 with IL-2 was explored in clinical trials. Increases in NK and LAK activity were observed and there were some responses in patients with colon cancer, melanoma and renal cancer [2026]. IL-1 has also been tested in combination with chemotherapy to enhance drug effectiveness and decrease blood cell suppression. IL-1 decreases the activity of drug metabolizing cytochrome P-450 [619]. In cultured cells, IL-1 had synergistic effects with certain forms of chemotherapy [1417, 2046]. Similar synergistic effects of IL-1 and chemotherapy were seen in animal tumor models [897, 1051]. Thus far the results in clinical trials have been rather disappointing. In ovarian cancer patients treated with carboplatin and in osteosarcoma patients treated with etoposide, the addition of IL-1 resulted in very modest increases in antitumor response [2072, 2199]. In preclinical studies, IL-1 offered some protection to progenitor hematopoietic cells undergoing irradiation or treatment with cytotoxic drugs [191, 1441, 1495, 1762]. When cancer patients were given IL-1 in phase 1 clinical trials, granulocytes and platelets were stimulated showing that IL-1 could be beneficial for blood cell recovery [357, 1842]. In children on chemotherapy (Ifosfamide-Carboplatin-Etoposide), unfortunately, treatment with IL-1α produced no significant hematoprotective benefit [576]. IL-1 produces significant though manageable side effects. Low dose IL-1 causes headache, myalgia, arthralgia, sleepiness and anorexia. Higher doses cause a rapid loss in blood pressure. These symptoms mimic septic shock. Highest doses produced grade IV hypotension and neurological signs [1842]. In summary, IL-1 stimulates T cells, B cells and hematopoiesis. Undoubtedly, it has a role in the development of immunity. But at present its toxicity and modest antitumor effects preclude approval for use in cancer patients. There is evidence that IL-1 stimulates the growth of certain types of cancer. In patients with chronic myelogenous leukemia, Il-1β levels were elevated and blocking

Walter M. Lewko and Robert K. Oldham IL-1 depressed cancer cell growth; IL-1 was determined to be a negative prognostic factor [2148]. IL-1 is an autocrine growth stimulator for certain human gastric cancers [864]. In capillary endothelial cells, IL-1 increased cell surface adhesion molecules and the binding of tumor cells [406]. Pretreatment of mice with IL-1 increased the metastasis of B16 [2074] and human melanoma cells [620]. When the mice were treated with IL-1Ra, there were fewer metastases, smaller metastases and the animals lived longer [620]. Perhaps therapies aimed at blocking IL-1 may be beneficial in controlling the growth and spread of certain cancers.

Interleukin-2 Growth And Activation of T, B and NK Cells; Activation-Induced Cell Death; Elimination of Self-Reactive T Cells IL-2 was originally described as a factor in the conditioned medium of mixed lymphocyte cultures that stimulated T cell growth [643, 936]. The following is a brief review of this very well studied cytokine. The early work is covered in greater depth in prior editions of this book [1110]. IL-2 is a member of the helical cytokine family, which includes IL-2, IL-4, IL-7 and IL-15 [1963]. IL-2 is produced mainly by T cells. Activated cytotoxic T cells, Th0 and Th1 cells secrete IL-2 but Th2 cells do not [1040, 1798]. Initial studies used natural IL-2 prepared from lymphoid cells [561, 1305, 1649, 2141]. It is a 133 amino acid glycoprotein with variable molecular weight due to the carbohydrate. The gene for IL-2 has since been cloned [1666, 1963]. The recombinant protein made in bacteria lacks carbohydrate. Certain forms of recombinant IL-2 have an amino acid change to facilitate production [210]. Recombinant IL-2 and the natural product have similar biological activities and stabilities [1663]. Both forms of IL-2 have relatively short half-lives of 1–2 h following intravenous injection [696, 1158, 1160] and 4 h following bolus sc injection [696]. IL-2 loss is mainly by renal clearance rather than hepatic metabolism or target cell binding [448]. Intraperitoneal injection has been used to treat abdominal malignancies. Injection i.p. produces sustained, locally high levels of IL-2; systemic toxicity is lower. Sometimes, repeat i.p. injections induced fibrosis, likely due to the release of secondary cytokines such as PDGF or TNF-α [145, 1091, 2044]. IL-2 has also been delivered by inhalation [818] or and by mini-osmotic pumps [1458].

169 The IL-2 receptor is composed of three subunits: IL2Rα, IL2Rβ and IL2Rγ (also called γC, common γ subunit). The α subunit has high affinity for IL-2. The β and γ chains are involved in signaling [968, 1002, 1097, 1823]. The three subunit complex exhibits highest affinity for IL-2 (Kd 10−10 M). In NK cells, the IL-2R is in the form of a βγ dimer; this two-subunit receptor has moderate affinity (Kd 10−9 M) and requires higher IL-2 concentrations to produce effects [734, 945, 1024, 1508, 1823]. Janus kinase activity is associated with the γ chain. The receptor complex is activated by phosphorylation. A major signal path involves the STAT transcription factors (signal transducer and activator of transcription), which bind to the activated receptor complex and are in turn activated by phosphorylation. The activated STATs translocate to the nucleus, bind an array of gene promoters and initiate transcription associated with IL-2 response. Immune suppressive drugs FK406 and Cyclosporin A act at the level of transcription factors, which are activated by IL-2 and other cytokines [491, 544, 656, 1262, 1263]. A peptide has been produced which comprises amino acids 1–30 of human IL-2. It forms a tetrameric structure that binds IL-2R β dimers. The peptide is an IL-2 agonist: it induces activation of CD8+ T cells and lymphokine-activated killer (LAK). Interestingly, it acts synergistically with IL-4, IL-9, IL-15 and with IL-2 itself. This peptide may have therapeutic potential [476, 477]. IL-2 was originally discovered by its capacity to stimulate proliferation in T cells. T cells produce IL-2 and it stimulates T cell growth. A defect in IL-2 production appears responsible for a type of severe combined immunodeficiency disease [2135]. IL-2−/− knockout mice have been developed which lack IL-2. In initial studies, proliferation response was weak in Con A or anti-CD3 Mab activated mononuclear leukocytes; otherwise the mice appeared rather normal [1754]. Further studies showed IL-2 −/− mice had increased autoimmune disease, ulcerative colitis, inflammatory bowel disease, anemia, progressive loss of B cells and altered bone marrow cell profiles [1185, 1710, 1711]. The animals suffered from uncontrolled T cell proliferation, apparently due to a defect in Fas-induced apoptosis [990, 1092]. Related observations have been made in mice lacking the IL-2 receptor α chain [2168] and β chain [1933]. In germfree IL-2 −/− mice, autoimmune disease developed but not colitis, which appeared to require enteric microbial antigen [341]. Interestingly, allograft rejection still occurred, not only in IL-2 −/− knockout mice [1886] but also in IL-2 −/− IL-4 −/− double knockout mice [1119]. These results suggested IL-2 is not an

170 absolute requirement for rejection. Other cytokines such as IL-7, IL-12, IL-15 or IL-21 may provide redundancy in certain IL-2 functions [1886]. Nonetheless, IL-2 appears necessary for the normal regulation of T-cell growth and involution. In particular, IL-2 has a role in tolerance by inducing T cell suicide [1706, 1933, 2168], in activation-induced T cell death [1092, 1626, 2062] and it is involved in the inhibition of T cell memory maintenance [1026]. Further, IL-2 induces regulatory T cells (Treg: Foxp3+ CD4+ CD25+), which are antigen specific cells that regulate immune response. In particular, they suppress effector T cells, which are self-antigen specific and autoimmunogenic. In many cancer patients, these regulatory T cells are enriched in the tumors and blood. Since many tumor antigens are essentially overexpressed self antigens, Treg cells may have a role in tumor immune escape and poor response to cancer biotherapy [165, 1309, 1806]. IL-2 stimulates cytotoxicity in NK cells [734, 1386, 1387], thymocytes [1800] and in cytotoxic T lymphocytes (CTL) [1799]. Peripheral blood lymphocytes treated with IL-2 generate LAK cells [176, 444]. Cytotoxic NK cells make up the major active component of LAK [1507, 1567, 1995, 2133]. NK cells lyse tumor cells by membrane channel forming perforin, granzymes and receptor mediated cell death by apoptosis. IL-2 also enhances antibody dependent cellular cytotoxicity (ADCC) mediated by lymphocytes against tumor cells [1810]. IL-2 stimulates B cell growth, differentiation and the production of Ig. IL-2 acts alone and together with other B cell stimulating cytokines such as IL-6 [886, 1419, 1872]. IL-2 also activates production of several cytokines including the interferons, tumor necrosis factor α (cachectin) and tumor necrosis factor β (lymphotoxin) [478, 611, 1016, 1599]. In this way IL-2 may induces a cascade of effects influencing immune response. Many tumor cell lines express the IL-2 receptor. Some of these cells respond directly to added IL-2 with decreased growth. Interestingly, IL-2 sometimes down regulates surface molecules such as ICAM and MHC, which are involved in antitumor immune response [2234]. Denileukin diftitox (ONTAK) is a recombinant cytotoxic chimeric protein composes of active domains of diphtheria toxin fused with the receptor-binding domain of IL-2. It binds to cells expressing the IL-2R, is internalized and kills susceptible cells. ONTAK has been approved for use in cutaneous T cell lymphoma and is being tested in other hematologic malignancies. It is also being studied as a means of controlling regulatory T cells in cancer patients [86, 380].

Cytokines IL-2 is used to treat renal cell carcinoma and melanoma patients [190, 1674, 1675, 2146]. Its effects generally appear due to T cells and NK cells. Response rates were related to IL-2 dose. Most patients tolerate IL-2, but in addition to the usual flu-like discomforts associated with cytokine therapy, high dose IL-2 induced hypotension and vascular leak syndrome (VLS) which may be life threatening [106, 1676]. VLS is due to capillary endothelial cell damage. Possible sources of damage include cytotoxic lymphocytes, neutrophils [717], complement [1982], TNF [458, 1607] and inflammatory factors such as histamine, serotonin and bradykinin [1211]. Toxicity may be due to IL-2-induced IL-1. In a phase I trial, cancer patients were treated with soluble IL-1 receptor, an antagonist, in an attempt to lessen IL-2 toxicity [1266]. Unfortunately, soluble IL-1R provided no benefit. The evidence for cytotoxic lymphocyte involvement is rather strong. Immune suppressive agents including IL-10 (discussed later) [1676, 1115] and inhibitors of immune cell outgrowth [1606] depress VLS. IL-2-induced LAK cells adhere to endothelial cells and kill them [375]. Blocking the binding of leukocytes to capillary endothelium [1481] and depletion of NK cells [1547] ameliorate VLS. Finally, IL-2-induced endothelial cell damage is significantly lower in mice, which are defective for perforin or Fas ligand (discussed later), both involved in lymphocyte-mediated cytotoxicity [1612]. IL-2 has been used ex vivo to stimulate patient peripheral blood lymphocytes to form LAK cells [1668, 1669]. Lymphocytes are collected by cytopheresis and cultured with IL-2 for several days. IL-2-treated lymphocytes are then reinfused back into the patient. Oldham and coworkers later developed a protocol, which only required a very short bedside incubation of lymphocytes with IL-2 prior to reinfusion [794]. LAK cells are preferentially cytotoxic to neoplastic cells, though lysis of normal capillary cells has been observed [76, 375, 1512, 1855]. LAK cells act in a non-MHC-restricted manner [673, 1677]. In fact, the presence of strong MHC on the tumor cell surface inhibits LAK. Initial reports indicated that LAK appeared effective in metastatic renal cancer and melanoma with response rates of 15–25% [474, 541, 1667]. But subsequent reports for patients with renal cancer questioned whether the cells provided any added benefit to the IL-2 given systemically at the time of LAK therapy [1669, 1674]. It appears that cancer patients treated with IL-2 develop LAK-like cells in vivo [1670]. IL-2 has also been used ex vivo to stimulate outgrowth of tumor derived T cells. These expanded T cells are called tumor infiltrating lymphocytes (TIL) [1018, 1671, 2011]. They are also referred to as tumor derived

Walter M. Lewko and Robert K. Oldham activated T cells (TDAC) [1108, 1109, 1207, 1208, 1493, 1494]. In the original work in mice, Rosenberg showed these cells were 100x more potent than LAK in anticancer activity and TIL were effective against certain LAK-resistant tumor cells [1672]. TIL are part of the adaptive immune response system; they are cytotoxic in an MHC restricted manner; antigen presenting cells were involved in their development. TIL are grown to greater than 1010 cells and then reinfused back into the patient. Response rates in melanoma and renal cancer are on the order of 10–25% with some durable remissions. Combinations with other cytokines are still being tested for increased treatment efficacy. Biochemotherapy, the combination of traditional chemotherapy with biological response modifiers, produces an unexpectedly high response in melanoma patients. The reason for this is not understood. Response rate was correlated with increased serum IL-6 and IL-10, both Th2 cytokines, in patients receiving cisplatin, vinblastine and dacarbazine followed by IL-2 and IFN-α-2b [674]. However, in spite of response rates exceeding 50%, it is not clear that biochemotherapy adds any long term survival benefit over IL-2 alone. IL-2 is an effective adjuvant, stimulating immune responses to tumor cells in mice. IL-2 is being tested as a drug and as a product of genetically engineered cells, in tumor cell and dendritic cell cancer vaccines [30, 587, 588, 1106]. In addition to cancer therapy, IL-2 has been used to treat patients infected with human immunodeficiency virus. CD4+ cell levels were increased with no increase in plasma virus [1017]. IL-2 has also been used ex vivo to generate antiviral T cells, which were reinfused into patients [200, 2095].

Interleukin-3 Hematopoietic Cytokine IL-3 is a 28,000 mw protein. It is produced mainly by activated T cells [323, 324] but also by mast cells, thymic epithelium, keratinocytes, neurons, monocytes, neutrophils and eosinophils [835, 857]. In the past, IL-3 has been referred to as CFU stimulating activity, mast cell growth factor, Thy1 inducing factor, multicolony stimulating factor, P cell stimulating factor, and hematopoietic growth factor [1327]. The gene for human IL-3 is on chromosome 5 in close linkage with IL-4, IL-5, IL-9 and IL-13 [2227, 2248]. IL-3 is released during immune response and serves as a bridge between the immune and hematopoietic systems.

171 The receptor for IL-3 has two subunits. The IL-3Rα binds IL-3 [977]. It has homology with the GM-CSFRα [1010]. The β subunit is shared with receptors for IL-5 and GM-CSF [726]. The β subunit does not bind cytokine but as part of each receptor complex it enhances affinity for the cytokine and it is responsible for signaling (Jak2-STAT5) [1315]. IL-3, IL-5 and GM-CSF generate similar signals [697, 1323]. There is also evidence that the IL-3Rα chain signals [1314] adding complexity and specificity to the response. IL-3 stimulates the growth of most early multipotential progenitor cells and early committed precursors. IL-3 also stimulates macrophages, granulocytes and mast cells through their most mature forms. IL-3 is a major developmental cytokine for basophils [1061, 1230, 2033, 1296]. Only the later stages of the erythroid [857] and megakaryocytic [2171] lines are no longer sensitive. IL-3 thus increases the production of macrophages, granulocytes, erythrocytes, and megakaryocytes. Effects on early erythroid and megakaryocyte growth are rather distinctive to IL-3. IL-3 serves as a primer and costimulator while other cytokines act later to induce differentiation. It stimulates both cell division and cell survival [407, 835, 2229, 924, 1919]. It acts synergistically with several cytokines including IL-6 [843], IL-11 [1405], G-CSF [842], thrombopoietin [1027], stem cell factor [2031] and Flt3 ligand [1791]. In murine cell cultures, IL-3 tended to depress the B lymphoid potential of lymphohemopoietic progenitors [767]. In human cells, IL-3 increased the production of B cell progenitors from uncommitted CD34+ CD38− cells [353]. Osteoclast development in hematopoietic tissue is influenced by colony stimulating factors. IL-3 has been reported to be stimulatory and regulatory. In RANK ligand mobilized osteoclast precursors, IL-3 inhibited osteoclast differentiation, diverting the precursors to macrophages [956]. Natural cytotoxic (NC) cells [1524, 1912] are mast cell-like morphologically and they participate in immune reactions such as tumor rejection and graft versus host disease. They differ from standard NK cells in origin, growth and kinetics of target cell lysis. Natural cytotoxic cells are IL-3 dependent. Unlike NK cells, they do not respond to IL-2 [875–876]. Natural cytotoxic cells appear to induce lysis by the release of TNF [876]. IL-3 has been studied extensively for possible clinical use [479]. It has been tested in combination with other cytokines such as GM-CSF, M-CSF, and EPO to stimulate hematopoietic recovery of chemotherapy patients and bone marrow transplant recipients. IL-3 increased platelets, leukocytes and reticulocytes. Side effects due

172 to IL-3 (fever, headache, myalgia) were generally mild to moderate and manageable [589]. There were some benefits but not sufficient to warrant becoming a part of standard therapy. PIXY321 is a GM-CSF:IL-3 fusion product, which has been tested in cancer patients with similar results and toxicities [899]. Unfortunately, most patients developed neutralizing antibodies suppressing the effects of subsequent PIXY321 treatments [1311]. At present, the main clinical use for IL-3 is in the production of cultured stem cells for patient reinfusion and gene transfer protocols [214, 467, 475, 997]. It has been reported that IL-3 may stimulate [90] or inhibit [1789] certain hematopoietic malignancies. DT(388)IL3 is a diphtheria toxin-IL-3 fusion protein, which is being tested in patients with acute myelogenous leukemia. It kills malignant progenitors while sparing normal progenitors [781]. There is interest in the use of particulate tumor antigens for anticancer vaccination. Particulate antigens tend to induce cytotoxic CD8+ cells while soluble antigens induce CD4+ helper T cells. IL-3 appeared to be beneficial in this type of vaccination. IL-3 increased the number of antigen presenting cells, the percent of these cells actually presenting particulate antigen and the number of CTLs produced [1594, 2239].

Interleukin-4 B Cell and T Helper Response Interleukin 4 (IL-4) is a 20 kDa glycoprotein. It was originally called B cell stimulatory factor-1 (BCSF-1) for its effects on B cell growth and Ig secretion [514, 801, 1544, 1608, 2249]. IL-4 is produced by activated CD4+ Th2 cells [132, 1036, 1040, 1127, 1375], mast cells [189, 204], basophils [1190], eosinophils [1463], NK cells [461, 1154], γ/δT cells [2307], CD4+ CD8+ T cells [1086] and NKT cells [1087, 1832, 2252]. The gene for IL-4 is located on human chromosome 5 in a region with other cytokines including IL3, IL-5, IL-9 and IL-13. The IL-4 receptor is composed of two chains, IL-4Rα and γc, the common γ chain found in several receptors including the IL-2R [1372]. The α chain has the IL-4 binding site and conveys specificity as an IL-4 receptor. The γ chain is involved in signaling. There is another configuration of the IL-4 receptor in which IL-13Rα replaces the γ chain, in which case the same receptor complex binds and responds to both IL-4 and IL-13 [736, 1439]. Signaling involves phosphorylation of certain proteins including the receptor itself and phosphati-

Cytokines dylinositol-3′ kinase [944]. In mononuclear and B cells, IL-4 receptor levels are increased by IL-4; IFNγ inhibits this increase [1853]. In addition to the membrane bound receptor, there is a soluble form found in body fluids and in the medium of cultured cells [526, 527]. The soluble receptor is smaller in size but it retains the capacity to bind IL-4 [1374]. The soluble receptor is not due to proteolysis of the membrane receptor, but rather differential mRNA splicing [122, 1374]. IL-4 stimulates soluble receptor production, apparently as a way of regulating IL-4 activity [307, 1638]. Soluble receptor competes with the membrane receptor for IL-4 [527, 1212]. The function of the soluble receptor appears to be mainly inhibitory [1212, 1730] but stimulatory [1730, 1871] effects have also been observed. Further, the soluble receptor may also serve as a carrier for IL-4, which protects IL-4 from metabolism and excretion, increasing its functional half-life [526, 457]. Dissociation of IL-4 from the soluble receptor would allow it to bind and activate the membrane receptor. IL-4 has a major role in immune response. It has direct effects on target cells, which are often synergististic with other cytokines. IL-4 also has a major regulatory function in immunity by its influence on the Th1/ Th2 system [2, 254, 1821]. IL-4 induces precursor Th0 differention to Th2 cells [2]. In part, regulation in the Th1/Th2 system involves apoptosis, programmed cell death. Glucocorticoids favor apoptosis in thymocytes and in mature T cells. Several cytokines including IL-4, IL-2 and IL-1 inhibit glucocorticoid-induced apoptosis. IL-4 specifically rescues Th2 cells from death while IL-2 rescues Th1 cells [1306, 2308]. As B cells depend on T cells for help, IL-4 is one of the major cytokines for this purpose. IL-4 acts directly on B cells and it is the main inducer of other Th2 cytokines, most of which also stimulate B cells. IL-4 increases pre-B cell growth, differentiation and Ig secretion [1015, 1597, 1850, 1851, 1871, 2082]. IL-4 stimulates class switching and the production IgG1 and IgE [321, 329, 409, 1552, 1851, 1871]. Among its other effects, IL-4 increases MHC II on B cells for antigen presentation [1459]. In bone marrow IL-4 has a synergistic effect with IL-11 on the induction and growth of hematopoietic progenitor cells [1407, 1537, 1637]. IL-4 increases the growth of monocytes [1974], macrophages [1447] and increases macrophage antitumor activity and antigen presentation [239, 351, 2302]. IL-4 enhances the growth of activated PBL but had little effect on unprimed resting cells. Timing of the cytokine appears to be a factor. While IL-4 stimulated

Walter M. Lewko and Robert K. Oldham PBLs previously activated with IL-2, IL-4 added to cultures simultaneously inhibited proliferation induced by IL-2 [707, 2021]. IL-4 tends to stimulate the growth of thymoctyes [2301] and T cells [2152]. But IL-4 effects are complex and in certain circumstances may be inhibitory [2153]. In cultures of TIL, addition of IL-4 in combination with IL-2, grew T cells from human tumors with specific activity against autologous tumor [933, 934, 1108, 2034]. IL-2 was required; TIL could not be generated using IL-4 only [934]. IL-4 induced the IL-2 receptor in mouse T cells [254]. IL-4 together with IL-2 produced better TIL growth and specific antitumor activity in many but not all tumors [534, 933]. In certain TIL cultures, early growth effects of IL-4 were lost or even reversed with time [534, 933]. In another study, IL-4 in combination with IL-2 tended to produce more T cells with increased antitumor cytotoxicity compared to TIL induced with IL-2 only (8,724). We developed an IL-4 dependent T cell line from a node specimen of a lymphoma patient. This tumor cell line required IL-4 in addition to IL-2 [1108]. Other reports did not show remarkable benefits of IL-4. In one study using melanoma, the addition of IL-4 to IL-2 promoted TIL growth in a minority of cases (5/24), but decreased growth in most cases (17/24). Likewise, specific lysis was enhanced by IL-4 in a minority of cases but depressed in the majority (13/19) [1132]. It may be that IL-4 stimulated those T cells, which had been optimally activated by tumor antigen, but inhibited those cells, which had not been properly activated. Treating PBLs with IL-2 typically induces LAK cells. In mice IL-4 could induce LAK in the absence of added IL-2 [1388]. When mouse PBLs were induced with IL-2, IL-4 tended to enhance LAK activity [1388]. In human PBLs, LAK may be induced by IL-4 in cancer patients pretreated with IL-2 but not in unprimed cells [752, 874, 932, 933]. In previously untreated cells, IL-4 added at the same time as IL-2 inhibited induction of LAK by IL-2 [203, 707, 752, 932, 1870, 2152]. Priming with IL-2 in vivo or in culture was necessary for IL-4 activity. Loss of the IL-2 receptor may be the mechanism by which IL-4 inhibited LAK [1133]. Knockout mice have been developed which are deficient in IL-4. IL-4 has many important and interrelated immune functions, but it is interesting that these mice had a relatively normal immune profile. Serum IgG1 was low. In response to nematode infection, the normal rise in IgE levels failed to occur. Otherwise, T and B cell development appeared relatively normal [1032]. More recent studies showed that IL-4 deficient knockout mice had impaired antitumor cellular immunity (Schuler T. 1999).

173 This may be related to the observations that long term IL-4 benefits IL-12 secretion that in turn stimulates cellular immunity [911]. IL-4 appears to have a role in several inflammatory diseases. IL-4 together with IL-3 stimulated proliferation of mast cells [1374, 2032, 2033], polymorphonuclear leukocytes [518] and fibroblasts [1342]. IL-4 also stimulates ICAM-1. In allergic airway inflammation, eosinophils accumulate and release IL-4 [1463]. IL-4 increases production of IgE [321, 329, 1552, 1852, 1872]. IL-4 also induces eotaxin, to further increase eosinophil infiltration [2298]. In TNF-activated keratinocytes, IL-4 enhances secretion of chemokines, which in turn attract and activate effector cells [19]. While IL-4 may have a role promoting inflammation, it also modulates inflammation directly, or by way of the Th system [1541]. In monocytes IL-4 inhibits production of IL-1, TNF-α and IL-6 while it stimulates IL-1Ra [525, 1973]. Conversely, IL-1 inhibits the production of IL-4 [1720]. Mouse autoimmune allergic encephalitis is a Th1-related disease. In knockout mice lacking IL-4, the disease was exacerbated; treatment with IL-4 had a protective effect [507]. There is, therefore, clinical interest in IL-4 for the management of allergy, inflammation, autoimmune disease and also cancer. The receptor for IL-4 is expressed in several types of cancer. In culture, IL-4 has inhibitory effects on certain hematological malignancies [16, 399, 452, 850, 1214, 1630]. IL-4 also inhibited the growth of certain solid tumor cells including breast [2004], stomach [1358, 1359], colon [2004, 2013] melanoma [790], lung [2012, 364], sarcoma [1602] and renal [791, 1473]. Antitumor effects in culture were generally IL-4 receptor-dependent [17, 1359, 1473, 1602]. IL-4 may be considered angiogenic in that normal fibroblasts and endothelial cells are stimulated by IL-4 [574, 1342, 2005]. On the other hand, it has also been reported IL-4 is antiangiogenic [2083]. In animal studies, IL-4 inhibited certain human head and neck, glioma and colon cell lines growing as xenografts in nude mice. No remarkable immune cell infiltration accompanied depressed growth [2012, 2013]. An acute lymphoblastic leukemia cell line from a child grew in nude mice. IL-7 stimulated growth while IL-4, TNF and IFN were inhibitory [664]. There are other reports of inhibited acute lymphoblastic leukemia, non-Hodgkin’s lymphoma and multiple myeloma cell growth [16]. These animal studies showed IL-4 may be able to inhibit tumor growth directly in addition to its immune effects. But the growth inhibition is in most cases just that; decreased growth rate; growth proceeds but at a slower pace.

174 Phase II trials have been carried out using IL-4 in cancer patients [85, 1157, 1171, 1226, 1971, 2021, 2150]. Unfortunately, response rates were very low and toxicities were significant, particularly in the gastrointestinal tract [1157]. IL-4 may cause cardiotoxicity [2020]. In vitro inhibitory effects of IL-4 on B cell chronic lymphocytic leukemia could not be confirmed in treated patients [1167] Vaccines have been tested using mouse tumor cells genetically engineered to produce IL-4. In mice vaccinated with IL-4 secreting tumor cells [637, 1979, 2264] or IL-4 secreting fibroblast cells mixed with tumor cells [1572], animals rejected the modified tumor cells and subsequent challenges of parental tumor cells [2264]. Cures of established tumors have also been reported [30, 636]. These approaches are now being tried in patients [67, 1485]. IL-4 stimulates cell growth and antigen presentation in dendritic cells, B cells and macrophages [239, 351, 1974, 2301, 2302]. Cancer patients, treated with daily sc IL-4 and GM-CSF had increased numbers of circulating antigen presenting cells [1683]. In current dendritic cell protocols for tumor vaccination, IL-4 is often used in culture together with GM-CSF to prepare and grow the dendritic cells for patient treatment [2039].

Interleukin-5 Eosinophil Growth/Differentiation; Inflammation; Augmentation of T, B, NK and Mast Cells IL-5 is a 12–18 kDa protein, which was originally known as B cell growth factor 2 (BCGF-2), T cell replacing factor, eosinophil colony stimulating factor, eosinophil differentiation factor and IgA enhancing factor. The human gene is located on chromosome 5 where it is linked with several other cytokines including IL-3, IL-4, IL-9 and IL-13 [969]. The molecule is a homodimer held together by disulfide bonds [96, 969]. IL-5 is produced by activated Th2 cells [1935, 1936], mast cells, eosinophils [202, 418, 459] and NK cells [2121]. In NK cells, IL-5 production is enhanced by IL-4 while IL-10 and IL-12 are inhibitory [2120]. The receptor for IL-5 is composed of two subunits, an α chain (IL-5Rα) and a β chain, which is identical to the β chain of IL-3R and GM-CSFR. The α chain binds IL-5. Its structure is quite distinct from that of the α chains of the IL-3R and GM-CSFR [1010, 1398, 1929, 1970, 2040]. The β subunit enhances the affinity of the α chain for IL-5 and it is responsible for signaling.

Cytokines Signaling is through the Jak-STAT pathway. IL-3, IL-5 and GM-CSF show similar patterns of signaling [1320, 1323]. There is evidence that the IL-5Rα chain also signals adding to the uniqueness and complexity of the IL-5R signaling system [1316]. There are membrane bound or soluble forms of the receptor, the result of alternative mRNA splicing [1970]. The soluble form appears to have a regulatory role in eosinophilia in that it binds IL-5 and neutralizes its activity [205, 421]. IL-5, IL-3 and GM-CSF downregulate the levels of IL-5Rα. This happens rather rapidly, within hours. This regulation occurs at the level of mRNA transcription as the gene is turned off [2105]. IL-5 stimulates growth and differentiation of eosinophils [1723, 2222]. It activates eosinophil function [1151] and potentiates chemotactic responses in eosinophils by IL-8 and RANTES [1769]. IL-5 also stimulates B cell growth and the production of IgA and IgM [1722, 1935, 2246, 2247, 1953]; as such it is one of the main factors from T cells, which provides help to B cells [96, 969]. IL-5 enhances IL-2 receptors and responsiveness in T cells and NK cells [935, 1953]. IL-5 augments IL-2-induced LAK activity in peripheral blood cells [58] and promotes, together with other factors, T cell differentiation and mast cells growth [2246]. Eosinophils have important proinflammatory effects, particularly in asthma [635, 783, 938]. For this reason there is interest in IL-5 and its antagonists in asthma and other inflammatory diseases [1723, 1278]. Though IL-5 influences several cells in immune response, significant antitumor activity in preclinical models has not been demonstrated. No clinical trials with IL-5 in cancer have been done.

Interleukin-6 Hematopoiesis, Thrombopoiesis, Inflammation IL-6 is the founding member of the IL-6 family of cytokines which includes leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, cardiotrophin-1, novel neurotrophin/B cell stimulating factor-3 and IL-11. These cytokines share structure and their receptors share a common gp130 subunit for signaling [183, 601, 906, 2273]. IL-6 is a 22–27 kDa glycoprotein. It is also referred to as B cell stimulating factor-2, B cell differentiation factor, cytotoxic T-cell differentiation factor, hepatocyte stimulating factor, IL-1 inducible 26 kDa protein, and

Walter M. Lewko and Robert K. Oldham interferon β2 [2052]. The gene for IL-6 is located on chromosome 7p21. Its primary structure has some homology with G-CSF [766]. IL-6 is produced by several types of activated cells including fibroblasts [1257], macrophages, B cells [1868], CD4+ Th2 cells [1043], CD8+ T cells [1708], epithelial cells [1023], endothelial cells [2213], eosinophils [1287], astrocytes [1079], neurons [1234, 1646], synovial cells [1324, 1940], megakaryocytes [1324], osteoblasts [859], mast cells [580], keratinocytes [365], Langerhans’ cells [365], neutrophils [496, 1287], colon epithelial cells [907] and certain tumors [743, 1164, 1749]. As with many other cytokines, the regulation of IL-6 is complex and disregulation may be involved in disease. IL-6 levels increase with bacterial and viral infection, inflammation and trauma [2053, 2102]. Wound fluids contain IL-6 and wound fibroblasts secrete IL-6 in culture [1238]. IL-6 levels may increase remarkably after surgery, up to several hundred-fold. Thrombin, which induces clotting and wound healing, stimulates the production of IL-6 by fibroblasts, epithelium and other types of cells [1865]. Endotoxin and TNF-induce the production of IL-6 and other inflammatory cytokines [550]. IL-1 increases expression of IL-6 and IL-8 in skin fibroblasts and arthritic synovial cells [612]; TGF-β induced the production of IL-6 in several cells including monocytes, keratinocytes and bone marrow stroma. In plamacytoma cells TNF and IL-1 were both stimulators of IL-6 production [2052]. IL-4 and IFN-γ acted synergistically to enhance IL-6 production in endothelial cells [803]. In lung fibroblasts, TGF-β induced IL-6 synthesis by a transient burst of H2O2 followed by an increase in cellular calcium, which activated the signal pathway for IL-6 expression [909]. And CD40 engagement stimulated IL-6 in B cells and several non-hematopoietic cells; activation of NF-κB was involved in the process [487, 743]. Heparin and heparin sulfate selectively bind IL-6 and several other cytokines. Bound cytokine, upon dissociation, is available to its receptor. Heparin protects the cytokine from proteolysis and may serve to keep the cytokine in high local concentration, close to where it was secreted, available for paracrine effect [1392]. The receptor for IL-6 is composed of two subunits, the IL-6Rα chain and gp130. IL-6 binds to the IL-6Rα subunit [2225], which in turn binds, dimerizes and activates gp130 subunits [1566, 1943]. This leads to the activation of tyrosine kinases, in particular Jak1. These kinases phosphorylate gp130; gp130 then binds and activates STAT transcription factors, in particular STAT3. Phosphorylated transcription factors translocate into the nucleus where target genes are activated [1566].

175 STAT 3 has different and even opposite functions depending on the cell type and physiological status. The gp130 subunit is shared with the receptor for oncostatin M and other members of the IL-6R cytokine family [602, 974, 1138]; the shared subunit appears responsible for the common effects brought about by these cytokines. Essentially all the cells of the body produce gp130, whereas IL-6Rα is expressed by IL-6 target cells including B cells [766] hepatocytes [610], monocytes, CD4 and CD8 T cells [2183], CD34+ stem cells [1946, 2183], neurons [1752], neutrophils [1331] and osteoblasts [2041]. There is a soluble form of IL-6Rα. It is interesting in that unlike most soluble cytokine receptors, it is active. The soluble IL-6Rα is generated in two ways, by proteolytic cleavage of cell surface receptor and by alternate splicing of mRNA [1391, 1665]. TACE, the protease involved in TNF-α production, appears to be responsible for proteolytic shedding of IL-6R [716]. The soluble form binds IL-6 and mediates IL-6 responses in target cells that express the gp130 but lack membrane bound IL-6Rα [1197, 1562]. Certain hematopoietic cells [1563], neurons [1234] and smooth muscle cells [987] were responsive to IL-6 only in the presence of the soluble receptor subunit. IL-6 has a major role in the regulation of hematopoiesis, inflammation and immunity. IL-6 −/− knockout mice were defective in immune and acute phase protein responses [991]. IL-6 stimulates proliferation of multilineage hematopoietic stem cells. Mice that over expressed both IL-6 and the soluble IL-6 receptor had grossly enlarged livers and spleens, and overproduced blood cells [1562]. IL-6 arrests the growth of cultured M1 cells while inducing differentiation into macrophages. IL-6 stimulates B cell differentiation and immunoglobulin production [766, 1394, 1872, 2064]. IL-6 is a costimulator of growth and cytokine production in thymocytes and T cells [1155, 2016, 2064]. IL-6 stimulates T cell activation, differentiation and antitumor activity [1385]. Fibroblasts and epithelial cells secrete IL-6 in response to blood clotting [1865]; along with other factors, IL-6 enhances megakaryocyte colony formation and the production of platelets [308, 755]. Natural killer cells are activated by IL-6 in peripheral blood lymphocytes [584, 838, 1847]. IL-6 has direct effects on several types of nonimmune cells. IL-6 is a hepatocyte stimulating factor [999]; it induced the production of acute phase proteins including fibrinogen [258] and the receptor for anaphylatoxin C5a [1740]. IL-6 also induced acute phase proteins in intestinal cells [1340]. IL-6 and the other inflammatory cytokines TNF-α and IL-1 induce ACTH and cortical release [1237]. IL-6 has a part in angiogenesis;

176 it stimulates endothelial cell growth and blood vessel formation [1378]. IL-6 also increases fibroblast outgrowth and collagen production for wound repair [468]. Physiological levels of IL-6 appear to protect neurons and support nervous system repair [682, 1037]. Mice that over expressed both IL-6 and its receptor showed accelerated nerve regeneration after damage [769]. In bone, IL-6 stimulated osteoclast production and bone resorption activity [1220]. IL-6 may be involved in epithelium-stroma signaling during normal development, for example, in breast tissue [831]. IL-6 appears to have a role in inflammatory conditions. The blood of patients with gram-negative septic shock contained elevated IL-6, TNF, IL-1 and LIF and the levels of each were correlated with disease severity [2077, 2118, 2119]. IL-6 and LIF were induced by TNF-α [880]. IL-6 and its soluble receptor were elevated in inflamed intestinal tissue [676, 1317]. IL-6 was one of the proinflammatory cytokines required for the induction of mouse colitis as a model for human Crohn’s disease [2223], for experimental antigen-induced rheumatoid arthritis [1510] and autoimmune encephalomyelitis [1718]. IL-6 is among the several proinflammatory cytokines expressed by synovial cells of arthritic patients [1324, 1963]. IL-6 is elevated blood and cerebral spinal fluid of patients with multiple sclerosis, Parkinson’s and Alzheimer’s disease [168, 682, 1434]. Overexpression of IL-6 in mouse brain induced acute phase proteins and neurodegeneration [235]. In scleroderma patients, IL-6 was one of the cytokines required for the continued production of autoimmune antibodies [1043]. IL-6 may play a part in cardiac disease. Fibrinogen is considered a cardiovascular risk factor [497]. Fibrinogen levels are elevated during inflammation; IL-6 is one of the main inducers of fibrinogen expression [1620]. IL-6 is generally referred to as a proinflammatory cytokine, but it appears to be involved in the resolution of inflammation as well. IL-6 attenuates the synthesis of proinflammatory cytokines while it has little effect on production of cytokines such as IL-10 and TGF-β. In IL-6 deficient mice, inflammation, eosinopilia, the secretion of chemokines and Th2 cytokines were increased. In mice that produced excess IL-6, inflammation and Th2 cytokine secretion were decreased [2102]. IL-6 has also been shown to induce the production of IL-1 receptor antagonist and the soluble TNF receptor, both of which inhibit inflammation [1998]. In astrocytes, IL-6 inhibited expression of TNF-α and down regulated levels of cell adhesion molecules [137, 1480, 1816]. One mechanism for the anti-inflammatory effects of IL-6 (and the related cytokine IL-11) may involve inhibition of transcription factor NF-κB [2023].

Cytokines Human herpes virus 8, which is associated with Kaposi’s sarcoma, produces a viral IL-6 molecule that has 25% homology with human IL-6 [1437]. It has certain effects that are similar to those of cellular IL-6 [226]. The viral IL-6 appears to be responsible for some of the virus’ pathology, including increased angiogenesis and hematopoiesis [60]. Interestingly, the viral IL-6 binds directly to gp130 and exerts its effects on cells independently of the IL-6 receptor, for which viral IL-6 appears to have little or no affinity [1390]. In preclinical studies, IL-6 showed promise as an anticancer agent. In vitro, IL-6 inhibited the growth of certain breast cancer cells, leukemia, lymphoma and other cell lines [296, 1357]. IL-6 increased ICAM-1 (CD54) in certain breast cancer and melanoma cell lines [831, 973]. IL-6 inhibited growth in several mouse tumor models; these mice developed tumor-specific CTLs, but not LAK cells. In mice treated with IL-6, growth and metastasis of Lewis lung [927] and melanoma [926, 1164, 1165] were inhibited. Resistance may develop in IL-6 sensitive cells. Loss of sensitivity was associated with loss of IL-6 receptors in some but not all cases [1825]. IL-6 is involved in the inflammatory response that produces tumor infiltrating lymphocytes [1385]. In cultures of TIL from human renal cell carcinoma, addition of IL-6 to the standard IL-2-containing medium stimulated TIL growth though not tumor cell kill; IL-6 alone did not induce TIL proliferation [1082]. In vaccination studies, IL-6 engineered mouse mammary tumor cells offered some protection to subsequent challenge by unmodified tumor cells. IL-6 and other IL-6 family members (IL-11, LIF, CNTF, NNT) induce appetite loss and wasting. In mice, colon adenocarcinoma cells produced elevated serum IL-6 and cachexia. The drug Suramin interfered with the binding of IL-6 to its receptor and reversed cachexia in tumor bearing mice [1900, 1901]. IL-6 is an autocrine growth factor in certain human cancer cells including multiple myeloma [115, 724, 1478], B-cell leukemia [754], cervical carcinoma [503], renal cell carcinoma and prostate cancer [1005, 1307, 1955]. In myeloma, growth stimulation appears to be linked with several of the IL-6 family cytokines (OSM, LIF, CNTF, IL-6) [689, 690, 2147]. In human melanoma, there is evidence that with tumor progression, there is a transition from IL-6 paracrine growth inhibition to autocrine growth stimulation. Serum IL-6 in melanoma patients was found to be a prognostic factor for poor survival and lack of response to IL-2 therapy [1966]. IL-6 was tested in cancer patients, including renal cancer, in phase I and II trials [2057, 2126, 2136].

Walter M. Lewko and Robert K. Oldham In addition to its potential anticancer activity, there was interest in IL-6 for the stimulation of platelet and blood cell levels. Patients treated with IL-6 demonstrated the usual cytokine flu-like side effects plus reversible anemia, leukosis, thrombocytosis, increased acute phase proteins, heart problems, elevated bilirubin and confusion indicative of neurotoxicity. There was a suggestion of benefit in certain studies but toxicity was significant. One report, a phase II study in advanced renal cell cancer, showed lack of efficacy [1758]. Anti-IL-6 monoclonal antibodies have been used to treat patients with myeloma [114, 2065] and AIDS associated Kaposi’s sarcoma [1610] without any apparent benefit. In a phase I trial involving pediatric patients given IL-6 plus G-CSF after myelosuppressive chemotherapy, there were increased hematological responses but with a high incidence of toxicity [188]. IL-6, IL-10 and TGF-β are cytokines reported to be secreted by tumors, which interfere with dendritic cell differentiation and antigen presentation, resulting in ineffective antitumor immune response [129].

Interleukin-7 T Cell, B Cell, Macrophage and Dendritic Cell Development IL-7 was first described as a factor produced by mouse bone marrow, which stimulated B-lymphocyte progenitor cells to multiply and develop [1422, 1423]. IL-7 is a 25 kDa glycoprotein. It is produced by bone marrow stroma [641, 1422, 1423], thymic stroma [1716], keratinocytes [69, 745, 1245], human intestinal epithelium [2115], B cells [134], follicular dendritic cells and vascular cells [1024]. IL-7 is also produced by tumors including certain carcinomas [910] leukemias [569, 1578] and lymphomas [973, 1001]. IL-7 is not detected in normal T cells [2263]. Upon secretion, IL-7 binds to integrin in the extracellular matrix; this may be the form utilized by thymocytes [980]. The IL-7 receptor is composed of two subunits, IL-7Rα and γc. The IL-7Rα chain contains the IL-7 binding site [640]. The γc is shared between IL-2R, IL-4R, IL-7R, IL-9R and IL-15 R [1002]. γc enhances receptor affinity of IL-7 and it participates in signaling [1002, 1461]. The activation response involves Jak1, pI-3 kinase, STAT3 and STAT5 [556]. Receptors that share γc have similar but not identical nuclear effects. Lack of active γ chain in patients with severe combined immunodeficiency disease results in loss of function in these five receptors. Loss of IL-7R function is a major reason for poor immune response in these individuals.

177 There is a soluble form of the receptor that is produced by a specific mRNA [641]. Three different IL-7 binding affinities have been observed [74, 558, 1523, 2017]. The highest affinity receptor is generally found in activated cells and appears to be the one responsible for cell division [73, 1523]. In human T cells, response to IL-7 depends on the activation state. In resting cells, IL-7 induces certain cellular proteins (CD25, IL2 R) but no proliferation. In activated cells, IL-7 is a potent inducer of cell division [648, 1365]. Immunosuppressive drugs cyclosporin and FK506 inhibit IL-7 stimulated growth, apparently by interfering with the function of the high affinity receptor [557]. IL-7 has important influences on several different immune cell types. Genetically deficient mice, lacking IL-7, were extremely lymphopenic [2084]; early lymphocyte development was severely impaired [1548]. In mice there is an absolute requirement for IL-7 in the development of B and T cells. Interestingly, it is unusual for the loss of any one cytokine to cause such impairment. This is probably due to redundancy of cytokine functions, which is not the case for IL-7 in mice [2084]. In humans, IL-7 is not so strictly required for the development of B cells [1592]. IL-7 induces the growth of early B lineage cells. In B lymphocytes, IL-7R was found on pre-B cells but not on mature B cells, consistent with its role in the early stages of B cell maturation [371, 372, 1918]. Stem cell factor and Flt3 ligand are costimulators [1229, 1424]. IFN-γ blocks the stimulatory effect of IL-7 on pre-B cells [592]. Mice deficient in IL-7 fail to support normal B cell development [2084]. For prolonged B cell outgrowth, additional IL-7 related signals appear to be required, which occur by way of physical contact with stromal cells. IL-7 supports outgrowth of pre-B and pro-B cells but the pre-pro-B cell population requires, pre-pro-B cell growth-stimulating factor. PPBSF is composed of at least two covalently associated subunits, one of which is an IL-7 chain. The other subunit is a protein that is attached to the stromal cell surface. Pre-pro-B cells bind PPBSF during stromal cell contact; PPBSF stimulates division and primes the cells for IL-7 [1273]. Thymic stromal lymphopoietin is another IL-7-related factor in early B cell development. It was discovered in the conditioned medium of a thymus cell line [568]. TSLP and IL-7 have overlapping actions [568]. TSLP binds the IL-7Rα with another receptor subunit; it does not use γc. TSLP induces the transcription factor STAT5 as does IL-7, but TSLP did not activate any known Janus kinases. Normal T cell development depends on IL-7. Mice lacking IL-7Rα [1205, 1223, 1548] and mice treated

178 with neutralizing antibodies to IL-7 [650] do not produce normal T cells. γδ T cells are missing entirely in IL-7Rα (−/−) mice [728, 1548]. αβ T cells are present, but they are not responsive [1548]. IL-7 is a growth/ differentiation factor for fetal pre-T cells, thymocytes [143, 735, 1492, 2080, 2153] and mature CD4+ and CD8+ T lymphocytes [73, 143, 293, 347, 564, 648, 1148, 1365, 2243]. An important effect of IL-7 in both T and B cells appears to be its stimulation of the bcl-2 antiapoptotic pathway, for the maintenance of lymphocyte viability [15, 1222, 2085]. IL-7 was the only cytokine among 16 factors tested capable of inducing rearrangement (diversity) in the T cell receptor V(D)J region. IL-7 activated expression of the RAG1 and RAG2 genes involved in gene rearrangement [306, 1383]. In activated human peripheral blood T cells, IL-7 induced the secretion of several cytokines including IL-2, IL-3, IL-4, IL-6, IFN-γ and GM-CSF [442, 443]. IL-7 also increased antitumor cytotoxicity in T cells [22, 143, 749, 776, 1180] and this cytotoxicity may persist long term in culture without frequent antigenic restimulation [1182]. IL-7 acts together with several cytokines in the development of T cells [2139]. Flt3 ligand and stem cell factor [1350, 1364] are co-stimulators with IL-7 for early T cells. IL-1 and GM-CSF induced outgrowth of mouse thymocytes; IL-7 was required [735]. IL-2 acts together with IL-7 to stimulate antigen-induced effector cells from memory CD8+ T cells. IL-7 may act alone or with IL-2 [125, 1283]. Together the effect is synergistic [1283]. IL-7 stimulates IL-2 receptor (CD25) levels in resting T cells [73]; this may explain the synergy. Among its many other functions, IL-7 induced proliferation and antitumor activity in human blood monocytes and macrophages [24, 871]. In treated mice, IL-7 mobilized myeloid progenitor cells from bone marrow to peripheral sites [686]. It supported eosinophil progenitors in human bone marrow cell culture [2071]. IL-7 induced lymphokine activated killer cells [1181, 1736, 1896]. While IL-2-LAK exhibited better melanoma cell kill, IL-7 LAK killed with a different pattern of cytokine release [1431, 1432], without TNF secretion and without toxicity towards normal cells [1731]. IL-7 appears to have a part in antigen presentation. IL-7 was a growth factor for mouse dendritic epidermal T cells [1245, 1246]. Dendritic cells produce IL-7 [1022]. Keratinocytes that surround the dendritic cells in skin produce IL-7 and TNF-α [1245, 1246]. Together these cytokines stimulated dendritic cell growth. IL-7 also increased levels of the co-stimulatory protein B7 on B cells [414]. Lymphopenia can occur in patients with HIV infection and chemotherapy. It has been shown that IL-7 may

Cytokines be administered to patients to expand CD4+ and CD8+ lymphocytes. This occurs with a decrease in the percentage of regulatory CD4+ T cells [1673]. Several preclinical studies suggested IL-7 may be useful in the treatment of cancer. Lung metastases of Renca renal cancer cells were reduced in mice treated with rhuIL-7; the mice showed increases in T cells, CD8/CD4 ratio, B cells, macrophages and NK cells [1001]. Nude mice with human colon cancer xenografts lived longer when treated with rhuIL-7 plus human T cells compared to mice treated with either alone. The antitumor activity appeared due to interferon induced in CD8+ cells, not cytolysis. Interestingly, injected interferon was not that effective suggesting local continuous release was better than systemic interferon therapy [1402]. IL-7 appears to be a good adjuvant during tumor vaccination. Tumorigenesis was decreased in mice inoculated with IL-7 gene engineered plasmacytoma cells [775], glioma cells [59] and fibrosarcoma cells [1261]. Animals that rejected engineered tumor cells developed immunity to subsequent injections of the parental cells [61, 1261]. The tumors were infiltrated with T cells. Complement receptor rich macrophages, eosinophils and basophils were also increased [776, 1261]. IL-7 was one of several cytokines which upregulated intercellular adhesion molecule-1 (ICAM-1) on human melanoma cells. This protein is involved in immune recognition and anticancer action [973]. Tumor infiltrating lymphocytes have been used to treat cancer patients. Interleukin-2 is used to activate and grow TIL in culture. IL-2 generally stimulates good initial growth and tumor cell kill, but antitumor activity is often difficult to maintain over the long culture time needed to grow sufficient cells for therapy, especially when tumor cells are not available as a source of antigen to restimulate the T cells. There are reports of difficulty maintaining CD4+ helper cells in IL-2-induced cultures. IL-7 has been examined to determine whether it provides any benefit in culture. IL-7 alone stimulated the growth of TIL cultures from certain renal cancer and enhanced IL-2-induced growth in others [1182]. Growth and antitumor activity persisted during long term culture. Antigen restimulation was not required to maintain antitumor activity. Others have reported IL-7 was not beneficial at the initiation of TIL culture but rather IL-7 stimulated growth and cytokine secretion in cultures that were already responding to IL-2 [1820]. There are reports that IL-7 stimulates certain types of cancer. IL-7 increased the growth of several leukemia cell lines [664, 1242, 1351, 1491, 1531, 2017]. Sezary lymphoma cell lines responded to IL-7 and

Walter M. Lewko and Robert K. Oldham IL-2 with increased and in some cases synergistic growth [374, 554]. The effect of IL-7 may be autocrine or paracrine [554, 664]. Abnormal cytokine secretion by tumor cells may be responsible for immune system abnormalities common in CLL patients [569]. Transgenic mice have been developed with increased IL-7 expression in lymphoid tissues. These animals had remarkable skin T cell infiltrates, the result of the growth stimulating effects of IL-7. These transgenic mice also produced T and B cell lymphomas. This model system shows that the IL-7 locus could behave as an oncogene. There have been preliminary studies using IL-7transfected autologous tumor cells as vaccines. Phase I trials in patients with melanoma showed that IL-7 genetransfected, irradiated cells administered subcutaneously were safe and produced tumor-specific CTL responses. One minor clinical response was reported [1338, 1339]. Another study, a phase I/II trial in ten patients with metastatic carcinoma, used autologous tumor cells transfected with IL-7, GM-CSF and double stem-loop immunomodulating oligodeoxyribonucleotides (d-SLIM). One CR, one PR and one MR were observed [2179].

Interleukin-8 Chemotaxis, Angiogenesis IL-8 is a potent chemotactic and proinflammatory cytokine [1250, 1384]. It is a member of the CXC (ELR+) chemokine family (see below). IL-8 is a relatively small, 6,000–8,000 mw glycoprotein [1755, 2096, 2257]. It is secreted by a variety of cell types including macrophages [1755], endothelial cells [623, 1455, 1603, 1756, 1905], neutrophils [255, 1949, 2130], epithelial cells [123, 913, 961, 1878], fibroblasts [1063], keratinocytes [1063, 2281], mast cells [1337] and eosinophils [2261]. Certain tumor cells also produce IL-8 including melanoma [1737], squamous cell carcinoma [2281] colon adenocarcinoma [1757]. IL-8 secretion is controlled by various factors such as IL-1 [1063, 1757], IL-3 [2121], IL-5 [1769], GM-CSF [1264, 2121], TNF [1063, 1904], vitamin D [2281], lipopolysaccharide [1757] and fibrin [1603]. Mast cell secretion of IL-8 was specifically stimulated by stromal cell factor-1 [1130]. There are two receptors for IL-8, CXCR1 and CXCR2 [5, 787, 1074, 1371, 1400]. These receptors bind IL-8 with high affinity and they also bind other members of the chemokine family [1074]. There are receptors for IL-8 on neutrophils, T cells, mast cells, macrophages,

179 endothelial cells and keratinocytes [950]. Like other chemokine receptors, the IL-8 receptors are members of the rhodopsin superfamily; these proteins characteristically contain seven transmembrane domains. Chemokine receptors are linked to phospholipase C through G proteins. Receptor activation results in increased diacylglycerol, inositol triphosphate and increased intracellular calcium released from cellular stores. IL-8 was first described as a macrophage-derived chemoattractant for neutrophils [1384, 1755, 2096]. IL-8 is also a chemoattractant for T cells [98]. It has little effect on B cells. In dogs injected with human IL-8, the site became infiltrated, mainly with neutrophils [1987]. IL-8 stimulates neutrophil production of superoxide anion, degranulation with the release of hydrolytic enzymes such as elastase, and migration through endothelium of capillaries [101, 252, 815, 1565]. Mast cell-dependent recruitment of neutrophils appears to be mediated by IL-8 [1130]. IL-8 also induces migration in IL-2 activated peripheral blood NK cells [1774]. In rabbits, IL-8 blocking antibodies had an inhibitory effect on inflammation, especially in the lung [1780]. IL-8 also stimulates angiogenesis [1346]. IL-8 is an attractant to antigen presenting cells such as macrophages and eosinophils [408, 1769, 1777]. As such, IL-8 may have an important role in the initiation of immune response as well as the recruitment of effector cells that carry out the response. Fibrin formation is associated with trauma, inflammation, wound healing and cancer. The addition of fibrin to cultured endothelial cells induced the release of IL-8 [1603]. The physical act of blood clotting may stimulate cytokine release and cell migration. Besides IL-8 there are several additional proteins and peptides that are chemotactic to immune cells; macrophage inflammatory protein α (MIP), IL-1α, and RANTES (see below) to mention a few. IL-8 appears to have a role in inflammatory diseases. Arthritic synovial fibroblasts constitutively oversecrete IL-8; IL-1β, itself elevated in synovial fluid, stimulates secretion further. Specific inhibitor studies showed that NF-κB, an important signal for inflammation genes, was involved in both spontaneous and IL-1-induced expression of IL-8 [612]. IL-8 or other chemokines released within tumors may have anticancer effects by recruitment of macrophages, granulocytes and lymphocytes. But there is also evidence that IL-8 contributes to tumor development and spread. In melanoma, IL-8 has been shown to be an autocrine growth factor [1737, 1833]. IL-8 stimulated tumor cell movement. Metastasis of melanoma cells in nude mice was correlated with the tumor’s capacity to produce IL-8 [1833]. Developing tumors require

180 adequate blood supply and may benefit from IL-8induced angiogenesis [994, 1905]. IL-8 secretion correlated with vascularity in human gastric cancer [976]. In a nude mouse model for human tumor progression, IL-8 was one of several angiogenesis factors that correlated with progression in ovarian cancer [2250]. Reduced IL-8 expression or blocking antibodies administered to nude mice decreased growth, invasiveness and angiogenesis in melanoma, breast cancer and transitional cell carcinoma cells [812, 966, 1299]. Therefore, IL-8 may act directly to stimulate tumor cells. Alternatively, IL-8 may activate neighboring normal cells to produce factors that tumors utilize for growth and metastasis.

Interleukin-9 Mast Cell, T Cell, Lymphoma Growth Factor; Inflammatory; Hematopoietic IL-9 is a 32–39 kDa, 144 amino acid protein. Its gene is located on chromosome 5 in a region near genes for GM-CSF, IL-3, IL-4 and IL-5. IL-9 is structurally related to IL-2, IL-4, IL-7 and IL-15 and some of their activities are similar. IL-9 was discovered as a factor in the medium of HTLV-transformed T cells that stimulated growth in a human leukemia cell line [2229]. IL-9 has also been referred to as P40 [2063, 2170], mast cell growth enhancing activity [1332] and T cell growth factor III [1748]. It is produced by activated Th2 cells [1634], by naive CD4+ cells [1747], and by mast cells [820]. In CD4+ cells, IL-9 secretion is stimulated by IL-1, IL-2, IL-4 and TGF-β and inhibited by IFN-γ [1745, 1746]. In mast cells, IL-1, IL-10 and kit ligand increase IL-9 secretion [820, 1899]. The receptor for IL-9 contains IL-9Rα and γc [968]. The signaling pathway involves Jak3, Jak1, the adaptor protein IRS-1 [2245] and the transcription factors STAT1, STAT2, and STAT5 [412, 116]. IL-9 usually acts together with other cytokines. With IL-3 or GM-CSF, IL-9 induced the growth of hematopoietic progenitor cells [784]. With erythropoietin, IL-9 supported erythroid colony (BFU-E) formation [447, 2170]. In mice, IL-9 with IL-2 stimulated fetal thymocyte proliferation [1917]. IL-9 increased growth in T cell lines and activated helper T cells [800]. Interestingly, quality of growth induced in certain Th cell lines by IL-9 was different from that of IL-2 in that IL-9 induced growth was hardly affected by glucocorticoids while IL-2-induced growth could be inhibited [1162]. IL-9 also increased granzyme B (protease involved in apoptosis and cytotoxicity) and high affinity receptors for

Cytokines IgE in several Th cell clones. These are mast cell-like characteristics that IL-9 induced in Th cells [1161]. In cultures of mast cells, IL-9 enhanced survival; IL-9 together with IL-3 and IL-4 stimulated mast cell growth and the secretion of IL-6 [819]. Proliferation of mast cells was one of the remarkable features of IL-9 transgenic mice [631]. In B cells, IL-9 potentiated IL-4induced immunoglobulin secretion [461]. IL-9 increased resistance to nematode infections [515]. Mice that oversecreted IL-9 showed increased allergeninduced inflammation and airway hyperresponsiveness [1279, 1977]. There is genetic evidence that IL-9 is involved in human asthma [451, 1450, 1460]. Related to cancer, IL-9 has been shown to stimulate growth and depress apoptosis in mouse lymphoma cells [1633, 2079]. Transgenic mice overexpressing IL-9 tended to develop lymphomas [1632]. HTLV-transformed T cells produce IL-9 [947]. IL-9 was an autocrine growth factor for cultured Hodgkin’s lymphoma and ReedSternberg cells [684]. It is possible that antagonists of IL-9 may be useful therapeutically.

Interleukin-10 Regulator of Immune Response IL-10 was first discovered in cultures of Th2 cells as a factor that inhibited IFN-γ production by activated Th1 cells [535]. It is a 17,000 mw protein with relatively little glycosylation [1177]. IL-10 is produced by several types of cells including regulatory T cells (Tr1 and nTreg) [2002, 2204], CD4+ Th2 cells [297, 535, 2262], thymocytes [1198], B cells [629, 1470], monocytes [423, 548], mast cells [1990], eosinophils [1416], and keratinocytes [494]. In CD4+ T cells, activation induces several cytokines including IL-2 as an early response and IL-10 as a relatively late regulatory response [332, 2262]. Blocking endogenous IL-2 prevented the increase in IL-10 [332]. IL-4, IL-7, IL-15 and IL-12 were costimulators of IL-10 production. IL-10 feeds back on the production of IL-2 and its own synthesis by deactivating T cells [332]. In macrophages, activation induced several cytokines including an early response for TNF-α (3.5 h) and a much later peak in IL-10 (48 h) [388, 423, 2116]. Cortisol (immunosuppressive) stimulated plasma IL-10 levels in treated normal volunteers [376]. IL-10 feeds back on its own production by deactivating macrophages and TNF-α secretion [388, 423, 2167]. The IL-10R structurally resembles the IFN receptor [773, 1014, 1869]. Signaling in monocytes and T cells

Walter M. Lewko and Robert K. Oldham involves tyk2, Jak 1, STAT1α, STAT3 and a different STAT3-like protein in monocytes, not observed in T cells [533]. IL-10 is a key regulator of immune response. A major effect of IL-10 is the suppression of Th1-dependent cellular immunity and the promotion of Th2-dependent humoral immunity [1347]. IL-10 deactivates a number of macrophage activities. It inhibits macrophage production of IL-12 and other cytokines that stimulate T cells [173, 369, 388, 536]. Further, IL-10 depresses macrophage production of peroxide [173] and nitrogen oxide [600]. In activated monocytes, IL-10 also inhibits the production of M-CSF. IL-10 regulates neutrophil activity. It inhibits endotoxininduced release of proinflammatory cytokines [920]. IL-10 depresses neutrophil production of MIP-1α, MIP-1β and IL-8 [920] and enhances the release of IL-1Ra [257]. IL-10 inhibits antigen presentation and T cell response [537]. In target cells, IL-10 down-regulates MHC class I [1243]. In monocytes, IL-10 depresses costimulatory and adhesion molecules involved in antigen presentation [2167]. IL-10 also inhibits dendritic cell-induced production of IFNγ by Th1 and CD8+ T cells [1188]. It decreases antigen presentation by macrophages to T cells, in particular, to Th1 cells [388]. IL-10 also depresses T cell growth directly [1942]. In mitogen stimulated T cells, IL-10 inhibits cell division and production of IL-2 and IFN-γ [1942]. In many ways IL-10 resembles IL-4, but interestingly, IFN-γ levels are stimulated by IL-4. This is the key difference in activities between IL-4 and IL-10 which otherwise act similarly as they tend to suppress cell mediated immune response. In NK cells, IL-10 depresses INF-γ secretion and cytotoxicity induced by IL-12 and TNF-α [2027]. In fibroblasts, IL-10 depresses the production of collagen, enhances the production of collagenase and stromelysin [1628]. In macrophages, it inhibits production of collagenase and stimulates the production of tissue inhibitor of metalloproteinases (TIMP) [1052]. As already noted, glucocorticoids, which are immunosuppressive, stimulate IL-10 levels in blood [376]. The overall effect of IL-10 is antiinflammatory and it tends to limit tissue damage associated with inflammation. IL-10 has been described as a growth and differentiation factor for CD8 T cells [297, 1198] and activated B cells [1687]. In T cells, IL-10 induced the IL-2R and thereby enhanced IL-2-dependent proliferation [1198, 331]. IL-10 is a chemotactic factor for CD8+ T cells (the first described) but it is an inhibitor of IL-8-induced migration for CD4+ cells [893, 895]. In a model for diabetes, IL-10 expression by islet cells caused the local accumulation of T cells [2181]. In B cells, IL-10 and

181 IL-2 acted together synergistically, as IL-2R levels were increased [547, 867]. IL-10 enhanced class II MHC expression [629] and stimulated the production of IgG, IgA and IgM [400, 867]. In B cells, IL-10 had opposite effects on apoptosis that appeared to depend on the activation state of the cells. IL-10 depressed apoptosis in germinal center B cells [1107] but it enhanced apoptosis in chronic lymphocytic leukemia B cells [546]. In Staphylococcus aureus-activated B cells, IL-10 added 3 days later inhibited apoptosis; when IL-10 was added at the time of S. aureus, apoptosis was stimulated [866]. In mast cells IL-10 acts together with IL-3 and IL-4, to stimulate growth [1990]. IL-10-deficient knockout mice exhibited a number of problems, in particular the mice developed enterocolitis due to unregulated immune response to microbes [1031]. IL-10 protects mice against the effects of endotoxin shock [613, 802]. Epstein Barr Virus genes code for vIL-10 (BCRF1). The protein is highly homologous with hIL-10 [806, 1348, 2076]. It appears that at some point in evolutionary time, EBV picked up this gene from a mammalian cell source. viral IL-10 is expressed soon after infection, within 2–3 h, whereas host IL-10 appears 20–30 h after infection. viral IL-10 expression interferes with antigen presentation and the antiviral immune response [423]. IL-10 functions during pregnancy. The uterus provides a site of immune privilege for the development of a fetus. Successful pregnancy is said to be a Th2 phenomenon [2127]. In an abortion-prone mouse model system, a defect in IL-10 production appeared to be responsible for fetal loss; treating the mice with IL-10 prevented fetal resorption [313]. The pregnancy hormone progesterone induces the production of IL-10 [709]. In human trophoblasts, IL-10 is an inhibitor of matrix metallopoteinase-9 production, a protease believed to have a role in parturition [1682]. IL-10 production in the placenta was shown to decrease at term. Lower IL-10 levels would allow the expression of MMP-9 during parturition and a shift from Th2 to Th1 immunity [709]. IL-10 may have a role in human diseases involving inflammation and inappropriate immune response. In volunteers treated with a single i.v. dose of IL-10, T cell levels were decreased. In endotoxin treated volunteers, pretreatment with IL-10 lowered fever and several proinflammatory cytokines [300, 1525]. IL-10 depresses lung granulocyte number and capacity for degranulation [1525]. IL-10 inhibits collagen production in fibroblasts. In patients with chronic hepatitis C, IL-10 normalized serum ALT (marker for hepatic inflammation), improved liver histology and reduced

182 fibrosis [1440]. Most patients with psoriasis benefited from treatment with IL-10 [77, 1627]. In Crohn’s disease, responses to IL-10 were observed in 23.5% patients. In another study, patients treated post surgically to prevent recurrence of Crohn’s disease did not show any benefit [336]. IL-10 treatments were well tolerated; observed toxicities, were moderate and reversible. In another approach, blocking antibodies to IL-10 were used to treat a small group of SLE patients. The patients were treated for 21 days; five/six patients benefitted from the antibody therapy [1142]. The immunosuppressive and antiinflammatory properties of IL-10 may be of value in tissue transplantation [125]. But it should be noted, certain mouse studies did not show any IL-10 benefit [1022]; in some cases IL-10 exacerbated graft vs. host disease [163]. Although IL-10 is generally antiinflammatory, it has certain proinflammatory effects, which may complicate its use in therapy. For example, one study showed that IL-10 potentiated IFN-γ release in LPS-treated volunteers [1066]. IL-10 may be elevated in cancer patients, naturally due to immune response or due to IL-10 production by tumors. Increased IL-10 could be involved in tumor growth and immune escape [882, 1173, 1574]. It has been shown that Epstein-Barr viral IL-10 is an autocrine growth factor for certain B cell lymphomas [121, 957]. IL-10 is also a growth factor for human myeloma cells, apparently by induction of a gp130 R dependent (e.g. LIF) process [689]. IL-10 may interfere with antigen presentation. Using antigen-pulsed DCs, repetitive vaccination induced CD4+ cells, which produced IL-4 and IL-10 [282]. IL-10 appeared to induce T cell anergy [282, 1173, 1934]. In tumor cells, IL-10 decreased expression of HLA class I, which would decrease recognition by effector cells [1243]. In Hodgkin’s disease, elevated IL-10 was determined to be an independent prognostic factor for treatment failure [174, 1727]. It thus appears IL-10 may have a role in tumor progression. It should be mentioned that during immunotherapy, IL-10 levels might rise [1996]. This could be a reason for lack of response. One might not expect IL-10 to be useful in a tumor vaccine. However, interesting results were obtained in mice vaccinated with mammary adenocarcinoma cells engineered to produce IL-10 [30, 624]. Some mice developed long term immunity. In another study, transgenic mice overexpressing IL-10 in antigen presenting cells rejected an immunogenic melanocytoma [678]. In another study, mice received tumor cells expressing both IL-10 and IL-12. 50–70% showed remission of metastases to lung, colon and breast tumors. Vaccination with

Cytokines cells expressing either IL-10 of IL-12 alone yielded little or no response. CD8 and CD4 cells were both involved and tumor specific antibodies were induced [677].

Interleukin-11 Hematopoietic Progenitor/ Thrombocytosis Stimulator; Regulator of Inflammation IL-11 was discovered as a factor produced by a bone marrow stromal cell line, which supported the growth of an IL-6-dependent plasmacytoma cell line [1543]. It is a 22 kDa glycoprotein [455, 456]. IL-11 is a member of the IL-6 cytokine family. It is produced by stromal cells in hematopoietic tissues [1543], fibroblasts [486, 1653, 2290], epithelial cells [485], chondrocytes and synovial cells [1200] and by airway smooth muscle cells [484]. IL-11 is induced by IL-1, TGF-β, histamine, eosinophil major basic protein and by certain viruses [484, 485, 481]. The receptor for IL-11 shares the gp130 subunit with receptors for the other IL-6 family members [1138, 1944]. IL-11 stimulates the growth and differentiation of hematopoietic progenitor cells and megakaryocytes [1404, 1405]. It influences very early stem cells and more committed precursor cells. In mouse cells, IL-11 acted synergistically with IL-3 and IL-4 to stimulate growth of primitive blast colony forming cells [1504, 1405]. IL-11 stimulates B cells and the production of immunoglobulin [1543, 2244]. IL-11 has a remarkable effect on platelet production and this is its main clinical use [456]. IL-11 inhibited the differentiation of fibroblast lines into adipocytes; IL-6 and TNF-α had similar effects [937]. IL-11 inhibited the production of IL-12 by macrophages [1104]. IL-11 is involved in bone metabolism; it is said to be critical for osteoclast development [625]. Also IL-11 is among the several cytokines capable of inducing acute phase protein release from hepatic and nonhepatic cells [1340]. IL-11 regulates Th2 responses and the production of inflammatory cytokines [2022, 2296]. IL-11 is secreted during respiratory tract infections, airway inflammation, and asthma [481, 1229, 2103] and certain other inflammatory responses such as Lyme disease [53]. IL-11 also ameliorates inflammatory bowel disease and oral mucositis [958]. IL-11 protects and restores damaged, inflamed tissue by the secretion of protease inhibitors [1202]. In an ovalbumin allergy model, lung overexpression of transgenic IL-11 decreased antigen-induced eosinophilia, inflammation and endothelial VCAM-1 in

Walter M. Lewko and Robert K. Oldham lung tissue. Th2 cytokines IL-4, IL-5 and IL-13 were diminished [2102]. IL-11 also has protective effects during radiationinduced thoracic injury [1623] and intestinal damage [455]. IL-11 protects against immune complex lung injury [1095] and oxygen-induced lung damage [2123]. In the care of cancer patients, IL-11 augments bone marrow recovery and platelet production. It is approved for use in patients with non-myeloid malignancies to prevent thrombocytopenia and to reduce the need for platelet transfusion following chemotherapy. It has been shown in breast cancer patients, for example, that treatment with rhIL-11 increased bone marrow megakaryocytes [1503]. Blood platelet counts were increased and patients experienced less thrombocytopenia [645]. In patients with thrombocytopenia requiring platelet transfusions, treatment with rh-IL-11 significantly decreased the need for transfusions with subsequent chemotherapy [1978]. RhIL-11 is well tolerated by adults and children. Adverse events are generally mild or moderate and reversible [1843]. Il-11 may have a role in progression of breast cancer. IL-11 and its receptors were higher in tumors than normal tissue and higher in node positive tumors than node negative tumors. Higher expression of IL-11 in tumors was linked to poorer survival of patients [708]. IL-11 also induces the formation of osteoclasts, which appear to be involved in the metastasis of breast cancer to bone [918, 1353, 1830].

Interleukin-12 Immune Stimulation, Inflammation, Hematopoiesis, Regulator of Innate and Adaptive Immunity IL-12 is a key cytokine with far reaching influences. It serves as a link between the innate and adaptive immune response systems [993, 2025, 2186]. IL-12 is a well studied cytokine. It was reviewed in some detail in a previous edition [1110]. The following is a brief overview with a discussion of certain new reports related to cancer. IL-12 was discovered in B cell lines as a factor, which acted with IL-2 to stimulate proliferation, IFN-γ secretion and cytotoxicity in NK cells and T cells [596, 737, 1149, 1560, 2054]. It has been referred to as natural killer cell stimulating factor [993], T cell stimulating factor-1 [649], and cytotoxic lymphocyte maturation factor [1891]. IL-12 is a 70,000 mw glycoprotein; it is composed of two chains, α (also called p35) and β (p40)

183 [680]. These chains are produced from two distinct genes, on separate chromosomes [680, 1822]. The α subunit has homology with IL-6 and G-CSF [1293]. The β subunit is unusual in that its structure is more like that of a receptor than a cytokine [603]. Expression of these two genes does not appear to be coordinated. Curiously, many cells in the body produce the α subunit without making β [368]. But both chains must be transcribed within the same cell for the production of active IL-12 [2185]. Regulation of IL-12 secretion generally occurs at the level of β chain synthesis [368] though this wasn’t the case in DCs prepared from newborns where low IL-12 production appeared to be due to lack of the α chain. Low DC IL-12 may be one reason why newborns have relatively poor cellular immune response [646]. Mouse cells secrete an IL-12 β-β homodimer that acts as a physiological antagonist of IL-12 [1252, 595, 731]. It is not clear whether the β-β homodimer has a function in humans. IL-12 is produced by macrophages, dendritic cells, neutrophils, microglial cells, keratinocytes and transformed B cells [368, 1189, 804, 1608]. IL-12 is not typically secreted by tumors except certain cancers of B cells origin [2185]. IL-12 production in macrophages is stimulated by bacteria, viruses, parasites [368, 804, 2185] and by contact with T cells through CD40–CD40L interaction [1817]. IL-12 production is enhanced by IFN-γ (Th1 cytokine) and inhibited by IL-10, IL-4 (Th2 cytokines), TGF-β and IFN-α/β [2108]. IL-12 receptor contains two chains, IL-12Rβ1 and IL-12β2. They have homology with the β chain (gp130) of the IL-6R family [318]. Both subunits are required for IL-12 function. β1 is responsible for binding. β2 is the subunit that signals [2200, 2201]. IL-12R is expressed mainly on NK cells and T cells [416]. It is not expressed on Th2 cells [1939]. In PBMC, IL-12R was upregulated by activation with PHA or IL-2 [416]. IL-12R signaling involves Tyk2, Jak2, STAT3 and STAT4 and p38 MAPK [97, 326, 873, 2282, 2303]. IL-12 promotes the development of cellular immune response. It does this by stimulating the production of IFN-γ and Th1-related cytokines in PBL, T cells and NK cells [284, 317, 824, 993, 1028, 1283, 1284, 1554, 2202]. IL-12 also stimulates hematopoiesis. It acts on progenitor cells together with growth factors such as GM-CSF [130, 1431, 1432, 1433]. IL-12 increases DC production of cytokines (GM-CSF, IL-1β, IL-6, IL-12, TNFα and IFN-γ) and antigen presentation [675, 1028, 1412]. IL-12, together with IL-2, stimulates the growth and differentiation of B cells [885]. IL-12 induces LAK cells when added to cultures of PBLs for 3–5 days. LAK cells are also induced by IL-2. At least part of the IL-2

184 effect on LAK appeared due to IL-12 for antibodies to IL-12 decreased the response [597, 1431]. In anti CD3 activated TIL, IL-12 stimulated growth and tumor cell lysis [50]. The effect of IL-12 was not prevented by antibodies to IL-2. When IL-12 was added to cultures with a low, suboptimal dose of IL-2, there was an additive effect on growth and cytotoxicity [50]. Growth induced by IL-12 alone was transient. Maintenance of growth after 72 h required IL-2. IL-2 appears to be more potent than IL-12. It remains to be seen whether IL-12-treated TIL cells have added clinical benefit. IL-12 appeared to be involved in inflammatory diseases such as multiple sclerosis, diabetes, and arthritis [2025]. However, more recent studies suggest IL-23, which is proinflammatory and structurally related to IL-12, may be responsible for certain effects attributed to IL-12, specifically in experimental allergic encephalitis. Further, IL-12 may be more regulatory, suppressing TNF and other proinflammatory cytokines [2279]. IL-12 secretion by antigen presenting cells may be responsible for differences in immune response between females and males [2161]. Females tend to have more vigorous humoral and cellular immunity. Females also have a higher incidence of autoimmune disease. An interesting study showed that activated AP cells from SJL female mice secreted IL-12 but not IL-10 and favored Th1 response. These female mice suffered a high incidence of experimental allergic encephalomyelitis. In males, AP cells produced IL-10 but not IL-12 and had a much lower susceptibility to the disease. Castration or estrogen treatment increased IL-12, decreased IL-10 and increased the incidence of this autoimmune disease [2161]. Anticancer activity of IL-12 may be related to any of a number of direct or extended effects that this cytokine has on Th1 differentiation, CTLs, dendritic cells, macrophages, NK cells, NK T cells, and vascular endothelial cells [1848]. IL-12 induces several cytokines; IFN-γ is a major down stream mediator of IL-12 antitumor activity [1898, 2297]. As a single agent, IL-12 inhibited a number of mouse model tumors [216, 1425]. Mice bearing B16 F10 melanoma, M5076 sarcoma and Renca renal carcinoma benefitted from treatment with IL-12. The antitumor effect did not require NK cells but did depend on T cells, specifically CD8+ T cells [216]. Mice with sarcomas, treated with IL-12, showed decreased tumor growth, increased longevity and in some cases complete regressions. Interferon γ was required for the IL-12 effect and it appeared to be mediated by CD4+ and CD8+ T cells [1425]. Downstream the chemokine IP-10 (interferon inducible protein) was responsible for

Cytokines IL-12-induced, CD8+ mediated immunity to mouse neuroblastoma [1559]. In mice with brain tumors, IL-12 induced a T cell response, decreased tumor size, and prolonged survival time [1690]. These studies suggest that systemic IL-12 may be of benefit in the treatment of CNS cancers. Several studies have shown that IL-12 inhibits angiogenesis in tumors [249, 349, 460, 606, 1898, 1964]. The process involves a complex interaction between several types of cells. IL-12 does not appear to act directly on endothelial cells [460, 1898]. Rather IL-12 induces IFN-γ, which in turn induces IP-10 and Mig; these two chemokines act directly on endothelial cells, to induce vascular damage, clotting and tumor necrosis [52, 64, 350, 1874, 1898, 1964]. Vasostatin is another antiangiogenic factor. In nude mouse studies, treatment with vasostatin or IL-12 inhibited tumor growth. Together they effectively blocked Burkitt’s lymphoma, colon carcinoma, and ovarian carcinoma [2230]. IL-12 engineered cells show promise as anticancer vaccines. IL-12 transfected tumor cells [264, 1152] or fibroblasts [2299] inhibited the growth of some established tumors and induced the rejection of subsequent tumor implants. In a model using an MHC I negative small cell lung cancer growing in nude mice (no capacity for CTL response), cells engineered to produce both IL-15 and IL-12 did not grow; coinjected wild type tumor was also rejected. This study showed that IL-12 could stimulate innate antitumor immunity and that macrophages appeared to be involved [427]. Dendritic cells transfected by bombardment with DNA-coated gold particles containing genes for tumor antigens express antigen and presented it. Co-transfection of these dendritic cells with IL-12 or IFN-α consistently enhanced the induction of specific CTLs in vaccinated mice [2039]. IL-2 and IL-12 interact; receptors are mutually stimulated [416] and they have additive or synergistic effects on NK cells [394], T cells [596, 1284] and macrophages [2157]. Administration of IL-12 with pulse IL-2 induced complete regression of established mammary carcinoma in treated mice [2158]. IL-12 has also been reported to potentiate the effects of tumor cell vaccines engineered to secrete IL-2 for colon cancer [2048] and glioma [981]. Time-release microcarriers have been used to deliver a single dose of adjuvant IL-12 by intratumoral injection. IL-12 alone and especially with GM-CSF, activated T effector/memory cells, killed regulatory T cells and increased CD8+ effector cells. Elimination of suppression and development of cytotoxic T lymphocytes eradicated metastatic tumors [960].

Walter M. Lewko and Robert K. Oldham The preclinical studies with IL-12 were quite encouraging. This cytokine had many effects similar to those of IL-2 and appeared to be less toxic. For these reasons and others, IL-2 and IL-12 may prove to be effective in combination or sequential biotherapy. Similarly, IL-12 gene insertion may prove an effective strategy for increasing tumor immunogenicity. Finally, ex vivo expansion of T cells with IL-12 in addition to IL-2 might be rendered more specific and more cytolytically effective. Studies from our laboratory and others indicate that IL-12 is effective in stimulating cytolytic populations of tumor derived T cells and may be of benefit in the development of T-cell based therapy. There have been several reports on phase I/II studies using IL-12. Patients with cutaneous T cell lymphoma, injected with rhIL-12, had a response rate (CR + PR) of 50% [1661]. IL-12 has also been tested in patients with renal cancer [84, 635, 1379, 1484, 1588, 1650], melanoma [84, 635, 1075, 1367, 1650, 1928] and ovarian cancer [829, 1096]. IL-12 has been used in combination with Herceptin in breast cancer patients [1534] and with IFNα2b in with renal cancer and melanoma patients [832]. Vaccination studies have been carried out using IL-12 as an adjuvant with peptide-pulsed antigen presenting cells [581] and using transfected, IL-12-secreting autologous tumor cells [1928]. Patients have also been injected intratumorally with transfected, IL-12-secreting fibroblasts [917]. Collectively, these studies showed that IL-12 induced immune response; NK cell and specific CTL activities were increased and serum cytokine levels were elevated, in particular, IFN-γ. Toxicities were significant but manageable. Lymphopenia was the most common problem. Clinical activity was observed but the response rates were disappointingly low (generally less than 10%) and not durable. In order to improve the response rate, IL-12 may have to be administered in combination with other cytokines, IL-2 for instance [635].

Interleukin-13 Regulation of Inflammation; Allergy IL-13 is a Th2 cytokine that has a role in parasite infections, allergy and the regulation of inflammation. Recombinant IL-13 has a molecular weight of about 14,000. It is related to IL-4 [1274, 2310]. Activated Th2 CD4+ T cells, CD8+ T cells [908, 1275, 1315, 1597], mast cells [225], eosinophils [2180], basophils, dendritic cells and certain B cell lines produce IL-13. The gene of IL-13 is located on human chromosome 5 together with genes for IL-3, IL-4, IL-5 and IL-9 [1271].

185 The receptor for IL-13 has two subunits, IL-4Rα and IL-13Rα1 [741, 758]. This complex also binds and responds to IL-4. Signaling involves Jak1 and Tyk2 [943, 944]. In addition, there is IL-13Rα2, which may be soluble or membrane bound [244, 445, 1940]. The soluble form appears to be a non-signaling decoy receptor [2197]; the membrane bound form may have certain functions [302, 531]. IL-13 appears to have remarkable effects on T cells; Th2 cells are stimulated and Th17 cells are inhibited. But the receptor for IL-13 is not found on T cells; these effects of IL-13 must be indirect. In DC for example, IL-13 inhibits expression of IL-6, a key factor necessary for Th17 differentiation [985, 1276, 2310]. In monocytes, IL-13 is anti-inflammatory and pro-allergy. IL-13 decreased production of several pro inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12, GM-CSF, G-CSF, MIP-1α, TNF-α, GROα and prostaglandin E2 [422, 1315, 1598, 2198]. IL-13 also inhibited antibody-dependent cell-mediated cytotoxicity [422]. IL-13 caused remarkable changes in monocyte phenotype and adherence. It stimulated levels of complement receptors CD11b and CD11c. IL-13 also stimulated MHC class II levels. IL-13 increased CD23 (an IgE receptor). IL-13 also increased CD49e and CD29, which together form VLA5 (integrin α5β1, a fibronectin receptor) [162]. IL-13 depressed levels of CD64, CD32 and CD16 (IgG receptors) [422]. IL-13 decreased CD14 (LPS receptor) [162, 339]. IL-13 protected animals from the lethal effects of LPS-endotoxemia [1382]. IL-13 increased production of IL-1 receptor antagonist [422, 1408]. IL-13 is also a potent suppressor of nitric oxide production in activated macrophages, epithelial cells and mesangial cells [139, 172, 286, 1733]. IL-13 does this by regulating levels of nitric oxide synthase and substrate arginine available for production [286, 1733]. Further, IL-13 inhibited the production of HIV-1 virus in cultured human macrophages [1343]. In B cells, IL-13 increased proliferation, the synthesis of immunoglobulin, and IgE class switching [328, 398, 1275, 1597]. IL-13 also enhanced the expression of MHCII and CD23 (an IgE receptor) [1277]. Essentially all of these effects of IL-13 in B cells are similar to those of IL-4. IL-13 has various additional activities. In neutrophils, IL-13 induced the secretion of interleukin-1 receptor antagonist [337]. IL-13 also stimulated the production of IL-6 in human keratinocytes [415] and in human microglial cells [1773]. In NK cells, IL-13 induced the production of IFN-γ and cytotoxicity [1315]. IL-13 is a chemotactic factor for human osteoblasts [1131]. In endothelial cells, IL-13 stimulates production of VCAM enhancing T cell adhesion [620]. IL-13 is a chemoattractant for monocytes

186 [1200] and stimulates production of monocyte chemoattractant protein-1 in vascular endothelial cells [633]. IL-13 is involved in inflammatory diseases. IL-13 regulates rheumatoid arthritis. Cultured explants of synovial tissue from patients contained elevated levels of IL-1β, TNF-α and PGE2. Transfection with IL-13 depressed the production of these inflammatory factors [2198]. While IL-13 is generally antiinflammatory, it does have certain proinflammatory effects. IL-13 has a central role in the development of allergic asthma [681, 2175]. IL-13 is a Th2 cytokine. It fosters humoral immune response and the production of IgE. Bronchoalveolar lavage cells from asthmatic patients challenged with ragweed allergen produced high levels of IL-13 [813]. Antigen stimulated CD4+ cells of patients with allergic rhinitis produced more IL-13 compared to normal controls. There are some reports on IL-13, relating to cancer. Renal cell carcinomas produce IL-13 and IL-13R [1474]. Addition of IL-13 to the medium of cultured cells inhibited growth up to 50% [1475]. Human glioma cells contained unusually high levels of the IL-13R. Tumor growth was inhibited by an IL-13-exotoxin conjugate [393, 830]. IL-13 is angiogenic. It stimulated the formation of vessel-like structures from endothelial cells in collagen culture and in vivo it stimulated outgrowth of vessels in the rat cornea assay [574, 703]. The mechanism appeared to involve the production of VCAM, which in its soluble form is angiogenic. IL-4 is used together with GM-CSF for in vitro production of dendritic cells in human vaccination studies. IL-13 substitutes for IL-4 in the production of dendritic cells [33]. It remains to be seen whether IL-13 may be of advantage in the development of anticancer vaccines.

Interleukin 14 B Cell Development and Memory IL-14 was discovered in the conditioned media of Burkitt’s lymphoma cell lines. It was originally referred to as high molecular weight B cell growth factor (HMWBCGF) [39–41]. It is a rather large glycosylated protein with a variable mw of 53–65 kDa, depending on carbohydrate content [42]. IL-14 is produced by PHA stimulated CD8+ T cells, CD4+ T cells (Th1 and Th2), NKT cells, follicular dendritic cells [342, 1704] and B cell lymphomas [552, 553]. Its mRNA has been detected in unstimulated vascular endothelial cells [1455]. IL-14 stimulates B cell growth and differentiation [39, 41]. It also appears to induce and maintain B cell

Cytokines memory [40, 1479]. Mice that were transgenic for IL-14 developed autoimmunity and B cell lymphomas, which resembled systemic lupus in patients [1803]. IL-14 is secreted by B cell malignancies and may have a role in the development of these diseases [552].

Interleukin 15 NK Cell Activity, Maintenance of T Cell Memory IL-15 was discovered as a factor in culture media of monkey kidney epithelium that supported IL-2 dependent growth in a T cell line. The same factor was identified in a human bone marrow stroma cell line [647]. IL-15 was also discovered independently, as the factor IL-T in a T cell leukemia cell line [108, 228]. IL-15 is a 14–15 kDa, 114 amino acid protein. It a member of the four α helix bundle cytokine family to which IL-2 belongs [647]. Many of its activities are similar to those of IL-2. While IL-2 is produced mainly by activated T cells, IL-15 is produced by a rather wide variety of cells types including peripheral blood mononuclear cells, epithelial cells, fibroblasts, placenta, skeletal muscle, heart, lung, liver, kidney [647], and keratinocytes [1334]. IL-15 was not detected in activated T cells [647]. While IL-2 synthesis is regulated at the level of transcription and mRNA stability, IL-15 is regulated at the translational level [107] which is less common but has been shown to occur for certain cytokines (e.g. IL-1β, TNF-α, TGF-β3, TGF-β1, GM-CSF). A pool of mRNA, readily available for translational activation, may allow rapid production of IL-15 when required, as in response to an intracellular infectious agent [107]. The IL-15R is composed of three subunits: IL-15Rα, IL-2Rβ and γC. The β and γc subunits are shared with IL-2 receptors; IL-2 and IL-15 have similar signaling patterns and cytokine activities [626]. And although the IL-15Rα chain binds IL-15 specifically, it is structurally related to IL-2Rα [46, 627]. IL-15R is expressed in a greater variety of cell types than the IL-2R [46, 627]. Interestingly, mast cells respond to IL-15; they have a different IL-15 receptor and signal transduction pathway [1945]. There is a soluble form of the IL-15Rα chain. It is released from cells by proteolysis. It binds IL-15 with high affinity and it acts as an IL-15 antagonist [1369] There is evidence that IL-15 can exist in a membrane-bound form, which is capable of reverse/bidirectional signaling when engaged by its receptor.

Walter M. Lewko and Robert K. Oldham This membrane bound IL-15 was upregulated in monocytes by IFN-γ [219]. IL-15 stimulates proliferation and differentiation of NK cells, B cells, and T cells. Knockout mice lacking IL-15Rα were deficient in NK cells, NK-T cells, CD8+ T cells and γδ T cells [1145, 1457, 2172]. In NK cells, IL-15 stimulated differentiation and activation [249, 1145, 1381, 1477, 2172]. IL-15 upregulated expression of NKGD2 receptors, which are involved in antitumor activity [1930]. IL-15 is also chemotactic for NK cells [29]. In B cells, IL-15 stimulated proliferation and secretion of IgM, IgG, and IgA [71]. In activated cytotoxic T lymphocytes, it increased growth and cytotoxicity [617]. IL-15 stimulated the growth of an IL-2 dependent CTL cell line [228]. IL-15 has been shown to rescue tolerant CD8+ T cells for use in adoptive anticancer therapy [1975]. IL-15 also increased growth and activity of γδ T cells [591, 1457]. IL-15, like IL-2, induced formation of LAK cells in cultures of peripheral blood lymphocytes [228, 617]. In primary cultures of tumor infiltrating lymphocytes, IL-15 replaced IL-2, inducing outgrowth and cytotoxicity of tumor derived activated T cells (TDAC) [1111]. In TDAC cultures that were initially induced with IL-2 and dependent on IL-2 for growth, IL-15 could replace IL-2 for the maintenance of growth [1111]. With increased awareness of the importance of IL-2 in Treg immunosuppressive function, there has been consideration for replacing IL-2, in the development of CTL for immunotherapy, with IL-15 alone or in combination with IL-21 [56, 2275]. IL-15 is synergistic with IL-12 in the induction of mouse Th1 clones [92]; IL-15 appears to have a strong pro-Th1 tendency. In transgenic mice, overexpression of IL-15 augmented Th1 response to infection with intracellular bacterium [1457]. Increased IL-15 is associated with inflammatory diseases including rheumatoid arthritis [1268, 1692], pulmonary sarcoidosis [12], multiple sclerosis [982] and inflammatory bowel disease [972, 1715]. On the other hand, IL-15 has been shown to inhibit allergy. IL-15 overproduction in transgenic mice had a negative effect on the development of allergic asthma, a Th2 disease; IL-15 depressed pulmonary eosinophilia and the production of Th2 cytokines [860]. But it should be noted IL-15 may have some pro Th2 effects; in mice primed with dust mite allergen, IL-15 stimulated IL-5 production by allergen-specific human Th2 clones and resulting eosinophil activation [1354]. While similar in function, there are differences between IL-2 and IL-15. IL-2 has a role in tolerance by inducing T cell suicide [1710, 1933, 2168], in activation-induced T cell death [1092, 1626, 2062] and it is involved in the inhibition of T cell memory maintenance

187 [1026]. On the other hand, IL-15 has anti-apoptotic effects [223] and increases the survival of CD8+ memory cells [1026, 2284]. In mast cells, IL-2 has little or no effect while IL-15 stimulates growth and response to IL-3 and stem cell factor [1945, 2091]. Human T cell lymphotropic virus codes for tax protein; when T cells were infected, tax expression increased the production of IL-15 [94, 95] and IL-15Rα [1227]. IL-15 appears to be responsible for the abnormal proliferation of T cells associated with infection by this virus [95]. The anti-apoptotic effects of IL-15 may have a role in psoriasis, a chronic proliferative inflammatory skin disease. Keratinocytes produce IL-15 and the IL-15 R. Compared with normal epidermis, biopsies of psoriatic skin lesions were high in IL-15 and IL-15 binding capacity [1693]. Cellular immunity is responsible for allograft rejection. A study showed that blocking the effects of IL-15 using soluble IL-15Rα chain prolonged the survival of heart grafts. This indicated that IL-15 has a role in graft rejection and that IL-15 antagonists may be therapeutically useful in the treatment of graft recipients [1845]. In a nude mouse xenograft tumor model, MHC I negative small cell lung cancer cells, engineered to secrete IL-15, grew more slowly and had a slightly reduced take rate; tumors were infiltrated with NK cells and the depletion of NK prevented the antitumor effect. Interestingly, when engineered to secrete both IL-12 and IL-15, the tumors were completely rejected, as were coinjected wild type tumors. Tumors were infiltrated with macrophages, NK cells and granulocytes. Activated macrophages appeared to be the major effector cells [426]. It has also been shown that in NK cells, IL-15 potentiates IL-12-induced secretion of IFN-γ, MIP-1, and IL-10 [521]. These studies show the importance of natural immunity and the synergistic action of IL-15 and IL-12 in the process. And in adoptive immunotherapy, Certain tumors express IL-15R and are candidates for cytokine-based therapies. An antibody (Mikbeta1) to the IL-2/IL-15β subunit has been tested in Phase I and found not beneficial in T cell large granular lymphocyte leukemia [1363].

Interleukin-16 Chemoattractive; Proinflammatory; Immunoregulatory; Anti-HIV IL-16 was originally described in 1982 as the lymphocyte chemoattractant factor (LCF) secreted by mitogenstimulated peripheral blood mononuclear cells [268,

188 270, 360]. IL-16 is produced mainly by CD8+ T cells. It is also produced by CD4+ T cells [2205], B cells [921, 1797], fibroblasts [1770], eosinophils [1129], mast cells [1697], dendritic cells [922] and epithelial cells [70]. IL-16 is also secreted by brain tissue; it may have a role in the interaction between the immune and nervous systems [1038]. IL-16 is synthesized as a proprotein. Nascent IL-16 lacks the usual signal peptide found on most secreted proteins [102]. This lack of a signal peptide is also a characteristic of IL-1 [89], IL-18 [1488] and FGF [3, 884]. Pro-IL-16 is cleaved to its active form by caspase-3, an enzyme in the same family as caspase 1 (ICE) which is responsible for the activation and secretion of IL-1β and IL-18 [2286]. Regulation of IL-16 release depends on the cell type. CD8+ T cells produce IL-16 constitutively and store it in the active form; histamine, serotonin, mitogen and antigen stimulate IL-16 release [271, 1047, 1048]. CD4+ cells, on the other hand, store inactive pro-IL-16; T-cell activation increases caspase-3 activity, pro-IL-16 processing and the secretion of active IL-16 [2203]. In bronchial epithelial cells, histamine, IL-1β and TNF-α stimulate secretion. Dexamethasone inhibits secretion; regulation of IL-16 may be part of the glucocorticoid antiinflammatory effect [70]. In fibroblasts, IL-16 mRNA is produced constitutively. Inflammatory cytokines such IL-1β induce the release of active IL-16 by a caspase-3 dependent mechanism [1770]. The amino acid sequence for IL-16 is distinct; it has little or no homology with other known cytokines. The monomer of IL-16 appears to be inactive; it aggregates to form a 56,000 mw tetramer, which is the active cytokine [102, 359, 360, 2286]. The C-terminal region is particularly well conserved between species and appears to be the region most critical for cell binding and activity [102, 941]. Interestingly, the N-terminal prodomain cleaved from pro-IL-16 by caspase-3 has a cellular function. It translocates into the nucleus and induces G0/G1 arrest, regulating cell division [269, 1631, 2287]. CD4 appears to be the primary receptor for IL-16. Cells that respond to IL-16 invariably express CD4. Anti-CD4 Fab fragments inhibit signaling by IL-16 [270, 358, 359, 360, 361, 1618]. Transfection of CD4 cDNA into CD4−cells enabled the cells to respond to IL-16 [358, 361]. The absence of CD4 in a mutant clone eliminated IL-16 response [1191]. IL-16 binds cell surface CD4 and induces signaling [1021, 1700]. A peptide of IL-16, based on the proposed CD4-binding sequence, blocked IL-16-induced chemotaxis in cultured splenocytes and decreased airway hyperresponsiveness in peptide-treated mice [385]. HIV-1 (gp120) binds CD4

Cytokines and inhibits chemokine signaling. In migrating lymphocytes, IL-16 induces protein kinase C; PKC inhibitors block migration induced by CD4 engagement [1532]. In macrophages, signaling involved the phosphorylation of two MAP kinase family members: the stress activated protein kinase/Jun N-terminal kinase (SAPK/JNK) and p38 MAP kinase. These two pathways are commonly activated by environmental stress signals such as UV irradiation and by certain other proinflammatory cytokines such as IL-1 and TNF-α [1021]. In T cells, the binding of IL-16 to CD4 resulted in activation of the src family tyrosine kinase p56 lck, which is associated with CD4 [1700]. In NK cells that expressed CD4, ligation induced cytokine secretion and cell migration [141]. The data favoring CD4 as the receptor are strong. But there is evidence in knockout mice genetically lacking CD4, that IL-16 was in fact still capable of inducing cytokine production and lymphocyte migration [1239]. In a mast cell line that lacks CD4, IL-16 mediated chemotaxis and signaling in a manner that could be blocked by antibodies to CD9. This suggested that CD9, a cell surface protein known to be involved in migration and cell adhesion, serves as an alternative receptor for IL-16 [1604]. IL-16 influences several cell types. The major effect is chemoattraction but it also has effects on immune response and HIV replication. IL-16 responsive cells are typically CD4 positive [269, 271]. IL-16 sensitive cells include CD4+ T lymphocytes [270, 357, 359, 1532], monocytes [357, 739, 1240], dendritic cells [739, 921, 922, 411], eosinophils [1618], mast cells [1604] and, interestingly, brain cells that are also CD4+ [1038]. In resting T-cells, IL-16 induced signaling and activation with migration and increased IL-2 receptor levels [357, 358, 360, 1532, 1700, 1981]. Several chemotactic agents induce migration in both CD4+ and CD8+ T cells; IL-16 is specific for CD4+ cells [2271]. It modulates and desensitizes certain chemokine receptors, thereby orchestrating T cell recruitment [1613]. Further, IL-16 appears to prime CD4+ cells for IL-2 and IL-15-induced growth [357, 1532]. IL-16 has several roles during antigen presentation. In monocytes and macrophages, IL-16 induced migration and the secretion of several cytokines including IL-1β, IL-6, IL-15 and TNF-α [1240]. In DC’s and macrophages, IL-16 stimulated levels of CD25 (IL-2R) and the costimulatory molecules CD80 and CD83 [739]. Further, IL-16 may act together with thrombopoietin during DC development to induce tolerogenic dendritic cells capable of inducing anergy in T cells [411]. And in lymphnodes, resident B cells and DC’s produce IL-16

Walter M. Lewko and Robert K. Oldham involved in the trafficking of Th cells and inward migration of additional dendritic cells [921, 922]. Delayed type hypersensitivity is mediated by antigen specific Th cells and involves cell movement. The role of IL-16 in DTH was studied in mice [2255]. IL-16 was expressed in DTH tissues but not in controls. Extracts of DTH tissue exhibited chemoattractant activity and IL-16 neutralizing antibodies inhibited this activity. When mice were pre-treated with antibodies, swelling, leukocyte infiltration and chemokine levels associated with DTH were depressed. These results suggested IL-16 had an important role in the recruitment of leukocytes and secretion of cytokines associated with DTH reactions [2255]. While IL-16 stimulates immunity it also has regulatory effects. CD4 is part of the TCR/CD3 complex responsible for antigen-induced activation of T cells. HIV-1 gp120 and antibodies that bind CD4 tend to interfere with activation [1476]. IL-16 also has immunosuppressive effects. It inhibited mixed lymphocyte reactions, deactivated T cells and lowered IL-2 secretion [1686]. Other studies have shown that skin cells transfected with IL-16 were immunosuppressive suggesting that IL-16 might be used to prevent graft rejection [572]. IL-16 induces migration and it also appears to regulate migration. When IL-16 bound CD4, there was a selective loss of chemokine receptor 5 activity. The two receptors were mutually inactivating. When MIP-1β bound CCR-5, T cell migration induced by IL-16 was inhibited. HIV-1 gp120 also appears to inhibit chemokine receptor signaling [1235]. IL-16 is one of several factors secreted by CD8+ T cells, which inhibit viruses. (Others antiviral factors include MIP-1α, MIP-1β and RANTES). Specifically, IL-16 interferes with HIV-1 replication in T cells [43, 103, 1191, 1734, 2294, 2295], macrophages and dendritic cells [2028]. The mechanism of inhibition appears to involve the interaction of IL-16 with CD4. A factor is produced which binds the core enhancer DNA, inhibits HIV-1 promoter activity and as a result blocks viral replication [1191]. IL-16 also blocks HIV-1 uptake by the cells [2028]. Decreased virus entry may be due to IL-16-induced CD-4 downregulation [739]. In a clinical study, HIV-1 infected patients were followed over an 8 year period. During the asymptomatic phase, serum IL-16 levels were maintained or increased. With disease progression, there was a drop in IL-16 [43]. These results support a natural role and potential therapeutic benefit of IL-16 in the control of HIV infection. IL-16 is involved in the development of several inflammatory diseases. IL-16 levels were elevated in fluid and tissue samples from patients with systemic

189 lupus erythematosus, asthma, inflammatory bowel disease and Crohn’s disease [1076, 362, 750, 942, 1236, 1049]. In related animal models, blocking antibodies to IL-16 ameliorated these diseases. On the other hand, IL-16 appeared to regulate inflammatory cytokines in a rheumatoid synovitis mouse model [986]. The synovial infiltrate contained activated CD4+ T cells secreting the proinflammatory cytokines IL-1β, IFN-γ, and TNF-α. CD8+ T cells produced a factor, which lowered the inflammatory cytokines. Anti IL-16 antibodies blocked the effects of this factor while treatment with rIL-16 mimicked the factor. It appeared that CD8+ T cells might have anti-inflammatory effects, which are at least in part mediated by IL-16 [986]. IL-16 has not been extensively studied in cancer. Nonetheless, its involvement in the activation and migration of several cell types including CD4+ T cells, dendritic cells and macrophages suggests it may have a role in antigen presentation, the development of tumor infiltrating lymphocytes and anticancer vaccination. Its antiviral effects may block HIV-related cancers.

Interleukin-17A Proinflammatory, Hematopoietic, Neutrophil Development The IL-17 cytokine family has six members. IL-17A, the first discovered, is a 155 amino acid, 20 kDa glycoprotein [555, 2232]. IL-17 family members have little or no homology with cytokines outside the family [555, 2232]. The gene is on chromosome 2q31. IL-17A was originally referred to as CTL-associated antigen-8 (CTLA-8) [1688]. Herpesvirus saimiri encodes a similar protein [2231]. IL-17 is produced by CD4 T cells, CD8 T cells and γδ T cells [1, 951, 555, 1144, 2232]. Bacterial endotoxin, IL1β, IL-6, IL-18, IL-23 and TGF-β stimulate IL-17A production [852, 1325, 712]. IL-17A is produced primarily by CD4 T-cells that are now referred to as Th17 cells [718]. These cells along with Th1, Th2 and Treg cells are major controllers of immune response. It is now believed that Th17 cells are responsible for many autoimmune diseases that were originally classified as Th1 diseases. The discovery of Th17 cells solved a problem in the observation that mice lacking the Th1 cytokine IFNγ suffered more, not less, autoimmunity than controls. The development of Th17 lineage appears to have two phases. In the first phase, Th0 cells differentiate into Th17 cells in response to TGFβ and IL-6 (or IL-1β; a proinflammatory signal is needed) and then there is an expansion/survival phase,

190 which is stimulated by IL-23. Th17 differentiation is inhibited by IL-2 and IFNγ (type 1 cytokines), IL-4 and IL-13 (type 2 cytokines), and IL-35 (Treg cytokine) [149, 718, 985, 1025, 1218, 1451, 1536, 2070]. Receptors for IL-17 have been found in most tissues tested. There are at least five members of the IL-17 receptor family. These receptors have little or no homology to other known cytokine receptors [2231, 2233, 1370]. IL-17 functions in host defenses against infection. Bacterial products induce IL-17 secretion [852]. IL-17 is required for defence against several types of infection, for example, bacterial pneumonia [2236]. IL-17 is involved in hematopoiesis. It appears to promote recovery in response to radiation damage [1961]. IL-17 is involved in inflammation. It stimulates production of molecules such as proinflammatory cytokines, chemokines, CSFs, prostaglandin, MMPs, NOS and antimicrobial peptides in a variety of cells including macrophages, fibroblasts, epithelial cells, keratinocytes, endothelial cells, mesothelial cells and dendritic cells [231, 513, 555, 1768, 878, 890, 1980, 2178]. IL-17 has particularly remarkable effects on neutrophil production and tissue infiltration. IL-17-treated fibroblasts release CSFs that stimulate neutrophil differentiation [555]. In transgenic mice that produced excess IL-17, there was enhanced hematopoiesis in general and granulopoiesis in particular. Peripheral WBC counts increased fivefold and neutrophils increased tenfold [1766]. In gastric mucosa, infection by H. pylori stimulated production of IL-17. IL-8 levels were increased and the tissue became infiltrated. Antibodies to IL-17 inhibited IL-8 secretion and antibodies to IL-8 blocked PMN leukocyte migration [1174]. Similar responses occurred in inflamed intestinal and bronchial epithelial cells [93, 1046]. Dysregulation of IL-17 appears to have a role in the pathology of allergic and autoimmune inflammatory diseases [18, 275, 276, 279, 1011, 1174]. In mice with collagen-induced arthritis, IL-1 and IL-17 levels were elevated; blocking antibodies for IL-17 alleviated joint destruction whereas IL-1 blockade had no effect, suggesting IL-17 was the more direct cause of arthritic damage [1169, 1414]. In patients with arthritis, synovial fluid IL-17 stimulated formation of osteoclasts, which caused tissue destruction [1011]. IL-17 also increased the production of gelatinase in human macrophages [879], aggrecanase and metalloproteinase in chondrocytes [230] and nitric oxide in osteoarthritic cartilage [88]. IL-17 has potential in cancer therapy. IL-17 stimulates bone marrow stromal cells to produce G-CSF, SCF and IL-8 for the production and mobilization of hematopoietic stem cells [1767]. This may be useful for stem cell transplantation. IL-17 promotes the maturation of

Cytokines dendritic cells [57]. This may have a role in cancer vaccination. It has also been shown that Chinese hamster ovary cells engineered to produce IL-17 were less invasive and metastatic [764]. There is also evidence that IL-17-transfected tumor cell vaccines can stimulate the generation of tumor specific cytotoxic T cells [133]. In a study on B16 melanoma in mice, tumor antigen specific CD4 Th cells that had been polarized in culture, were adoptively transferred into mice with growing tumors; interestingly, the Th17 cells had superior antitumor activity compared to Th1 cells, the type traditionally associated with anticancer activity [1395]. However, there are reports that by its proinflammatory properties, IL-17 may stimulate development of cancer. It has been shown that IL-17 increased the growth of cervical tumors transplanted in nude mice [1967]. In prostate, normal tissues produced little IL-17, but most benign prostate hyperplasia specimens and prostate cancers showed high IL-17 expression, mainly in T-cells but also in epithelial cells [1887]. In lung cancer cell lines, IL-17 had no direct effect on tumor cell growth but had a remarkable stimulatory effect on secretion of several angiogenic CXC chemokines. In SCID mice, lung cancer cells engineered to produce IL-17 grew larger and were more vascular than controls suggesting a role for IL-17-induced chemokines and angiogenesis in tumor growth [1467].

Interleukin-17B Proinflammatory Cytokine IL-17B has 27% amino acid identity with IL-17A. The gene is on chromosome 5q32–34 [1113]. IL-17B mRNA is produced by cells in the pancreas, small intestine, and stomach but not by activated T cells. IL-17B does not bind the IL-17 receptor. Rather it specifically binds the protein IL-17Rh1 (IL-17 receptor homolog 1). At least one additional IL-17 family member, IL-25, also binds IL-17Rh1 [1073, 1807]. The mRNA for this receptor has been detected in pancreas, kidney, thyroid, liver, brain and intestines. Receptor levels in intestine were increased during inflammation [1807]. In a monocyte cell line, IL-17B stimulated production of IL-1β and TNF-α while IL-17 had little effect. In activated fibroblasts, IL-17 stimulated production of IL-6 while IL-17B had no effect [1113]. These results showed that though the two cytokines are structurally related, they have distinct activities. In mice, IP injection of rIL-17B induced influx of neutrophils. This was probably not a direct effect but due to stimulated release of chemotactic factors [1807].

Walter M. Lewko and Robert K. Oldham

Interleukin-17C Proinflammatory Cytokine IL-17C has approximately 27% homology with IL-17. The distribution of IL-17C mRNA appears to be rather restricted. It was found in prostate and fetal kidney. Activated T cells did not produce it. The gene for IL-17C is on chromosome 16q24. The receptor used by IL-17C is not known; it does not appear to bind the IL-17 receptor [1113]. In a monocyte cell line, IL-17C stimulated production of IL-1β and TNF-α while IL-17 had no effect. In activated fibroblasts, IL-17 stimulated IL-6 secretion while IL-17C had no effect. These results showed that while IL-17 and IL-17C are related, they have distinct activities [1113]. Expression of IL-17C in mouse lung by adenovirus-IL-17C infection promoted neutrophilia and inflammatory gene expression such as IL-6 and IFNγ [825].

Interleukin-17D Proinflammatory, Inhibitor of Hematopoiesis IL-17D has 202 amino acids and as such it is the largest member of the IL-17 family. IL-17D is most homologous to IL-17B with 27% identity. The gene is on chromosome 13p11. IL-17D mRNA is expressed in a number of tissues including skeletal muscle, brain, adipose, heart, lung and pancreas. It was poorly expressed on activated CD4 T cells, CD8 T cells, monocytes and activated B cells [1880]. In cultured endothelial cells, Il-17D stimulated production of IL-6, IL-8 and GM-CSF. IL-17D had a suppressive effect of myeloid colony formation in agar gel assays [1880].

Interleukin-17E: see Interleukin-25 Interleukin-17F Proinflammatory, Asthma, Inhibitor of Angiogenesis Interleukin-17F is also referred to as ML-1 [930]. It has high homology with IL-17. The gene is on chromosome 6p. Activated CD4 T cells, monocytes, basophils and

191 mast cells produce IL-17F. It was not expressed by CD8 T cells or B cells [1881]. IL-17F is a homodimer; heterodimers with IL17A chain occur in mouse Th17 cells. Potency was intermediate of the two homodimeric forms in a chemokine release assay [1122]. The receptor for IL-17F is the protein IL-17RC. This receptor is related to IL-17R and IL-17A also binds it [1030]. IL-17F has a remarkable effect on vascular endothelial cells. It stimulated the production of endothelial IL-2, TGF-β and monocyte chemoattractant protein-1. In an in vitro assay for angiogenesis, IL-17F inhibited the formation of tubular, vessel-like structures [1881]. IL-17F may be acting directly or through the induction of cytokines such as TGF-β, which inhibit angiogenesis [1219, 1881]. IL-17F also stimulates the production of IL-6, IL-8 and GM-CSF in bronchial epithelium and it regulates cartilage matrix turnover [833, 929]. IL-17F is involved in asthma. In mice, expression of IL-17F in lung by adenovirus-IL-17F infection promoted neutrophilia and the expression of inflammatory genes such as IL-6 and IFNγ [825]. In humans, expression of IL-17F (but not IL-17) was increased in lung biopsies of asthma patients after allergen challenge. When added to cultured bronchial epithelial cells, IL-17F stimulated the production of IL-6 and IL-8 and cell surface ICAM-1; it recruits neutrophils into bronchial airways [930]. Polymorphism in the IL-17F gene appears to influence susceptibility to asthma. Japanese subjects with arg substituted at position 161 were protected from the disease; this variant blocked the induction of IL-8 by wild type IL-17F [931]. There are no reports on cancer for this relatively new cytokine, though IL-17F is of interest for its proinflammatory and antiangiogenic effects.

Interleukin-18 Interferon γ, NK and Th1 Response, Regulation of Inflammation IL-18 was discovered in mice with severe hepatitis induced by Propiobacterium acnes and the endotoxin LPS [1417]. IL-18 was originally called interferon-γ inducing factor (IGIF) for its remarkable stimulation IFNγ secretion. It is synthesized as a 192 amino acid proprotein, which is processed to the 156 amino acid active form [2045]. IL-18 is similar to IL-1β in that its nascent protein lacks the signal peptide usually found on secreted proteins [120, 1489] and proteolytic activation of the proprotein is carried out by caspase-1 (ICE) [688].

192 Activation is necessary for secretion; caspase-1-deficient macrophages do not secrete IL-18 as usual when treated with LPS [512, 618, 688, 1779]. IL-18 is produced by macrophages, dendritic cells [1489, 1600] and Kupffer cells [1779, 2045]; levels are increased by microbial infection or LPS treatment. IL-18 is also produced by adrenal cortex (in the area of cortisol production) [340], by skin keratinocytes [1895] and by airway epithelium [234]. In adrenal cortex, expression was increased by cold stress as a possible link between the environment and immune response [340]. Treating mice with contact allergens stimulated IL-18 expression in keratinocytes [1895]. In airway epithelial cells, IL-18 levels were related to Th1 status [234]. IL-18BP is a binding protein found in circulation, which has relatively high affinity for this cytokine. As a feedback loop, IL-18 induces IFNγ and interferon induces IL-18BP. It competes with the receptor for free IL-18 and in this way regulates IL-18 activity [963, 1465, 1640]. A relative lack of IL-18BP occurs in patients with hemophagocytic syndrome, a disease with elevated free IL-18 and characterized by a strong Th1 response [1260]. The IL-18R contains two subunits [180, 965, 2014]. The α subunit binds IL-18 and the β subunit performs signal transduction. Although the β subunit does not bind IL-18, as part of the receptor complex it enhances affinity for IL-18 [965]. Interestingly, the IL-18Rα subunit was originally discovered and named IL-1 receptor related protein (IL-1Rrp) for its molecular similarity to the IL-1 receptor, though this protein did not actually bind IL-1 [439, 1488, 1538]. At the time, it was referred to as an orphan receptor for lack of a known ligand; later it was determined to be the receptor for IL-18 [795, 1986, 2014]. The IL-18Rβ subunit is also a member of the IL-1R family. It is related to the IL-1R accessory protein. When IL-18 binds, it activates a pattern of cell signaling which is similar to that of IL-1 [392, 1000]. In knockout mice genetically defective for the IL-18 receptor, development of Th1 cells was impaired. NK cells had decreased cytotoxicity and IFN-γ production [796]. Overexpression of IL-18 in transgenic mice resulted in high levels of IgE, IgG1, IL-4, IL-5, IL-13 and IFN-γ, reflecting both Th1 and Th2 cell activation [796]. IL-18 and IL-12 have similar effects on several types of cells. These effects are often synergistic. In certain cases, IL-12 appeared to be required for IL-18 response. This seems to be due to the ability of IL-12 to increase IL-18 receptor levels. IL-12 upregulates both α and β subunits. Increased IL-18 receptor levels enhanced the capacity for response [521, 963, 1724, 2214, 2253]. IL-18 and IL-12 synergistically induce IFN-γ in T cells [998,

Cytokines 1300, 1652, 1894, 2253], B cells [2253], NK cells [1954, 2009], macrophages [1393, 1741] and in DCs [573]. IL-18 stimulates activated Th1 cells to grow and produce IFN-γ [1489]. It is well known that Th1 cell differentiation requires IL-12. Il-18 acts synergistically with IL-12 [1300]. IL-12 initiates differentiation; IL-18 intensifies the response. IL-2 is a costimulator of Th1 differentiation and growth [998]. IL-18 and IL-12 both increase IL-2 receptor levels. IL-18 stimulates Th1 cells but it did not appear to influence Th2 cell growth or production of IL-4 and IL-10 [998]. With Th2 differentiation, IL-18 receptor expression is lost and with it capacity to respond to IL-18 [2214]. Although IL-18 and IL-12 have related activities, there are differences. IL-12 initiates differentiation of Th1 cells; IL-18 does not initiate but enhances Th1 differentiation [1487, 1652]. IL-18 induced Th1 cells to produce IL-2; IL-12 did not [998]. In mouse activated PBLs, IL-18 stimulated GM-CSF production and depressed IL-10; IL-12 did not [2046]. IL-18 is generally considered a proTh1 cytokine, but IL-18 does stimulate Th2-related responses [995, 1033, 1199, 1684, 2162]. B cells contain receptors for IL-18 and IL-12. These two cytokines synergize in the production of immunoglobulin and IFN-γ [13]. IL-18 also stimulates IL-4 secretion in NKT cells [1085, 2251, 2254] and IL-13 secretion by NK cells [797]. Overexpression of IL-18 by B cells and T cells in transgenic mice induced high levels of both Th1 and Th2 cytokines [796]. IL-18 also acts synergistically with interferon-α/β in the secretion of IFN-γ by T cells. Virus infection induced macrophages to produce IFN-α/β and IL-18 (but not IL-12). Conditioned media from these macrophages induced IFN-γ in T-cells. Neutralizing antibody to IFNα/β blocked secretion of IFN-γ; antibody to IL-12 had no effect. These studies showed that the type I interferons acted synergistically with IL-18 in the production of immune interferon [1725]. IFN-α increased the levels of an adapter molecule (MyD88), which facilitated IL-18 receptor signaling [1724]. IL-18 has remarkable effects on NK cells. IL-12 and IL-2 are also well known stimulators of NK cells. IL-18 in combination with IL-12 or IL-2 induced much higher NK cell proliferation, IFN-γ secretion and antitumor activity than IL-12 or IL-2 induced when added alone or together [2009]. These results suggested an obligatory role for IL-18 in NK proliferation and function [2009]. Cell killing by Fas-mediated apoptosis is stimulated by IL-18. IL-18 upregulates Fas ligand (discussed below) in NK cells [2035] and in helper T-cells [381].

Walter M. Lewko and Robert K. Oldham In NK cells, IL-18 acts together with IL-2 and IL-12. The IL-18 treated NK cells were positive for perforin but killed target cells exclusively by FAS engagement [2035]. By its ability to stimulate interferon-γ secretion, IL-18 also stimulates Fas levels in many target cells. IL-18 has a role in the induction and regulation of inflammation. Neutrophils respond to IL-18 with increased production of cytokines, CD11b (complement receptor) and granule release. In a mouse model, IL-18 neutralizing antibodies suppressed footpad inflammation caused by carragean injection [1102]. PBLs treated with IL-18 produce pro-inflammatory TNF-α and in turn IL-1β and the chemokines IL-8 and MIP-1α [1601]. IL-18 also brings about regulation of inflammation. In T cells and NK cells, IL-18 together with IL-2 stimulated the production of IL-13, an inhibitor of inflammation [797]. IL-18 appears to be involved in rheumatoid arthritis [652, 1103]. Elevated levels of IL-18 are found in arthritic synovial fluid and serum [652]. In a collageninduced arthritis model, mice lacking IL-18 had a greatly reduced incidence and severity of arthritis compared with control mice. T cells from IL-18 deficient mice had a reduced capacity for antigen-induced cell division and secretion of proinflammatory cytokines [2131]. IL-18 has also been implicated in other inflammatory diseases including Crohn’s disease of the bowel [1577] and in sarcoidosis [662]. IL-18 appears to have a role as both a mediator and regulator of angiogenesis. In culture, IL-18 stimulated vascular endothelial cells to migrate and form capillary-like structures. In animals, implants containing small amounts of IL-18 stimulated the growth of new vessels [1535]. As a regulator, it has been shown that IL-18 suppressed angiogenesis and it did this by blocking bFGF-stimulated cell division in endothelial cells [240]. IL-18 has anticancer activity in many tumor models. Elevated IFNγ and NK cells are generally required, but depending on the system CD4+ or CD8+ T cells may be involved [1301, 1302, 1509]. Combinations of IL-2 plus IL-18 [1854, 2156] and IL-12 plus IL-18 [1117, 1509, 1624] generally have synergistic effects. These studies have been extended to an IL-2/IL-18 fusion protein [4], which produce NK and T cell responses with lower toxicity. Additionally, IL-18 is being tested, with some success, in cancer vaccines [840, 1228, 1852, 1969, 2210, 2224]. Further, it has been reported that NKT (generally considered to be a regulatory and immunosuppressive cell type) may have anticancer activity. NKT cells isolated and cultured with IL-12 plus IL-18 produced activated cells that inhibited tumors when transferred to mice [118].

193 It has been suggested that IL-18 may be involved in cytotoxic T cell development by acting as a bridge between the innate and adoptive immune systems [1962]. IL-18 stimulates IFN-γ levels and activates NK cells (Fas L) causing tumor cell apoptosis. This provides apoptotic bodies for dendritic cell antigen presentation and the induction of specific CTLs [1962]. IL-18 is being tested in cancer patients. In a phase I trial, IL-18 was administered to 28 patients, mainly renal cancer and melanoma. Increases in serum IFNγ, GM-CSF and other indications of cell activation were observed. There were two partial responses; further studies are warranted [1651]. It should be mentioned that while IL-18 has anticancer activity, certain cancers may be able to exploit the IL-18 system. For example, IL-18 binding protein is naturally secreted in response to IFNγ and it downregulates IL-18 activity. Many tumors secrete IL-18 BP. In this way the anticancer effect of IL-18 may be inhibited by the tumor [1545]. In certain cancers, it has been reported that IL-18 stimulates growth, angiogenesis, adhesion to endothelium and invasion [246, 962, 1535, 2075]. In these tumors IL-18 may contribute to progression. Further, IL-18 may have a role in immune escape. IL-18 upregulates Fas ligand in B16F10 melanoma cells. It is possible that tumor cell-Fas ligand may induce apoptotic death of effector cells resulting in immune suppression [311].

Interleukin-19 Inflammation IL-19 was discovered in LPS-stimulated monocytes [582]. It is a member of the IL-10 cytokine family (IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, IL-29). The gene for IL-19 is located together with IL-10 on chromosome 1q31–32. A homodimer appears to be the active form [582]. LPS and GM-CSF stimulate monocytes to produce IL-19. In LPS activated monocytes, IL-4 and IL-13 each enhanced [584] and IFNγ depressed IL-19 secretion [583]. Il-19 is also produced by keratinocytes of psoriasis lesions [1660]. IL-19 binds and signals through the IL-20 receptor (IL-20Rα/IL20Rβ). IL-19 does not bind the IL-10 receptor. IL-20R is expressed in skin, lungs, reproductive organs and various glands. Receptor levels are in inflamed tissues [462, 583, 1540]. Monocytes treated with IL-19 secrete IL-6, TNF-α, and reactive oxygen species [1125]. In T cells, IL-19 appears to favor Th2 differentiation and IL-4 secretion.

194 [583]. In PBMCs IL-19 induces IL-10 secretion and its own production. IL-19 production is in turn downregulated by IL-10 [902]. IL-19 appears to be involved in asthma [1124] and psoriasis [1114, 1660]. Asthmatic patients had twice the serum levels of IL-19 compared with controls; levels of IL-4 and IL-13 were also elevated. In culture, activated T cells treated with IL-19 secreted Th2 cytokines IL4, IL-5, IL-10 and IL-13 [1124].

Interleukin-20 Inflammation, Skin Development and Immunity IL-20 is a member of the IL-10 family. The gene for IL-20 is located on chromosome 1q31–32 together with the genes for IL-10, IL-19 and IL-24 [582]. IL-20 is produced by keratinocytes [1641], monocytes [2187] and glial cells [799]. IL-20 binds two receptors (IL-20Rα/IL20Rβ) and (IL-22Rα/IL20Rβ) [169, 1540]. IL-20R levels are elevated in psoriatic skin [169]. IL-20 signals by STAT3 activation [462, 463]. IL-20 appears to regulate keratinocyte involvement in skin inflammation [1641]. Overexpression of IL-20 in transgenic mice resulted in aberrant differentiation of epidermis, keratinocyte proliferation and abnormal skin resembling psoriasis. These mice also lack adipose tissue and their T lymphocytes show high apoptosis; they die soon after birth [169]. IL-20 stimulates the growth of CD34+ multipotential progenitors in a unique way, without imparting any tendency towards a particular developmental pathway, erythroid, granulocytemacrophage or megakaryocyte [1139]. In humans, IL-20 appears to be involved in psoriasis and skin inflammation [2128]. In lesions from patients, IL-20 and its receptor were elevated. IL-20-treated T cells produced increased levels of keratinocyte growth factor [2128]. Glial cells can produce IL-20 and LPS stimulates release. This suggests that IL-20 may have a role in inflammation of the brain [799]. In arthritis patients, synovial fluid contains elevated IL-20; synovial fibroblasts treated with IL-20 secreted IL-6 and IL-8. In rat model, IL-20 blockade decreased severity of arthritis. It appears that IL-20 is involved in joint inflammation [809]. There is evidence for IL-20 being pro-angiogenic and anti-angiogenic. Endothelial cells contain IL-20 receptors. When treated with IL-20 these cells proliferate, migrate and form tubular structures; bFGF, VEGF and

Cytokines IL-8 are released. However, in studies of lung cancer, IL-20 inhibited COX-2 and prostaglandin E2 production in epithelial and endothelial cells, and it inhibited PMA-induced angiogenesis [805, 746].

Interleukin-21 NK, T and B Cell Development, Immunomodulation IL-21 is a pleiotropic cytokine, which is structurally related to IL-2, IL-4, and IL-15 [78, 1520, 1540]. IL-21 is produced mainly by Th2, Th17 and follicular T cells [316]. The receptor for IL-21 has two subunits, IL-21Rα and IL-2Rγ. IL-21Rα is related to IL-2Rβ and IL-4Rα; IL-2Rγ is the subunit common to several IL-2 family receptors. The IL-21 receptor is expressed on T, B, and NK cells [78, 699, 1520]. T cells are stimulated and regulated by IL-21. Typical Th2 responses (e.g. to schistosomes, airway inflammation) depend on IL-21 [570, 1561]. IL-21 also supports the clonal expansion of virus and tumor antigen specific effector CD8+ T cells [2059, 1120, 1121]. IL-21 sustains the expression of CD28, an important costimulatory receptor [34, 1134]. It increases or decreases IFN-γ production depending on conditions [1903, 1932, 2208]. But IL-21 also regulates CD8+ T cell expansion when required by inducing apoptosis [113]. In mice, plasmid DNA vaccines expressing HIV-1 env glycoprotein showed increased T cell response when combined with IL-21 [177]. At least in part, increased generation of CD8+ cytotoxic T cells may be the due to a suppressive effect IL-21 has on Foxp3 regulatory T cells (Li 08) or the response of Th cells to Tregs [1550]. IL-21 is involved in NK cell expansion, function and regulation. In human bone marrow cultures, IL-21 stimulated NK cell proliferation and maturation [1539]. In cultured mouse cells, IL-21 increased NK function even as it depressed growth [919]. IL-21 appears to be able to modulate cytotoxicity towards certain target cells. In human NK, IL-21 depressed the NKG2D receptors while enhancing others [227]. When NK cells were treated with IL-21, human breast cancer cells coated with Trastuzumab were more effectively killed by ADCC [1655]. IL-21 influences development of B cells and Ig production. IL-21 induces differentiation of naive and memory B cells into antibody-secreting plasma cells [501, 1521]. Depending on the conditions, IL-21 may increase or decrease growth or induce apoptosis [1540, 1285]. IL-21 regulates Ig class switching. It is a switch

Walter M. Lewko and Robert K. Oldham factor for IgG1 and IgG3; IL-21 depresses IgE [1522, 1551, 1931, 2196]. IL-21 inhibits dendritic cell activation and maturation [192, 1902]. It is part of a negative feedback loop between dendritic cells and T cells in which activated dendritic cells stimulate T cells, inducing them to produce cytokines including IL-21 which in turn inhibits dendritic cells [1902]. IL-21 is involved in the Th system and regulation of inflammation. IL-21 is produced by Th17 cells and in an autocrine manner IL-21 sustains expression of the Th17 transcription factor RORγT and secretion of IL-17 [2129]. IL-21 has been studied in a number of different experimental tumors. It has been shown to have anticancer activity related to T cells [338, 427, 577, 1361, 1420], B cells [1835], NK cells [338, 577, 1420, 1849, 1951, 2101] and NKT cells [1849]. Several of these studies used IL-21 as an adjuvant in vaccines. Addition of IL-15 to IL-21 improved the response [177, 1420, 2275]. Regulatory T cells may limit vaccine immunogenicity. In one mouse study, use of IL-21 secreting tumor cells together with anti-CD25 antibodies to block regulatory T cells increased cure rate from 17% to 70% [338]. IL-21 has direct effects on the growth of some hematologic malignancies. In B cell chronic lymphocytic leukemia, IL-21 is able to induce apoptosis in CpG-treated [883] or CD40-stimulated cells [387]. On the other hand, IL-21 is a growth factor for certain adult T-cell leukemia [2042] and melanoma cells [198]. IL-21 is in clinical studies. Melanoma and renal cell carcinoma patients, treated with IL-21 in phase I, tolerated the cytokine reasonably well and showed evidence of anticancer activity [383, 1187]. IL-21 is also being used to develop more effective CD8+ T cells for adoptive immunotherapy [760]

195 There is a soluble IL-22 binding protein, which antagonizes IL-22 activity. The IL-22BP has homology with IL-22R, but it lacks the transmembrane cell-binding region. In effect, the IL-22BP is a decoy receptor [463, 1013]. IL-22 levels rise in LPS-treated animals. IL-22 appears to function during inflammation and immune response. IL-22 mainly targets non-circulating cells involved in innate immunity including epithelium, keratinocytes, and hepatocytes. In keratinocytes, IL-22 induces hyperplasia, migration, antimicrobial proteins and production of proinflammatory molecules [178, 2191]. In liver cells, IL-22 increases the production of acute phase proteins [466]; it also induces LPS binding protein, which neutralizes this proinflammatory and Th17-inducing microbial product [2189]. IL-22 also has a mild inhibitory effect on IL-4 production by Th2 cells [2212]. IL-22 levels are elevated and it appears to be involved in certain inflammatory diseases including rheumatoid arthritis [847], psoriasis [1186, 2191, 2291], inflammatory bowel disease and Crohn’s disease [49]. IL-22 does stimulate inflammatory molecules, and may cause pathology but it also appears to be involved in resolution of inflammation and tissue regeneration. In mice, IL-22 has been shown to ameliorate experimental autoimmune myocarditis [287] and ulcerative colitis [1922]. In pancreatic acinar cells, IL-22 induces pancreatitis-associated protein [11]. IL-22 has a protective effect during liver injury. IL-22 induced antiapoptotic factors in hepatocytes and stimulated growth suggesting a role for IL-22 in liver cell survival and tissue repair [1611]. With regard to cancer, there is a report that IL-22 inhibited the growth of EMT6 breast cancer in treated mice by cell cycle arrest [2125].

Interleukin-23 Interleukin-22 Inflammation IL-22 is a member of the IL-10 cytokine family. It was originally referred to as IL-10-related T cell-derived inducible factor (IL-TIF). Activated Th17, Th1 and Th2 cells produce IL-22 [464, 465, 1123, 1186, 2188, 2212]. IL-22 is also expressed by IL-9 stimulated mast cells [464] and by activated NK cells [2190] The IL-22 receptor consists of two chains, IL22Rα and IL-10Rβ. Each chain separated binds IL-22 but both chains, associated, are necessary for activity. Signaling is through STAT 1, 3 and 5 [1012, 2212].

T cell Stimulation, Inflammation, Regulator of Adaptive and Innate Immunity IL-23 is composed of two subunits. The p19 subunit is related to p35 of IL-12. And p40 is identical to p40 of IL-12. Activated dendritic cells, macrophages and keratinocytes produce IL-23 [1502, 1533, 1575]. The IL-23 receptor is composed of IL-23 Rα and IL-12Rβ1. IL-23 activates Jak-STAT signaling characteristic of the IL-12Rβ1 subunit [1573]. The IL-23 receptor is expressed on DC, macrophages and T cells [1533]. The effects of IL-23 are similar to but distinct form IL-12. In mouse cells, IL-23 induces proliferation of memory T cells (CD4+ CD45RB low). IL-12 does not [1502].

196 IL-23 does not stimulate IFN in mouse T cells. In human T cells, IL-23, like IL-12, increases IFN-γ production, though to a lesser degree than IL-12. IL-23 also stimulates growth in human memory CD45RO T cells [1502]. T cells express ICOS (inducible costimulator) protein, which is a CD28 family member. ICOS is important for enhancement of Th and CD8+ T cell responses. In human T cells, ICOS is increased by IL-23 and IL-12; it is inhibited by IL-4 [2122]. This may provide a powerful way for IL-23 and IL-12 to amplify T cell responses. IL-23 induces inflammation [1502]. IL-23 appears to be involved in encephalomyelitis, arthritis and inflammatory bowel disease [298, 363, 1399, 2242, 2279]. Transgenic mice overexpressing IL-23 p19, had severe multiorgan inflammation, impaired growth, infertility and early death. Blood contained increased levels of neutrophils, IL-1α, TNF and acute phase proteins [2132]. TGF-β and IL6 induce Th17 differentiation; IL-23 stimulates and maintains the Th17 cells [10, 1058]. The reason for heightened inflammation and pathology may be explained by the lack of regulatory IL-10 in IL-23differentiated T cells. When induced with TGF-β and IL6 only, T cells secrete IL-10 in addition to IL-17 and the other proinflammatory cytokines [1267]. Microbial products induce macrophages and DCs to secrete IL-23. IL-23 is required for neutrophil production. Mice that were p40-deficient lacked neutrophils; treatment with IL-23 (but not IL-12) restored neutrophil levels [1841]. In dendritic cells, IL-23 stimulated IL-12 production, as did IL-12 itself. Further, IL-23 treated DC’s promoted immunogenic antigen presentation of an otherwise tolerogenic peptide, which IL-12 did not do [128]. IL-23 has been shown to be an effective adjuvant in anticancer vaccines for colon carcinoma [1143, 1795, 2112], melanoma [1143, 1497, 1516], and glioma [2267]. Prior depletion of regulatory T cells enhanced antitumor response. IL-23 induced IFNγ; CD8+ T cells mediated antitumor immunity [1497]. There is also evidence for non-T cell mediated antitumor immune effects of IL-23 in human pancreatic and esophageal tumors in nude mice [1794, 2043]. Some human tumors have elevated IL-23 levels. There is evidence that the proinflammatory nature of IL-23, compared with IL-12, may actually be responsible for the tumor growth and progression in certain cancers [1057].

Interleukin-24 IL-24 was discovered in melanoma cells that had been treated to induce differention, using IFNβ plus mezerein (a protein kinase c activator) [889]. IL-24 was originally

Cytokines called mda-7 (melanoma differentiation antigen-7). Its counterpart in mice is called FISP [1738, 1790]. In human cancer, Mda-7 is a tumor suppressor; expression of the gene in melanoma cells inhibited proliferation and induced apoptosis [889]. Loss of mda-7 expression was associated with disease progression [488]. Later, when the cytokine nature of mda-7 became clear, it was designated IL-24 [261, 278, 810, 1731]. IL-24 is a member of the IL-10 family [261]. The gene is located in the IL-10 cluster on chromosome 1q31–32 [810]. Activated T cells and macrophages produce IL-24. It is also expressed by many tumors [1582, 2187]. Expression is stimulated by IL-1, IL-2 and several other cytokines but not by Th2-related cytokines [1582]. IL-24 signals through two receptors IL-20R1/IL-20R2 and IL-22R1/ IL-20R2 [2104]. IL-24 is a pro-inflammatory cytokine; it stimulates the production of IL-6 and TNF [261]. Overexpression of MDA-7 in tumor cells, by adenovirus-IL-24 gene transfer, suppresses a variety of cancer types including breast, cervical, colorectal, glioma, lung, mesothelioma, ovarian, pancreas, prostate and sarcoma [242, 277, 278, 810, 1071, 1201, 1712, 1731, 1914, 1915, 1916, 2289]. Susceptible tumors exhibit decreased growth and increased apoptotic death; IL-24 also inhibits angiogenesis [295, 1617, 1731]. Radiation resistance may be reversed by IL-24 treatment [1914, 1915, 2218]. Conversely, IL-24 resistance, as due to overexpression of antiapoptotic survival factors, may be reversed by irradiation [1914]. There is much interest in IL-24 for the treatment of cancer [540, 854, 1070]. IL-24 depresses growth, induces apoptosis, inhibits angiogenesis and increases radiosensitivity in a variety of tumors, with little toxicity towards normal cells. There is particular interest in the use of mda-7 for gene therapy [854]. Adenovirus-IL24 infected cells which produce IL-24 are inhibited; bystander tumor cells are also affected [1913]. Several experimental adenovirus constructs have been developed to optimize the process. INGN 241 has successfully completed phase I trials [366, 2010].

Interleukin-25 Proinflammatory; Regulation of Th17 IL-25 is a member of the IL-17 family. It is also referred to as IL-17E. Th2 cells, eosinophils, mast cells, macrophages and microglial cells produce IL-25 [844, 915, 985, 1807, 2109]. IL-25 binds IL-17Rh1, the same receptor used by IL-17B [1073, 1807]. There are high levels of this receptor in kidney and moderate levels in other organs. IL-25 stimulates production of type 2 cytokines that protect against intestinal helminths [508, 1518, 1519].

Walter M. Lewko and Robert K. Oldham Transgenic mice that overproduce IL-25 had increased expression of Th2 cytokines IL-4, IL-5, IL-10 and IL-13, G-CSF and the Th1 cytokines IFN-γ and TNF-α as well. The TG mice had eosinophilia, elevated IgE and IgG1, and immune cell infiltration in many organs. These mice also suffered epithelial hyperplasia, hypertrophy in several organs and jaundice [1526]. IL25 appears to be involved in several inflammatory diseases. In mice, overexpression of IL-25 in lung by adenovirus-IL-25 infection induced airway hyperreactivity with Th2-like responses including IL-4, IL-5, IL-13 and eotaxin production, eosinophil infiltration, mucus production [825]. In eosinophils, IL-25 enhanced survival, increased expression of surface adhesion molecules [301] and stimulated production of proinflammatory IL-6, IL-8, MIP-1α, and MCP-1 [2195]. Lung tissue from asthmatic patients and atopic skin lesions show elevated expression of IL-25 and its receptor [2109]. IL-25 induces catabolic activity in articular cartilage and may be involved in arthritis [230]. It appears that during inflammation, Th2 cells and eosinophils/basophils may collaboratively reinforce one another to cause the disease state [2109]. While IL-25 seems to cause inflammation, IL-25 also appears to regulate it. For example, IL-25 controls Th17 inflammation. IL-25 −/− mice are much more sensitive than normal mice to the debilitating effects of experimental autoimmune encephalitis. In the CNS, microglial cells produce IL-25, which in turn induces IL-13 in Th2 cells. IL-13 downregulates DC activity and lowers the cytokines IL-1β and IL-6, which drive Th17 differentiation. In this way, IL-25 secretion in brain may be responsible for alleviation of damage associated with IL-17 in experimental encephalitis [985].

Interleukin-26 Epithelial Immune Response IL-26 is a member of the IL-10 family. It is also referred to as AK155 [532, 988]. IL-26 was discovered as an overexpressed protein in cultured T cells transformed by herpesvirus saimiri [792, 988]. The gene for IL-26 is located on chromosome 12q15. T cells, in particular activated memory T cells, and NK cells produce IL-26 [2187]. Secreted IL-26 binds heparin where it is stored in an inactive form until it is released [792]. The receptor for IL-26 is IL20R1/IL-10R2 [792, 1802]. Epithelium is a major target for IL-26. Treatment with IL-26 enhances expression of ICAM-1 and secretion of IL-8 and IL-10. By targeting colon epithelial

197 cells and skin keratinocytes, IL-26 may have a role in mucosal and cutaneous immunology [792].

Interleukin-27 Adaptive and Innate Immune Response; Regulation of Inflammation/Th17 IL-27 is a member of the IL-12 family. This cytokine has two subunits, p28 and EBI3 (Epstein-Barr virus induced gene 3). Activated dendritic cells and monocytes produce IL-27. Microbial and host factors stimulate production. The IL-27 receptor is WSX-1. The major targets of IL-27 are CD4+ T cells and NK cells [1713]. In naive T cells, IL-27 supports proliferation and increases IL-12R levels. IL-27 stimulates IFNγ production in NK cells and in activated T cells; in naïve T cells, IL-27 induces T-bet, IL-12R and Th1 differentiation [747, 2258]. Il-27 regulates inflammation. In mice where the IL-27/IL-27R system was defective, infection resulted in severe chronic inflammation. IL-17 inflammatory effects were exaggerated [115, 1663, 1911, 2176]. IL-27 regulates inflammation at several levels. It stimulates regulatory cells to secrete IL-10 [115]. IL-27 also appears to act directly on Th cells to suppress Th17 cell development [1911, 2258]. IL-27 also acts directly on activated granulocytes and macrophages to suppress production of reactive oxygen [2176]. IL-27 has anticancer activity. Expression of IL-27 in colon cancer cells has been shown to be immunogenic [309, 772]. Tumors secreting IL-27 were rejected. Mice developed tumor specific protective immunity. Interferon γ, CD8+ T cells and NK cells appeared to be involved [309, 772]. Poorly immunogenic B16F1 melanoma cells, engineered to secrete IL-27, were also inhibited. This growth inhibition was due to NK cells; added IL-12 was synergistic [1497]. IL-27 has also been found to be antiangiogenic [1811].

Interleukin-28A, Interleukin-28B, Interleukin-29 Type III Interferons; Antiviral, Antitumor IL-28A (IFN-λ2), IL-28B (IFN-λ3) and IL-29 (IFN-λ1) are three recently discovered cytokines in a new class of interferons, referred to as the lambda or Type III interferons. They are produced by activated (e.g. viral RNAtreated) dendritic cells, monocytes and macrophages. IFNα and TNFα enhance the λ interferon response to

198 viruses by increasing expression of toll-like receptors and signaling molecules. The λ interferons are related to the type I interferons and to IL-10. The receptor used by these cytokines is IL-28RαIL-10Rβ. The λ interferons activate typical IFNinducible genes including MHC I and II, oligoadenylate synthetase and MxA. λ interferons inhibit replication of certain viruses in vitro and stimulate antiviral immune response in treated mice [54, 1241, 1288, 1514]. IFN-λs are involved in the exquisite cell interactions involved in immune regulation. Monocytes, macrophages and dendritic cells produce IFN-λs and these cells are influenced by them, IFN-λ secretion increases with LPS-induced dendritic cell differentiation and maturation. Keratinocytes may be activated; Th1/Th2 ratios and cytotoxic T cell activities may be enhanced [2190]. And it has also been shown that IFN-λ-treated dendritic cells induce proliferation of Treg cells, which eventually suppress effector T cell activity [1292]. IFN-λs also have anticancer activity. In cultured neuroendocrine carcinoma cells, IL-28A and IL-29 inhibited cell proliferation and induced apoptosis (2300). IFN-λ secreting tumor cells implanted in animals stimulated NK and CD8 T cells immune response, resulting in decreased growth and metastasis in melanoma, fibrosarcoma and colon tumor models [1065, 1466, 1728].

Interleukin-31 Pruritis, Allergic Inflammation IL-31 is a cytokine that is produced by T cells, in particular, Th2 cells. IL-31 receptor is composed of two chains, IL-31 Rα and oncostatin M Rβ. The IL-31R is found in activated monocytes, epithelial cells and in sensory neuron bodies associated with skin. IL-31 appears to be responsible for itch sensation. Transgenic mice overexpressing IL-31 develop itching skin lesions analogous to human atopic dermatitis. IL-31 is elevated in mouse and human dermatitis lesions, but not in human psoriatic skin lesions. Pruritis in a mouse model appeared to be related to IL-31 secretion [431, 1438, 1859, 1952]. IL-31 is involved in asthma. In lung epithelium, IL-31 induced EGF, VEGF and inflammatory chemokines [855] and inhibited cell proliferation [291]. IL-31 also has regulatory functions. Mice that are IL31R−/−have exaggerated Th2 responses; when injected with schistosome eggs, these mice developed severe lung inflammation, granulomas and fibrosis. Levels of

Cytokines IL-4, IL-5 and IL-13 were elevated. IL-31 signaling appears to limit the type 2 inflammatory process [1558].

Interleukin-32 Inflammation IL-32 was discovered as the transcript NK4 in activated immune cells. Many types of cells produce IL-33 when exposed to inflammatory conditions. IL-33 has no homology with other known cytokines. IL-32 has six isoforms. Activated NK cells, T cells, and IFNγ-treated epithelium and monocytes produce IL-32 [438, 900, 964]. IL-32 induces inflammation by way of p38 MAP kinase and NF-κB signaling [1444]. IL-32 induces monocytes to produce TNFα, IL-8, PGE2 and other inflammatory factors. It signals through NFκB and MAP kinase [692]. IL-32 synergizes with the microbe sensing NOD receptor system for the induction of IL-1 and IL-6 by muramyl dipeptide. But IL-32 had no such stimulating effect on the TLR system [1443]. In addition to inflammation, IL-32 also influences specific immunity. IL-32 induced differentiation to macrophages from blood monocytes and, interestingly, from dendritic cells [1444]. Apoptotic T cells were high in IL-32. Forced expression of IL-32 induced apoptosis. This suggested that IL-32 is involved in AICD. This effect of IL-32 on T cells may be by a non-secreted, intracrine mechanism [630] There is evidence IL-32 is involved in several diseases including rheumatoid arthritis, psoriasis, Crohn’s disease and chronic obstructive pulmonary disease [438]. In tissue biopsies from patients with rheumatoid arthritis compared with osteoarthritis, IL-32 was increased and levels correlated with disease severity [900]. Of 171 genes tested, four genes were overexpressed in RA fibroblasts, and the gene for IL-32 was most pronounced [229].

Interleukin-33 Inflammation, Allergy IL-33 is an IL-1 family member. It is produced by vascular endothelium and certain fibroblasts. IL-31 is synthesized as a precursor and cleaved by caspase-1, in a manner similar to IL-1 processing. The IL-33 receptor is the IL-1 receptor-related protein ST2. It signals through NF-κB and MAP kinases.

Walter M. Lewko and Robert K. Oldham IL-33 enhances expression of Th2 cytokines IL-4, IL-5 and IL-13. In mast cells, IL-33 stimulates maturation, survival and the production of IL-8 and Th2 cytokines. In treated mice, IL-33 induces severe mucosal inflammation [28, 434, 839, 1750]. IL-33 and Th2 cytokines were required for expulsion of intestinal nematodes [821]. In mice, Il-33 has been shown to protect against the development of atherosclerosis [1310]. In endothelial cells, IL-33 appears to also have an intracellular role, apart from its receptor. It translocates into the nucleus where it binds chromatin and as a nuclear protein it represses transcription [247].

Interleukin-35 Treg Cytokine, Regulation of Th17 IL-35 is a heterodimeric cytokine that is composed of IL-12α and IL-27β chains. nTreg cells (CD4+ CD25+ Foxp3+) produce IL-35. The secretion of this cytokine is upregulated when Treg cells are activated by contact with effector T cells. IL-35 appears to have a major role in regulation for IL-27β−/− Treg cells show loss of function. For example, they do not control homeostatic proliferation as wild type cells do; they do not cure inflammatory bowel disease. Recombinant IL-35 has been shown to induce Treg cell proliferation and production of IL-10. It suppressed the expansion of CD4+ CD25−effector T cells and it inhibited differentiation and IL-17 production by Th17 cells. And in treated mice, rIL-35 decreased the severity of collagen-induced arthritis [335, 1451].

4-1BB Iigand Costimulation of T Cells, T Cell Memory 4-1BB ligand (4-1BBL) is a 30 kDa transmembrane glycoprotein. T cells, stromal cells of thymus and spleen, and antigen presenting cells including monocytes, B cells and dendritic cells express this ligand [23, 389, 639, 1059, 1584]. The receptor is 4-1BB (CD137). It is a 30–35 kDa transmembrane protein, which is related to the TNFR [1585, 1761, 1763]. The human gene is located on chromosome 1p36 in a cluster of related genes [1763]. There is a soluble form of this receptor, the product of alternative mRNA splicing [1787]. 4-1BB was originally discovered as an activation-induced antigen on T cells [23, 639, 1044, 1045, 1765]. 4-1BB is also produced by B cells, dendritic cells, monocytes and

199 NK cells [1761, 2160]. It is also found on epithelial, endothelial, and neural cells [184, 1622]. In T cells, the engagement of 4-1BB induces proliferation, cytokine secretion, cell survival and the suppression of AICD. 4-1BB stimulates memory response to second antigen challenges [147, 238, 317, 389, 639, 827, 1585, 1764, 1948]. 4-1BB is one of several costimulatory signaling systems, which may supplement, modify and, to a degree, replace costimulation through the B7/CD28 system [213, 389, 390, 391, 715, 828, 833]. 4-1BB is stimulatory but it is also regulatory; it preferentially induces proliferation of CD8 T cells over CD4 T cells [1818]. 4-1BB signaling tends to suppress Th2 responses and humoral immunity [789, 1319]. In this way, 4-1BB signaling can ameliorate Th2-related allergy and autoimmunity [312, 1927]. In NK and NKT cells, stimulation of 4-1BB induces activation and the secretion of cytokines. Mice lacking 4-1BB have depressed NK numbers and activity [1289, 2078]. NK cells are well known as cytotoxic effector cells but they also appear to help CTL differentiation. When stimulated with antibodies to 4-1BB, NK cells produce IL-2 and IFNγ, which increase the growth and cytotoxicity of antigen-specific T cells [1289, 2160]. 4-1BB ligation likewise stimulates dendritic cells to secrete IL-6 and IL-12, which enhance antigen-specific T cell response [2159]. In this way 4-1BB costimulates T cells directly and indirectly by its cytokine stimulating effects in several types of cells. On binding its receptor, 4-1BB ligand is capable of reverse signaling. In T cells, reverse signaling suppresses immune response and induces apoptosis [1764]. In monocytes, reverse signaling induces IL-6, IL-8, TNF-α and ICAM-1 but inhibits the production of IL-10 [1059]. Several studies show that 4-1BB stimulation serves as an adjuvant in vaccines against cancer [693, 862, 1029, 1116, 1233, 1290, 1312, 1333, 1718, 2138, 2211, 2215, 2280] and viruses [146, 390, 1958]. For example, in mice with melanoma, survival was shorter in mice that lacked 4-1BB compared to normal 4-1BB+ mice. Treatment of normal mice with agonistic antibodies to 4-1BB further increased survival [906]. Agonistic antibodies to 4-1BB enhanced the efficacy of a tumor lysatedendritic cell based vaccination [1866]. 4-1BB ligand has been detected in several carcinoma cells. These tumors cells costimulate T cells and IFNγ production. Reverse signaling stimulated tumor cell IL-8 production and depressed growth [1717]. Several mouse studies showed the benefit of injected mabs to 4-1BB suggesting it may be another receptor for which antibodies may be useful in the treatment of human cancer.

200

CD27 ligand Costimulation of T and B Cells; B Cell Differention; Apoptosis CD27 ligand (CD27L, CD70) is a cell surface glycoprotein. It is a member of the TNF family [638]. Activated B cells [1093], T cells [8, 1505], NK cells [2226], dendritic cells [224] and certain B cell malignancies [1619, 928] produce CD-27L. It is expressed preferentially on CD45RO memory CD4 T cells [8]. In dendritic cells, CD27L is induced by CD40L or by TLR stimulation [224]. Upon binding its receptor, CD27L may back-signal to increase cytotoxicity in γδ T cells [1505], antibody production in B cells [1094] and growth of certain B cell malignancies [928]. CD27 is the receptor for CD27L [187, 638, 763]. CD27 is a 50 kDa transmembrane glycoprotein. It is a member of the TNFR family [233, 762, 2060]. CD27 is expressed on naive and memory T cells [8, 155, 1924, 2060], germinal center and circulating memory B cells [984, 1253, 2015], B cell leukemias [2061] and on NK cells [1923]. CD27 is considered a memory marker for T cells [2015] and B cells [985]. Activation of T cells markedly increases CD27 and induces the release of a soluble form of the receptor, which can be found in blood and urine. The soluble form appears to be a proteolytic product [761, 762, 1146]. Elevated levels are characteristic of infection, autoimmunity and B cell malignancies. Soluble recombinant CD27 is an antagonist [87]. The CD27–CD27L system is costimulatory for the induction of T cells. Stimulating antibodies to CD27 induce T cell activation, proliferation and cytotoxicity [66, 638, 992, 1924, 2060]. Naive (CD45+) T cells and CD45RO memory cells respond to CD27 ligation [8, 992]. In NK cells, CD27 expression is upregulated by IL-2. CD27 ligation stimulates NK activation and cytotoxicity [1923]. For memory B cells, CD27 appears to be part of the switch between memory and activated plasma cells. engagement inhibits differentiation of B cells into Ig-secreting plasma cells [1616]. It favors the development of memory B cells with high antigen affinity [1615]. CD27 engagement induces apoptosis in activated T and B cells. The cytoplasmic segment of CD27 is rather short. Unlike TNF and Fas, CD27 does not have a death domain. However, this receptor does associate with a cytoplasmic protein, Siva, which has a death domain segment and Siva mediates CD27-induced apoptosis [1591]. In the treatment of cancer, there is interest in the CD27 system for its ability to stimulate T, B, and NK

Cytokines cells. When vaccine tumor cells are transfected with CD27L, there is enhanced immune response [348, 1153, 1453]. CD27 ligation supports the clonal outgrowth of tumor-specific T cells [638, 762]. Further, the CD27 system appears to be important in the production of T cells for adoptive cell immunotherapy [811, 1589]. IL-2 induces growth of tumor-specific T cells and part of its stimulatory effect is due to upregulation of T cell CD27L. Blocking antibodies to CD27L depressed IL-2-induced growth. It appears that CD27 signaling mediates IL-2-induced T cell proliferation [811, 1589]. It should be noted, CD27L [928, 1093] and CD27 [2061] are found on B cell malignancies and these molecules can have a role in tumor progression. Also, CD27L has been observed on certain non-hematological tumors including renal cell carcinoma [429] and brain tumors [732, 2177]. Tumor cell-CD27L causes apoptosis in T and B cells and might have a role in immune escape [429, 2177].

CD30 ligand T Cell Costimulation, Selection; Apoptosis; Regulation of Ig Class Switching CD30 ligand (CD153) is a 40 kDa transmembrane glycoprotein in the TNF family. It is found on macrophages [1840], B cells [598, 2259, 2260], CD4 and CD8 T cells [2259], megakaryocytes, neutrophils, erythrocyte precursors [598], eosinophils [1570], mast cells [541] and many though not all leukemias [599]. On binding its receptor, CD30L has the capacity to signal back into its parental cell [2165]. Reverse signaling in neutrophils stimulates an oxidative burst and IL-8 production. In T cells, reverse signaling stimulates proliferation and IL-6 production [2165]. CD30 is the receptor for CD30L. It is a 105–120 kDa transmembrane protein and a member of the TNFR family [473, 605, 1840]. The intracellular segment contains two binding sites for TNFR-associated factors (TRAFs 1, 2, 3) which function in signaling [601]. The CD30 antigen was originally discovered as Ki-1, a marker for Reed Sternberg cells in Hodgkin’s lymphoma [1759]. CD30 is found on activated T, B, and NK cells [35, 186, 489, 2088]. CD30 expression is transient; in T cells it depends on activation and CD28 signaling [621]. CD30 is a marker for activated, cytokine secreting helper T cells (Th0, Th1 and Th2) [704]. In particular, CD30 expression is associated with Th2 response [1255]; CD30 is stimulated by IL-4 and inhibited by IFN-γ [621]. CD30 is expressed on subsets of activated

Walter M. Lewko and Robert K. Oldham CD45RO+ memory T cells [35]. CD30+ cells produced IFN-γ and IL-5 while CD30− cells produced more IL-2. CD30+ T cells showed enhanced ability to provide help for B cells producing Ig [35]. Soluble CD30 is shed from cells and found in blood. CD30 shedding is increased by activation of CD30 and by treating cells with PMA. The protease responsible is TACE, the enzyme that processes and releases TNF-α [711]. Elevated levels of sCD30 are found in the blood of patients with immune disorders such as lupus erythematosis and rheumatoid arthritis and in patients with colon cancer and Hodgkin’s disease. In Hodgkin’s lymphoma patients, sCD30 correlates with poor prognosis [2256]. Changes in levels may be used to monitor a disease [711, 868, 904]. The specific biological effects of the CD30L–CD30 system are difficult to discern. Expression of the receptor is transient and certain effects appear to be opposed. CD30 engagement is costimulatory for the growth and differentiation of specific T cells [621, 683, 1840]. But the CD30L–CD30 system also inhibits function and induces apoptosis [1407, 1840]. CD30-deficient mice, have impaired negative selection of thymocytes suggesting a fault in apoptosis [36]. CD30 transgenic mice overexpressing CD30 in the thymus showed enhanced thymic negative selection [303]. Moreover, CD30 signaling induced TCR-dependent apoptosis in a T cell hybridoma cell line [186]. Lack of CD30 expression is also associated with the development of autoimmune T cells in experimental diabetes [280, 729, 1041]. On the other hand, in NOD mice that are genetically prone to diabetes, neutralizing antibodies to CD30L interfered with the development of the disease [280]. In a cytotoxic large granular lymphocyte cell line, CD30 activation depressed effector functions including FasL, perforin and granzyme production while inducing the effector cell’s own apoptotic program [1407]. CD30 thus appears be able to coordinate gene expression in NK and T cells to control effector selection, growth and differentiation and then downregulate cytotoxic functions, inhibit growth and induce effector cell death. T cells influence B cell antibody production and class switching by way of the CD30L–CD30 and CD40L–CD40 systems. CD40 ligation stimulates Ig class switching. CD30L reverse signaling inhibits class switching at the level of CD40 signaling [273, 274]. CD30 is also required for the development of B cell memory responses [593]. Mast cells expressing CD30L appear to have a role in Hodgkin’s disease. Mast cells were the predominant CD30L-positive cell type in tumors and these cells were capable of stimulating growth of Hodgkin and Reed

201 Sternberg cells [1336]. CD30L also appears to have a role in cutaneous inflammation. In atopic dermatitis and psoriatic skin lesions, mast cells were the predominant CD30L-positive cell type. On binding CD30, back signaling in these mast cells induced the secretion of IL-8 and other inflammatory chemokines. This mast cell activation occurred in an IgE-independent manner [539]. Eosinophils express CD30L and they stimulate the growth of co-cultured CD30-Hodgkin’s lymphoma cells [1570]. Eosinophils also express CD30; treatment with agonistic Mabs to CD30 induced eosinophil apoptosis [142]. A relatively small subset of normal lymphocytes express CD30 but it is overexpressed in several diseases making it an attractive target for therapy. In T cells CD30 is increased in autoimmune disease and in allergy [614]. The CD30 antigen is also found on virally transformed and HIV-infected lymphocytes [729]. It is highly expressed in Hodgkin’s disease, anaplastic large cell lymphoma and certain other malignant lymphoid disorders [1759]. In many lymphoid cell lines, CD30 ligation induces apoptosis but there are cases in which CD30 has no effect or stimulates growth [683, 1081, 1185]. Where CD30 ligation induces apoptosis in Hodgkin’s lymphoma cells, the presence of soluble CD30 was found to interfere. This may explain why sCD30 correlates with poor prognosis in these patients [2259]. Antibodies are being developed which bind specifically to cellular CD30 epitopes that are lacking on the shed receptor [1411]. In patients with Hodgkin’s disease, bispecific (Anti-CD16/CD30) antibodies have been tested with some success in a phase I/II trial. This construct was designed to activate NK cells and target the tumor cells [721]. SGN-30 is a humanized monoclonal antibody that inhibits Hodgkin’s disease tumors in nude mice [2088]; it is currently undergoing clinical trials.

CD40 ligand Costimulatory, Proinflammatory; B, and T Cell Stimulation CD40 ligand (CD154) is a 35 kDa membrane protein. It is expressed by CD4 T cells [1691] and by CD8 cells [1707]. The levels of this powerful cytokine are rather well controlled. It is produced during CD4 cell activation and down regulated upon binding its receptor [109, 260, 2058, 2240, 2241]. A soluble form of CD40L is released from CD4 T cells upon activation [653]. Soluble CD40L is biologically active and will substitute for cell-bound CD40L [955, 1055].

202 CD40 is the receptor for CD40L. It is a 50 kDa membrane protein, constitutively expressed, mainly on B cells [266], and to a lesser extent on dendritic cells, macrophages [21], T cells [511], fibroblasts [2241], thymic epithelial cells [585] and endothelial cells [786]. CD40 is also present on some tumor cells. The CD40/ CD40L system stimulates immune response, inflammation and apoptosis depending on the cell type and the conditions. In B cells, CD40 signaling causes activation, maturation and survival [400, 946, 1294]. The triggering of B cells through CD40 is critical for the induction of Ig production [72]. In the absence of CD40, B cells were tolerogenic [221, 952]. Binding Th2 cell CD40L induces reverse signaling and the secretion of IL-4 that in turn stimulates B cells [167]. The CD40 system provides help during CTL development. CD40L on CD4 T cell interacts with CD40 on AP cells (DC, B cell, virus-cell or tumor cell) to induce antigen processing, presentation and cytokine secretion [136, 756, 1645]. CD40 engagement also increases DC survival, costimulatory molecules B7.1 and B7.2, ICAM-1, and cytokines such as IL-12, which stimulate T cells and NK cells [136, 159, 263, 267, 1645, 1753]. The CD40L–CD40 system is necessary for a good immune response. CD40L-deficient mice were defective in antiviral immunity and memory CTL response [181]. Mice defective in the CD40 system were very susceptible to infection with Leishmania [236, 912, 1861]. Treating mice with stimulatory anti-CD40 Mabs produced a good T cell immune response to an otherwise weak Listeria monocytogenes immunogen [1658]. CD40 activation also appears to be required for allograft rejection; blockade of the CD40 system generally prevents rejection and induces tolerance [470, 651, 863, 1064]. CD40L is a proinflammatory cytokine. Engagement of CD40 induces the production of inflammatory cytokines in fibroblasts and macrophages [21, 2241], and the upregulation of surface adhesion molecules such as ICAM and VCAM on fibroblasts and endothelial cells [786, 1062, 2241]. Treating animals with soluble CD40L intranasally induced lung inflammation with infiltrating neutrophils and macrophages. IFN-γ intensifies CD40Linduced inflammation [2163]. Platelets contain both CD40 and its ligand and may be involved in CD40related inflammation and immune responses [377, 953]. CD40 on lung fibroblasts appears to have a role in pulmonary fibrosis [2241]. CD40–CD40L interaction is also required for a number of experimental autoimmune diseases. In animal models for myasthenia gravis [848], multiple sclerosis [615, 667], arthritis [471] and diabetes [105],

Cytokines CD40 activation exacerbated the disease while blocking CD40 depressed it. Development of autoimmunity is complex and sometimes paradoxical. The absence of CD40–CD40L interactions might induce autoimmune disease [1034]. Patients with rare X-linked mutations in the CD40L gene develop hyper-IgM syndrome, show defects in thymus-dependent immune responses, antibody production and germinal center formation. These patients suffer recurrent infection, increased cancer and autoimmune disease [441, 727]. The CD40 system regulates apoptosis, positively or negatively, depending on the target cells. In dendritic cells, for example, CD40 ligation on immature DCs induced survival and maturation, whereas CD40 ligation on LPS-matured DCs induced apoptosis [405]. Where CD40 induces apoptosis, it up-regulates Fas. Where CD40 inhibits apoptosis, it increases levels of the survival protein bcl-X [858, 2113]. The CD40–CD40L system has some remarkable antitumor effects. In normal B cells, CD40 ligation stimulated growth. In neoplastic B cells CD40 ligation tended to inhibit growth and induce apoptosis [575]. Certain solid tumors express CD40. When this is the case, CD40L might act directly on the tumor cells to induce death by apoptosis [756, 765, 2086]. CD40 ligation stimulates chemokine release. In human cervical carcinoma cells, CD40L induced macrophage chemoattractant protein-1 (MCP-1) and IFN-γ-inducible protein 10 (IP-10). The addition of IFN-γ had a synergistic effect [32]. These chemokines are involved in the recruitment of effector T cells. IP-10 is also angiostatic [1788]. As mentioned, CD40 ligation activates antigenpresenting cells and secretion of IL-12 by DCs. Good cytotoxic T cell responses were observed in animals treated with stimulating anti-CD40 mabs, or immunized with tumor cells engineered to produce CD40L [430, 433, 446, 567, 695, 740, 1415, 1863]. CD40 ligation enhanced the outgrowth and longevity of T cells [1256]. CD40–CD40L interactions were required for protective anticancer immunity by vaccination [1196, 695]. When CD40 signaling was blocked, no systemic antitumor immunity developed [1195]. CD40 signaling stimulates NK activity and related antitumor effects [245, 1415, 2038]. P815 mastocytoma cells engineered to produce CD40L were promptly rejected when injected into mice. NK cells mediated the anticancer effect. Production of IL-12 was required but effector CTL cells were not [1415]. In another study, mice were treated with stimulatory anti-CD40 mabs to activate signaling [2038]. Three different types of tumors were tested; growth and metastasis were inhibited. NK cells were required. The effect of CD40 was not

Walter M. Lewko and Robert K. Oldham direct but probably through APCs that produced NK cell stimulating IL-12 [2038]. A phase I trial of rhuCD40L in patients with solid tumors and NHL, encouraging preliminary results were seen including one long-term complete response [2087]. In a phase I vaccine trial, patients with CLL were treated with CD40L-producing autologous tumor cells. Leukemia cell counts decreased and lymph node size was reduced again suggesting that CD40L may be therapeutically useful [2154].

Chemokines Cell Activation, Migration, and Recruitment; Inflammation; Angiogenesis Chemokines include a rather large number of proteins, which have a variety of activities but function primarily as chemoattractants and activators of leukocytes [1656]. They induce cell migration, recruitment and infiltration into sites of inflammation, homing to sites of immune cell differentiation and passage across cellular barriers. They are involved in T cell activation and the polarization of Th1/Th2 cells [687, 1656]. Chemokines may also act on non-hematopoietic cells. They facilitate cell movement during inflammation, wound repair and morphogenesis. Chemokines are also involved in pathology, tumor growth and metastasis. HIV viruses use certain chemokine receptors (CXCR4) for entry into cells [524]. Chemokines are relatively small 7–14 kDa proteins. Most chemokines contain four conserved cysteines in the amino terminal region. Chemokines are classified based on the sequence in the region of these cysteines. 1. CXC chemokines (α chemokines): the first two conserved cysteines are separated by a single nonconserved amino acid. CXC chemokines may be classified further based on the presence or absence of Glu-Leu-Arg (ELR) just preceding the first conserved cysteine. There are at least five receptors for CXC chemokines. These receptors are members of the rhodopsin-like, seven transmembrane domain receptor family [1147]. As examples of chemokine– receptor interactions, CXCR1 and CXCR2 bind IL-8 and GCP-2 (granulocyte chemotactic protein-2) [5]. CXCR3 binds IP-10 (interferon-induced protein-10) and Mig (monokine induced by γ interferon) [894]. SDF-1 (Stromal derived factor-1), a pre-B-cell growth factor as well as a chemokine,

203 appears to be the only ligand for CXCR4 [1409]. ELR CXC chemokines tend to activate migration in neutrophils, the non-ELR CXC chemokines tend to activate lymphocytes. 2. CC chemokines (β chemokines): the first two conserved cysteines are together. There are nine known receptors for these chemokines termed CCR1, CCR2, etc. Well studied CC chemokines include MIP-1α (macrophage inflammatory protein-1α), MIP-1β, MCP (Monocyte chemoattractant proteins) 1, 2, 3, RANTES and eotaxin. CC chemokines are chemotactic for most types of leukocytes (T, B, DC, NK, eosinophils, basophils, DC), but not for neutrophils. [1656]. 3. C chemokines: two of the four conserved cysteines are missing, the first and the third. Lymphotactin is a C chemokine. NK cells produce it. Lymphotactin is specifically attractive for lymphocytes [949]. 4. CX3C chemokines: the first two conserved cysteines have three intervening non-conserved amino acids. There is a specific receptor Cx3CR-1 for these cytokines [849]. Fractalkine (neurotactin) is a Cx3C chemokine. It has two active forms; one is a membrane bound protein. The other is a soluble protein released from the membrane by the protease TACE [2030]. Fractalkine is expressed in neurons, microglial cells, fibroblasts and endothelial cells. It is upregulated during inflammation. Fractalkine is a chemoattractant for T cells, NK cells and monocytes and it induces adhesion [119, 1530]. In the brain fractalkine appears to be antiapoptotic, a survival factor for microglial cells [171]. ELR+ CXC chemokines are angiogenic; they stimulate endothelial cell migration and tube formation; these include IL-8, GRO (growth-related oncogene)-α, GRO-β, GRO-γ, GCP-2 and ENA-78 (epithelial neutrophil-activating protein 78) [1906]. The receptor CXCR2 is responsible for ELR+ angiogenesis [5]. Non-ELR CXC chemokines tend to be inhibitors of angiogenesis. IP-10 and Mig are angiostatic [1204, 1906]. Both cytokines bind receptor CXCR3 [2142]. The anticancer activities of several cytokines may involve the downstream production of angiostatic chemokines such as IP-10 and Mig [32, 52, 222, 1788]. Ephrins and ephs, their receptors, are also involved in cell migration. They act as chemodirectants during morphogenesis, inflammation and immune response. Ephrins and ephs are found on a wide variety of cells. In contrast with the chemokines, which mainly stimulate migration and adhesion, the ephrins are chemorepulsive agents which tend to be transiently expressed

204 at times when the movement of cells needs to be redirected. Chemokines are involved in many inflammatory diseases and in the development of cancer. Cell movement and angiogenesis are hallmarks of malignancy. Elevated levels of angiogenic chemokines are associated with tumor progression and metastasis [63, 976, 1167]. IL-8 production has been correlated with tumor vascularity and progression [976, 2250]. Chemokines are of interest in the design of therapeutic agents, including antitumor vaccines. For example, lymphotactin is produced by NK cells and attracts T cells [949]. A vaccine using tumor cells co-transfected to produce lymphotactin and IL-2, induced potent antitumor immunity in mice [432]. In another study, a vaccine with Lewis lung carcinoma antigen-pulsed dendritic cells, transfected to produce lymphotactin, induced a much more potent antitumor response than a control vaccine lacking lymphotactin. In mice with established tumors, vaccination with the antigen-pulsed lymphotactin-DCs inhibited metastasis. Only one relatively small 104 DC dose was required to immunize the mice [241].

Fas ligand Inducer of Apoptosis Fas ligand (FasL) is a transmembrane protein in the TNF family. It is expressed on activated lymphocytes and on a variety of other cells including certain tumor cells. In T cells, FasL is induced by TCR activation. Its production is inhibited by retinoids and glucocorticoids [2107, 2228], by inhibitors of protein kinase C and calcium mobilization [2107], and by cyclosporin [217]. Fas is the receptor for FasL [1410, 1839, 1976]. Fas (also known as Apo 1, CD95) is expressed on lymphocytes and many different types of cells including tumor cells. There are soluble forms of Fas; they are generated by alternative mRNA splicing [253]. The FasL– Fas system is responsible for the induction and regulation of programmed cell death in the peripheral immune system. Apoptosis is a way to regulate and terminate an ongoing immune response, or to eliminate useless or potentially damaging immune cells [1019, 1184].The importance of the FasL/Fas system in the regulation of lymphocytes can be seen in the autoimmune diseases and lymphoproliferative disorders in mice, associated with mutations in the FasL (gld) and Fas (lpr) genes [330, 1410, 1698, 1976]. Various types of cancer and tumor cell lines express FasL [135, 700, 1452, 1469, 1703, 1814, 1897]. The Fas–FasL

Cytokines system is involved in activation-induced cell death in lymphocytes. The tumor cells that express FasL are generally negative for Fas and tend to be resistant to apoptosis [700, 1468, 1469]. The expression of FasL may explain the general phenomenon of immune resistance (immune privilege), by which certain normal tissues (eye chamber, testis) and tumors are able to evade immune effector cells. Cells that express surface FasL may induce apoptotic death in lymphocytes [670, 700]. In tumor biology this idea has been referred to as “FasL counterattack.” in which infiltrating lymphocytes may be killed upon contact with FasL+ tumor cells [135, 1468, 1469]. This theory is interesting but controversial. Certain reports show that tumors expressing FasL may be rejected [61, 124, 519, 1778]. In transplantation research, some have tried to engineer graft tissues with FASL to make them less immunogenic but with they have met with variable success [31, 916]. Tumor cells may contain Fas. When this is the case, tumor development and therapy may be influenced by Fas-induced apoptosis. Dendritic cells express FASL, as well as other proapoptotic cytokines TNF, LTα1β2, TRAIL, and may kill tumor cells directly [1166]. CTLs express FASL. Irradiation was found to increase Fas on tumor cells and prior irradiation enhanced apoptotic response to adoptive CTL therapy [281]. Vitamin E stimulated Fas and Fas ligand levels in certain cultured breast cancer cells and induced death by apoptosis [2037]. In some leukemias and solid tumors, drugs such as adriamycin and methotrexate induce Fas and FasL causing death [566]. Capillary leak syndrome involves endothelial cell damage. It is associated with chronic infection and it is a dose-limiting toxicity in the use of IL-2 and certain other cytokines to treat cancer patients. Capillary damage appears to be due to cytotoxic lymphocytes [375]. Studies using perforin knockout mice and mice with defective FasL and Fas genes showed that both perforin and Fas-FasL were responsible [1612].

FLT-3 ligand Stimulator of Early Hematopoietic Stem Cells and Dendritic Cells Flt-3 ligand is a membrane-bound cytokine. In structure and activity, it is related to macrophage colony stimulating factor and stem cell factor [710, 1175]. The human gene is located on chromosome 19 [1176, 1265]. It is expressed by a wide variety of hematopoietic and nonhematopoietic tissues [710, 1176]. Flt-3 ligand is produced in both membrane bound and soluble forms, both active.

Walter M. Lewko and Robert K. Oldham Soluble forms are produced by proteolysis [1177] or alternative mRNA splicing [1176, 1265]. Soluble Flt-3 ligand is elevated in serum of patients with certain anemias [1179]. In patients on myelosuppressive chemotherapy, elevated plasma Flt-3 ligand is prognostic for poor recovery from thrombocytopenia [170]. Flt-3 (fms-like tyrosine kinase-3, CD135) is the receptor for Flt-3 ligand. It is a transmembrane protein. Flt-3 was named for its similarity to fms, the receptor for M-CSF [1679, 1680]. Flt-3 is also referred to as flk-2 (fetal liver kinase 2) [1251] and Stk-1 (stem cell tyrosine kinase-1) [1837]. It is a 158 kDa glycoprotein. The human gene is on chromosome 13q12 [1680]. This receptor is expressed mainly by hematopoietic stem cells and progenitor cells [1248]; it is low or missing on most mature cells [370, 1251, 1286, 1837]. Flt-3 ligand binds and activates the receptor. Dimerization of Flt-3 ligand appears to be required for receptor dimerization and signaling [710, 1177, 1178]. Flt-3 ligand has a role in the proliferation, survival and differentiation of hematopoietic progenitor cells [1269]. Mice lacking Flt-3 ligand have reduced numbers of myeloid progenitors, B-lymphoid progenitors, NK cells, and dendritic cells [1272]. Mice lacking Flt-3 appear healthy. However, their stem cells are deficient in their ability to reconstitute lymphoid and myeloid cells when transplanted into irradiated mice [1192]. Mice injected sc with Flt-3 ligand show a remarkable increase in hematopoietic precursors [193, 194]. Flt-3 ligand stimulates the growth and survival of early progenitor cells [909, 1298, 1448]. GM-CSF, IL-3, SCF, IL-11 and IL-12 are synergistic [909, 1298] while TNF-α and TGFβ are inhibitory [2068]. Flt3 ligand acts together with IL-7 or SCF to stimulate B cell development from progenitors in bone marrow and thymus [768, 823, 1272, 1424, 2067]. Flt-3 ligand has a remarkable influence on the development of dendritic cells. Mice treated with Flt-3 ligand produce greater numbers of dendritic cells [452, 1224, 1819, 1890]. Spleen cells with markers for dendritic cells went from less than 1% of total in controls to 20% in Flt-3 treated mice [1224]. DCs from treated mice expressed more costimulatory CD80 and CD86 [1890]. Patients treated with Flt-3 ligand, also showed a remarkable increase in dendritic cells [1221]. Flt-3 ligand and stem cell factor cooperate to induce recruitment and expansion of CD34+ CD14+ dendritic cell precursors (CFU-DC). Flt-3 ligand and SCF are not required for the differentiation step. Other cytokines, including GM-CSF, IL-4, TGF-β and TNF-α stimulate maturation of the expanded precursors [367]. Flt-3 ligand also stimulates the production of NK cells [194]. Treatment of mice increased the number of

205 NK cells in blood, bone marrow, spleen and liver [1555, 1801]. It appears to do this by stimulating proliferation of pro-NK cells and the production of mature, non-activated NK. Flt-3 ligand increases NK cell responsiveness. IL-2 rapidly induces proliferation, cytotoxicity and LAK activity in Flt3 ligand-treated cells [1801]. Flt3 ligand influences certain non-hematopoietic cells. Osteoclasts respond to M-CSF with increased differentiation; Flt-3 ligand can substitute for M-CSF [1069]. Neural stem cells express Flt-3; Flt-3 ligand inhibited EGF and FGF-2 induced proliferation. Neurons are also Flt-3+. In culture, Flt-3 ligand synergized with nerve growth factor to promote neuron survival [195]. Flt-3 is expressed by many myeloid and lymphocytic leukemias [157, 370, 1286, 453, 1271, 1678]. When cells are positive for the receptor, flt-3 ligand may stimulate growth in culture. In AML, 20–25% of adult patients have a mutation in flt3. This mutation is an internal tandem duplication. The presence of this mutation is an adverse prognostic factor. This is a gain of function mutation that confers growth advantage on the cells [2151]; signaling is constitutively activated [725]. Selective tyrosine kinase inhibitors are being tested in patients with flt3+ tumors. In a trial on elderly patients with AML, responses were observed for lestaurtinib (CEP701) in both mutated and wild type Flt3 tumors [989]. Tumor bearing mice treated with Flt3 ligand generate antitumor responses. Several preclinical studies have shown this in lymphoma, leukemia, and several types of solid tumors [283, 320, 499, 1183, 2097]. Soluble and membrane bound forms of Flt-3L were effective. Dendritic cells and CD8 T cells appeared to be responsible for antitumor activity though NK cells might also be involved [1546, 1555, 1804, 1826]. In a mouse model for metastasis, local irradiation plus subsequent Flt-3 ligand treatment enhanced survival. This appeared to be due to increased presentation of antigen derived from dying tumor cells. In vivo manipulation of dendritic cells was used to induce immune response in three mouse model tumors. Mice were treated with several daily Flt3 ligand injections to induce DCs, then with CG oligonucleotide to mature the DCs in the presence of tumor peptide antigen. Existing tumors were inhibited and the mice were protected against a new challenge [1490]. It should be noted again that Flt-3 ligand acts together with other cytokines. For example, Flt-3 ligand and CD40 ligand were shown to be synergistic in the induction of antitumor immunity. Blocking CD40 obviated the effect of Flt3 ligand [179]. Flt3 ligand does not directly influence the growth of most types of non-hematological tumor cells in culture, although it has been reported that neural crest tumors

206 express Flt-3 and that cell lines treated with Flt-3 ligand showed increased growth and decreased apoptosis [1999]. Flt-3 is an apparent site of immune suppression. Vascular endothelial cell growth factor is produced by many tumors; it is an angiogenic factor and it is also immunosuppressive. VEGF interferes with Flt-3mediated DC production. It appears to do so by inhibiting the activation of the transcription factor NF-κB [1483]. There is interest in Flt-3 ligand for the treatment of cancer patients undergoing stem cell transplants. Patients may be pre-treated with Flt-3 ligand to mobilize stem cells to increase the number available for harvest. Flt-3 may also be used to expand these cells in culture. Studies in normal volunteers and in cancer patients showed that Flt-3 ligand is relatively safe. Few side effects were observed [1098, 1221]. As seen in mice and other animals, Flt-3 ligand caused a remarkable increase in stem cells and dendritic cells in blood and other tissue sites [1221]. Flt-3 ligand is effective alone but most likely it will be used in combination with other cytokines such as G-CSF and GM-CSF [193, 194, 1920]. In culture, stem cells and progenitor cells may proliferate for several weeks when Flt-3 ligand is added alone or together with other cytokines such as IL-1, Il-3, Il-6, IL-11, IL-12, erythropoietin, SCF and thrombopoietin [381, 578, 872, 1564, 1568, 1860]. Flt-3 ligand appears to maintain the stem cells, to sustain long-term hematopoiesis after reimplantation [381, 1564]. Trials have begun in cancer patients, using Flt-3 ligand to treat patients or to generate dendritic cells for vaccination. In a preliminary study, colon cancer patients injected with Flt3 ligand showed increased lymphocytes in blood, increased percent dendritic cells in PBMCs and increased dendritic cells infiltrating tumors [1366]. In another report, patients were vaccinated with antigen-pulsed autologous dendritic cells. The patients were pre-treated with Flt-3 ligand to mobilize DCs. The DCs were harvested, loaded ex vivo with a peptide of carcinoembryonic antigen and then reinfused. The preliminary results were promising. Two of 12 patients showed dramatic regressions; one had a mixed response. Anticancer activity correlated with the generation of specific CD8 T cells [549].

Leukemia inhibitory factor T Cell Development, Regulator of Inflammation, Embryonic Development Leukemia inhibitory factor (LIF) is a member of the IL-6 cytokine family [2273]. It is a pleiotropic cytokine; many of its effects overlap with those of IL-6. LIF is produced by fibroblasts [2008], macrophages [51] endothelial cells

Cytokines [1168] thymic epithelium [1067] and synoviocytes [395, 249]. The gene for LIF is on chromosome 22q12 where it colocalizes with the gene of OSM. In immune cells, secretion of LIF is stimulated by endotoxin, IL-1 and TNF [1297]. In thymic epithelial cells, EGF and TGF-β stimulate LIF production [1743]. The receptor for LIF has two subunits, LIFRβ and the common gp130 utilized by IL-6 family cytokines [602]. LIF has a role in the production of CD4 and CD8 T cells. LIF is secreted by thymic epithelium [1067]. Too much or too little LIF causes abnormal T cell development [1295]. In transgenic mice that overproduced LIF, thymic epithelial architecture was disrupted and cortical thymocytes were decreased [1804]. In LIF deficient mice, thymocytes were produced but they were insensitive to ConA activation [498]. LIF regulates inflammation. Depending on the setting, LIF may be pro- or anti-inflammatory. LIF knockout mice showed abnormal inflammatory response in nervous tissues [579, 1926, 2003]. Injection of rLIF into skin and joints induces swelling and infiltration [248, 1277]. LIF is elevated in ulcerative colitis; LIF stimulates the growth of cancer cells and may thus have a role in the development of colon cancer associated with this inflammatory disease [622]. LIF is proinflammatory, but there is also evidence LIF can inhibit inflammation. In knockout mice lacking LIF, inflammation is exacerbated in footpads injected with Freund’s complete adjuvant. Delivery of LIF using an adenoviral vector suppressed inflammation [2296]. In psoriasis, LIF and IL-11 have similar depressive effects on inflammation and cytokine production [2022]. LIF is involved in septic shock. The blood of patients had elevated LIF as well as TNF, IL-1 and IL-6; the level of these cytokines was correlated with the severity of the disease [2077, 2118, 2119]. LIF and IL-6 appear to be induced by TNF-α [880]. Blocking antibodies for LIF depress the lethality of endotoxemia [166]. Interestingly, treatment of mice with LIF before a lethal dose of endotoxin favored survival of the mice [26, 2117]. This may be due to induction of acute phase proteins that have antiinflammatory, protective effects [1258]. LIF has growth, differentiation and metabolic effects in non-hematopoietic tissues [759, 1042, 1295, 1297]. LIF reversibly inhibits the differentiation of embryonic stem cells [1449]. In fetal pituitary corticotropes, LIF inhibits cell division, stimulates differention and increases ACTH production [1884]. LIF regulates neuronal differentiation and increases survival [1345]. It stimulates bone development and extracellular matrix metabolism. LIF regulates mammary cell growth and development [1137]. Maternal expression of LIF is required for blastocyst implantation [1892].

Walter M. Lewko and Robert K. Oldham Mice injected with LIF suffer weight loss associated with cachexia. Weight loss is a characteristic of several gp130 signaling cytokines, IL-6, IL-11, CNTF and NNT [1784]. LIF has some remarkable effects in cancer. As mentioned, it is required for normal immune and inflammatory response. In tumor cells, LIF has both negative and positive direct effects on growth. LIF was discovered based on its ability to arrest growth and induce differentiation in murine myeloid leukemia cells [604]. In cultured human glioma cells, LIF inhibits growth and induces astrocytelike differentiation [701]. But LIF can stimulate growth in many types of tumor cells including human multiple myeloma [2285] colon cancer [964], breast cancer [425, 500, 948] and prostate cancer [948]. Other cytokines in the IL-6 family similarly influence tumor cell growth based on gp130 signaling [2285]. There is interest in agents that regulate growth by this signaling pathway.

LIGHT Costimulatory Molecule; T Cell Activator; Inducer of Apoptosis LIGHT is a 240 amino acid cytokine that was first discovered in the cDNA library of activated T cells. It is a member of the TNF family [1044, 1254]. LIGHT is homotrimeric and it is produced in both soluble and membrane bound forms, both active. LIGHT is expressed by T cells, granulocytes, monocytes, immature dendritic cells and certain tumors [719, 1368, 1957, 1959, 2278]. LIGHT binds two receptors, HVEM (herpes virus entry mediator) and lymphotoxin β receptor (LTβR) [2278]. HVEM is a member of the TNFR family [1044, 1352, 1958]. It is the cell receptor for herpes simplex infection [1344, 1254]. HVEM is also referred to as TR2 (TNF-related receptor 2) [1044] and ATAR (another TRAF-associated receptor) [808]. It is a 283 amino acid transmembrane protein. Signaling involves TRAF binding and the activation of NF-κB, Jun N-terminal kinase and AP1 [808, 1231, 1254]. HVEM is localized on T cells, B cells, NK cells, monocytes, endothelial cells and immature dendritic cells [720, 1044, 1344, 1958]. Its levels are up regulated by cell activation and down regulated by LIGHT [1958]. HVEM is also a receptor for lymphotoxin-α [1254]. In T cells, LIGHT costimulates proliferation, cytokine secretion and surface protein levels [720, 1044]. Disruption of LIGHT, interferes with lymphnode formation, depresses T cell proliferation in a MLR, the secretion of IL-2, IFN-γ, IL-4 and TNF-α [720, 1739]. Disruption of LIGHT also prolongs allograft survival [2237].

207 As typical of TNF-family members, LIGHT induces apoptosis. Normally, only cells that express both HVEM and LT-β receptors are susceptible to LIGHT-induced cell death. No apoptosis was observed in cells expressing only one of them [719, 2278]. Because lymphocytes do not express LT-β receptor, LIGHT does not cause apoptosis in these cells. TR6 (also called DcR3) is another member of the TNF receptor family. TR6 is a soluble, non-signaling protein that binds both LIGHT and FasL and is, therefore, a decoy receptor. TR6 appears to have a regulatory role in LIGHT and FasL mediated cell death [2265]. LIGHT is expressed on immature dendritic cells. Engagement of LIGHT costimulates T cell proliferation. Blockade of LIGHT, using soluble recombinant receptors, inhibits DC-mediated allogeneic T cell response [1957]. LIGHT stimulates inflammatory response. Triggering HVEM together with toll-like receptors on neutrophils synergistically stimulates respiratory burst, degranulation, secretion of IL-8 and opsonization of particles [722]. At an early stage in liver regeneration, lymphocytes infiltrate the hepatic tissue. Hepatocyte cell division is stimulated and this depends on the expression of T cellLIGHT and liver cell-LTβR [45]. LIGHT costimulates T cell anticancer immunity. Typically immune cells express LIGHT but it is also found in melanoma cells and on microvesicles released from melanoma; co-culture of microvesicles with lymphocytes in the presence of IL-2 costimulated CD8 T cell proliferation and apoptotic death in the melanoma cells [1368]. Human breast cancer cells transfected with LIGHT cDNA triggered immune response and regression in an in vivo model system [2278]. NK cells express HVEM; tumor cells that express LIGHT activate NK cells to kill tumors in a manner that requires IFNγ and CD8 cells. The NK cells provided help (IFN) in the activation of CTLs [509]. With respect to apoptosis, LIGHT triggers cell death in tumor cells. Normally, two receptors (HVEM and LTβR) are required for LIGHT-induced apoptosis but in tumor cells only one, the LTβR, was necessary [1662].

Lymphotoxin-a (Tumor Necrosis Factor b) Lymphoid Organogenesis; Inflammation; T Cell, B Cell, Bone Cell Development Lymphotoxin α (LT α) was discovered in the conditioned medium of activated T cells [505, 1078, 1695]. It was named for its lymphocyte origin and its toxicity to certain cells. The gene for human LTα is on chromosome 6,

208 close to the TNF-α gene. LTα is a TNF family member; it is also referred to as TNF-β [9, 1553]. LTα is produced by Th1 cells, CD8 T cells, early B cells, NK cells and also by astrocytes. LTα is synthesized as a 202 amino acid precursor with a signal peptide and it is cleaved to 171 and 194 amino acid forms [9]. LTα selfassociates to form homotrimers (LTα3) with a conformation similar to TNF-α. It is a soluble cytokine. The homotrimer binds both TNFRI and TNFRII [209, 212, 777, 1112]. Because the TNFRII-LTα3 complex does not signal, for LTα3 TNFRII is a decoy receptor and an antagonist [1281]. LTα3 also binds herpesvirus entry mediator (HVEM), a receptor for LIGHT [1254]. Lymphoid organ development depends on LT-α and other cytokines such as LT-β, TNF-α and LIGHT. LT-α(−/−) mice, were born without lymph nodes or with defective nodes. Peyer’s patches were also lacking. Spleen histology was abnormal [110, 419, 1004, 1706]. Both LT-α and LT-β appeared to be required for normal lymphoid organogenesis [1249, 1445, 1636]. LT-α and TNF-α are involved in the formation of B cell follicles, T and B cell segregation, follicular dendritic cell clustering and the formation of germinal centers. Mice deficient in either LT-α or TNF-α failed to form germinal centers [502, 2110]. LT-α (−/−) mice have impaired effector T cells. These mice were much more susceptible than controls to HSV infection. CD8 T cells were induced normally but their effector functions were depressed. The cells failed to become CTL and did not secrete INF-γ when stimulated with antigen [1035]. LT-α was also required for the development of memory B cells and their capacity to respond to antigen [571]. LT-α has a major part in inflammation. It induces the expression of leukocyte adhesion molecules VCAM-1 and ICAM-1 on endothelial cells [265, 1581]. In a mouse model, antibodies to LT-α and TNF-α prevented allergic encephalitis [1694]. Transgenic mice overexpressing LT-α have remarkable inflammation in the sites of targeted expression. The infiltrate consists of T cells, B cells, macrophages, and dendritic cells [1020, 1569]. TNF receptors were studied in this system. Mice lacking TNFRI failed to develop inflammation. TNFRI appears to be the primary receptor involved in LTα-induced inflammation. Lack of TNFRII did not did not influence inflammatory response. Lack of the related cytokine lymphotoxin β did not prevent inflammation but altered the response [1705]. LT-α is involved in bone and tooth metabolism. TNF-α and LT-α stimulate osteoblast and osteoclast activity [1988]. IL-1 acts synergistically with TNF

Cytokines to stimulate bone resorption [1883]. In a model for periodontal disease, soluble recombinant receptors for IL-1 and LT-α, acted as antagonists and depressed recruitment of inflammatory cells, osteoclast activity, bone loss and periodontal destruction [83].

Lymphotoxin-b Lymphoid Organogenesis; Inflammatory; NK, Dendritic Cell Development Lymphotoxin-β (LT-β) is a 33 kDa transmembrane protein that was originally cloned from a T cell hybridoma [212, 685]. It is related to LT-α in structure and function. LT-β is expressed mainly on activated T cells, B cells and NK cells [2116]. LT-β chains form homotrimers and heterotrimers with LTα; LTα1-LT-β2 is the major form [209, 212, 777, 2116]. LT-β binds the LT-β receptor (LT-βR). This receptor is related to the TNF receptors. It binds LT-β in its trimer and heterotrimeric forms; it does not bind TNF-α or the LT-α homotrimer [209, 211, 212, 355, 766]. LIGHT is also a ligand for the LT-βR [2278]. Follicular stromal cells, monocytes, fibroblasts, smooth muscle and skeletal muscle cells express the LT-βR. It is also on human melanoma and adenocarcinoma cell lines [211, 402, 2051]. LT-β, LT-α, TNF-α and LIGHT are all critical in the development of lymphoid organs [1249, 1445, 1636, 1739]. LT-β was also required for the homing of dendritic cells to lymph nodes [2206]. LT-β induces IFN-β and this promoted survival of lymphocytes during murine cytomegalovirus infection [110]. LT-α and LT-β stimulate the development of NK cells. LT-β(−/−) mice had fewer NK cells [841, 861, 1846] and LT-βR was required for normal NK development [841, 2205]. The effect of LT-β was not dependent on IL-15, another essential cytokine in NK maturation [841]. It appeared that an effect of LT-β on marrow stromal cells was critical for an early step in NK development [2205]. LT-α and LT-β are required for the normal development of NK T cells. These cells are important in that they may be stimulated to quickly produce large amounts of IL-4, IFN-γ, and IL-10. They are involved in certain types of antitumor activity, the prevention of autoimmunity and protection against bacterial and parasitic infections. Mice that are LT-α(−/−) and LT-β(−/−) have reduced NK T cells and they are low in IL-4 and IL-10. Both LT-α and LT-β are needed suggesting LT-α1β2 (not the homotrimer) is the active form [483].

Walter M. Lewko and Robert K. Oldham LT-β has both cell mediated and direct effects on cancer. It is required for normal production of dendritic, NK, NK T and LAK cells. Mice lacking the LT-βR showed enhanced tumor growth and metastasis [861]. Direct LT effects have also been observed in certain human adenocarcinoma cells that have the LT-β receptor. Signaling through the LT-βR induced cell death in culture and arrested tumor growth in vivo [211, 1194]. Human melanoma cell lines also express the LT-βR. When the receptor was activated, growth was inhibited and proinflammatory cytokines were secreted [402].

Novel neurotrophin B Cell Development; Regulator of Inflammation Novel neurotrophin (NNT) is a member of the IL-6 cytokine family. It is also referred to as B cell stimulating factor-3 and cardiotrophin-like cytokine. NNT is a 225 amino acid protein. It is homologous with IL-6 family members, in particular cardiotrophin-1 (CT-1) and ciliary neurotrophic factor (CNTF). The gene for NNT is on chromosome 11q13 where it is close to the gene for CNTF (11q12). mRNA for NNT is produced by lymph nodes and spleen and to a lesser extent by other tissues [1783, 1808]. The receptor for NNT contains ciliary neurotrophin Rα, gp130 and LIFRβ. Signaling, involves the JAK/STAT pathway and the activation of STAT-3. In this way NNT resembles other IL-6 family members [384, 1088]. NNT stimulates B cells. Increased B cell production and secretion of IgM, IgE, IgG was observed in mice treated with NNT [1784, 1783]. These mice suffered weight loss. NNT, like certain other IL-6 family members, induces cachexia. As its name implies, this cytokine is stimulates nerve growth. In culture, NNT increased the survival of chick embryo neurons [1784]. Gene deficiency studies showed that NNT and the ciliary neurotrophic factor receptor were required for motor neuron development [396, 1099]. In mice, NNT induced the production of serum amyloid A protein. It also potentiated IL-1-induced secretion of glucocorticoids. Many of these effects are shared with certain other members of the IL-6 family, but there are differences. NNT was characteristic in that it did not induce hematopoiesis; NNT induced growth of M1 macrophage cells whereas IL-6. LIF, OSM and CT-1 inhibited M1 growth and induced differentiation [1784].

209

Oncostatin M Lymph Node/T Cell Development; Inflammation; Growth and Wound Repair Oncostatin M (OSM) is a 28 kDa cytokine. It was discovered in the conditioned medium of lymphocytes as a factor capable of inhibiting certain cancer cells [207, 1210, 2274]. Activated T cells, monocytes and neutrophils produce OSM [207, 213, 666, 826]. The gene for OSM is on chromosome 22q12 where it colocalizes with the gene for LIF. It is a member of the IL-6 cytokine family that utilizes gp130 as a receptor component [1664, 2273]. In humans, OSM binds and signals through two receptors. The first is the receptor for LIF, gp130-LIFRβ. It is responsible for effects induced in common by OSM and LIF [601, 602]. The second is gp130-OSMRβ, which is a specific OSM receptor and is responsible for the effects particular to OSM [1373, 1984]. OSM receptors are on a variety of cells including tumor cells. The OSM mechanism of signaling involves MAP kinase, JAK-STAT and the PIP3-kinase pathways [38, 99, 837, 1009]. Oncostatin M has remarkable effects on lymph node development and the production of lymphocytes. Thymus is a key site for T cell processing. But even in athymic animals a certain amount of T cell production occurs in bone marrow, liver, intestine and in lymphnodes. In transgenic mice overexpressing oncostatin M, thymus atrophies and T cells (immature and mature) accumulate in the lymphnodes [175, 325, 1209]. High OSM endows lymph nodes with the ability to sustain T cell development. OSM also mobilizes lymphocytes causing them to move into lymphnodes and to recycle into circulation. OSM has multiple effects related to wound repair and inflammation. It is a growth stimulator for fibroblasts [793] and vascular smooth muscle cells [679]. OSM is a fibroblast activator. It stimulates growth of 3T3 cells, dermal fibroblasts and synoviocytes [694]. OSM increases the production of extracellular matrix collagen [836, 837] and glycosaminoglycan [469]. It stimulates the production and activation of collagenases that degrade the extracellular matrix [705, 1644]. Lytic enzymes and collagen metabolism are part of the growth process in connective tissue. OSM also increases IL-6 production in fibroblasts [208] which itself stimulates fibroblast mitosis and collagen production [468]. Transgenic mice that overproduce OSM have developmental abnormalities and visceral fibrosis [1209].

210 OSM has both stimulatory and regulatory effects on inflammation. It is increased in the synovial fluids of rheumatoid arthritis patients [817]. In a mouse model for RA, OSM appeared to suppress inflammation and tissue damage [2092]. It stimulated the production of acute phase proteins, which are known to have antiinflammatory activity. [1642]. In fibroblasts, OSM inhibited the production of IL-1-stimulated proinflammatory cytokines IL-8 and GM-CSF [1643]. OSM also increases fibroblast production of tissue inhibitor of metalloprotease (TIMP) [1644], which inhibits collagenase activity and tissue destruction. On the other hand, OSM has been shown to have proinflammatory effects. When injected into joints, it caused cartilage resorption and inhibited proteoglycan production resulting in tissue damage [127]. And OSM alone, and together with IL-1 and TNF-α, stimulates production of several matrix metalloproteinases in cartilage [356, 1009]. As implied by its name, OSM has been shown to inhibit growth in several cancers including melanoma [207, 2274], myeloid leukemia [213], glioma cells [702] and breast cancer [1137]. There is interest in OSM for these cytostatic effects and for its role in immune response. But OSM has not been tested clinically. It should be noted, OSM is a growth factor for Kaposi’s sarcoma [1308]. Certain of its proinflammatory effects might exacerbate infiltration and angiogenesis [1605]

Osteopontin Pro Th1, Nitric Oxide Synthetase Inhibiting Cytokine Osteopontin (OPN) is a secreted phosphoglycoprotein. It is also referred to as early T lymphocyte activation protein-1 (Eta-1). OPN is produced by activated T cells, macrophages, osteoblasts, endothelial cells, brain and certain epithelial cells. Its expression is increased by IL-1, IFN-γ TNF-α, bFGF, phorbol esters, glucocorticoids and 1,25 dihydroxyvitamin D3 [79]. OPN is also produced by transformed cells [1785]. There are two receptors for osteopontin, integrin αVβ3 on endothelial cells, fibroblasts and other nonhematopoietic cells, and CD44 that is on leukocytes [79, 694]. The synthetic peptide GRGDSP blocks integrin binding and activation [694]. Integrin αVβ3, CD44 and osteopontin are cell surface extracellular matrix components which function structurally and in signaling. OPN has an important role in bone growth and remodeling. OPN binds osteoclasts and functions in osteoclast recruitment. OPN is a regulator of hydroxyapatite depo-

Cytokines sition for bone calcification [82, 182, 413]. OPN is not normally present in soft tissues, but it does accumulate at sites of abnormal calcium deposition, such as atherosclerotic lesions and calcified heart valves [542, 1875]. OSN appears to be controlling calcium deposition, for in OSN deficient mice, vascular calcification is exacerbated [1867]. In bone marrow, OPN appears to control stem cell migration and the size of the stem cell pool to prevent excessive expansion [1456, 1893]. Osteopontin controls the activation of T cells. It favors Th1 immune response [79]. Mice deficient in OPN fail to develop Th1 immunity during viral and bacterial infection; production of IL-12 and IFN-γ is decreased while IL-10 is increased [79]. Attenuation of experimental autoimmune encephalitis, a Th1-related disease, was observed in OPN deficient mice [881]. OPN appears to stimulate Th1 response at the level of dendritic cells. OPN promotes migration of dendritic cells from tissue sites to lymphnodes; it induces TNF and IL-12 (Th1) cytokines; and it increases adhesion and costimulatory molecules [1635]. Though it favors the Th1 pathway, OPN stimulates B cell proliferation and Ig production. Osteopontin is involved in acute and chronic inflammation. It has chemotactic, pro-inflammatory effects but it also has prominent regulatory functions. Elevated OPN levels have been observed in atherosclerosis [87], glomerulonephritis [816] and in granulomatous diseases [1430]. During heart failure, increased OPN levels are observed in myocardial cells [1831]. In a mouse model for rheumatoid arthritis, OPN appeared to cause joint destruction by promoting angiogenesis and cartilage cell apoptosis [2268]. However, OPN depresses nitric oxide synthetase and NO production in macrophages [694, 1657], kidney epithelium [814] and endothelial cells [1771]. Inhibition of nitric oxide appears to be a major regulatory function of OPN limiting damage during inflammation. In endothelial cells, OPN acts as a survival, cell adhesive and chemotactic factor. It organizes angiogenesis. Fibroblast growth factor 2 (3, 1068) stimulates OPN production by endothelial cells. In turn OPN induces monocyte chemotaxis. Tissue becomes infiltrated with monocytes. OPN stimulates monocytes to produce TNF-α and IL-8, which are both angiogenic [1068]. This mechanism could have a role in normal wound repair or in pathology such as cancer. Several properties of OPN may contribute to cancer cell growth and behavior. Variable osteopontin levels are observed in cancer. Increased OPN secretion was associated with decreased nitric oxide production and decreased killing of tumor cells by macrophages and

Walter M. Lewko and Robert K. Oldham endothelial cells. In this way, OPN may have a role in immune escape by cancer cells [413]. OPN also induces cyclooxygenase, and the progression of prostatic cancer [877]. OPN induces monocyte secretion of IL-1β [1421] and other growth and proangiogenic factors. OPN is expressed by normal brain and astrocytoma cells; it increases astrocytoma cell migration [440]. Hepatocyte growth factor activates a genetic program in epithelial cells for cell dissociation, growth and invasion. This program is upregulated during progression in many tumors. HGF induces osteopontin; antibody studies suggest that OPN mediates HGF-induced invasive behavior [1280]. Melanoma produces OPN; in melanocytes OPN functions as an antiapoptotic survival factor [608]. Osteopontin has been observed in many breast cancers. Its presence is often associated with calcification. Several studies have related osteopontin accumulation in breast cancer with decreased survival [770, 1696, 1834, 2036].

OX40 ligand Costimulation in T Cell, B Cell Development/Memory; Inflammation OX40 ligand (OX40L) is a member of the TNF family. It is a 32–34 kDa, 183 amino acid, membrane bound glycoprotein. OX40L is expressed mainly on dendritic cells [632, 1511], B cells [1908], endothelium and activated T cells [117, 851, 1571]. OX40 (CD134) is the receptor for OX40L. It is a 48 kDa, 250 amino acid, membrane glycoprotein. OX40 was originally described in CD4 T cells as a surface antigen, which was similar to the nerve growth factor receptor [1213, 1542]. It is found only on activated T cells [232, 2000]. Normally, its levels are very low and rise following activation [232, 655]. OX40 signaling involves TRAF-2, TRAF-3 and NF-κB [62]. The OX40L–OX40 system functions as a costimulator during antigen presentation for the activation and increased longevity of T cells, in particular CD4+ Th cells. Mice deficient in OX40 signaling show reduced T cell response to infection. B cell response appears to be normal [294, 1006, 1396, 1571, 2000]. OX40L–OX40 interactions occur in addition to B7-CD28 signaling [111, 660, 386, 887, 2243] between AP and T cells. OX40L acts synergistically with B7 to costimulate CD4 Th cell expansion and the secretion of IL-2, IL-4 and IL-5 [655]. OX40L was required for priming [1436] and sustained later stage CD4 T cell proliferation and memory [654, 655].

211 OX40L is capable of reverse signaling back into the cells that express it [1511, 1908]. In intermediate stage dendritic cells, OX40L ligation, by interaction with T cells or by agonist Mabs, caused reverse signaling to the dendritic cell, enhancing maturation and the secretion of cytokines TNF-α, IL-12, IL-1β and IL-6 [1511]. B cell-deficient mice produce a poor T cell response [1141]. B cells function during antigen presentation; they appear to stimulate T cells after their initial contact with DCs. OX40L has a role in this process. OX40L is expressed on splenic B cells. When B and T cells interact, OX40 engagement stimulates T cell growth and IL-2 release [1908, 1909]; reverse signaling through OX40L on the B cells induces B cell growth and differentiation. OX40 ligand is found mainly on antigen presenting cells. However, OX40L is also present on CD4 T cells and it appears to have a role in T cell response [1862]. When activated in culture, T cells that were genetically deficient in OX40L proliferated less than normal T cells and when they were transferred back into mice, survival was reduced [1862]. It thus appears that T cell OX40L provides additional signals to sustain CD4 T cell longevity. OX40 ligand has been shown to shut down IL-10 producing regulatory T cells. OX40 L inhibited the generation of the regulatory cells and additionally it inhibited IL-10 production in differentiated IL-10 producing regulatory T cells [865]. OX40L is expressed on vascular endothelial cells. It may function during inflammation in the binding and extravasation of activated OX40-expressing T cells [851]. The OX40–OX40L system appears to be involved in several inflammatory diseases including allergic encephalomyelitis [1436, 2134], graft versus host disease [1909, 1910, 2000], inflammatory bowel disease [751] and rheumatoid arthritis [215]. In certain cases, amelioration of the disease has been demonstrated when the animals were treated with anti-OX40 antibodies [751, 1910, 2134]. Exacerbation of inflammatory disease has been shown in transgenic animals overexpressing OX40 [1436]. Tumor infiltrating lymphocytes were also positive suggesting a role for OX40 in host response to cancer [472, 2073]. There is interest in costimulatory cytokines as adjuvants during vaccination. OX40 ligation by agonistic antibodies promoted memory with increased survival of specific T cells [655]. OX40 costimulation could even reverse established anergy [112]. It has been shown that OX40 signaling by several means enhanced immune response during cancer vaccination [27, 44, 379, 669, 836, 914, 1362, 1529] OX40 costimulation together

212 with GM-CSF was able to produce a strong response to the naturally occurring Her2/neu tumor antigen, capable of inducing regression in established tumors [1397]. OX40L and OX40 are on HTLV-infected T cell leukemias and the virus regulates expression. OX40L and OX40 may be involved in the development and growth of this cancer [117, 1321].

RANKL/TRANCE B Cell, DC, Osteoclast Development, Lymph Node Organogenesis RANKL (receptor activator of NF-κB ligand) is a membrane bound, TNF-related protein, It functions in the development of osteoclasts, lymphocytes and in lymph node organogenesis [450, 1003]. RANKL is also referred to as TRANCE (TNF-related activation-induced cytokine), osteoclast differentiation factor [2235] and osteoprotegerin ligand. RANKL is expressed on T cells [48, 903, 1003, 2193, 2194], B cells [2269], dendritic cells [48, 2192] and bone marrow stromal/osteoblast cells [48, 1950, 2269]. In T cells RANKL levels increase with TCR activation [2106]. RANK and osteoprotegerin (OPG) are receptors for RANKL. They are membrane-bound proteins and members of the TNFR family. RANK is found on a variety of cell types, in particular, osteoclast precursors [1003] dendritic cells [48, 1003, 2193, 2221] and B cells [2269, 2270]. OPG is also referred to as follicular dendritic cell-derived receptor-1 and osteoclastogenesis inhibitory factor [218, 825, 2221, 2235]. OPG has membrane bound and soluble forms [2221, 2269]. OPG and RANK are both upregulated by CD40L, an important cytokine in germinal center and B cell development [2269]. RANK is the active receptor, which induces response in target cells; OPG is a non-signaling decoy receptor that competes with RANK [1813, 2270]. Inhibitory activity is mainly associated with soluble form of OPG. The function of the bound form is not certain. There is a report that it may be able to induce apoptosis [2221]. Other investigators were unable to detect any signaling [2269]. RANKL has several functions in the immune system. In dendritic cells, it stimulates antigen presentation [48, 903, 1003, 2192], Bcl-XL antiapoptotic activity and survival [2193]. OPG suppresses this [2270]. The dendritic cells from OPG-deficient mice are altered in that they are activated and show increased capacity to stimulate T cells [2270]. RANKL stimulates normal B cell development. OPG also regulates this process. Mice lacking

Cytokines OPG have altered B cells; transitional B cells are increased and proB cells are more sensitive to IL-7 growth stimulation [2270]. RANKL is a morphogenesis factor; it is involved in the early development of lymphnodes. Mice deficient in RANKL and RANK had abnormal lymphnode development and B cell production [416, 1003]. Estrogens tend to inhibit B cell lymphopoiesis during pregnancy [970]. Estrogens stimulate stromal cell secretion of factors that are inhibitory [1845]. Estrogens also stimulate the production of OPG [778]. RANKL stimulates osteoclast maturation and bone development. Osteoprotegerin regulates osteoclast activity [1050, 1828, 2221]. Macrophages and osteoclasts share a common lineage [517]. Peripheral blood monocytes cultured with RANKL, G-CSF and TGF-β formed osteoclasts [807, 913, 1793]. B cells may also be involved in osteoclast development. B cells are a source of stimulatory RANKL. B-lymphoid progenitor cells are also a source of osteoclast precursor cells [1215]. Mice lacking the RANKL and RANK have problems with bone and vascular development. The mice lack osteoclasts and develop osteoporosis [218]. Mice deficient in OPG are viable but osteoclast activity goes unchecked and as they age, the mice develop severe osteoporosis [1328, 2270]. While little has been done with the RANK/RANKL system in cancer, there is interest in costimulatory molecules that may enhance anticancer immune response. It has been shown that DNA vaccines that include the RANKL gene and dendritic cell vaccines that express RANK/RANKL produce more potent T cell responses [1322, 2155]. It may be that controlling OPG will be useful during immune therapy and vaccination [2270].

Stem cell factor Early Progenitor and Mast Cell Growth Factor Stem cell factor (SCF) was discovered in rat liver as a protein responsible for the outgrowth of very early progenitor cells in bone marrow [2305]. SCF is also referred to as kit ligand, steel factor and mast cell growth factor [47, 345, 2006, 2169]. There are two forms of SCF, soluble and membrane bound, produced by alternative splicing of the same pre-mRNA [47, 345, 2006, 2007]. Several types of cells express SCF, including bone marrow stroma, fibroblasts, liver and spleen [1244, 1248]. The receptor for SCF turned out to be the product of the protooncogene c-kit [47, 345, 543, 2169, 2304].

Walter M. Lewko and Robert K. Oldham This receptor is a protein tyrosine kinase [2219]. It is found on stem cells, progenitor cells and mast cells [1947, 2033, 2143]. A ligand-induced dimerization is part of the receptor activation process. SCF induces the downregulation of its own receptor by internalization [2238]. SCF stimulates growth of early progenitor cells (hematopoietic, lymphoid, and myeloid). Other cytokines induce differentiation and SCF may act synergistically with them [373, 2007, 2033]. SCF has a particularly remarkable effect on mast cells. Mice genetically deficient for SCF suffer anemia and are very deficient in tissue mast cells [609, 977, 978]. SCF acts on early mast cell progenitors to stimulate outgrowth. IL-3 acts at a later stage to stimulate further growth and antiparasite activity [2266]. SCF depresses apoptosis and thus serves as a mast cell survival factor [834]. rhuSCF injected sc into patients activates mast cell degranulation and proliferation with a wheal and flare response at the injection site [346]. Mast cells and SCF may be involved in fibrosis. Fibroblasts are a major source of SCF. Mast cells stimulated by fibroblast SCF release histamine and eotaxin, the eosinophil chemokine [780]. SCF also stimulates mast cell adhesion to fibronectin of fibroblasts and other cells [382]. Mast cells induce fibroblasts to produce collagen. Protracted inflammation produces fibrosis [322, 1105]. In this way SCF may be involved in the development of fibrotic reactions characteristic of chronic inflammatory disease and tumors. In humans, IL-4 depresses the growth and survival of mast cells by down regulating the expression of c-kit [1824]. γδ T cells appear to have a role in innate and adaptive response to viruses and other parasites [1782]. The proliferation of γδ T cells appears to depend on SCF. Mice lacking SCF receptor lacked intestinal γδ T cells while αβ T cells were not affected [1054]. SCF and its receptor are produced by several different types of tumors [1244, 1427] and may stimulate growth. Tumors that have tested positive for c-kit and for SCF include small cell lung carcinoma [748, 1701, 1781], breast [1426], testicular [1907], uterine, cervical and ovarian cancer [853] and melanoma [1428]. While it is not unusual to have the same cell express both the cytokine and its receptor, SCF will usually influence tumor growth by a paracrine mechanism. In this way the normal fibroblast component of a tumor could serve as the source of SCF for c-kit+ carcinoma cells. For therapy, it is possible that SCF blockade may inhibit tumor growth or that presence of this cytokine or c-kit on tumor cells may be used to target antibodies, drugs or toxins. Interestingly, loss of c-kit expression has been observed

213 with progression in breast cancer [1426] and melanoma [1428] reminiscent of the loss of estrogen receptor, which occurs with progression in breast cancer. SCF is being tested for its ability to synergize with G-CSF (filgrastim) to mobilize CD34+ progenitor cells into peripheral blood. It has been shown that SCF in combination with G-CSF may be useful to stimulate hematopoietic recovery after chemotherapy [506, 2124]. SCF with G-CSF also decreased the number of aphereses needed to obtain progenitor cells for autologous transplantation [562].

Tumor Necrosis Factor a Inflammation, Immune Regulation, Apoptosis, Endothelial Damage TNF-α was first described in 1975 as a factor found in animals treated with BCG or LPS. It was named for its ability to induce hemorrhagic necrosis in tumors [250, 661, 2174]. TNF-α was later found to be identical to cachectin, a factor responsible for metabolic wasting in patients with advanced cancer or infection [150–153]. Human TNF-α exists in both soluble and membrane bound forms [1549]. The soluble 17 kDa protein is generated from the 26 kDa membrane-bound form by a protease, TNF-α converting enzyme (TACE) [161, 1377]. TNF-α forms a trimer in solution [75]. The soluble form is more potent and appears to be responsible for most TNF-α bioactivity. The membrane-bound form is also active; it binds and signals through its receptor and by reverse signaling back into its parental cell [91, 714, 753]. TNF-α is produced by a number of different types of activated cells including macrophages, T cells (CD4+ Th1 and CD8+) [634] and B cells [2029, 2116], dendritic cells [2293], neutrophils [2069], adipocytes [954], keratinocytes [1135], mast cells [158, 644], mammary epithelium [2049], colon epithelium [907], pancreatic β cells [2220], osteoblasts [1330], astrocytes [1077], neurons [1972] and steroid-producing adrenal cells [637]. Pathogen molecules such as LPS and bacterial DNA stimulate TNF production in macrophages [241, 1304, 1866, 1876]. TNF-α expression increases with activation of T cell receptors [634] and B cell receptors [2029]. TNF-α production is well regulated. In macrophages, for example, LPS induces TNF-α; then TNF induces IL-10 [1579, 2114] and IL-10 feeds back on TNF-α and other inflammatory cytokines [613, 802] in a regulatory loop. Release of TNF-α from macrophages is inhibited by glucocorticoids, progesterone [1309], and

214 by estrogens [288, 1614, 1796]. Glucocorticoids are well known antiinflammatory agents. Progesterone has some glucocorticoid activity; antiinflammatory effects may be beneficial during pregnancy. There are at least three receptors for TNF-α. TNFRI (55 kDa) and TNFRII (75 kDa) bind both TNF-α and LT-α. A third receptor, described in liver, binds TNF-α but not LT-α [1760]. Nearly all mammalian cells express TNFRI [659]. It appears to be responsible for most TNF-α effects [2094]. The cytoplasmic part of TNFRI contains a death domain that is involved in apoptosis. TNF-α signaling also involves activation of NF-κB and p38 MAP kinase pathways, which induce inflammation and other non-apoptosis responses [2066]. TNFRII preferentially binds membrane bound TNF-α [665]. TNFRII lacks a death domain though clearly it stimulates apoptosis [1976, 2288]. It seems that TNFRII does not induce apoptosis directly but rather it cooperates with TNFRI; a complex is formed which induces apoptosis (397). Mice lacking TNFRII appeared to develop normally but were less sensitive to TNF-α and its toxicity [495]. TNF receptors I and II are shed from cells and found in tissue fluids [493, 1060]. The soluble receptors are generated by the protease TNF receptor releasing enzyme [659, 738, 925, 1389, 1464, 1844]. Production of shed TNFR is specific and regulated. Activation by LPS or by TNF-α itself induces TNFR shedding from many types of cells [131, 344, 1083, 1587, 1889]. In patients with inflamed livers, soluble TNFR levels were correlated with disease severity [2184]. Shed receptors bind TNF-α and act as competitive antagonists. The soluble receptor thus has an antiinflammatory effect [892, 1889]. IL-10, an antiinflammatory and immunosuppressive cytokine, stimulates the release of soluble TNFR from monocytes as part of its mechanism of action [905]. TNF stimulates the secretion of a number of cytokines including TNF-α itself, IL-1, IL-6, IL-8, IFN-γ, GM-CSF, M-CSF, PDGF, IL-10 and NGF. Directly or indirectly, it increases the expression of several growth factor receptors, adhesion molecules, collagenases and plasminogen activator. TNF-α and IL-1 are key proinflammatory cytokines. They share several biological functions [150, 152, 1498] even though they are not related molecularly nor do they share receptors. It appears that both cytokines, on binding their receptors, activate several downstream protein kinases in common [691, 698]. TNF-α has a remarkable ability to induce death by apoptosis in many different types of cells for its role in the regulation of morphogenesis, inflammation and immune response [1726]. When TNF binds RI and RII, death domains are activated [1965, 2094]. p38

Cytokines MAPK and NFκB are also activated. Apoptosis is induced by a caspase protease cascade, starting with caspase-8. The caspases clip and activate a series of enzymes and other proteins, producing the morphology and DNA fragmentation observed in apoptotic cells [75, 2094]. TNF-α also induces Fas expression in cells such as CD4+ T cells, another way in which it causes cell death by apoptosis [2292]. TNF-α not only induces apoptosis but it also regulates it. Activation of NF-κB by TNF-α induces antiapoptotic survival factors that interfere with caspase activation (691). TNF signaling induces death and NF-κB, independently. Sensitivity to TNF-induced apoptosis was enhanced in cells that were not able to activate NF-κB. Thus TNF-α stimulates apoptosis and later suppresses it [2050, 2099]. TNF-α influences many processes in immune response. In dendritic cells, TNF stimulates maturation, activation and antigen presentation for the induction of specific T cells [1648]. TNF stimulates IL-12 secretion in macrophages and other cells; IL-12 enhances T cell development. In T cells, TNF-α and IL-1 together induce Th1 development and secretion IFN-γ [1809]. In many target cells, TNF-α and IFN-γ stimulate MHCI expression to enhance antigen presentation and susceptibility to CTL-induced killing [1515]. TNF-α is a major stimulator of outgrowth in γδ T cells. This response was correlated with TNFRII levels [1053]. TNF-α has a major part in the control of certain infections. For example, TNF-α works together with γ-IFN and IL-4 on macrophages to control tuberculosis [545]. In part, this is due to the induction of nitric oxide synthetase [126, 1729]. TNF-α also induces apoptosis in the mycobacteria-laden macrophages, sequestering the pathogens in apoptotic bodies [140, 457]. In neutrophils, TNF-α increased complement receptor CD11b, adhesion to endothelium, release of reactive oxygen, degranulation, phagocytosis and ADCC [586, 983, 1429, 1792]. TNF-α also increased cellular leukocyte adhesion molecules such as ICAM-1, VCAM-1 and E-Selectin in endothelium [1193], renal tubule epithelium [2209] and in liver [2184]. These adhesion molecules function during tissue infiltration to direct effector cell migration and activity. In capillary endothelium, TNFα is angiogenic and functions in wound repair [1084]. TNF stimulates fibroblast growth and enzyme secretion associated with vessel formation [698]. However TNF-α also causes damage and it is believed to be responsible for vascular leak syndrome [458, 1607]. TNF-α is involved in bone metabolism. TNF-α is produced by osteoblasts; it stimulates osteoblast mitosis [1330] and bone resorption by osteoclasts [1988].

Walter M. Lewko and Robert K. Oldham TNF-α is produced by lipocytes and it may have a role in obesity. TNF-α mRNA levels are increased in the lipocytes of obese patients; levels decrease with weight loss [954]. TNF-α is involved in inflammatory and autoimmune diseases. Most of the deleterious TNF effects appear to be mediated by TNFRI [37, 1685]. In septic shock, LPS and bacterial DNA act synergistically to stimulate TNF-α production [590]. Mice treated with recombinant soluble TNF receptor are protected from toxicity and death [1100]. Collagen-induced arthritis was attenuated in mice treated with antibodies to TNFRI and in mice genetically deficient for TNFRI [1355, 1991]. A protease inhibitor which blocks both TACE and matrix metalloproteases (induced by TNF) produced good responses in rat arthritis models [343]. Antibodies to TNF-α (Infliximab, Etanercept) have been approved for use in patients with rheumatoid arthritis and are being tested in patients with Crohn’s disease and psoriasis [292, 1203, 1454, 1721]. Interestingly, in experimental diabetes and in certain other inflammatory diseases, TNF-α appears to have a two-part influence. TNF-α expressed early was required for disease progression. With prolonged exposure, TNF-α suppressed inflammatory and autoimmune responses [315, 642, 1609]. Certain autoimmune diseases were exacerbated in mice lacking TNF and TNFR. The antiinflammatory effects of long term TNF appear to be due to the suppression of T cell growth and cytokine secretion, associated with downregulation of the T cell receptor [668, 730, 870, 1864]. TNF inhibits tumor growth. It does this several ways. In culture, TNF has a direct effect on many tumor cell lines [250, 1921]. The addition of IFN-γ often has a synergistic effect [563, 2174]. In animals, TNF acts systemically to bring about tumor regression [250]. TNF may act directly on the tumor cells, but its most remarkable effect is on tumor’s vascular endothelium [1435]. Vessels break down and clotting occurs, shutting off the tumor’s blood supply. TNF also stimulates the immune system and upregulates surface antigens involved in tumor cell recognition. TNF and IL-2 synergistically increased cytotoxicity in LAK cells [1517], NK cells [1513] and TIL [1118, 2047, 2111]. TNF may be useful as an adjuvant in tumor vaccines. In animal models, tumor cells engineered to produce TNF induced immune response [30, 162, 775]. The engineered cells were not as tumorigenic; animals with regressed tumors were immune to subsequent tumor challenge. Further, TNF-α has been used to produce dendritic cells for immunization. Dendritic cells prepared from bone marrow were grown in culture with

215 GM-CSF and TNF-α and then pulsed with specific tumor antigens prior to inoculating mice [262, 1259]. TNF-α generally inhibits tumor growth, however, there are cases in which it stimulates growth. TNF appears to be an autocrine and paracrine growth factor for certain ovarian cancers. TNF-α induced its own production. IL-1 also stimulated TNF-α levels and ovarian cancer growth [2207]. In the B16 mouse melanoma model, TNF-α stimulated tumor metastasis to lung. The induction of VCAM-1 by TNF-α may have been responsible [1486]. TNF-α causes thrombocytopenia. Studies in mice showed this depended on TNFRI. Since platelets did not have the TNFR, TNF-α appeared to be stimulating platelet activation and consumption indirectly, possibly by increasing thrombin, plasmin or 5-hydroxytryptamine which are platelet agonists [1941]. Clinical studies with tumor necrosis factor in cancer patients have been extensive. Unfortunately, the results are disappointing. TNF-α has many functions, perhaps too many to be useful clinically. At effective doses, TNF-α causes substantial multiorgan toxicity, hypotension and flu-like symptoms [1318]. As a single agent, it has not shown much antitumor effect [220, 522, 1836, 2149]. By administering TNF-α in isolated limb perfusion [559, 1128, 1496], in organ perfusion [25] and intravesicularly (bladder cancer) [1786], higher doses may be used while containing the toxicity. In these settings, TNF-α has been given in combination with INFγ and chemotherapy (e.g. melphalan). This largely avoids the systemic toxicities. More substantial effects have been observed. Unfortunately, TNF-α administered intrapleurally and intraperitoneally have not been very useful in the control of malignant effusions [771, 1877]. Current strategies with TNF-α emphasize in vitro use to produce dendritic cells and TNF gene transfected tumor cells for vaccines. The TNF gene is also being inserted into activated lymphocytes to exploit the capacity for tumor-specific T cells to home into the target site where TNF is released locally. Recombinant soluble TNF receptor and antibodies to TNF are being tested for control of TNF-related toxicities during immunotherapy. In one study, soluble receptor was administered in combination with IL-2. IL-2-related toxicity (due to TNF release) was modulated while the IL-2 antitumor response was preserved [2020]. Thalidomide, the infamous sedative and teratogen [1291], is once again being prescribed as an immunomodulatory agent and it is being tested in cancer patients. Thalidomide suppresses TNF-α production, inflammation and certain cell surface adhesion molecules [1356, 1462, 1719]. It is also antiangiogenic [953].

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TRAIL Apoptosis in Neoplastic Cells; Regulation of Inflammation Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, also called apo-2 ligand) is a 281 amino acid transmembrane protein and a member of the tumor necrosis factor family [1576, 2166]. Most leukocytes and certain tumor cells express TRAIL [2166]. In T cells, TRAIL is induced by activation and by IFN-α/β [939, 1776]. Cyclosporin and glucocorticoids inhibit its expression [1406, 2107]. There are at least five receptors for TRAIL; TRAIL-R1 (DR4) [1528], TRAIL-R2 (DR5) [1555, 1751, 1772, 2089], TRAIL-R3 (DcR1) [404, 1341, 1805], TRAIL-R4 (DcR2) [403, 1232, 1527] and osteoprotegerin [490]. TRAIL receptors are found on many cells, often together with TRAIL. TRAIL-R1 and TRAIL-R2 contain cytoplasmic death domains and induce apoptosis in cells, mainly neoplastic cells. TRAIL may induce apoptosis or regulate cell growth in normal T cells, mast cells and hepatocytes [138, 401, 888, 896, 1856]. By comparison with TNF-α and FasL, TRAIL has little normal tissue toxicity [81, 19]. Two possible explanations for this lack of toxicity are competitive antagonism and induction of antiapoptotic proteins. TRAIL-R3 and TRAIL-R4 bind TRAIL but do not induce apoptosis. They lack cytoplasmic death domains and thus serve as decoy receptors [80, 1227, 1555]. In a similar way, osteoprotegerin is a soluble receptor that binds TRAIL and competitively antagonizes its apoptotic activity [490]. Cancer cells may produce OPG and for these tumors OPG can function as a paracrine survival factor [785, 1809]. Normal cells and TRAIL-resistant tumor cells generally express antiapoptotic proteins such as FLIP [671, 856, 2283] and IAP [403, 404, 420, 560, 2100]. These interfere with caspase activation and function as survival factors. Drugs that control survival factors potentiate anticancer effects of TRAIL [1140]. TRAIL is responsible for cell contact-induced tumoricidal activities of CD4 T cells [1985], NK cells [939], monocytes [672] and dendritic cells [289, 510]. In CD4 cells, for example, studies using blocking and activating antibodies showed that TRAIL-induced apoptosis is one of the ways cytotoxic CD4 T cells kill melanoma [1985]. The upregulation of TRAIL on T cells may be responsible for certain antitumor effects of interferons α and β [939]. Dendritic cells can kill tumor cells directly by TRAIL-induced apoptosis. In addition to decreased tumor burden, DC-TRAIL provides apoptotic bodies

Cytokines that stimulate dendritic cell maturation [1689] and serve as antigen for immune response [20, 779]. TRAIL regulates inflammation and autoimmune disease. This has been studied in mouse models for autoimmune arthritis [1856] and encephalomyelitis [757]. Blockade using soluble receptor to TRAIL exacerbated these diseases while TRAIL expression diminished them. TRAIL apparently decreases the activation of autoimmune T cells. The anticancer effects of TRAIL are remarkable but complex. Mice with genetic TRAIL deficiencies had increased risk for hematological malignancies [2276] and methylcholanthrene-induced sarcomas [352]. TRAILinduces apoptosis in most melanoma cell lines [1985, 2283]. In human colon and breast cancers growing in nude mice, activation of TRAIL receptors induced apoptosis and inhibited growth [81, 319, 2090, 2106]. These studies show that TRAIL and TRAIL R1 and R2 may have therapeutic potential in the treatment of cancer. But not all cancers respond. And in certain tumors TRAIL induces NF-κB, which promotes survival and growth [480]. Drugs that inhibited NF-κB signaling sensitized cells to apoptosis and increased anticancer response to TRAIL [104, 1140, 1172, 2098]. Clinical trials have been initiated in cancer patients using apoptosis-inducing TRAIL receptor antibodies [1580, 2001].

TWEAK Cell Growth, Survival; Angiogenesis; Inflammation TWEAK (tumor necrosis factor-like weak inducer of apoptosis) is a member of the TNF cytokine family. It is expressed in many types of cells. Tweak is synthesized as a membrane-bound protein that is cleaved to release a soluble, active, trimeric cytokine [304]. The TWEAK receptor is Fn14. It is a TNF receptor family member. Fn14 was discovered as a cell surface protein that was induced by fibroblast growth factor [523, 2164]. Fn14 is upregulated in injured blood vessels [2164] and in regenerating liver [523]. The cytoplasmic tail binds and signals through TNF associated factors-1, 2, 3 and 5 [206, 2164]. TWEAK activates NFκB, which translocates to the nucleus to induce genes; TWEAK activates caspases 3 and 8 in those cells that undergo Tweakinduced apoptotic death [206, 2019]. In a cultured monocyte line, TWEAK induced the cells to differentiate into osteoclasts. Fn14 was not the receptor for this differentiation since the cell line lacks

Walter M. Lewko and Robert K. Oldham Fn14 and inactivating mabs to Fn14 did not block the Tweak effect. This study suggested the existence of a second TWEAK receptor [1583]. It was recently reported that CD163 is an additional binding protein for TWEAK. It is present on monocytes and macrophages where it is known to function as a scavenger for hemoglobin. CD163 has been shown to compete with Fn14 for TWEAK; may serve as an alternative receptor [185]. TWEAK is a remarkably proinflammatory cytokine. It induces the expression of factors such as IL-6, IL-8, RANTES, MCP-1, IP-10 and ICAM-1 in a number of cell types including fibroblasts [305], keratinocytes [891], synoviocytes [305], bronchial epithelial cells [2216], macrophages [967], mesangial cells [237], astrocytes [1702] and endothelial cells [713]. Typically, the addition of IFN-γ has a synergistic effect. TWEAK levels are elevated in tissues of mice with experimental autoimmune encephalitis [417] and collagen-induced arthritis [1556]. TWEAK is angiogenic. It induces mitosis and migration in human endothelial and aortic smooth muscle cells. FGF-2 and VEGF, which are also angiogenic, stimulate TWEAK receptor levels. TWEAK induced strong vessel growth in the rat cornea angiogenesis assay [449, 713, 1180, 2164]. TWEAK appears to be involved in neoplastic progression. The Fn14 gene is rapidly induced during liver regeneration; Fn14 is overexpressed in hepatocellular carcinomas [523]. When human embryonic kidney cells were transfected to express TWEAK, these cells formed larger and more vascular tumors in nude mice [774]. Glioblastoma specimens and glioma cells in culture overexpress Fn14. And when glioma cells were treated with TWEAK, the cells became resistant to apoptosisbased therapy. Tweak activates the NFκB path for growth and survival. All told, blocking Fn14 may be of benefit in the management of glioblastoma and other TWEAK/Fn14 expressing cancers [2018].

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Abbreviations ADC; antibody dependent cytotoxicity; AICD, activation-induced cell death; AP, antigen presentation; baso, basophil; BCGF, B cell growth factor; BCSF, B cell stimulatory factor; BM, bone marrow; CFR, cytokine family receptor; CFU, colony forming unit; CNTF, ciliary neurotrophic factor; CTL, cytotoxic T lymphocyte; DC, dendritic cell; DcR, decoy receptor; DR, death receptor; EAE, experimental autoimmune encephalitis; EGF, epidermal growth factor; ELR, glutamic acid-leucine-arginine (a chemokine designation); eos, eosinophil; EPO, erythropoietin; ETA-1, early T lymphocyte activation protein (OPN); FISP, IL-4-induced secreted protein; γc, common gamma chain (IL-2Rγ chain); G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte macrophage colony stimulating factor; gp, glycoprotein w/mw (x 10−3); HLA, human leukocyte antigen; HMW-BCGF, high molec wt-B cell growth factor (IL-14); HVEM, Herpes virus entry mediator (LIGHT R); IAP, inhibitor of apoptosis; ICAM, intercellular adhesion molecule; ICE, IL-1β cleavage enzyme = caspase 1; IFN, interferon; Ig, immunoglobulin; IL,

Cytokines interleukin; ILA, induced by lymphocyte activation (4-1BB); IL-1Ra, IL-1 receptor antagonist; IL-1RAcP, IL-1 receptor accessory protein; IL-1RAK, IL-1 receptor associated kinase; IL-1Rrp, IL-1 receptor related protein (IL-18Rα); IL-TIF, interleukin 10-related T cell-derived inducible factor (IL-22); IP-10, interferoninduced protein-10; JAK, Janus kinase; -L (as a suffix), ligand; LAK, lymphokine activated killer; LIF, leukemia inhibitory factor; LIGHT, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells; LIT, lymphocyte inhibitor of TRAIL; LN, lymphnode; LPS, lipopolysaccharide; LT-α, lymphotoxin-α (TNF-β), LT-β, lymphotoxin-β; Macro, macrophage; MAPK, mitogen-activated protein kinase; MCP, macrophage chemoattractant protein; M-CSF, macrophage colony stimulating factor; MHC, major histocompatibility complex; Mig, monokine induced by IFN-γ; MIP, macrophage inflammatory protein; MMP, matrix metalloprotease; Mono, monocyte; neut, neutrophil; NF-, nuclear (transcription) factor as in NF-κB; NGF, nerve growth factor; NK, natural killer cell; NNT, novel neurotrophin; nTreg, natural regulatory T cell; OSM, oncostatin M; PAMP, pathogen-associated molecular patterns; OPG, osteoprotegerin; OPN, osteopontin; PBL, peripheral blood lymphocyte; PDGF, platelet derived growth factor; PG, prostaglandin; p, protein w/ mw (x10−3); PMA, phorbol myristate acetate; -R (as a suffix), receptor; RANKL, receptor activator of NF-κB ligand (TRANCE); RANTES, regulated on activation, normal T cell expressed and secreted; rhu- (as a prefix) recombinant human; SAPK, stress activated protein kinase; s- (as a prefix) soluble (not membrane bound); SCF, stem cell factor; SCID, severe combined immunodeficiency disease; SDF, stromal derived factor; SLE, systemic lupus erythematosus; SOCS, suppressors of cytokine signaling; STAT, signal transducers and activators of transcription; Stk, stem cell tyrosine kinase (flt-3), TACE, TNF-α converting enzyme; TCR, T cell receptor; TDAC, tumor derived activated T cell (TIL); TGFβ, transforming growth factor-β; Th, helper T cell; TIL, tumor infiltrating lymphocyte; TIMP, tissue inhibitor of metalloprotease; TLR, toll-like receptor; TNF, tumor necrosis factor; TNFR, TNF receptor; TPO, thrombopoietin; TRADD, TNF Receptor 1-associated death domain; TRAF, TNF receptor-associated factor; TRAIL, TNF-related apoptosis-inducing ligand; TRANCE, TNF-related activation induced cytokine (RANKL); Treg, regulatory T cell; TWEAK, Tumor necrosis factor-like weak inducer of apoptosis; tyk, tyrosine kinase; VCAM; vascular cell adhesion molecule; VLS, vascular leak syndrome.

9

Interferons: therapy for cancer DAVID GOLDSTEIN, ROBERT JONES, RICHARD V. SMALLEY, AND ERNEST C. BORDEN

Stimulate the phagocytes. Drugs are a delusion. Find the germ of the disease; prepare from it a suitable antitoxin; inject it three times a day, a quarter of an hour before meals and what is the result? The phagocytes are stimulated; they devour the disease and the patient recovers-unless, of course, he’s too far gone. – Sir Ralph Bloomfield Bonnington in The Doctors Dilemma George Bernard Shaw, 1902.

Isaacs and Lindemann, in England, first characterised interferon (IFN) in 1957 and coined the word to signify a protein, elaborated by virus-infected cells, that functions to prevent their infection by a second virus [108]. However, difficulties with chemical isolation and characterization led to great skepticism about the molecule’s existence; indeed, “the scientific community dubbed the discovery ‘imaginon’ ” [192]. Time and effort have proven Isaacs and Lindemann right. Interferons are now a well-characterized group of proteins. They are a large family of four major immunological types, alpha, beta, gamma, and omega [199]. The alpha and beta interferons were collectively known as the type I interferons while gamma interferon was referred to as type II interferon. There are multiple natural alpha interferon molecules and one natural beta interferon molecule. These type I interferons share a common receptor which is present on nearly all cells in the body. The alpha interferons, originally derived from leucocytes, and beta interferon, originally derived from fibroblasts, are actually secreted by nearly all mammalian cells. Even though they share a common receptor, their activation pathway must differ since the transfection of the human type I receptor gene into murine cells allows alpha but not beta expression [266]. Gamma interferon, on the other hand, is produced by T lymphocytes upon specific antigen recognition. All interferon molecules have antiviral and antiproliferative capacity, and to some extent immunomodulatory activity, although gamma is a stronger immunomodulatory molecule than the type I interferons [19]. The enormous amount of clinical study of the interferons in malignancy established the field of biologic therapy of cancer. The many new agents currently under investigation to inhibit angiogenesis and the aberrant growth factor networks associated with malignancy have all taken advantage of the insights provided during the clinical

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

study of the role of interferons as anti neoplastic agents. A review of these interferon studies remains a valuable resource of guidelines on how to optimise clinical studies in malignancy using novel non-traditional agents.

Nomenclature Alpha interferon is produced following viral stimulation. There are at least 14 subtypes of alpha interferon, each immunologically related but differing by a number of amino acids [290]. Several nomenclature systems exist for identifying these subtypes. The two commonly used, but unrelated, systems use either lettering A, B, C, D, etc., or numbering 1, 2, 3, etc. to define the subtypes. A newer proposal, approved by the Nomenclature Committee of the International Society for Interferon Research, has been recently published [199]. A corresponding proposal for nomenclature of the interferon-gene-induced proteins exists. Three natural alpha interferon pharmaceutical products are currently available for use in patients in various countries in the world. The original Finnish Red Cross partially purified material developed by Cantell and subsequently also produced by others from the buffy coat of peripheral blood is available as Finnferon® in some European countries. A highly purified natural alpha interferon produced by virally stimulated human lymphoma (Namalva) cells has been produced in pharmacologic quantity by Burroughs Wellcome Co (now Glaxo Wellcome) and is known by the trade name Wellferon®. It is marketed in Japan, Canada, Mexico and Europe but not the United States for treatment of hairy cell leukemia (HCL) and some viral disorders. Alferon N® is an aqueous formulation of human alpha interferon proteins with a specific activity of 2 × 108 IU/mg of protein and is manufactured in the United States by the Purdue Frederick Company. It has been approved in the United States for the intralesional treatment of refractory or recurring external condylomata acuminata in patients over 18 years of age. These natural alpha interferons contain most, if not all, of the multiple alpha molecules.

277

278 In addition to the natural alpha interferon products, there are several pure single alpha molecules obtained by recombinant technology that are available for clinical use. Interferon alpha A (alpha 2) has been pharmacologically produced as recombinant alpha interferon by three companies, Hoffman-LaRoche in Nutley, NJ; Schering Corporation based in Kenilworth, NJ, and Boehringer Ingelheim in Germany. These products are known as Roferon A® (rIFN alfa 2a), Intron A® (rIFN alfa 2b) and Berofor® (rIFN alfa 2c), respectively. The alpha subtype, interferon alpha C (alpha 10) has been produced in pharmacologic quantity in Israel. Roferon A® is manufactured by Roche Laboratories using recombinant DNA technology employing a genetically engineered E coli bacteria containing an interferon alpha 2 gene obtained from a human myeloid leukemia cell line; it has an approximate MW of 19,000 daltons and a specific activity of 2 × 108 IU/mg protein and is approved for use in the United States for the treatment of HCL and AIDS-related Kaposi’s sarcoma in patients 18 years of age or older. Intron® A is produced by Schering Corporation and is obtained from the bacterial fermentation of a strain of E coli which bears a genetically engineered plasmid containing an interferon alpha 2 gene from human leukocytes. It has a specific activity of 2 × 108 IU/mg protein and is approved in the United States for treatment of patients 18 years of age or older with HCL and for selected patients with condylomata acuminata, AIDS related Kaposi’s sarcoma, chronic hepatitis non-A, non-B/C and with chronic hepatitis B. Pegylated interferon alpha has been extensively studied in hepatitis [140, 152] and initially in renal carcinoma [165] and chronic myeloid leukemia [111]. It reduces frequency of administration (once/week versus three) and has an improved toxicity profile. A recombinant IFN beta, with a single amino acid substitution separating it from the natural beta molecule, was developed by Triton Biosciences, Inc., Berkeley, CA, and is currently manufactured by Berlex and marketed by Chiron Corporation (US) and Schering AG (Germany) under the trade name Betaseron® for treatment of multiple sclerosis. It is obtained by bacterial fermentation of a strain of E coli that bears a genetically engineered plasmid containing the altered gene for human interferon beta. The native gene was obtained from human fibroblasts and altered in a way that substitutes serine for the cysteine residue found in position 17 in the native molecule (interferon betaser17). The recombinant protein is a highly purified molecule with 165 amino acids, a MW of approximately 18,500 daltons and a specific activity of approximately 32 MU/mg protein; it does not include the carbohydrate side chains

Interferons: therapy for cancer found in the native molecule. Betaseron® is approved in the United States for the treatment of ambulatory patients with relapsing-remitting multiple sclerosis. Biogen, Inc has produced a recombinant molecule from the natural beta interferon gene, which, since it is produced in eukaryotic cells, is glycosylated. This product, with the trade name Avonex®, has been approved in the US for treatment of relapsing forms of multiple sclerosis. Serono also has a beta interferon molecule interferon beta-1a for multiple sclerosis. Two recombinant gamma interferon molecules have been cloned and were clinically developed, one by Genentech, Inc., and one by Biogen, Inc. These will be referred to, respectively, as rIFN gamma (Genentech) with a trade name of Actimmune® and rIFN gamma (Biogen) with a trade name of Immuneron. Actimmune® is manufactured by bacterial fermentation of a strain of E coli containing the DNA, which encodes for the human gamma interferon molecule. It has a specific activity of 30 million units/mg protein, and is approved for clinical use in the United States for reducing the frequency and severity of serious infections associated with chronic granulomatous disease. Immuneron is not yet approved for prescription use in the US. These various products are listed in Table 1. The generic designations will be used throughout this chapter. Intracellular signaling in response to binding of type one and two interferons to their respective receptors has been carefully dissected out. Both subtypes of interferon result in binding and phosphorylation of Janus activated kinase molecules (Tyk2, Jak 1 and 2) followed by phosphorylation of signal transducers and activators of transcription (Stats) which translocate to the nucleus and activate interferon stimulated genes [198, 113].

Table 1. Interferon nomenclature IFN

Generic

Natural alpha interferon Cantell IFN IFN alfa (Le) Lymphoblastoid IFN alfa N1 Leukocyte derived IFN alfa N3 Recombinant alpha interferon Alpha A rIFN alfa 2a Alpha A rIFN alfa 2b Alpha A rIFN alfa 2c Alpha C rIFN alfa 10 Recombinant beta interferon rIFN beta 1B rIFN beta (Biogen) Recombinant gamma interferon rIFN gamma (Genentech) rIFN gamma (Biogen)

Trade name Finnferon® Wellferon® Alferon N® Roferon A® Intron A® Berofor®

Betaseron® Avonex® Actimmune® Immuneron‫‮‬

D. Goldstein et al.

Clinical use In the early 1970s, work by Kari Cantell and co-workers led to the production of sufficient quantities of alpha interferon, made from buffy cell layers, to support limited clinical trials in patients with several types of malignancies [253, 254, 50]. This work was expanded in North America by the U.S. National Cancer Institute (NCI) which sponsored trials beginning in 1975 and by the American Cancer Society (ACS) which supported trials beginning in 1978. Despite very limited clinical information, interferon was heralded in the popular press at the time as a significant new drug for the cure of cancer. The early clinical trials with this partially purified natural alpha interferon demonstrated response rates in breast cancer, osteosarcoma and lymphoma, comparable to those achievable with chemotherapeutic agents. The wave of enthusiasm and outpouring of venture capital rapidly led to the development of DNA recombinant technology and, following the successful cloning in 1979 by Taniguchi et al. of beta-interferon [259] and the subsequent cloning of the alpha interferon subtypes and gamma interferon, the production of recombinant interferon molecules. Furthermore, the isolation and purification of natural alpha Interferon from Namalva cell cultures led to far greater availability of natural alpha interferon and the number of clinical trials increased dramatically. The partially purified IFN alfa (Le), used in the early trials sponsored by the NCI and ACS, had a relatively low specific activity and, since it was a supernate, was composed of not only a variety of subtypes of alpha interferon but multiple other cytokines as well. [95, 18] working with patients with breast cancer in the US [50] working with patients with ovarian carcinoma in Scandinavia, reported response rates of over 20% using this natural alpha interferon product. These results have not been reproduced in these tumors using rIFN alfa 2a or 2b, raising the question of what other active constituent(s) in this interferon preparation may have induced these responses.

B Cell Malignancy Several B cell malignancies have been shown to be therapeutically sensitive to alpha interferon. Multiple myeloma and non-Hodgkin’s lymphoma (NHL) were among the first malignancies to respond in the early trials with the Cantell interferon. Hairy cell leukemia (HCL) was shown in the early 1980s to be exquisitely sensitive to alpha interferon and

279 this biologic was rapidly approved in most countries for the treatment of this previously untreatable malignancy.

Hairy Cell Ieukemia Hairy cell leukemia, a B cell malignancy, is a disease with an exquisite sensitivity to alpha interferon and was the first human malignancy to be so identified. Prior to the development of the interferons, no adequate therapy existed for this relatively rare disorder. Splenectomy was the standard approach when treatment was necessary and was useful for controlling pancytopenia for a period of time but had no effect on the leukemic process. Quesada was the first to demonstrate the beneficial effect of alpha interferon in this disease in 1982 [205]. After finding that a dose of 12 MU/m2 was poorly tolerated, he and his colleagues at the MD Anderson Cancer Center, arbitrarily settled on a regimen of 3 MU (1 MU = 106 units) administered daily subcutaneously. Several other investigators followed suit and have shown that high response rates are attainable with the potential for a prolonged response duration, even with lower doses. Seventy-five percent to 80% of patients will obtain major clinical benefit with improvement in hematologic parameters and a decrease in the leukemic (tumor cell) population. Treatment of several months’ duration is required for maximal benefit and continued treatment is necessary to maintain clinical benefit. Equivalent efficacy has been shown with each of the alpha interferon products and with beta interferon [69, 82, 207, 284, 285, 76, 77, 83, 84, 72, 280, 242]. Gamma interferon is ineffective in this disease [209]. Following Quesada’s lead, multiple studies by Golomb et al. at the University of Chicago, established an objective anti-leukemic response (CR or PR) rate of 20–25% and an improvement in hematologic parameters in another 60% for an overall major clinical benefit in 80–85% of patients [82, 83, 84]. Following cessation of therapy, patients relapsed but remission could be successfully reinduced [208, 84]. The studies by the groups at MD Anderson and the University of Chicago with recombinant alpha interferon led to the approval of alpha interferon in the United States for the treatment of HCL. A significant cost benefit associated with interferon treatment was demonstrated [190] and additional studies with natural alpha interferon demonstrated its superiority over splenectomy, the inadequate standard of therapy prior to the development of alpha interferon [244]. During the mid-1980s, investigators in Innsbruck began to explore the question of dose effect in patients

280 with HCL and determined that a dose as low as 0.5 MU (500,000 U) was biologically active and immunologically stimulating, as measured by the production of neopterin [105, 69]. Subsequent comparative studies with rIFN alfa 2c [72] and IFN alfa N1, showed the low dose was as effective as the standard dose in inducing a return to normal of the platelet count (within 36–38 days median) and of the neutrophil count (within 90–130 days median), as well as reducing the need for red cell transfusion support and the incidence of infection. The standard dose, however, was clinically and statistically more effective in its antileukemic effect, i.e., reducing hairy cell infiltration in the marrow [242]. However alpha interferon has been supplanted by cladribine [289, 26]and pentostatin [112] as the treatment of choice, just 1 decade after it was shown to be the first effective systemic agent for the treatment of this disease. Its role as an additional agent has not proven to be significant [153]. Of interest a new biologic agent – anti cD22 monoclonal antibody combined with an exotoxin has shown activity in cladribine resistant disease [128]. Thus the promise shown by interferon that a biologic agent could have an anti tumour impact and modify the natural history of a malignant disease has been kept. It is likely that more targeted biologic therapy will remain an integral part of the treatment of this disease.

Multiple Myeloma Interest in the treatment of multiple myeloma with alpha interferon was initially stimulated by the pilot studies of Mellstedt et al. in 1979 and the ACS-supported trial reported in 1980 [160, 187]. Each study demonstrated objective anti-tumor responses in a small number of previously heavily treated patients. A number of phase II trials of both natural and recombinant alpha interferon were subsequently performed and cumulatively suggested that about 20% of patients with multiple myeloma, refractory to cytotoxic chemotherapy, might obtain an objective response to a variety of doses and schedules [38, 276, 181, 25, 36, 180, 206, 37]. Because of these encouraging results in previously treated patients, alpha interferon was compared with cytotoxic chemotherapy in previously untreated patients but chemotherapy proved superior in terms of both response rate and duration of response [4, 147]. Several trials were organized to evaluate the addition of alpha interferon to various combinations of cytotoxic chemotherapy in terms of response induction [35, 227, 114, 182]. One of these a large Swedish trial involving over 300 patients evaluated the addition of IFN alfa (Le) administered on days 1–5 and days 22–26 of every 4-week

Interferons: therapy for cancer cycle to melphalan/prednisone which were given on days 1–4 and demonstrated a benefit in overall response and survival for patients with either IgA or Bence-Jones myeloma but not IgG myeloma [188]. This study was the third in a series of trials by this group to demonstrate a clinical benefit from this natural alpha interferon in patients with IgA myeloma. The explanation for benefit only for patients with IgA myeloma in this series of trials is not apparent [227]. A study of VMCP with or without alpha interferon randomised 240 patients, there was no difference in response rate but fewer patients receiving interferon progressed and the median duration of response was 6 months longer (12.4 months versus 18.3 months, p < 02) for patients receiving alpha interferon [146]. A subsequent study evaluated the addition of alpha interferon to a five-drug combination (VBMCP) [182]. In 628 patients the complete response rate was increased 18% versus 10% and there was an increased time to progression with rIFN alfa2, 30 months versus 25. Of interest the best response was seen in the IgA subgroup. A small Australian study of adding interferon alpha 2a to intensive combined chemotherapy failed to show a benefit in either response rate or overall survival [114]. Two meta-analyses have now examined the clinical advantage of adding interferon to chemotherapy. Ludwig et al. performed a global analysis on 30 trials evaluating a total of 3,948 randomized patients, 17 of which added interferon and concluded there was a slight (6.6%) advantage in response rate and median relapse free survival (4.8 months) and overall survival 3.1 months [146]. The subsequent individual patient meta-analysis of 24 trials with 4,012 patients confirmed the slightly higher response rates (57.5% versus 53.1%) and progression free survival – 33% versus 24% at 3 years, a 22% reduction in risk. The median time to progression was increased by 6 months and the risk of death decreased by 8% [92]. However, any advantage of adding interferon to induction chemotherapy in this disease is too small to be clinically meaningful and is probably not worth the added financial cost (US$42,236.19/life year saved) or side effects. Open to further exploration however is the persistent intriguing observation of the possible benefits in patients with IgA/Bence-Jones myeloma. Several trials were next established to evaluate the use of alpha interferon as a maintenance agent in patients with myeloma. Following the induction of a response with cytotoxic chemotherapy, various groups randomized patients to receive alpha interferon or no further therapy until the onset of progressive disease [151, 223, 195, 279, 20, 16, 146, 225].

D. Goldstein et al.

281

In addition, the Nordic Study Group randomized patients at the start of treatment; those randomized to receive alpha interferon received it continuously [91]. These studies are summarized in Table 2. The US cooperative group trial and the trial in Germany showed no benefit from interferon therapy. The other seven trials have demonstrated either a prolonged time to progression or duration of plateau phase in the interferon maintained group. Ludwig et al. also performed a global analysis for these maintenance studies also and concluded that alpha interferon both relapse free survival by 4.4. months and overall survival by 7.9 months at a cost of US$18,114.95 (Figures 1 and 2) [146]. The individual patient meta analysis showed an increase in progression free survival at 3 years of 27% versus 19%, a 34% reduction in risk of progression and a 12% decreased risk of death [92]. Median survival is increased by 4 months. Such an effect also needs to be balanced against not only cost but also quality of life. In that regard a recent study suggests that after 12 months there is no significant difference in quality of life in two Nordic maintenance studies [282]. A recent study with pegylated interferon suggests it may further improve quality of life in this setting [240]. A randomised trial of maintenance alpha interferon, following high dose melphalan consolidation with bone marrow transplant rescue has also been performed. rIFN alfa 2b had a beneficial effect on prolonging remission, especially in those patients who achieve a complete remission 46 versus 27 months at 52 months of follow up and in improving survival at 52 months 95% versus 75%. However by 77 months most patients had died and the benefit was not sustained [40]. In addition the European marrow transplant registry has reported in a

Table 2. IFN versus CT in CML Group/reference

Comparison

IFN effect on Dur of Resp survival

Italian study group (202) German study group (75, 76)

Alpha IFN versus CT

+

+

Alpha IFN versus busulfan Alpha IFN versus HU Alpha IFN versus busulfan Maintenance with α IFN versus no maintenance

+

+

+ +

Neg +

+

+

Japanese study group (128) UK study group (7) HU = Hydroxyurea

retrospective analysis of 473 patients that received maintenance IFN compared to 419 patients who did not that overall survival and progression free survival were significantly better – 78 months versus 47 months and 29 versus 20 months. The retrospective nature of the report requires cautious interpretation [15]. A third study from the Nordic Group also used maintenance interferon following high dose therapy and showed a benefit for this program over a historic control but the role of interferon itself in prolonging survival cannot be determined [137]. In summary, alpha interferon is an active agent in myeloma. Current data support its greatest usefulness when used as an agent capable of prolonging remission in patients with low tumor burden; in some of these patients it may also improve survival.

Non-Hodgkin’s lymphoma (NHL) Several clinical trials conducted in the late 1970s with the Cantell interferon suggested efficacy in patients with low and, to a lesser extent, intermediate grade NHL [161, 95, 144, 104]. Single agent trials in the 1980s with the more highly purified recombinant and natural alpha interferons confirmed the efficacy of alpha interferon in patients with NHL [64, 177, 136]. Two single institutions and the CALGB, in three separate trials, combined alpha interferon with an alkylating agent in patients with low grade NHL and demonstrated the combination to be relatively non-toxic and well tolerated. The combination induced a response in 50–75% of patients, more readily in previously untreated individuals [29, 28, 189, 30]. The group at MD Anderson, in a single institution non-randomized study, evaluated the use of IFN alfa N1 as maintenance therapy, inducing a response with CHOP plus Bleomycin and then treating patients with IFN alfa N1 until relapse [157]. They concluded, based on historical controls, that alpha interferon prolonged the duration of response but did not improve survival. Subsequently a large number of randomised trials have been reported in complete or abstract form evaluating, by direct comparison, the addition of alpha interferon to cytotoxic chemotherapy in the initial treatment of the disease. In studies using less intensive regimens such as chlorambucil or CVP there has been no benefit on overall survival and at best a marginal improvement in progression free survival in one study [197, 96, 216]. There have been two large prospectively randomized trials

282

Interferons: therapy for cancer

Study start year, code and name

Prog’sions/Patients IFN None

Statistics (O-E) Var.

O.R. & Cl* (IFN : None)

Odds Redn. (SD)

Interferon in Induction: 14/15 16/19 61/69

-2.6 -4.2 -2.9

6.3 6.5 37.9

34% (33): P = 0.5

86J MGCS 1986

13/16 15/21 97/105

87E EMSG 2 (1)

32/70

37/56

-7.9

16.7

35% (19): P = 0.6

88A EOOG 9486

123/161

121/153

-13.9

59.2

21% (12): P = 0.07

89H KIF, Avicenne

43/97

42/95

1.3

21.2

-7% (22): P = 0.8

90D NMSG 04–90

83/125

89/134

-16.4

44.6

31% (15): P = 0.01

90H ALSG Myeloma II

14/24

21/26

-8.0

7.9

64% (22): P = 0.016

90J Ital NHLSG (I)

11/28

15/21

-4.7

6.0

54% (20): P = 0.04

91B GMM (I), Mexico

20/40

14/40

0.7

6.9

-51% (40): P = 0.6

451/685 (65.8%)

440/638 (69.0%)

-52.7

213.4

22% (6) reduction P = 0.0003

85G GATLA 3-M-85 86I Rome IFN 1

Subtotal

45% (29): P = 0.1 -5% (17): P = 0.6

Test for heterogeneity between trails: c20 = 15.8; P = 0.07 Interferon in maintenance: 85D Ital. MMSG M84 87C SWOG 8624

35/50

44/51

-14.0

18.0

54% (15): P = 0.001

78/107

83/104

-8.1

39.9

15% (14): P = 0.2

33.9

45% (15): P = 0.0003

87D NCI-C MY6

66/89

80/92

-21.1

87H MGWS (extended)

45/51

62/64

-23.5

23.6

63% (15): P < 0.0003

89B EMSG 2 (M)

22/46

37/54

-8.8

14.6

45% (15): P = 0.09

89E GMTG MM02

41/52

56/65

-5.0

24.0

10% (16): P = 0.3

89F Royal Marsden

31/42

33/42

-6.0

15.5

20% (21): P = 0.1

89A MRC-MYEL-6e

118/143

116/140

-12.8

57.8

20% (12): P = 0.00

89E CMN (M). Mexico

-21% (120): P = 0.5

9/13

7/8

0.6

1.1

90B PETHEMA

38/50

40/42

-8.7

18.4

39% (19): P = 0.01

90K Ital. NHLSG (M)

30/44

26/48

0.5

14.3

-4% (27): P = 0.9

91E GERM

36/70

46/66

-10.0

19.4

40% (16): P = 0.02

549/787 (71.6%)

635/776 (81.8%)

-116.7

-260.5

34% (5) reduction P < 0.00001

493.9

29% (4) reduction

Subtotal

Test for heterogeneity between trials: c211= 21.7; P = 0.03

Total

1000/1452 (68.9%)

1075/1414 (76.0%)

-169.4

95% CI for total 98% CI for individual trials

0.0

0.5 IFN better

Test for heterogeneity (22 trials): c231 = 41.0; P = 0.008

1.0

1.5

2.0

None better

Effect P < 0.0001

Test for heterogeneity between subtotals: c21 = 3.5; P = 0.08

Figure 1. Meta-analysis of impact of interferon on progression free survival (adapted from report in the Br J Haematol, ref. 227)

Estimated percentage still progression free

D. Goldstein et al.

283

100

0

1

2

3

4

5

6+

years

90 80 70 60 50 40

32.6%

30

22.9%

20

- allocated IFN (% ± s.d.) - allocated none (% ± s.d.)

10 0

20.0%

23.8%

0

1

Progressions/period–years IFN 412/1214 None 561/1102

16.2%

14.7%

6+

2

3

4

5

237/770 319/558

184/450 115/300

57/287 45/129

27/176 16/113

5.3 % SD 2.0 (logrank 2P < 0.00001)

years

28/224 16/114

Figure 2. Progression free survival for the addition of interferon to standard treatment of multiple myeloma (adapted from report in the Br J Haematol, ref. 227)

performed by cooperative groups in the United States and Europe evaluating the effect of alpha interferon added to four-drug induction cytotoxic chemotherapy [244, 246]. Both trials added alpha interferon to a CHOP-like induction regimen; the Group d’Etude des Lymphomes Folliculaires (GELF) treated only patients with follicular lymphoma but with bulky (>7 cm) disease while the ECOG in the United States studied patients with bulky or symptomatic low grade and intermediate grade NHL. The ECOG used cyclophosphamide, Oncovin®, prednisone and Adriamycin® (COPA) as the chemotherapy regimen, administering alpha interferon for 5 days (D22–26) of every 28 day cycle for a total of 8–10 months. The GELF used cyclophosphamide, VM 26, prednisone and Adriamycin® monthly for 6 months and then every 2 months for an additional 12 months giving alpha interferon three times a week for the entire 18 months. Although the two trials were very similar in design, there were some important differences. Both groups treated patients with bulky, symptomatic disease, all follicular lymphoma in one, and follicular and diffuse in the other while the aggressiveness and length of treatment with both chemotherapy and alpha interferon was different. The GELF demonstrated a better response rate in the group receiving interferon, but had an unusually low response rate in the chemotherapy-only arm. The GELF study demonstrated an improved duration of response and time to treatment failure 2.9 years versus 1.5 years

and an improved overall survival (not reached versus 5.6 years, 10% of patients withdrew because of IFN related toxicity and 28% required dose reduction). The initial ECOG publication reported an improvement in survival but a subsequent analysis a year later, although demonstrating a consistent 10% increase in survival over a 5 year period, did not show statistical significance [8]. A study of quality of life has similar to the myeloma study supported a benefit in terms of quality adjusted life years for IFN treatment [32]. By contrast a large SWOG study in 571 patients comparing eight cycles of ProMACE 9 day 1-MOPP (day 8) with and without interferon alpha as maintenance for 2 years showed no benefit in either progression free or overall survival [62]. In addition to these reported trials, there have been two small trials examining the issue of dose, one showing a dose response [68, Gams, 1990, personal communication] the other no statistical difference in response rates. The question of influence of dose and schedule remains unanswered because of the very small numbers of patients. A meta-analysis only reported in abstract form [215] – summarised eight randomised trails involving 1,756 patients. It suggested that maintenance treatment in trials with more intense initial therapy showed a 14% survival advantage at 5 years (74% versus 60%) and 19% at 8 years, but no benefit for lower initial intensity treatment. Thus Alpha interferon may be an active therapeutic agent in patients with NHL, low grade, but its role has

284 yet to be clarified. Optimal dose and schedule have yet to be defined and the question of whether higher, less tolerable doses are needed for maximal effect remains unanswered. Additionally, alpha interferon may be most active in patients with lower tumor burdens, i.e. with or following CHOP-like regimens. In high grade NHL a small randomised study showed no benefit to 1 year of maintenance therapy following initial therapy [10]. However a recent survey has shown some evidence of activity in relapsed high grade disease [9] and trials in the post transplant setting for relapsed high grade patients are ongoing. The whole role of IFN is now uncertain following the approval of anti CD-20 monoclonal antibody (mabthera – see chapter). Its activity and excellent toxicity profile for relapsed low grade lymphoma both alone and conjugated with radionucleide and the increasing data on its use as initial therapy in both low grade and high grade NHL [88] make it a very attractive biologic agent. However given that patients may ultimately relapse following mabthera therapy the role of IFN alpha to enhance the effectiveness of this therapy requires further study [41]. In addition novel directions in combination to avoid chemotherapy are being examined [120].

Other lymphomas Several small phase II studies have been performed in-patients with cutaneous T cell lymphoma (CTCL), chronic lymphatic leukemia (CLL), and adult T cell leukemia- lymphoma (ATLL). Alpha interferon has been shown to have an anti-tumor effect in patients with these disorders. Bunn et al. initially showed that rIFN alfa 2a induced responses in patients with CTCL. Responses were seen in both cutaneous and extracutaneous sites [21, 22]. These encouraging results were followed by a confirmatory report in a small series from Duke and Northwestern [185]. Three complete and ten partial responses (response rate-59%) were induced and the suggestion of a dose–response relationship was demonstrated. Investigators from Northwestern University then combined rIFN alfa 2a with psoralen plus ultraviolet light irradiation (PUVA) in a Phase I dose escalation trial in which the dose of alpha interferon was escalated from 6 to 30 MU IM tiw [132]. This escalation was based on the steep dose–response relationship observed in the earlier trials. A complete response was obtained in 12 patients (80%). Several phase II trials in patients with cutaneous T cell lymphoma have confirmed this high response rate [133, 222, 283]. In one long term follow up report the best

Interferons: therapy for cancer response was seen in earlier stages (Ia, b, Iia), frequency of complete response was maximal by 6 months. 57% relapsed within 1 year but 175 had a very prolonged complete response with a mean of 31 months [115]. One randomized trial has been reported comparing the relative value of the combination of PUVA and IFN to retinoic acid and IFN [249]. The PUVA combination had a higher complete response 70% versus 38% and overall response 80% versus 60%. Once again the recent reporting of an IL-2 toxin conjugate with efficacy in the resistant disease setting, although at a cost in terms of toxicity [249] suggests that biologic therapy will have an ongoing role. Combination of IFN with emerging new therapeutics is continuing [226]. Several small pilot trials have evaluated the use of alpha interferon in patients with CLL [64, 229, 177, 193]. Alpha interferon appears to have a mild to moderate ability to decrease the population of circulating leukemic cells, especially in patients with early or minimal disease who have not received prior therapy. However, none of these studies strongly suggest a clinically significant benefit from alpha interferon as a single agent. ATLL has been etiologically associated with infection with HTLV-1, a human retrovirus endemic in southern Japan and the Caribbean basin [201]. All three interferons, alpha, beta, and gamma have been shown to have an anti-tumor effect occasionally durable, in a small percentage of patients with the acute form of this disease. A multi-institutional group studied the two drug combination of rIFN alfa 2b and AZT in 19 patients and induced a major response in 11 patients, including a complete response in five [78]. Some of these patients had disease resistant to cytotoxic chemotherapy. Several of the responses were durable.

Chronic Myeloid leukemia (CML) CML is a triphasic disease, consisting of chronic, accelerated, and blastic elements. Management options include stem cell transplants, alpha-interferon, the tyrosine kinase inhibitor (STI571) and chemotherapy. The choice of therapy depends on the phase of the disease and the characteristics of the patient. Interferon has most effect in the chronic phase as initially found by Talpaz et al. at MD Anderson [256]. This group conducted a series of uncontrolled trials, which initially demonstrated the ability of interferon alphã2a to control leucocytosis and subsequently demonstrated its ability to reduce the size of the Ph positive clone and to induce a clinical and histologic CR. Large multi-institutional randomized trials have since demonstrated the superior efficacy of alpha interferon compared to cytotoxic treatment in terms of haematological response and survival

D. Goldstein et al. and, until recently, it was established as the treatment of choice for patients with CML who were not eligible for a bone marrow transplant.

Interferon Monotherapy; Early Studies Based upon the demonstration of in vitro antiproliferative activity of alpha interferon against CFUs [183, 174, 210, 12]. Talpaz and co-investigators initiated a series of trials in the early 1980s initially using partially purified alpha interferon in patients with CML and subsequently with recombinant alpha interferon molecules. Their initial observations that alpha interferon could reduce the size of the malignant clone of cells was subsequently confirmed; treatment with alpha interferon reduced the number of Ph positive cells in over half of the responding patients. About 20% of patients obtaining a conversion to a cytogenetically normal marrow, confirmed by molecular studies [255, 256, 257, 288]. This is a true pathologic CR. Such an effect had met with only limited success using aggressive cytotoxic chemotherapy [241]. It is now clear that patients gaining a major partial or complete cytogenetic response have improved survival.

Randomized Studies of Interferon Monotherapy Versus Chemotherapy A number of prospectively randomized studies have confirmed the superiority of interferon monotherapy over chemotherapy in the treatment of early chronic phase CML (Table 2). The Italian Cooperative Group first demonstrated alpha interferon’s superiority over hydroxyurea with a karyotypic response rate of 30% in the interferon group versus 5% in the chemotherapy group and significantly improved median survival in patients given interferon [265]. A prospectively randomized study by the German CML Study Group compared the use of IFN alpha 2a, given at a daily dose of up to 9 MU SC, with standard cytotoxic chemotherapy either busulfan or hydroxyurea [98, 99]. The number of patients with a reduction in the Ph1 positive clone and the time to progression to accelerated or blast crises were both increased in the interferon group compared to either cytotoxic agent. The overall survival of patients was superior in the group of patients treated with alpha interferon therapy but statistical significance was demonstrable only when interferon was compared to busulfan. This high daily dose was poorly tolerated, however, and 16% of patients had to discontinue alpha interferon therapy. The financial cost of interferon therapy was also substantially higher than for chemotherapy. Further

285 studies from Japan and the UK have reached similar conclusions [179]. In the Japanese study, newly diagnosed patients with CML in chronic phase were randomized to receive either alpha Interferon or busulfan. Although a complete cytogenetic response occurred in two patients receiving busulfan (not previously noted with this agent), there was a statistically and clinically significant difference in favor of alpha interferon in terms of major cytogenetic response (13 of 80 patients) and in predicted 5-year survival rate. The UK study using a different approach demonstrated superiority of alpha interferon over no maintenance therapy after induction of a response by either busulfan or hydroxyurea [6].

Interferon in combination with cytotoxic drugs in the treatment of CML Interferon has been used in combination with cytarabine (ara-C) following work showing ARA-C could selectively suppress CML clones in vitro and also demonstrate antitumour activity when given as a low dose continuous infusion in vivo. The MD Anderson experience of combining interferon alpha with ara-C has been addressed by Kantajaran et al. They initially reported on a group of 60 patients with advanced phases of CML in 1992 showing that a study group receiving daily alpha interferon and intermittent ara-C had a better CHR compared to historical controls receiving interferon alone. A subsequent report on 140 patients with Ph positive early chronic phase CML in 1999 [117] showed that the schedule of ara-C may also be important. The study group received combination treatment of IFN-alpha (5 × 106 U/m2 daily) and low dose ara-C (10 mg/m2 daily), compared to historical controls receiving interferon (5 × 106 U/m2 daily). A significantly improved CHR (92% versus 84%) was seen with low dose continuous versus intermittent ara-C and although there was a trend to an improved major cytogenetic response this was not statistically significant (50% versus 38%, p = 0.06). Two randomized studies using a combination of interferon with cytotoxic drugs have also been reported. Guilholt et al. (240) reported on 721 patients with previously untreated early phase CML. Patients received hydroxyurea (50 mg/kg/day) with interferon (5 × 106 U/ m2 daily) with or without cytarabine (20 mg/m2/day for 10 days each month). There was a significantly improved CHR (66% versus 55%) and also a survival advantage at 3 years (86% versus 79%) in the patients given the ara-C. An Italian study [220] randomized 540 patients with Ph positive chronic phase CML to receive daily interferon alone or in combination with ara-C (40 mg/

286 kg/day subcutaneously for 10 days/month). The combined treatment group had a significantly increased major and complete cytogenetic response rate (28% versus 19%) and Kaplan Meier calculated survival benefit of 85% versus 80% at 3 years. Interferon is clearly highly active in the treatment of chronic phase CML being able to induce major cytogenetic responses reflecting a real survival benefit over conventional cytotoxic therapy. Combining it with ara-C appears to increases activity further. However there are a number of issues that should be considered. Firstly one should assess the relative benefits of carrying out a stem cell transplant. The full details required cannot be covered in the context of this chapter but in young patients with a good performance status a transplant should still be regarded as the treatment of choice. In addition the dosing and duration of interferon treatment to achieve maximal anti-tumour efficacy must be balanced with its toxic side effects. Although their studies were uncontrolled, the MD Anderson group has amassed a significant amount of experience and believes that relatively high doses, administered daily, are required for maximum beneficial effect [118]. However, a number of their patients require dose reduction because of lassitude, neurologic problems and/or thrombocytopenia and neutropenia. By contrast a subsequent analysis of randomised trials of the MRC and HOVON showed no benefit for high dose versus low dose [127]. With regard to duration it appears that interferon only very rarely elicits a complete molecular response even when a complete cytogenetic response is achieved, so there was a real motivation to obtain a more efficacious but less toxic therapy. The advent of the tyrosine kinase inhibitor Glivec has filled that niche [45] reported in patients with chronic phase CML that had failed interferon therapy showing that of 54 patients receiving more than 300 mg 53 had complete haematological responses, with 29 obtaining cytogenetic responses (17 of which were major or complete) with very little in the way of toxicity. This success has lead to the rapid FDA approval of Glivec in the treatment of interferon resistant disease. Subsequent studies in first line therapy led to its adoption as the standard of care [176]. However the development of resistance to Glivec therapy occurs both in chronic phase disease and blast crisis and the mechanisms of resistance have been analysed at a molecular level [85]. Despite new targeted agents, interferon may well still have an important role as an additional therapeutic option. Alpha interferon also has clinical utility in patients with other myeloproliferative disorders including essen-

Interferons: therapy for cancer tial thrombocytosis and polycythemia rubra vera [238, 79, 94, 135, 258, 261]. Control of the markedly elevated platelet count, decreasing the risk of resultant lifethreatening complications can be obtained in nearly all patients with this disorder. The red cell mass, in patients with PRV, can also be reduced leading to symptomatic improvement [236]. Pegylated interferon can provide these benefits with some reduction in toxicity [224].

Solid Tumors Alpha interferon has a demonstrable beneficial effect in patients with some solid tumors; patients with malignant melanoma, renal cell carcinoma, and AIDS related Kaposi’s sarcoma have benefited by treatment with alpha interferon. Alpha interferon has been approved in the US for the treatment of two of these tumors, AIDS-related Kaposi’s sarcoma and for high risk patients with malignant melanoma following surgical removal of the primary lesion.

Kaposi’s Sarcoma Trials in patients with Kaposi’s sarcoma (AIDS) have indicated that about 40% of patients will obtain an anti-tumor response [130]. There has been the suggestion of a dose–response effect, with better results coming with treatment in the range of 20–50 MU/m2 [43, 90, 212, 270]. Most of these studies indicate benefit is most likely in patients with stage II and III disease, in patients with the absence of a history of opportunistic infection, and in patients with a relatively good lymphocyte (>15,000/ mm3) and helper lymphocyte (CD4) count (>400/mm3). Patients with advanced stage disease and/or poor immune status are less likely to respond [73]. An analysis of 364 patients has suggested prolonged survival for patients with the highest CD4 counts [56]. Studies of interferon in combination with chemotherapy have not shown any benefit over interferon alone [129]. Studies evaluated the combination of interferon with AZT and suggested synergy against the HIV virus and possibly also against the tumor, which is itself virally induced by HHV-8 [155]. A prospective randomized trial has shown that moderate doses of IFN, at least 8 mU/m2 are needed [232]. A small randomised trial against chemotherapy has further supported its use with an increase in survival of 24 versus 13 months [186]. Unfortunately with the exception of those patients with high CD4 counts, the length of response is only approximately 6 months and, a significant beneficial effect on survival in the majority of patients remains undefined.

D. Goldstein et al. Once again this indication may be of limited use as the dramatic impact of effective combination antiretroviral therapy has sharply reduced the incidence of kaposis sarcoma and should always be the first approach in a new patient with Kaposis Sarcoma. Furthermore excellent palliation without the toxicity of IFN is now possible with liposome encapsulated doxorubicin (Doxil).

Melanoma The role of interferon 2 alpha in the treatment of melanoma has been investigated in the context of metastatic disease and also as adjuvant therapy following the removal of lesions at high risk of recurrence. Although there are a number of trials that have addressed the role of interferon in metastatic disease most of the current emphasis has been on defining the exact role of interferon in the adjuvant setting and this will form the main point of discussion in this section.

Advanced Disease Response rates in patients with advanced melanoma vary from 5% to 27%, with an average of 15% [39, 122] and an intravenous schedule has allowed higher doses to be used with less associated toxicity [122]. Metastatic malignant melanoma is a difficult malignancy to treat. The response rates for single agent alpha interferon compare favorably with those of single agent cytotoxic agents, but response rates with single cytotoxic agent therapy are low compared to combinations of cytotoxic drugs [156]. Thus there has been interest in combining interferon with cytotoxic drugs. An early study randomizing patients to receive DTIC 200 mg/m2 intravenously for 5 days every 4 weeks with or without interferon (given as 15 MU/m2 for 5 days a week over 3 weeks and then 10 MU/m2 sc three times a week) showed a statistically significant improvement in response rate and median survival in the interferon group. Unfortunately a larger follow up study as well as a number of other trials have failed to demonstrate a statistically significant advantage of adding interferon to a variety of cytotoxic regimens [58]. Considering the additional toxicity afforded by interferon it would therefore appear it has relatively little to offer in the metastatic setting. However a recent metaanalysis of 3,273 patients in 20 randomised trials was recently carried out assessing single-agent DTIC versus combination chemotherapy with or without immunotherapy in metastatic melanoma [107]. This included 926 patients in five trials that utilized interferon and showed that the combination of DTIC

287 with interferon produced a tumour response rate 53% greater than with DTIC alone (95% CI 1.10–2.13) but this was not translated into a survival benefit. There have also been studies that have used interferon in combination with interleukin 2 as additional therapy to conventional cytotoxic drugs. Although there is frequently an increased response with the addition of Immunotherapy, statistically significant survival advantages are again lacking [119, 217, 55]. This conclusion is also supported by an additional meta-analysis which also examined chemotherapy combined with both interferon and interleukin-2 similarly showing improved response rates without a survival gain [110]. Thus, considering the considerable toxicity afforded by such therapy, further studies are unlikely to be of value.

Adjuvant Therapy A number of trials in the last few years have addressed the role of alpha interferon as adjuvant therapy after surgical removal of high risk lesions. These have been designed to test whether interferon improves survival and if so which disease stage will benefit and how intense the treatment must be. The most highly influential of these was the ECOG E1684 trial which included patients with a T4 lesion, with or without the presence of local lymph nodes, and any other primary lesion with proven lymph node involvement (resected IIB or III) [125]. After surgery patients were randomized to receive high dose interferon, consisting of an induction phase of 20 MU/m2 for 5 days every week for 4 weeks followed by a maintenance phase of 10 MU/m2 for 3 days a week over 48 weeks in total, or observation. The Kaplan-Meier analysis showed a statistically significant improvement in disease free survival (26% versus 37%) and overall survival (37% versus 46%) in the treatment group and led the FDA to approve the use of interferon alpha in this patient group. However there was substantial toxicity experienced in the treatment group and it was hoped that a less toxic equally efficacious protocol could be generated. Thus there was considerable interest in the results of the WHO melanoma program where lower less toxic doses were employed. This study included patients with stage III disease using a 3 MU dose of drug three given times weekly compared with observation. Unfortunately, although data is still being generated from this trial, no global overall or relapse free survival benefit has yet been demonstrated indicating a low dose protocol may be ineffective in this patient group [24]. This has been further confirmed in two studies of low

288 dose interferon alone [97, 126] and two in combination with interleukin-2 and retinoic acid respectively. This has been reinforced by a further three arm ECOG study, E1690, evaluating the efficacy of high dose interferon, as documented in the 1684 study, compared with a lower dose (3 MU/day three times weekly for 2 years) and simple observation [123]. This study again showed a significant improvement in relapse free survival between the high dose treatment and observation alone. However there was no relapse free survival advantage in the low dose treatment compared to observation. Further support for high dose adjuvant interferon was forthcoming in the recently published ECOG study, E1694. This trial was designed to compare a high dose interferon with a markedly less toxic GM2 ganglioside vaccine, thought to be active in melanoma, to see if the vaccine offered a more tolerable alternative. Here the interferon arm was clearly superior after an interim analysis thus leading to premature closure of the study [124]. All these studies therefore advocate the use of high dose interferon in the adjuvant setting for patients with positive nodal disease. However there are a number of issues that are cause for discussion. Firstly in the 1690 study where high dose, low dose and observation arms were compared there was no statistically significant overall survival advantage in having the high dose treatment compared to observation alone. This was is in contrast to the earlier E1684 study where a clear overall survival benefit was apparent. Of note the overall survival in the observation arm in the later E1690 study approached that of the treatment arm in the E1684 study. This has not been completely rationalised but one possibility considers the availability of salvage interferon treatment in those patients relapsing in the observation arm of E1690. This was not available for patients relapsing in the E1684 study and thus most of the observation arm patients in the E1690 study differed from those in E1684 as they would have received interferon at some point. It is possible therefore that any overall survival benefit apparent for the adjuvant treatment in E1690 may have been diluted. Intuitively this may argue that the use of interferon could be delayed until relapse considering the toxicity it causes in the adjuvant setting. However one must remember that although other studies show interferon does have activity in metastatic melanoma this has not been translated into a survival benefit (see previous section). In addition a follow up study addressing quality of life issues in the 1684 trial using Q-TWIST analysis revealed that in spite of toxicity the adjuvant treatment group had more quality of life adjusted survival time than the observation group [33]. This is

Interferons: therapy for cancer clearly highly persuasive and if such data is also made available from the E1690 trial it would reinforce the advantages of adjuvant high dose interferon treatment. In spite of the above data there are two studies using low dose interferon have indicated some therapeutic efficacy in certain patient groups. For example Grob et al. [89] compared 18 months of low dose interferon with observation alone in 489 patients with resected disease of >1.5 cm depth but with no nodal involvement. They showed a statistically significant improvement in disease free survival and a trend towards overall survival. Similar results were also published by Pehamberger et al. using a year of interferon treatment (196). Those patients without nodal involvement may therefore represent a group that could be treated with a less aggressive protocol. The length of treatment required is uncertain as a shorter course would clearly improve the toxicity. Although no randomised trials are testing node positive patients with very short interferon courses the E1697 is currently looking at 1 month of HDI versus observation in T3 N0 resected disease. By contrast a trial – EORTC 18952 comparing two intermediate doses of IFN alpha 2b in high risk melanoma patients showed no benefit [49a]. Recent data on pegylated interferon alfa-2b shows similar benefits but with possible reduced toxicity [49b] A recent meta-analysis concluded that the use of HDI should be considered in the adjuvant treatment of resected stage IIB and stage III melanoma because of improvements in disease free and 2 year mortality [268]. The important questions as to whether the full extended course is required, and whether one may be able to better select patients who will particularly benefit from treatment remain unanswered. However for stage IIA disease a low dose protocol may be appropriate. In metastatic disease the additional issue of at best very modest survival benefits versus additional toxicity mean that treatment should be tailored on an individual patient basis and clearly selection must be careful.

Renal Carcinoma Alpha interferon role in this malignancy may well now be only of historical significance. It has an anti-tumor effect in about 12–15% of patients with metastatic renal cell carcinoma (RCC) [281, 204] and may be more effective in patients with less bulky disease and when given in higher doses [154, 61, 168, 273, 228, 231, 211]. A sufficient number of randomised trials have now been reported to allow a clear picture of the role of

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single agent interferon. Interferon either alone or in combination with vinblastine is more effective than medroxyprogesterone [34] or vinblastine [204]. As a result a Cochrane Meta analysis [281] analysed 42 studies involving 4,216 patients and showed an average response rate of 10.2% with 3.2 5 CR, a median survival time of 11.6 months and 2 year survival of 22%. They estimated a pooled survival hazard ratio of 0.78 for IFN treated patients. They suggested that IFN alfa 10 MU S.C. three times per week should be the control arm for future studies (Table 3). A large randomised study of interferon gamma [80] showed that this cytokine is inactive. It also demonstrated that spontaneous remissions do occur with significant frequency 6% (CI 2.5–13.2%), higher than previously reported and a 15.7 month median survival was noted for this early stage metastatic group [53]. This suggested that the activity of interferon alfa in this disease needs to be assessed against that standard, at least in good performance early stage disease. The role of interferon as adjuvant therapy post nephrectomy showed no survival benefit [203, 264, 200]. The benefit of nephrectomy as part of a combined surgical-biotherapeutic approach has been examined. 241 patients were randomised to either interferon alone 5 mU three times per week or nephrectomy plus interferon. The survival in patients allocated to nephrectomy was superior 12.5 months versus 8.1 months [63].

Table 3. Recent randomised Trials of IFN alpha in renal cell carcinoma

VBL #260 IFN + VBL IFN #121 IFN + VBL IFN #263 IFN + VBL MEGACE IFN #261 IFN #269 IFN + Cisretinoicacid IFN #275 IFN + Nephrectomy IFN #267 IFN + IL-2 MEAN IFN Response

12 months survival (%)

N

RR (%)

MST (weeks)

81 79 53 66 82 83 176 174 139 145

2.5 16.5 11 24 12 8 7 14 6 12

37.8 67.6 34 24 24 36 88 128

123 123

4 4

34 50

N/A

147 140

8 19 11

56 72 36

12 20

38 56 N/A N/A N/A N/A 32 43 22 28

24 months (%) 19 38

13 22 10 19

The response rate to interferon of 3% was surprisingly low however. The benefits of combining interferon with any other agent appear limited [173, 66, 204, 7, 164, 46, 165, 166]. The role of interleukin-2 is addressed in great detail in another chapter. However it is instructive to review the results of a recent randomised trial of interferon alfa versus IL-2 monotherapy versus the combination compared to medroxyprogesterone [171]. In 492 patients no survival benefit was shown for either drug [172]. The recent very impressive results with the antiangiogenic agents such as the VEGF inhibitors and their consistent superiority over interferon has made them the standard of care [167, 54, 106]. However at least one study showed a benefit to the combination of bevacizumab and interferon which means it remains an option for exploration but the lack of efficacy of the combination of temsirolimus and interferon compared to temsirolimus alone means that issues of toxicity may differ with each drug combination. Other approaches such as dendritic cell vaccines [103, 131] and autologous transplantation [27] may still be the subject of combined therapy also.

Other Solid Tumors Aside from the three solid tumors listed above, there have been trials in many other types of malignancy. There are some encouraging leads. rIFN alfa 2a has been shown to enhance the cytotoxic effect of 5-FU in vitro and, in pilot clinical trials, the combination induced a partial response in over 60% of patients [5, 274, 275]. However, a prospective randomized trial evaluating the addition of alpha interferon to 5 FU in patients with advanced colorectal cancer has demonstrated no benefit, only added toxicity, from the addition of alpha interferon [102]. By contrast the development of combination therapy with oxaliplatin, irinotecan and the addition of bevacizumab and cetuximab to them has opened new avenues and will be covered in other chapters. Other trials have demonstrated clinical benefit from interferon therapy in patients with carcinoid tumors [163, 178]. Improvement in symptomatology associated with a decrease in 5-hydroxyindoleacetic acid levels has been reported in approximately 50% of patients but objective regression is much less frequent. In addition trials using interferon with other agents such as octreotride have indicated that combined treatment may improve efficacy [67]. The role of other targeted agents such as bevacizumab is being explored and combination with these VEGF targeting agents may need to be explored. There has also been encouraging

290 results using interferon with 13-cis retinoic acid as bioadjuvant therapy in locally advanced head and neck squamous cell carcinoma following experimental evidence that these agents may work synergistically in this tumour [141, 233]. Once again the positive results with cetuximab and chemotherapy may overtake this work (review elsewhere). Anti-tumor activity has also been demonstrated with a similar drug combination in patients with squamous cell carcinoma of the cervix [142]. Although it is relatively ineffective in patients previously treated with radiation therapy [278].

Mode of Action Alpha interferon has an antiproliferative effect, antiviral effect, an immune augmenting effect, and a differentiation effect and an anti-angiogenic effect. Any one or combination of these effects may induce an anti-tumor response. Gamma interferon (Type II IFN) is a more potent immune stimulator of monocyte function and class II HLA activity than are either alpha or beta interferon (Type I IFNs) [269]; however, on a weight basis, the Type I IFNs are ten times as potent as gamma interferon in their antiviral effect.

Antiproliferative In vitro studies and murine models have been used to demonstrate the anti-proliferative action of interferons [87, 262]. Decreased tumorigenicity has also been shown in cells pretreated with human interferon [17, 87]. Whether these models are applicable to human tumors in vivo remains conjectural. Cell cycle analysis has shown that interferon causes extension of all phases of the cell cycle and prolongation of the overall cell generation time [121, 194, 252]. In some cases, an accumulation of cells in G0 has been observed, accompanied by a decrease in transition to G1. This decrease in growth rate may be incompatible with cell life [262]. The critical antiproliferative mechanisms operative at the cellular level have not been elucidated, but it is possible that they may be mediated by the same inhibitors of DNA and RNA synthesis that are found in virus-infected cells. Specifically, induction of 2′5′-oligoadenylate (2,5 A) synthetase leads to endoribonuclease activation, which in turn inhibits RNA transcription by degrading mRNA linked to dsRNA [213, 230]. In addition, a protein kinase and a phosphodiesterase pathway represent mechanisms of inhibition of protein synthesis which are parallel to,

Interferons: therapy for cancer but independent of, 2,5 A synthetase [230]. Whether these are, in fact, central to antiproliferative as well as antiviral activity remains speculative [260]. Because of its high degree of sensitivity to the Type I interferons, HCL would appear to be an ideal disease in which to determine a mechanism of action for interferon’s antitumor effect. Type I interferon receptors are present on hairy cells and are down regulated with therapy [13, 59]. A lack of demonstrable down regulation in vivo has been associated with lack of response [14]. Immunologic recovery as manifested by a return of NK activity and normalization of T and B cell counts has been documented [175] but responses occur without improvement in NK activity [70,71]. Further, hairy cells were not susceptible to NK cytotoxicity in vitro [235]. It has also been shown that alpha interferon, when cultured with peripheral blood mononuclear cells of patients with HCL, gives rise to multi-lineage colonies composed of myeloid and erythroid progenitors – evidence for the existence of circulating hematopoietic stem cells responsive to a differentiation effect of interferon [162]. It has also been shown that the mononuclear cells of HCL are defective in their ability to release tumor necrosis factor (TNF) and that interferon overcomes this – perhaps interferon and TNF then act synergistically as antiproliferative or cytotoxic agents [2, 3]. The low dose – standard dose randomized study with IFN alpha N-1 in patients with HCL supports both of these theories; both doses were comparable in their ability to induce an increase in platelet and neutrophil counts (differentiation) while the standard dose was more effective as an antileukemic (antiproliferative) agent [242]. This is the only prospectively randomized clinical evidence available to support a dose-response effect. Ford et al. and subsequently Pagannelli et al. have shown that HCL proliferation in vitro required B cell growth factor (BCGF) and Pagannelli et al. have shown that Type I but not Type II interferon inhibits the effect of BCGF [191, 65]. These observations strongly suggest that interferon’s primary anti-tumor effect is an antiproliferative one, at least in this tumor setting. Most recently a down regulation of telomerase activity in human malignant heamatopoietic cell lines may have identified a novel antiproliferative mechanism [286].

Antiviral Activity That interferons have antiviral properties is well established and alpha interferon is approved in many countries for the treatment of several forms of viral hepatitis. Whether this effect is the basis for interferon’s antitumor effect in tumors possibly related to viral etiologies,

D. Goldstein et al. such as Kaposi’s sarcoma associated with AIDS and ATLL, is unknown. Although other tumors may be of viral origin, most prominently cervical carcinoma, there has not been a major investigative effort mounted in this area and currently the antiviral effect of alpha interferon remains unexploited in the treatment of malignancy.

Immune Modulation Support for immune stimulation as a mechanism of action is found in experiments with animals, in which tumors known to be interferon-resistant in vitro have regressed when the animal received systemic treatment with interferon [42, 87]. A variety of immune changes have been described, but the most relevant appear to be the effects on natural killer (NK)-cells and macrophages. In vitro work with human lymphocytes shows that interferon increases the cytotoxic potential of NK-cells by recruitment of pre-NK-cells, increasing activity of activated cells, and by augmenting NK-cell-mediated antibody-dependent cell-mediated cytotoxicity [100, 194]. Alpha and beta interferons also appear to cause NK activation at a lower dose and over a shorter time interval than does gamma interferon [263]. In clinical trials, both the dose and route of interferon administration have been shown to effect NK-cell activity significantly. Several trials have suggested that low-dose interferon results in more marked NK-cell activity [51, 134, 47, 48]. This is a confusing issue, however, since others have reported widely differing effects on NK-cell activity, varying from a consistent increase [52, 202, 145] to a consistent decrease [143, 148, 149, 149, 247] in addition to individual variation without discernable pattern [170]. Much of the controversy may be related to the dose of interferon used. Unpublished work from the NCI by Varesio et al. [267] has suggested a bellshaped curve for both the antiproliferative effect and immune augmentation. These curves do not precisely overlap. A few clinical studies have examined the long-term immunologic effects of alpha interferon treatment; again, these differ with respect to whether there is an increase [134, 237] or decrease [148, 149, 150] in NK activity. A study by Silver et al. [237] (p. 175) showed that low dose alpha interferon led to an increase in NK activity within 48 h, which was, however, not sustained despite continued administration. Repeated high-dose interferon gave a more sustained increase of NK activity in the long term, even though it did not lead to the same initial rise as low-dose inter-

291 feron. However, intravenous administration was used for the high dose, and intramuscular administration for the low dose, and the schedule of therapy was also different (alternate weeks for low-dose versus monthly for highdose). Both uncontrolled factors, i.e., route of administration and schedule, might have caused the differences noted. An earlier study with lymphoblastoid interferon showed a dose-related decrease in NK activity over a 6-week period, after an initial stimulatory effect; but in that instance the treatment was given three times a week [134]. This suggests that an intermittent schedule may result in different patterns of response. Since interferon consistently stimulates NK-cells in vitro, the inability to reproduce consistent effects of interferon on NK-cell activity in clinical practice suggests a need to explore confounding in vivo effects, such as scheduling, dose, or sampling method (peripheral blood versus tumor site). Furthermore, both Type I and Type II interferons have been shown to induce resistance to NK activity in vitro. The controversy surrounding NK-cell function after interferon treatment suggests that it may not be a useful immunologic marker. Macrophages, more consistently than NK-cells, are affected by gamma interferon; indeed, gamma interferon has all the activities of a macrophage-activation factor (MAF) [42, 269]. Gamma interferon acts to increase the number and density of Fc receptors, which in turn are associated with an increase in antigen-presenting function. Enhancements of phagocytosis and also of antiviral activity and cytotoxicity occur [194, 252]. Thus, assays of macrophage/monocyte function may be of more relevance for assessment of gamma interferon action, whereas NK or antiviral activity may be the best choice for alpha or beta interferons. In addition to functional studies, changes in serum molecules, particularly neopterin, tryptophan and beta 2 microglobulin have been found to be very consistent markers of biologic response to interferons. Their relationship to the degree of immune modulation in vivo is unclear but recent studies with alpha, beta and gamma interferons have shown the optimal biologic dose to be well below the maximum tolerated dose [71, 72, 81]. The ability to increase cell-surface antigen expression presents another aspect of interferon action that may be important in tumor control. Augmentation has been demonstrated for the expression of Fc receptors on lymphocytes and of Class I and II (Ia) MHC antigens [42, 194, 252] on several other cell types as well. In addition to augmentation of expression, interferon has also been shown to induce HLA expression in a variety of normal cells [219]. In neoplastic cells, interferon has been shown to induce HLA antigen expression

292 and augment tumor-associated antigen (TAA) expression in several cell lines [74, 86, 75, 23]. This work has been expanded and augmentation has been demonstrated in vitro in tumor cells obtained from malignant effusions and following the intraperitoneal administration of gamma interferon to patients [81, 93]. The increased expression of TAA occurring concurrently with enhancement of macrophage antigen-presenting function, may improve the endogenous antitumor activity of macrophages. The actions of interferon in promoting tumor antigen expression suggest another major line of inquiry, namely, the combination of interferon with monoclonal antibody (MoAb) therapy, with or without linkage to a toxin [218]. One of the major stumbling blocks to MoAb therapy has been modulation or poor expression of tumor antigens; if pretreatment with interferon can increase antigen expression, then the effectiveness of subsequent MoAb treatment might be greatly enhanced. However, the risk of similar expression in nontarget areas must be kept in mind, since it is often the level of expression rather than the novel nature of the antigen which characterizes the tumor cell. At least one early trial has demonstrated potential therapeutic effect [277]. The group at Stanford University and IDEC Pharmaceutical Corporation in California has performed an interesting trial. These investigators previously reported the use of anti-idiotype antibodies in patients with B cell lymphomas, obtaining a response in five of ten patients including 1 CR [158, 159]. Patients ultimately failed because of the emergence of a dominant clone of antigen (idiotype) negative cells [158, 159]. An animal model was developed to evaluate therapeutic modalities in this setting. Using this model, it was shown that a synergistic effect was obtained with the combination of anti-idiotype antibodies and interferon [11]. rIFN alfa 2a, 12 MU/m2 SC tiw, was combined with anti-idiotype therapy and administered to twelve patients. Responses were obtained in nine (with 2 CR), not substantially different from the results with anti-idiotype therapy alone. Interferon failed to prevent the emergence of idiotype negative clones. Currently ongoing studies evaluating the combination of alpha interferon and anti CD-20 monoclonal antibody may be clinically more encouraging [41].

Differentiation Interferons have been shown to have a variety of effects on differentiation. In vitro differentiation has been enhanced in mouse myeloid leukemia cells and in erythroblasts of the Friend leukemia system, whereas conversion of mouse

Interferons: therapy for cancer 3T3-Li cells into adipocytes and of human monocytes to macrophages has been inhibited [219, 252]. Other evidence supporting an effect on differentiation includes an increased expression of HLA antigens, enhanced excitability of nerve cells, and decreased beating frequency of myocardial cells after interferon treatment [219]. Interestingly, sodium butyrate, a well known differentiating agent, enhances this effect of interferons in vitro [219]. An important finding – which may suggest one cellular mechanism for inducing differentiation – is the evidence of decreased c-myc and c-Ha-ras gene expression following interferon treatment, with as much as a 60% decrease in mRNA formation [31]. With 3T3 fibroblasts, this decrease in mRNA has been associated with a reversion to normal cell type. Thus, interferoninduced differentiation appears to be a significant effect and may be part of the antineoplastic action of interferon.

Anti-Angiogenesis Inhibition of experimental angiogenesis by interferons was first demonstrated in a mouse tumor model [234]. This observation was confirmed in infants with lifethreatening hemangiomas [57]. Hemangioma regression and significant clinical benefit was demonstrated in more than 80% of seriously effected infants. Successful angiogenesis is essential for the expansion of any malignant tumour. It is a complex process involving extensive interplay between cells, soluble factors and extracellular matrix component. The mechanism through which interferons mediate this inhibition is unclear but inhibition fibroblast growth factor (FGF) pathways appears to play some part. For example IFNalpha can inhibit FGF induced endothelial cell proliferation and both IFN-alpha and beta can down-regulate the expression of FGF in human carcinoma cells [101, 239]. In addition experiments in nude mice have shown that IFN-alpha can lower the expression of FGF and this correlates with a decreased blood vessel density and an inhibition of growth of ectopically implanted tumours in these animals [44]. In the clinical setting interferons have been shown to be effective in producing antiangiogenic effects in a number of tumours. These include Kaposi’s sarcoma [56], bladder carcinoma [248] and giant cell tumour of the mandible [116]. However the intense interest in angiogenesis has lead to the development of a number of newer antiagiogenic agents [139]. Although a number of these have become standard of care, the pleiotropic activities of interferon in combination may still need to be understood to add to the overall antineoplastic effect of some targeted agents [287]

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Side Effects While side effects of the Interferons can be debilitating, most appear to be reversible upon cessation of therapy. As one might expect, high doses give rise to more severe manifestations than low doses. The major documented short term side effects are fevers, headache, and myalgia, while fatigue, a long term side effect, can be severe enough to be dose-limiting. Gastrointestinal side effects, in particular, anorexia, nausea, vomiting and/or diarrhea, are neither universal nor severe, but may lead to weight loss, which can be profound with high-dose or prolonged moderate-dose administration. Elevated serum transaminase (but not clinical hepatitis) has also been noted [134, 245, 237]. Hypotension with higher doses of both alpha and gamma interferons can be dose limiting. Both granulocytopenia and thrombocytopenia have occurred, but are rapidly reversible [237, 15] and rarely dose limiting unless the patient has had extensive prior radiation. Margination of leukocytes rather than direct marrow suppression appears responsible. Central nervous system toxicities, including paresthesias, weakness, somnolence, decreased attention span, short-term memory impairment, confusion, depression, personality change, and even coma at very high doses, have all been seen. EEG abnormalities, including a slowing of the dominant alpha rhythm and diffuse slow waves, have been documented [214, 245, 1, 60]. The potential of depression to limit beneficial therapy such as adjuvant treatment of melanoma has led to a randomised study of paroxetine, which substantially reduced the incidence from 45% to 11%. Although only a small number of patients were studied, its prophylactic use may well improve the therapeutic index for higher dose IFN therapy [169]. Exacerbation of coronary artery disease has been reported. Although cardiac toxicity may be life-threatening, serious cardiac toxicity due to interferon is unusual. A significant history of coronary artery disease is a relative contraindication for interferon therapy, particularly at high doses. Life-threatening acute pulmonary toxicity, renal failure, and unexplained sudden death have also been noted on rare occasions. Evidence has increased indicating that long term treatment with interferons results in the production of antibodies to interferon. In a study of 51 patients with hairy cell leukemia, neutralizing antibody was detected in 16 patients, six of whom developed some degree of clinical resistance [250]. In a previous review of more than 617 patients who received intramuscular interferon alfa 2a, 25% were

293 reported to have neutralizing antibody [109]. Antibody formation is a significantly less frequent phenomenon in patients treated with either rIFN alfa 2b or IFN alfa N1 [251, 271, 272]. Of the three allelic genes used in the development of recombinant interferon alfa 2, the gene used to produce rIFN alfa 2b is by far the more common allele in North American individuals [138].

Summary This review of the clinical effects of the interferons indicates that alpha interferon, when used as a single agent, has antitumor effects in a large number of malignancies, perhaps more than any single chemotherapeutic agent does. However, like many cytotoxic agents, the use of interferons in combination with cytotoxic drugs, other biologics, or antiviral agents remains under explored. Clinically, the interferons proved to be a significant addition to our therapeutic armamentarium and served well as the prototype for subsequent more targeted biological therapeutics. Interferon has overcome two misleading labels at opposite ends of the spectrum: of “imaginon” when most scientists doubted its existence, and more recently that of “magic bullet,” when the public was led to believe it might be the cure-all for cancer. In the context of cancer treatment, interferons have been the prototype of the so-called “fourth arm” of therapy; surgery, radiotherapy, chemotherapy and biotherapy [184].

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10 Monoclonal antibody therapy ROBERT O. DILLMAN

Abstract Monoclonal antibodies were the first successful targeted anti-cancer therapeutics in the modern era. In the 1970s elucidation of hybridoma and molecular biology technologies established mechanisms to produce and modify monoclonal antibodies with therapeutic anti-cancer potential. In the 1980s early investigation with murine monoclonal antibodies in animal models and patients established most of the principles of antibody therapy including immunologic and regulatory mechanisms of anti-cancer effects, and the use of immunoconjugates in which antibodies act as carriers. These early studies also showed that most of the toxicities associated with monoclonal antibodies relate to the target antigen, and less often immune interactions with the antibody itself. In 1997 the United States Food and Drug Administration approved rituximab (Rituxan) for the treatment of B-cell lymphoma, making it the first monoclonal antibody to receive regulatory approval for the treatment of human malignancy. As of early 2008, there were six commercially available unconjugated monoclonal antibodies with marketing indications for treatment of human malignancy. In addition to rituximab for B cell malignancies, this list includes alemtuzumab (Campath) for B and T cell malignancies, trastuzumab (Herceptin) which inhibits Her-2 overexpressing breast cancers, anti-epidermal growth factor receptor (EGFR) antibodies cetuximab (Erbitux) and panitumumab (Vectibix) which inhibit various epithelial solid tumors including colorectal cancer, and bevacizumab (Avastin) which binds to vascular endothelial growth factor (VEGF) and is probably useful to treat most malignancies, including colorectal, lung, and breast cancers. These products have been most useful when combined with chemotherapy because of additive or synergistic anti-tumor effects. Keywords Alemtuzumab • bevacizumab • cetuximab • panitumumab • rituximab • trastuzumab

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

Background and Rationale for Antibody Therapy Historical aspects 1900–1980 Monoclonal antibodies (Mab) were the first successful anti-cancer targeted therapeutics in the modern era [168]. A chronological history of monoclonal antibody development is shown in Table 1. At the end of the nineteenth century antisera and antibodies were discovered as part of the independent work of Emil Behring and Kitasato Shibegan that led to developing diptheria antitoxin. By immunizing an animal with foreign antigens contained on cells, early immunologists recognized that they could produce antisera against antigens on the surface of cells. The potential to utilize antibodies as therapeutic agents was first espoused in the early twentieth century. The German immunologist, bacteriologist, and 1908 Nobel Prize winner Paul Ehrlich used the term “passive immunization” to describe the use of antisera and antibodies in the treatment of disease as opposed to the “active immunization” using cell or antigen-based vaccines. Ehrlich, whose 150th birthday was 14 March 2004, is generally considered the father of antibody therapy and is credited for the term “magic bullets” to describe the potential for antibodies to specifically target bacteria or cancer cells as opposed to normal cells [201, 644]. Before 1975, it was virtually impossible to isolate a specific antibody. Initial therapeutic endeavors were with “antisera”, a collection of heterogeneous antibodies from multiple clones, typically derived from animal serum. Animals were repeatedly immunized with preparations of human cells which induced an immune response. Animal blood was collected several weeks later at a time when a primary immune response was estimated to occur, and the serum fraction isolated.

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Table 1. Milestones in the development of monoclonal antibodies for the treatment of cancer 1908 1927 1975 1980 1982 1984 1992 1995 1997 1998 2000 2002 2003 2004 2004 2006

Paul Ehrlich, German immunologist states concept of “magic bullets” for seeking out cancer cells, awarded Nobel Prize Publication describing serotherapy of 10 patients with chronic myelogenous leukemia Kohler and Milstein report “hybridoma” methodology to produce monoclonal antibodies; awarded Nobel Prize in 1986 First publication of treatment of a cancer patient (lymphoma) with a monoclonal antibody First published report of a complete clinical remission in a patient (lymphoma) treated with a monoclonal antibody, murine anti-idiotype First U.S. FDA approval of a therapeutic monoclonal antibody, murine anti-CD3 muromonab-CD3 (Orthoclone OKT3) to prevent renal allograft rejection First U.S. FDA approval of a radiolabeled anti-cancer monoclonal antibody, murine anti-B72.3 indium-labeled monoclonal antibody, 111In-satumomab pendetide (Oncoscint), for radioimmunodetection of colon and ovarian cancer Approval in Germany of murine monoclonal antibody edrecolomab (Panorex) for the adjuvant treatment of colon cancer in Germany U.S. FDA approval of chimeric anti-CD20 monoclonal antibody rituximab (Rituxan) for the treatment of B-cell lymphoma, first indication to treat human malignancy FDA approval of humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin) for the treatment of breast cancer, first indication to treat a solid tumor U.S. FDA approval of humanized anti-CD33 immunotoxin, gemtuzumab ozgomicin (Mylotarg), for the treatment of acute myelogenous leukemia, first approval of antibody-targeted immunotoxin U.S. FDA approval of murine anti-CD20 radiolabeled monoclonal antibody Y-90 ibritumomab tiuxetan (Zevalin) for B-cell lymphoma, first approval of a radiolabeled antibody for treatment of malignancy U.S. FDA approval of murine anti-CD20 radiolabeled monoclonal antibody I-131-tositumomab (Bexxar) for B-cell lymphoma U.S. FDA approval of bevacizumab (Avastin) for colorectal cancer U.S. FDA approval of cetuximab (Erbitux) for colorectal cancer U.S. FDA approval of panitumumab (Vectibix) for colorectal cancer

Such sera were then modified by repeated absorption with normal human tissues that were presumed to not contain tumor associated antigens. This process increased the specity of the final product which constituted an antiserum. The degree of reactivity of this immunoglobulin collection was tested in bioassays using serial dilution titrations, and the product was characterized on the basis of the dilutional titers, such as 1:64 or 1:256. Typically, large animals such as horse, sheep, or goats were immunized in order to yield large volumes of anti-serum. Because each fourlegged biofactory was biologically unique, it was impossible to reproduce a given antiserum exactly, even in the same animal. There were always issues of purity and the impossibility of exactly reproducing any given serum that inevitably consisted of multiple antibodies with various specificities and affinities. In as much as antibodies were genetically determined for an individual animal, the supply of any given antiserum was limited, and therefore there was no ability to perform large clinical trials with any specific antiserum. Some antisera, such as anti-thymocyte globulin, are still in clinical use today, but the biotechnology industry is trying to replace as many as possible with single or combinations of Mabs.

In 1975 Hans Kohler and Caesar Milstein published a paper in which they described the hybridoma technology, which allowed isolation of cloned cells that could secrete a single type of antibody [400]. For this work they were awarded Nobel Prizes in 1986. Following publication of this paper numerous academic laboratories and early biotechnology companies began making Mab by immunizing rodents with human cells and antigens. The potential advantages of Mab over antiserum as a therapeutic product were readily apparent as summarized in Table 2.

Table 2. Comparative advantages of monoclonal Antibodies vs. Antisera Monoclonal Antibody

Antisera

Purified antigen to optimize

Cell or crude antigen satisfactory Screening for specificity Homogeneous Reproducible lots Uniform affinity Unique specificity Unlimited production

Absorptions for specificity Heterogeneous Variable lots Variable affinities Variable specificities Limited quantities

Robert O. Dillman

Antisera Trials 1925–1980 Between 1925 and 1980 numerous clinical studies were performed using antisera. These pioneering studies with heterologous and isologous antisera have been reviewed in more detail elsewhere [137, 607, 795]. Such trials were limited by the quantity of purified anti-tumor antisera available, the purity of such preparations, and difficulties producing similar lots of new antibody. Nevertheless, some antitumor effects were noted. One of the earliest reports by Lindstrom in 1927 described ten chronic myelogenous leukemia (CML) patients whom he treated with 15 courses of rabbit antisera. A decline in peripheral blood myeloid cells was noted in five patients, but there were also significant side effects that were attributed to impurities in the preparation, although these may well have been secondary to antibody binding to circulating CML cells. In the 1970s anti-thymocyte globulin (ATG) was used by several investigators to treat patients with T-cell malignancies [198, 224, 815]. All eight patients described in these reports had some improvement in their Sezary syndrome and/or lymphadenopathy. Therapy was complicated by hypotension and chills. Prophylaxis with diphenhydramine and steroids seemed to prevent these side effects during subsequent treatments, but this may have been because circulating T lymphocytes had already been eliminated from the circulation. Interpretation of antisera studies was complicated by issues of antibody source, purity and specificity. Certain toxicities were identified, but it was unclear whether these were due to impurities, hypersensitivity reactions to foreign proteins, or to antitumor effects. Although side effects were typically attributed to allergic reaction to foreign proteins, it is likely that most of the toxicity observed was actually due to specific interaction with leukocytes and the release of cytokines [170].

Animal Models and Antibody Therapy The chapters on antibody therapy in the first three editions of Principles of Cancer Biotherapy [162], [165], [169] summarized some of the important animal work with antisera and Mab that laid the foundation for the use of such products in man. The demonstration of safety, tumor targeting, and anti-tumor effects demonstrated in various animal models justified and guided human clinical trials that eventually established Mab as an effective anti-cancer treatment modality [816]. The following conclusions can be drawn from the heterologous antisera and studies in animal models. First, antitumor effects can be mediated by Mab in vivo. Second,

305 with some antibodies, antitumor effects are observed in immunodeficient mice even in the absence of an interaction with complement and/or effector cells, suggesting that antitumor effects can be mediated by regulatory effects. Third, immune-mediated antitumor effects vary to some extent with the Ig subclass and isotype of the Mab. Fourth, just as with chemotherapy, antitumor effects are more difficult to achieve in the presence of large tumor burdens. Fifth, combining antibodies to different antigens is a promising strategy to overcome problems of heterogeneity [14, 16, 534]. Sixth, animal models are of little use in predicting toxicities in humans for Mabs that react with antigens that are more prevalent in humans or only expressed in humans.

Human Trials with Murine Monoclonal Antibodies 1980–1989 Clinical trials in man with murine Mab began in 1980 in hematopoietic malignancies and subsequently in solid tumors. Most of the clinical investigation during the 1980s involved pilot and phase I trials with mouse Mab, and relatively few patients were actually studied. Perhaps the first written report of monoclonal antibody therapy was that by Nadler et al. who treated a man with B cell lymphoma with a murine Mab called AB89 [518]. On successive days the patient received slow infusions of AB89 at doses of 25, 75, and 150 mg; 1 month later a 1.5 gm dose was administered. There was a transient decrease in the lymphocyte count after each treatment, but no significant antitumor effect was noted. During 1980 and 1989 a number of clinical studies were conducted with Mab directed against antigens found on cells from hematologic malignancies [19, 38, 161, 177, 179, 180, 227, 228, 446, 478, 485–488, 518, 569, 600, 666] and solid tumor cancers [109, 161, 205, 240, 262, 263, 328, 375, 443, 513, 535, 625, 651–653, 666, 738, 776]. In 1982, the first report of achieving complete regression of tumor in a patient with B cell lymphoma was published [487]. Most of the principles of antibody therapy, including immunologic and regulatory mechanisms of anti-cancer effects, and the use of immunoconjugates in which antibodies act as carriers were established during these early trials. These early trials also established that most toxicities and adverse events associated with monoclonal antibody infusions are the results of interactions with the target antigens, and less often due to immune interactions with the antibody itself. The potential of Mab for the treatment of human malignancy was clearly recognized [163, 533]. In the early 1990s two radiolabeled Mab received regulatory approval for imaging and radioimmunodetection

306 of tumors. In 1995, Germany granted the first approval of a mouse monoclonal antibody for cancer therapy (Panorex) for colorectal cancer, although this product was never approved in the United States. In the early 1990s trials began with murine Mab that had been modified using molecular biology technologies to produce antibodies that consisted mostly of human amino acid sequences. In 1997 the United States Food and Drug Administration (FDA) approved the first monoclonal antibody (rituximab) for the treatment of malignancy in the United States [168]. During the next decade several unconjugated and conjugated antibody products received regulatory approval for commercial use.

Antibodies and Antigens When foreign cells are injected into an animal, they induce an immune response that results in the production of immunoglobulin (Ig) proteins, called antibodies, each of which binds specifically to certain cell surface molecules, termed antigens. Collectively these antibodies are antisera. The antigens are characterized as glycoproteins, glycolipids, polysaccharides, etc. A tumorspecific antigen is one that is found only on cancer cells. A tumor-associated antigen is an antigen expressed on tumor cells, but also known to be present on normal cells. Antibodies consist of sequences of amino acids that are linked by disulfide bonds that yield two heavy chains and two light chains. Each light chain is connected to a heavy chain by a disulfide bridge, and the two heavy chains also are connected via disulfide bridges. As shown in Fig. 1, enzymatic cleavage of these various sites by papain or pepsin yields antibody fragments termed Fab, F(ab’)2, and Fc. The basis for specificity of antibody binding resides in the variable regions of the heavy and light chains (Fv), and more specifically

Figure 1. Structure of immunoglobulins and fragments. Two heavy chains are connected by a disulfide bond, and each in turn is connected to a light chain by a disulfide bond

Monoclonal antibody therapy in the sequence of peptides that constitute the hypervariable region (Fhv) [418, 643]. The Fhv region constitutes the “idiotype” of the Ig, and the specific peptide sequences of the idiotype are termed “idiotopes.” The six hypervariable subregions of the variable region are also referred to as the complementarity-determining regions (CDR). “Lock-and-key” is a popular metaphor for the unique chemical relationship and structural binding between a specific antibody and its antigen target. The affinity of antibody for its antigen is a measure of how tightly the antibody binds to the antigen. The diversity of humeral immunity is made possible by the various combinations and permutations of arrangements and combinations of Fv regions, heavy chains, and light chains [419, 643]. More specifically there are variable (V), diversity (D), and joining (J) genes that code for sequences on the heavy chain (V,D, and J), and light chain (V and J) that provide nearly infinite combinatorial potential for rearrangement. Based on their chemical composition, the light chains of Ig are classified as kappa or lambda, and heavy chains as immunoglobulins IgG, IgM, IgA, IgD, or IgE. Other differences in heavy chains allow subclass characterization such as IgG1 IgG2a, IgG2b, IgG3, IgG4, etc. Human chromosomes 2, 14, and 22 contain various loci for light and heavy chains. The basis for antibody interaction with other components of the immune system, such as complement proteins and effector cells, resides in carbohydrate residues that are present on the Fc portion of the Ig molecule and Fc receptors (FcR) on effector cells [250, 524, 746]. The interaction between Fc and FcR is influenced by several factors including the affinity between the antibody isotype and the specific receptor, the composition of the sugar side chain on the Fc-portion of the antibody, and inhibitory characteristics of the FcR. Subclasses of Fc and receptors have been defined that determine whether a specific antibody will bind complement or not. Antibody-dependent cell cytotoxicity (ADCC) depends in part on the specific N-glycosylation of the Fc domain of the monoclonal antibody. Cross-linking of FcR triggers various immune reactions including phagocytosis after opsinization, ADCC, and release of various cytokines that act as inflammatory mediators [117, 701]. FcR that bind human IgG Fc are referred to as FcγR and these are subclassed based on their binding characteristics and function. From a functional perspective, there are two main types of FcγR found on the surfaces of immune effector cells, activating FcγR, and inhibiting FcγR. Based on genetic polymorphisms the activating FcγR include the high-affinity receptor CD64 (FcγRI), and the low affinity receptors CD32a (FcγRIIa) and CD16 (FcγRIIIa). CD64, which is expressed on monocytes,

Robert O. Dillman macrophages, and dendritic cells, is the only FcγR with a high affinity to Mab monomeric IgG. Cells with a CD64 Fc receptor can bind circulating Mab that is not bound to antigen, while the lower affinity Fc bind better to Mab that is bound to antigen. CD32a is expressed on these cells, and also on neutrophils, eosinophils, and platelets. CD16a is highly expressed on NK cells and macrophages. There is some evidence that these polymorphisms are predictive of the clinical efficacy of certain antibodies. This is leading to specific strategies to modify the Fc portion of antibodies in order to optimize the anti-tumor effector functions [667, 695]. Theoretically antibody therapy is the most tumor specific approach to systemic cancer treatment that has been discovered. As noted earlier, antibodies directed against a tumor antigen are tumor specific to the degree to which that antigen is found only on tumor cells. At one time it was believed that all cancers were foreign and that tumor-specific antigens would be readily discovered. Unfortunately, phenotypically and genetically, cancer cells are more like normal cells from that host, and as a result, most tumor antigens are only relatively tumor specific because they are found in certain normal tissues as well. However, many tumor-associated antigens are expressed much more heavily only during embryonic development; and, therefore are relatively tumor specific. Also, certain viruses produce or induce antigens that are relatively tumor specific. The idiotype encoded by B cells of a specific B-cell malignancy is an example of a tumor specific antigen.

Unconjugated Antibodies as Therapeutic Agents As illustrated in Fig. 2 and Table 3, there are many ways in which antibodies may be used in cancer therapy [161, 163, 533]. This chapter covers the rationale for the use of unconjugated (or “naked”) antibodies in cancer therapy and the current role of such Mab in the treatment of human malignancy. In subsequent chapters of this book, the use of conjugated Mab as carriers of cytotoxic substances including chemotherapy agents, radioisotopes, and natural toxins are reviewed.

Manufacturing of Monoclonal Antibodies Hybridoma Technology In 1975 Kohler and Milstein, published a landmark paper in which they described the “hybridoma technology,” which opened the doors to reproducible and large-

307 scale production of Mab [400]. Various laboratories utilized this technology to identify large numbers of mouse Mab that reacted with tumor-associated antigens [158]. As shown in Fig. 3, in this methodology mice or rats are injected with whole tumor cells or purified tumor associated antigens (such as carcinoembryonic

C’ COMPLEMENT

EFFECTOR CELL

ACTIVATED T-CELL γ - IFN

TUMOR CELL

IL-2

RICIN A (Toxin)

α-, β-IFN

TNF

DNR (Drug) Y90 (Isotope)

Figure 2. Schematic of potential applications of monoclonal antibodies for cancer therapy. Various antigens, representing the heterogeneity of cancer, are depicted on the tumor cell surface. Bifunctional antibodies are depicted with one Fab directed to a tumor antigen and the other to a biologic response modifier. One antibody has a Fab directed to a CD2 or CD3 receptor on T cells, which results in their activation and enhanced cytotoxicity. The Fc portion of the antibodies are attached to various cytotoxic substances including complement, effector cells (granulocytes, monocytes, killer T cells) of the immune system, and cytotoxic agents such as toxins, radioisotopes, and chemotherapy agents. IFN, interferon; TNF, tumor necrosis factor; IL-2, interleukin-2; DNR, daunorubicin. Ann Intern Med 1989, 111:592–603. By permission of the American College of Physicians

Table 3. Strategies for in vivo use of monoclonal antibodies as anti-cancer therapy Antibody alone Complement mediated cytotoxicity (CMC) Antibody dependent cell-mediated cytotoxicity (ADCC) Regulatory interactions (ligands, receptors) Anti-idiotype vaccines Immunoconjugates Radiolabeled antibodies Immunotoxins Chemotherapy-antibody conjugates Cytokine immunoconjugates Cellular immunoconjugates Multi-targeted immunoconjugates

308

Monoclonal antibody therapy

Immunization

HPRT+ spleen cells

Nonsecretory HPRT −

B-cell culture

Fusion in PEG

Selection in HAT medium

Screening of supernatants for antibody and selection of clones

Recloning and subcloning Supernatant (contains 10 to 100 mg /mL MOAb) Purified ascites (contains 10 to 20 mg/mL MoAb)

Figure 3. Schema for murine monoclonal antibody production. From [176]. By permission of Biomedical Information Corporation

antigen) to induce a polyclonal immune response against the foreign human proteins. The animal’s immune system responds with production of antibodies directed against the tumor antigens. The blueprints for the antibodies are contained in by B lymphocytes that are found in large quantities in the spleen. The spleen of an immunized animal is removed and minced into a single-cell suspension. If placed in tissue cultures, such cells typically die off in a few days and are unable to proliferate. For this reason, the B lymphocytes are combined with other mouse B cells, typically mouse myeloma cells, that have been specifically selected for certain characteristics, including (1) ability to grow in culture as a

single clone, (2) inability to produce and secrete immunoglobulin themselves, and (3) inability to grow in the presence or absence of specific medium constituents. In the presence of a cell-fusing substance, such as polyethylene glycol, individual spleen cells with the genetic machinery for making antibody against the tumor fuse with cells from the immortal cell line, thus producing hybrid cells, or hybridomas that retain the ability to secrete antibodies. By the plating process of limiting dilution, a single hybridoma clone of cells can be isolated that retains the ability to secrete the antibody encoded in the DNA of the mouse spleen cells, hence the term “monoclonal antibody.” The antibodies are isolated

Robert O. Dillman from culture media and screened for specificity against various cells and tissues. A major limitation of this approach is that antigens are defined by the mouse immune system, and tumor antigens are defined by differential expression on normal as opposed to cancer cells. The first large-scale production factories for Mab for clinical trials were the peritoneal cavities of Balb-C and athymic mice. Injection of the hybridomas into the peritoneal cavity resulted in a malignant ascites that was rich in the monoclonal antibody. This technology helped launch the biotechnology industry, and made possible mass production of promising antibodies [14, 94, 157]. The techniques of mass production included the in vivo expansion of hybridomas as ascites in mice, and in vitro expansion of such hybrid cells in various large tissue culture vessels and bioreactors.

Recombinant Technology and Engineering Monoclonal Antibodies Murine hybridomas that produced desirable antibodies were used as a source for genetic material for mass production in unicellular factories, and to make chimeric or humanized Mab. Recombinant DNA technology was used to introduce antibody-encoding genes into other cells to facilitate antibody production by cells such as Escherichia coli, yeast, and Chinese Hamster Ovary (CHO) cells [42, 44, 58, 508]. Certain mouse plasmacytoma lines also glycosylate proteins in a similar manner. These unicellular organisms are currently the factories of choice for Mab production because they produce human-like N-glycosylation of the proteins, which is crucial for proper folding of the Ig protein for the stereochemistry that is needed for optimal binding to antigen. Recombinant DNA technology has displaced hybridoma technology as the method of choice for monoclonal antibody production. Recombinant techniques have made it possible to engineer a variety of Mab constructs [6, 157, 631]. Furthermore, recombinant DNA biotechnology has made it possible to modify mouse Mab into partially humanized forms. As discussed later in this chapter, this has led to the production of mouse–human chimeric Mab and humanized Mab as diagrammed in Fig. 4, as well as antibodies designed to react with more than one antigen as shown in Fig. 2. As of 2007, of the 18 different therapeutic antibody products approved for human use in various diseases, ten are manufactured in CHO cell lines and eight are made in murine plasmacytoma cells. Additional modifications include engineering the Fc portion to optimize interactions with complement, or binding to FcR to

309

Figure 4. Comparison of different types of monoclonal antibodies mouse antibodies illustrating the component of murine amino acid sequences in each. Shown are mouse antibodies (omabs), mouse/human chimeric antibodies (-ximabs) in which the murine variable region is combined with a human constant region, “humanized” antibodies (-zumabs) in which only the hypervariable peptide sequences are retained from the original mouse antibody, and human antibodies (umabs)

optimize ADCC. Other genetic engineering modifications have focused on making smaller antibody molecules as single chain Fv, Fab, and F(ab’)2 [325, 584].

Human Monoclonal Antibodies In terms of therapeutic potential, there are several reasons why human Mab offer advantages over those of other species [21, 258, 416]. First, they are less immunogenic and less allergenic than non-human antibodies. Second, certain subclasses of human antibodies are more effective than murine antibodies in facilitating complement and/or antibody-mediated cytotoxicity in vitro. Third, by dose-to-dose comparisons, human antibodies have longer half-lives and prolonged serum levels because there is less non-specific uptake, destruction and metabolism. There is evidence that antibodies are taken up by FcR receptors, called neonatal or Brambell receptors (FcRn), which bind the Fc portion of antibodies and remove them from the circulation [357]; [442]. Human IgG1, IgG2, and IgG4 have high affinities for FcRn which transport endocytosed antibodies from the lysosome back to the serum, while murine antibodies and human IgG3 have a low affinity for FcRn. For antibodies with a low affinity for FcRn, instead of being transported back to the cell surface, they are catabolized [527, 808]. Several approaches have been used for isolating human Mab [189, 258, 671]. In one approach, it is presumed that patients with cancer have developed at least a limited immune response to their tumor. Regional draining lymph nodes, which are the first line of regional immunologic defense against tumor, are excised to obtain a source of B lymphocytes, some of which make antibodies against the tumor. Regional lymph nodes that drain known cancer sites are potential sources of B lymphocytes that may

310 have been programmed to make antibodies against the primary cancer. Immunization with tumor cells or antigen, followed by excision of draining lymph nodes, especially sentinel lymph nodes, has also been used to isolate antibody producing B lymphocytes [282, 299]. Another source of immune reactive B cells is the peripheral blood, and the frequency of such cells may be enhanced by immunization. For example, researchers have injected irradiated autologous tumor cells into the skin with an immune stimulant such as Bacillus Calmette-Guerin (BCG) or granulocyte macrophage colony stimulating factor (GM-CSF). Rather than harvesting lymph nodes, 2 to 3 weeks after immunization peripheral blood B lymphocytes are collected, some of which secrete human antitumor antibodies Once antibody-producing B cells have been isolated, they can be fused with other human cells of B cell lineage that have been selected for similar characteristics as those immortal mouse B-cell lines described above, to produce antibody secreting clones [51, 123, 129, 256, 257, 670]. It is also possible to fuse human lymph node B cells with cells from immortal mouse B-cell lines, and still get secretion of human antibody, although such cross-species hybridomas are usually unstable [67, 635, 802]. Other investigators have isolated B lymphocytes from lymph nodes and then infected these cells with Epstein-Barr virus to immortalize cells producing human Ig [96, 113, 189]. Using various techniques, human Mab have been produced that react with breast cancer [67, 395, 635], lung cancer [669], colorectal carcinoma [299], brain tumors [670], stomach cancer [802] and melanoma [241, 364, 686]. However, human Mab were more difficult to develop than murine Mab because of the instability of human hybridomas and immunoglobulin secreting cells, low secretion rates, and difficulties in mass producing and screening. These inherent problems in human Mab production have been overcome by recent advances in transfection technology and molecular immunology that have made it feasible to integrate DNA for human Ig into cells that can easily produce large quantities of human Mab. Newer technologies such as polymerase chain reaction (PCR) [202] and transgenic mice [271] have led to identification of vast libraries of human antibodies that may be superior in terms of antigen and antibody selection. In recent years various laboratories have been able to clone cDNA for VH and VL contained in hybridomas and B lymphocytes that were producing low levels of desirable antibodies. These were then linked to human hinge and constant genes to produce totally human Mab in more efficient cellular factories. Today one can take Ig producing genes from B-lymphocytes and produce

Monoclonal antibody therapy the entire repertoire of antibodies which can then be screened for their specificity, and the Fv isolated to produce fully human recombinatorial products [6, 326, 631]. Using such libraries, fully human Mabs can be produced by linking Fv from the display library with human Ig hinge and constant regions. Human Mab can also be produced by immunizing transgeneic mice whose immune system has been replaced with a human immune system engineered to produce human Mab of a specific class and subclass. Immunization of these animals results in human B cells that produce human Mab.

Chimeric and Humanized Monoclonal Antibodies Because of the difficulties in producing and manufacturing human Mab, the extensive characterization of murine Mab that react against human tumor associated antigens, and the clinical disadvantages of murine Mab, other strategies have been developed to create mouse/human chimeric Mab and genetically engineered “humanized” Mab (Fig. 4) [1, 44, 58, 124, 325, 386, 440, 508, 510, 788]. One way to improve the activity of a Mab is to class-switch the Mab so that it has an Fc portion that is more suited for interaction with human complement and/or effector cells [44, 157, 508]. Human IgG1 is usually considered most desirable because it effects both CMC and ADCC. IgG3 also facilitate favorable CMC and ADCC effects, but these proteins tend to aggregate which is problematic from a production standpoint. It has generally been taught that IgG4 is the least useful, especially in terms of CMC, because of a lack of Fc receptors for this IgG subclass. In the chimeric approach, the variable domains of the light and heavy chains of a desirable murine Mab, are linked to hinge and constant domains of a human Ig. This is most often done with recombinant techniques in which the mouse genes for the variable domains of amino acids are combined with the human domains for the hinge and constant region amino acids. The genes are then inserted into bacteria or yeast for large-scale production. The bacterial preparations are typically not glycosylated while those grown in yeast are glycosylated, which is important for interaction via the Fc portion of the antibody. It is also possible to isolate the genes specific for the six hypervariable amino acid sequences on the light and heavy chains (CDR) of a desirable murine antibody, and these can then be inserted into the framework of human Ig to create a “humanized” Mab via what is called “CDR grafting”. In this

Robert O. Dillman construct the only residual mouse amino acid sequences remaining are those that give the antibody its three-dimensional binding specificity. All of the unconjugated monoclonal antibody products approved for cancer treatment are either chimeric (cetuximab, rituximab), humanized (alemtuzumab, trastuzumab, bevacizumab) or human (panitumumab). Because of the human Fc portion of the Ig molecule, products, they possess superior cytotoxic potential based on interactions with complement and/or effector cells, and are much less immunogenic, which is associated with a longer bioavailability [662]. The availability of “chimeric,” “humanized,” and fully human Mabs has greatly reduced the limitations resulting from the production of endogenous human anti-Ig antibodies such as human anti-mouse antibody (HAMA), human anti-chimera antibody (HACA), and human antihuman antibody (HAHA) in man, and facilitated the use of repeated treatments with specific Mab for extended periods of time.

Antibodies with Multiple Specificities and/or Functions It is also possible to make Mab with specificity to more than one antigen-binding site, and more than one functional capability. Some examples of bispecific and bifunctional Mab, in which the antibody construct reacts with more than one antigen, are illustrated in Fig. 2 [124, 223, 711, 772, 793]. This can be done by chemically linking either two different Fab pairs or F(ab’)2 pairs, or two intact antibodies. It is also possible to make quadromas (a hybridoma of two monoclonal antibody-secreting hybridomas), which secrete Mab that react with two different antigenic binding characteristics [123]. One can then select the appropriate quadroma that is producing antibodies with two different antigenic binding characteristics. In recent years these approaches have been replaced by recombinant DNA techniques in which the genes for different murine variable or hypervariable regions of interest are linked to a humanized constant region and then manufactured in cells [223].

Monoclonal Antibody Nomenclature Because of the tremendous number of Mabs that are under development, a nomenclature system has been developed that provides information about the construct and the target. These names are reserved until the product is being tested in trials that are considered pivotal for

311 Table 4. Components that precede “mab” in the nomenclature for naming a monoclonal antibody Infix

Target

Inflx

Origin

Tu (m) Ci (r) Li (m)

Tumor Cardiovascular Immune

-a-o-xi-

Co (l) Me (l) Go (v)

Colon tumor Melanoma Ovarian tumor

-zu-u-axo-

Rat Mouse Chimeric human/mouse Humanized Human Rat/murine hybrid

Pr (o)

Prostate

regulatory approval. The suffice “mab” indicates that the product is a monoclonal antibody. The syllable directly in front of mab indicates the source such as mouse, chimeric, humanized, or human. The prefix of the name has no specific meaning. A second syllable may be added if there is something else linked or attached to the product. The key components that appear before “mab” for names adopted for the United States are shown in Table 4. Most of the Mab are either mouse (-omab), chimeric (-ximab), humanized (-zumab), or human (-umab). Thus, rituximab is a chimeric Mab that targets tumor (as is cetuximab). Trastuzumab is a humanized mab that targets tumor (as is alemtuzumab). Bevacizumab is a humanized mab that has a cardiovascular target (vascular endothelial growth factor). Panitumumab is a human mab that targets tumor. Limilimumab and tremelimumab are human mabs that that have an immune target (CTLA4) while daclizumab is a humanized mab with an immune target. Tositumomab and ibritumomab are murine mab that target tumor while oregovomab is a mouse mab that targets ovary. Catumaxomab is a rat/mouse hybrid anti-tumor mab.

Mechanisms of AntibodyMediated Anti-Tumor Effects As outlined in Table 1, there are at least three different rationales for using antibodies alone in vivo as cancer treatment. The first rationale involves two different immune mediated effects involving the Fc portion of the immunoglobulin and interaction with other components of the host immune system. The second rationale encompasses concepts of regulating tumor cells or their microenvironment. The third concept relates to the antiidiotype cascade as a means of immunization to induce endogenous antibodies.

312

Monoclonal antibody therapy

Complement Mediated Cytotoxicity

macrophages, granulocytes and eosinophils have such receptors and can precipitate tumor cell lysis when linked via Ig to a cell. Collectively, such cells are referred to as “effector cells.” The Fc portion of Ig is crucial for linking antibody to effector cells via their FcR as shown in Fig. 6; the specific carbohydrates on the Fc are crucial for this interaction [117, 524]. The numbers of Fc receptors and affinity for Fc predict the degree of ADCC activity [447, 796]. Antibody may first bind to its cellular target followed by effector cell binding via the Fc, or alternatively, effector cells may first bind to the Fc of the antibody, and then be carried to the tumor target. Ex vivo incubation of Mab and effector cells followed by in vivo delivery has produced anti-tumor responses in animal models [308]. Circulating target cells coated with antibody are removed in the liver and spleen, presumably because of Fc receptors on tissue macrophages in these sites. For human Mab, the ability to bind specifically to macrophages of the reticuloendothelial (RE) system is restricted to IgG1 and IgG3 antibodies and is absent from IgM and other IgG subclasses. There is evidence that mouse Mab differ in their subclass binding to human effector cells. Various studies have confirmed that among murine Mab, the hierarchy for effecting ADCC is IgG2a, followed by IgG3, apparently because of enhanced Fc receptor

Complement mediated cytotoxicity (CMC) or complement dependent cytotoxicity (CDC) involves the fixation of complement via the Fc portion of Ig and activation of the complement protein cascade resulting in membrane damage and cell destruction as shown in Fig. 5 [238, 768]. Murine antibodies are usually ineffective in fixing human complement in vitro. Exceptions to this are IgM and certain IgG3 murine antibodies [109, 794]. In man, studies of hemolytic anemia have confirmed that human IgM is the most efficient human Ig class for complement fixation followed by IgG1, IgG3, and IgG2. Because complement fixation occurs at the Fc portion of antibody, this approach requires whole intact Ig or an Fc segment, rather than antibody subunits. As noted earlier in this chapter, in the era of chimeric, humanized, and human Mab, human Fc constructs can be selected to optimize for complement binding if that is considered desirable.

Antibody Dependent Cell-Mediated Cytotoxicity The second immunological cytotoxic mechanism is ADCC [304, 305]. Many lymphocytes, monocytes,

COMPLEMENT-DEPENDENT ANTIBODY MEDIATED TUMOR CELL LYSIS

Na+ K+ H2O

C

IgG

Figure 5. Diagram of complement mediated cytotoxicty (CMC) featuring antibody binding, fixation of complement, and cell death

TUMOR CELL & ANTIBODY

MEMBRANE DAMAGE

TUMOR CELL LYSIS

ANTIBODY-DEPENDENT CELL - MEDIATED CYTOTOXICITY (ADCC)

Fc RECEPTOR

Figure 6. Diagram of antibody dependent cell-mediated cytotoxicity featuring antibody binding, attachment of effector cells via the Fc receptor to the Fc tail of the antibody molecule, and cell death

LYMPHOCYTE

LYSIS NOT C’ DEPENDENT

Robert O. Dillman binding [308, 340]. In addition, different antibodies of the same subclass and isotype, directed against the same antigen, exhibit different degrees of Fc binding [179, 660]. Murine IgG3 anti-disialoganglioside antibodies have been cytotoxic with human effector cells in vitro [108, 305, 720]. Among commercially available chimeric and humanized antibodies, most have been engineered with a human IgG1 Fc portion that can effect ADCC [691]. For both ADCC and CMC the in vivo anti-tumor effect is also dependent on the ability of the host immune system to provide sufficient complement proteins or effector cells to produce cytotoxicity. The use of various biological response modifiers may be useful in augmenting such an immune response. To maximize efficiency of effector cell-antibody binding, some investigators have utilized leukopheresis to harvest leukocytes, and then incubated these with antibodies to enrich the antibody-effector cell population. Various lymphokines, such as interleukins-2, -4, -12, and -15 may be useful to enhance the cytotoxic activity of the effector cells. It has been demonstrated that cytotoxicity, either by ADCC or CMC, involves a threshold of antigen binding sites. Lymphokines, such as alpha and gamma interferon, can enhance target antigen expression in some tissues [180, 274, 275, 432, 519]. Gamma interferon increases Fc receptors on effector cells and thereby can enhance ADCC [181, 519, 767, 775]. There are good examples from animal models suggesting that various biologicals may be used in combination to maximize an anti-tumor effect. The agents most commonly used for this purpose in human trials are interferon alpha, IL-2 and GM-CSF. These other biologicals might be used systemically, or targeted specifically by other antibodies.

Regulatory Approaches Malignancy may result from the overproliferation of cells, the failure of cells to undergo programmed cell death (apoptosis), or a combination of these. Antibody modification of these events constitutes a second mechanism of antibody therapy, and is encompassed by the term “regulatory,” in contrast to “immunologic,” to characterize this potential mechanism [163]. Such receptor targets can also be associated with cytotoxic effects mediated by complement and/or effector cells. In contrast, ligand or microenvironment targets may produce anti-tumor effects purely by regulatory biologic effects. It is known that tumor cells have a variety of receptors that are important for growth or proliferative

313 advantages and for preventing or delaying apoptosis that might be targeted for antibody therapy [269, 480, 568, 731]. Advances in understanding of cellular physiology have led to identification of a number of ligand–receptor–signal transduction systems in which a ligand binds to a receptor which initiates a series of enzymatic reactions that signals the nucleus of the cell to activate genes, translation, and transcription to produce proteins that are important in cell function, differentiation, proliferation, and apoptosis. Epithelial growth factor (EGF) and vascular endothelial growth factor (VEGF) and their receptors are some of the best known examples of such interactions. Increased levels of these receptors have been found in increased quantities in cancer cells and/or other rapidly proliferating cells. Conceptually, Mab directed against growth factor receptors may either block or down-regulate the number of receptors on the cell surface, and thus impair a cell’s ability to differentiate and/or divide, ultimately resulting in cell death or apoptosis. Such antigens may also serve as targets for Mab that can induce CMC and/or ADCC, or as targets for cytotoxic immunoconjugates. Many receptors internalize after their ligand or an antibody bind to them resulting in what is sometimes referred to as “antigen modulaton.” Rapid internalization limits the potential for CMC or ADCC, but may be an advantage for the internalization of conjugated cytotoxic chemotherapy agents or toxins.

Idiotype Network One of the first targets proposed for a regulatory approach was the Ig idiotype of B cell lymphoid malignancies based on the cascade of anti-idiotype antibodies involved in regulating B cell proliferation [65, 246, 247, 351]. Cells from a malignant B-cell clone express and sometimes secrete a specific antibody with a unique binding ability. The peptide sequences in the hypervariable region of the light and heavy chains of the antibody are the idiotopes that collectively constitute the idiotype of that Ig, and account for its specificity. Under normal conditions memory T cells signal B cells to produce Ig via surface idiotype receptors. Other B cells may produce anti-idiotype antibodies that may be important in negative feedback control of a given B-cell clone that has been activated. A detailed description of the “network theory” of anti-idiotype regulation is very complex, and beyond the scope of this chapter. In its simplest construct, infusion of an antibody directed against a B-cell lymphoma idiotype might suppress (growth regulate) that clone back to its baseline state by inducing

314

Monoclonal antibody therapy Antigenic stimulation

B-Cell proliferation and differentiation

Ig

Antigenic stimulatioin Anti-idiotype

Figure 7. Diagram of anti-idiotype feedback inhibition of B-cell clonal proliferation, based on the specificity of the hypervariable region of the antibodies produced by that clone of cells

apoptosis and/or limiting proliferation. How this might relate to B-cell malignancy is depicted simplistically in Fig. 7. It is also important to note that the idiotype of a given B-cell tumor is perhaps the most tumor specific antigen that has been identified. Idiotype network theory has led to a concept of taking advantage of idiotype targets as a form of passive immunization [404]. According to the “idiotype network” theory, the idiotype of an Ig is very immunogenic and important in negative-feedback self-regulation. Injection of a mouse antibody (AB1) directed against a patient’s tumor antigen will lead to production of many different antibodies, but some will be anti-idiotype antibodies (AB2) directed against the hypervariable determinants of the AB1. Some of these AB2 may present the same three-dimensional antigenic structure as the original tumor antigen. Subsequently, such AB2 anti-idiotype antibodies may induce anti-anti-idiotype antibody (AB3), which because of the “lock-and-key” structural relationship between antigen and antibody, will have the same binding specificity as the original mouse antibody (AB1), except that it will be a human antibody (AB3) [316, 781]. A more direct approach using this same principle involves simply taking a well-characterized anti-anti-idiotype antibody (AB2) and directly injecting it as an immunogen to try to induce a human AB3 that will react with the tumor antigen. This theoretical approach has been demonstrated in animals and may have application in both cancer and autoimmune diseases [314, 378]. Early clinical trials testing this approach have been associated with successful production of sufficient antibodies to produce immunologic and anti-tumor effect in man. [39, 104, 229, 230, 493, 494]. This concept and results of clinical trials testing

such products are discussed in more detail in this book in the chapter on vaccines.

Epidermal Growth Factor and its Receptors (EGF and EGFR) Another heavily studied regulatory system is the human epidermal growth factor receptor (EGFR) family of four transmembrane receptors abbreviated as Her1, Her2, Her3, and Her 4 or Erb-B1, Erb-B2, Erb-B3, and ErbB4 respectively), which are involved in regulation of cell proliferation and survival. EGFR are overexpressed on the tumor cells of subsets of patients with most epithelial solid tumors [608, 611]. The prototype for this family of receptors is Her1 (a.k.a. EGFR or EGFR1) which binds to a variety of ligand growth factors including epidermal growth factor (EGF), transforming growth factor beta (TGF-β), epiregulin, betacellulin, and amphiregulin, which activate the cytoplasmic tyrosine kinase domain of the receptor, resulting in downstream activation of various signal transduction intermediates that result in cell proliferation and other functions associated with malignancy [471, 480]. This is the target of the commercially available antibodies cetuximab (Erbitux) and panitumumab (Vectibix). Her2 (a.k.a. Her-2 neu) originally attracted interest when it was found to have oncogenic properties and to be over expressed on tumor cells from about 25% of breast cancer patients. [632, 673]. Her2 is unique among the EGFR family in that it has no known ligand, but instead can bind with each of the other EGFR (a process called “dimerization”) which also induces signal transduction. When over expressed, Her2 can dimerize with itself to induce the tyrosine kinase activity that leads to signal transduction.

Robert O. Dillman Heregulin is a known ligand for Her3, which unlike the other heregulin receptors, has no tyrosine kinase activity. Neuroregulins 1, 3, and 4 and epiregulin are ligands for Her4.

Vascular Endothelial Growth Factor and its Receptors Judah Folkman hypothesized that angiogenesis was critical for growth and metastasis of tumors. [226]. The angiogenesis-inducing ligand vascular endothelial growth factor (VEGF) and its cell membrane receptor, vascular endothelial growth factor receptor (VEGFR) were predicted to be good targets for anticancer therapy [218, 634]. The commercially available monoclonal antibody bevacizumab (Avastin) binds VEGF. VEGF (a.k.a. VEGF-A) is one of a family of related proteins that induce angiogenesis and/or lymphoangiogenesis via binding one of several related receptors. VEGF is the central mediator of tumor angiogenesis, which is crucial for proliferation, invasion, and metastasis. It is produced by cells from many tissues and messenger RNA levels of the VEGF gene are over expressed in most tumors. Elevated levels of VEGF are predictive of poor prognosis. A variety of experiments suggested that this ligand would be a useful target for monotherapy, or in combination with other therapeutic modalities [12, 55, 248, 420, 681].

Considerations for Clinical use of Monoclonal Antibodies as Cancer Therapy As summarized in Table 5, there are a number of factors that must be considered, when choosing an antibody for clinical development, and there are a number of specific issues associated with therapeutic application. This section focuses on the general principles of antibody therapy that were, for the most part, established during clinical trials conducted between 1980 and 1989 with Mab directed against antigens found on cells from hematologic malignancies [19, 38, 161, 177, 179, 180, 227, 328, 446, 478, 485–488, 518, 569, 600], and solid tumor cancers [109, 161, 205, 240, 262, 263, 375, 439, 513, 535, 625, 651–653, 666, 738, 776]. Many of the studies used products that reacted with CD5 [38, 177, 179, 227, 228, 488, 486, 488], or anti-idiotype antibodies [446, 478, 487], or the antibody 17-1A against adenocarcinomas [109, 240, 439, 651–653, 738].

315 Table 5. Issues for clinical application of monoclonal antibodies Affinity and avidity of antibody for tumor antigen Ability to effect complement and/or antibody-dependent cell mediated lysis Biological significance of the antigen to the malignant cell Biological behavior of the antigen (secreted, internalized, rate of production) Density of antigen expression Dose and schedule of administration Expression of antigen on normal tissues Human anti-antibody response (HAMA, HACA, HATA, etc.) Heterogeneity of antigen expression Location, size, and vascularity of tumor mass Therapeutic index: efficacy vs. toxicity Total body burden of tumor and antigen

Antitumor Effects In Vivo Binding to Malignant Cells In association with intravenous (i.v.) infusions of antibodies, several studies used fluorescein conjugated antimouse antibodies to show that many of these products, especially those resulting from immunizing animals with malignant hematologic cells, did bind to circulating blood tumor cells and cells in the bone marrow in man [19, 38, 161, 177, 488, 518, 600]. Mab binding to tumor cells located in lymph nodes, tumor masses, and skin infiltrates were directly demonstrated using immunofluorescence and immunoperoxidase techniques [179, 328, 375, 513, 535], and indirectly with low doses of Mab conjugated to technetium, indium or iodine, as radioactive tracers [92, 183, 286–289, 417, 427, 451, 496, 514, 515, 719]. The relative specificity of this uptake was established by failure to image any “false positive” lymph node sites, and the successful imaging of nonpalpable nodes that subsequently were proven to contain cancer by surgical excision and histopathology evaluation [183, 719]. Further proof of binding specificity was shown in a patient with T-cell lymphoma, in whom a radiolabeled anti-CD5 murine Mab was concentrated in lymph nodes, but injection of a radiolabeled murine anti-melanoma Mab of the same subclass was not associated with lymph node uptake [92]. Various studies showed that antibody binding was more easily demonstrated in cutaneous tumors than in solid tumors located in viscera or in lymph nodes [535]. This may relate to cutaneous blood supply, but it could also relate to the relatively small size at which cutaneous tumors can be recognized. Some investigators have suggested that direct infusion into the lymphatic system may be superior to i.v. infusions in certain disease settings

316 [719], but this is not a practical option because of the technical difficulties associated with such an approach. In general, after an i.v. infusion of a large quantity of Mab, the hierarchy of binding to antigen-bearing cells is circulating blood cells > bone marrow > skin > lymph nodes > small tumor nodules > large tumor masses. There has been great interest in finding ways to overcome the interstitial pressure effects found in larger tumor lesions, which impair penetration of antibodies into the core areas of such tumors [52, 345].

Clinical Efficacy and Mechanisms Significant antitumor responses were relatively uncommon in early trials with mouse Mab, but most of these involved relatively limited dosing in pilot, phase I, or limited phase I/II trials, and were not definitive tests of Mab as cancer treatment. Nevertheless, it was encouraging that tumor responses following murine Mab therapy were reported for both hematologic malignancies and solid tumor cancers. Some of the best responses in these early trials took place in patients with follicular (nodular) lymphoma and malignant melanoma, two diseases in which the frequency of spontaneous regressions had suggested an existing antitumor immune response [699]. As a general observation, when Mab bind in sufficient number or density to cells in the peripheral blood, those targeted cells are removed in the reticuloendothelial system. Human trials showed that infusions of 51Cr or 111I-labeled autologous tumor cells resulted in marked uptake of the isotope label first in the lung, then in the liver and spleen [161, 488, 518]. Investigators rarely could detect associated decreases in complement levels, although Ritz et al. [600] found deposits of C3 on monoclonal antibody-coated cells in one ALL patient, and complement deposition was demonstrated in tissues in colon cancer patients receiving KS1/4 [205], and in melanoma patients receiving the R24 antibody [738]. Changes in the viability of circulating target cells have been observed following monoclonal antibody binding, although this is difficult to demonstrate because of timing and technical reasons [19, 518]. These studies suggest that binding of Mab to peripheral blood cells may actually damage the cell membrane and that such cells are then removed in the reticuloendothelial system rather than lysing in the intravascular compartment. The best evidence of cell destruction, rather than sequestration, comes from studies following radiolabeled autologous tumor cells [177, 488, 518], and the finding of an increased lactate dehydrogenase (LDH) associated with decreased AML cells [19].

Monoclonal antibody therapy In patients in whom tumor regressions have been seen, the precise mechanism of antibody-mediated tumor regression is difficult to ascertain. Anti-leukemic effects are dependent on the Mab used, the rate and dose of Mab infused, the density of antigen expression, and whether there is circulating antigen or endogenous antibodies that bind to the therapeutic antibody. In earlier trials of mouse Mab, given in limited quantities, in most instances the anti-leukemic responses were relatively transient, but administration of chimeric and humanized Mab in large quantities sufficient to sustain a large pharmacokinetic “area-under-the curve” for prolonged periods of time, have been associated with meaningful durable clinical response. In the absence of sustained levels of Mab, antibody-coated target cells are removed from the circulation, but are rapidly replaced by cells from other organs such as bone marrow, lymph nodes, and possibly spleen. Some cells may survive by trafficking to other sites, and then return to the circulation. For non-modulating antigens, the leukemia cell count remains depressed as long as Mab levels persist in the circulation. However, for modulating (internalizing) antigens, the cell count begins to recover in association with entry into the circulation of cells with a lower density of antigens. Such modulated leukemia cells subsequently re-express the target antigen in vitro or in vivo once the antibody concentration has dropped to a negligible level [639, 663]. Antigen modulation (cycling, internalizing) has been a limiting factor for many antibodies directed against hematopoietic malignancies, but is not an issue for Mab that react with stable membrane antigens, especially when the target antigen is important as a receptor for cell proliferation, or helps a cell delay or avoid programmed cell death. The precise explanation for the responses seen in B-cell lymphoma patients treated with anti-idiotype Mab has not been clearly defined. Idiotype is a modulating target, which led investigators to adopt therapeutic strategies of repeated low dose therapy so that target cells would have a sufficient density of antigen to effect CMC or ADCC. Investigators postulated either a direct cytotoxic effect or a regulatory effect via the idiotype network [65, 246, 247, 351]. The most responsive group was follicular lymphoma. Analyses suggested that those follicular lymphoma patients who responded had a greater infiltration with T cells prior to therapy [478]. Responses were more readily achieved in patients with very low levels of circulating idiotype, which is probably indicative of dose threshold related to antigen burden. As discussed later in this chapter, even a decade after its approval, it is still not clear whether immunolgic and/or regulatory effects are more important for

Robert O. Dillman the clinical effects observed with the anti-CD20 chimeric Mab rituximab. Although the best responses in patients with solid tumors have been achieved with Mab that effect both ADCC and CMC in vitro, regulatory mechanisms related to the function of the target antigen (receptor) may be more important. For example, the mechanism for the anti-tumor effects that leads to the excellent clinical results seen with antibodies to human EGFR are probably related to the importance of those receptors in sustaining proliferation. Secondary immunologic effects may also be important. Sequential biopsies from melanoma patients who exhibited a clinical response to the R24 mouse antibody revealed increasing infiltration with CD3+, CD8+, Ia+ T cells in the presence of degranulated tissue mast cells [779]. Complement deposition was also noted. Even though responses were seen within 2 weeks of beginning therapy, tumors continued to recede well beyond the treatment period despite the presence of HAMA. These responses may involve a more complex interaction with the host immune system, perhaps triggering a cascade of inflammatory events including activation of T lymphocytes that persist for a long period of time [318], or they could be inducing anti-apopotic and/or anti-proliferative effects. It has been suggested that mast cells not only have local inflammatory effects, but also may function as immunoregulatory cells in a an immune response [243]. Unfortunately, most advanced cancer patients do not have tumors that are as easy to analyze sequentially as melanoma. Selection of patients with primarily lymph node or soft tissue disease facilitate prospective studies, but they also result in unintentional bias because soft tissue and lymph node metastases tend to be more responsive to any intervention than bone, liver, brain, or other visceral metastases. Other interesting observations raise questions regarding the possible mechanisms of antitumor affects against microscopic disease [183, 287]. A young patient underwent a left radical neck dissection for regional spread of melanoma. Subsequently an anti-p97 antibody labeled with 111Indium illuminated three apparent lesions in the right neck, although repeated physical examinations and a computerized tomographic scan of the region were negative for tumor. No other lesions or lymph nodes were visualized, but 3 weeks later he developed three palpable lymph nodes in the right neck in sites consistent with the scan. A neck resection was recommended but he declined surgery, and the neck lesions subsequently resolved. He was known to remain free of disease for over 3 years. A second male patient had had a melanoma resected from his upper back and later had a

317 right axillary recurrence. There was uptake of 111Indium anti-p240 in the right axilla but there was also uptake in the left axilla, which was clinically negative. Subsequently, a small left axillary node was palpated but spontaneously regressed. No other lymphadenopathy was visualized by imaging and no other lymphadenopathy was palpated by examination. Both axillae were explored with confirmation of tumor in the right axilla, but no tumor was found on the left. There are at least four possible explanations for these intriguing observations of what clearly were antigen-specific interactions. First, there may have been a local antitumor effect against microscopic tumor cells. Second, there may have been antibody binding to residual antigen retained in draining regional lymph nodes. Third, there may have been antibody reaction with regional B lymphocytes that were expressing anti-idiotypic antibody to endogenous anti-p240 or into p97 antibodies. A fourth possibility is that the uptake was in macrophages or dendritic cells that had been loaded with tumor antigen. The mechanism for the beneficial clinical effects of anti-VEGF antibodies is also more complex than originally thought. It was hypothesized that blocking VEGF, so that it could not bind to VEGFR, would interfere with tumor vascular supply and starve the tumor of oxygen, stimulating factors, and other nutrients. There is now some evidence that changes in afferent and efferent blood vessels themselves may be important more important in concentrating chemotherapy at the tumor site than antitumor effects from altered tumor blood supply.

Adverse Events: Toxicity and side Effects The various adverse events observed with commercially available anti-cancer Mab are shown in Table 6. These can be characterized as infusion reactions, acute or delayed hypersensitivity reactions, or non-immunologic biologic effects. It should be emphasized that the major adverse events associated with Mab are secondary to the biologic effects induced by antibody–antigen binding rather than immune reactions to the antibody itself [170, 175, 184]. This explains why most of the toxic effects described for murine Mab are also seen with the chimeric, humanized, and human forms of Mab directed to the same antigen. Table 6 clearly shows that alemtuzumab, is associated with the most toxicity, even though it is a humanized antibody, because it reacts with circulating T cells and other mononuclear cells that release large amounts of cytokines in response to the Mab binding to CD52 and the destruction of the cells by CMC and/or ADCC. Rituximab reacts with circulating B cells that are not as rich in inflammatory cytokines.

318

Monoclonal antibody therapy

Table 6. Percent of patients experiencing specific acute toxicities in clinical trials of monotherapy with a potpourri of murine and human antibodies (177 patients with 20 different malignancies), rituximab, trastuzumab, alemtuzumab, bevacizumab, cetuximab and panitumumab Antibody Target # of Patients Tumor target Abdominal Pain Angioedema Arthralgia Asthenia/malaise Bronchospasm Chills/rigors Cough Diaphoresis Diarrhea Dizziness Dyspnea Fever Headache Hypertension Hypotension Myalgia Nausea Edema Pruritus Rash Rhinitis Throat irritation Urticaria Vomiting

19 Mo 3 Hum

Ritux

Tras

Alem

Beva

Cetux

Panitu

Mo/Hu Many 177 Several <5 – – – – 13 – 10 – – – 15 – – – – 7 – 12 12 – – 12 6

Ximab CD20 356 Lymph 14 11 10 26 8 33 13 15 – 7 7 53 19 – 10 10 23 8 14 15 12 6 8 10

Zumab EGFR-2 352 Breast 22 – 6 42 – 32 26 –

Zumab CD52 149 CLL 11 – – 13 9 86 25 19 – 12 26 85 24 – 32 11 54 13 24 40 7 12 30 41

Zumab VEGF 234 Colo 8a – – 5a – – – – 2a – – – – 8a – – 6a – – – – – – 6a

Ximab EGFR1 420 Colo 26 – – 48 – 21 11 21 25 – 17 27 26 – – – 29 10 11 90 – – – 25

Humab EGFR1 229 Colo 25 – – – – <5 14 <5 21 – <5 <5 <5 – <5 – 23 12 57 90 – – – 19

13 22 36 26 – – – 33 10 – 18 14 12 – 23

Ritux = rituximab, Tras = trastuzumab, Alem = alemtuzumab, Bev = bevacizumab, Cetux = cetuximab, Panitu = panitumumab a Bevacizumab data in this table reflects only grade 3 to 5 toxicity.

Because of the individual regulatory development of these products, it was not always clear which adverse events were product-related as opposed to target-related, and this has led physicians to attribute toxicity to the Mabs themselves, when, in fact, the effects were predictable because of the significance of the antigen target, and the cascade of events that follow CMC and ADCC, many of which are due to the subsequent release of cytokines, as opposed to a hypersensitivity reaction to the antibody. However, there are allergic or hypersensitive differences as well. For instance, cetuximab and panitumumab both target EGFR1, and have certain toxicities in common that relate to biologic effects associated with blocking EGFR, such as acneiform skin rash and gastrointestinal symptoms including abdominal pain, nausea, vomiting and diarrhea. However, the chimeric Mab that is made in

murine plasmacytoma cells has a higher rate of infusionassociated flu-like symptoms, such as fever, headache, chills, and dyspnea, compared to the human antibody that is manufactured in CHO cells.

Infusion Reactions These include a constellation of symptoms that predictably occur during or shortly after infusion of a Mab. The typical symptom complex includes fever, chills, sweats, prostration, nausea, and sometimes dyspnea and hypotension, which occur within a matter of minutes to hours after an i.v. antibody infusion is initiated. They are due to direct effects on tumor and non-tumor cells which express the antigen, and indirect effects mediated by the secondary release of various cytokines as a result

Robert O. Dillman of antibody binding to the target antigen, or formation of immune complexes with circulating soluble antigen. Most infusion reactions associated with Mab are nonallergic in nature, and rather are due to the interaction of the antibody with circulating cells via antigen or Fc receptors, and perhaps endothelial cells. However, because of the effects of cytokines, it can be clinically difficult to distinguish between allergic and non-allergic infusion reactions. For many years it has been clear that most infusion-related adverse events are non-allergic in nature, and are directly related to antibody-antigen interactions and the interaction with complement and effector cells rather than being IgE, mast cell or eosinophil mediated reactions [170, 175, 184]. The antigen– antibody binding to circulating cells in immunocompetent patients, and subsequent interaction with complement or effector cells, is predictably followed by the release of cytokines and/or the removal of those circulating cells via macrophages in the reticuloendothelial system [19, 38, 79, 177–179, 180, 227, 228, 435, 446, 478, 485–488, 518, 569, 600, 787]. The onset of these reactions is typically within 15–30 min after initiating an antibody infusion, but depends on the dose and rate of infusion, and the distribution of the target antigen. Typical adverse events associated with binding to circulating cells are the same as those seen with high doses of various cytokines, such as interferon, interleukin-2, or high doses of GM-CSF. These include fever, rigors/ chills, sweats, maculopapular erythematous skin rash, urticaria, pruritus, edema, hypotension, tachycardia, headache, nausea, vomiting, diarrhea, fatigue, elevated hepatic transaminases, throat tightness, pain, thrombocytopenia, dyspnea, bronchospasm, anaphylactic shock, and even death [435]. The severity of infusion reactions vary greatly depending on the nature of the antibody (mouse or human), the distribution and density of the target antigen on circulating cells, and the immunocompetence of the patient. Nearly all patients who have received antilymphocyte and/or antigranulocyte antibodies have experienced such side effects if they had levels of circulating target cells at the time of infusion. The reactions are predictable whether a patient is receiving murine, chimeric, or humanized antibodies, suggesting that the antigenic target is the most important variable, although differences related to the Fc portion of the antibody are also important. The only time these reactions were not seen was in patients repeatedly treated with murine Mab who had high titers of endogenous antimouse antibodies that apparently neutralized the infused antibody [179]. Thus, the presence of endogenous human antimouse antibodies actually prevented many of these side effects by altering the pharmacology

319 and bioavailability of the murine Mab which limited binding to the target antigen. The observation that some F(ab’)2 can cause the same reaction suggests that antibody binding to antigen may be sufficient to induce cytokine release even without interaction of the Fc portion of the antibody in an ADCC reaction [721]. Antibodies that react with circulating T cells, which are more numerous and contain more cytokines than B lymphocytes are associated with more severe infusion reactions. Binding to granulocytes also triggers severe infusion reactions. As described earlier in this chapter, studies with radiolabeled cells showed that once antibody binds to circulating cells, they are removed in the reticuloendothelial system including the lung, liver, and spleen [177, 485, 518]. When large numbers of cells are removed in the lungs, this may be associated with dyspnea and hypotension. For this reason, when it is known that an antibody will react with circulating cells, the initial infusion rate needs to be slow, and high-dose bolus administration is avoided. Once circulating target cells are no longer present, subsequent infusions may be safely given at more rapid rates. For instance, the author has administered full dose rituximab over 30 min during subsequent weekly treatments once B lymphocytes have been cleared from the circulation, and others have confirmed the safety of this approach in formal trials with delivery over 45 to 90 min [82, 655]. For patients who are initiating therapy with a monoclonal antibody that reacts with circulating cells, premedication with steroids, acetamenophen and/or diphenhydramine decrease the severity of symptoms associated with an infusion reaction, but does not typically prevent such a reaction [175]. Once the target cells are gone, there is no need to give such pre-medication. It is possible that combined prophylactic therapy with histamine blockers and steroids starting 24 h before treatment would reduce many of these toxicities, much as they reduce the immune reactions associated with infusion of paclitaxel chemotherapy.

Tumor lysis Syndrome Tumor lysis syndrome is a constellation of symptoms and complications associated with the rapid cytotoxic death of rapidly proliferating tumor cells [817, 818]. It has been well described in association with systemic chemotherapy for rapidly proliferating malignancies such as acute leukemia, and patients with bulky tumor loads of Hodgkin’s disease, large B cell lymphoma, and testicular cancer in which highly effective systemic chemotherapy destroys large number of proliferating cells in a short time period. This is followed by increases

320 in serum potassium, uric acid, and phosphates released by the destroyed cells, with subsequent hypotension, renal failure, and other medical complications that can be life-threatening or lethal. For single-agent unconjugated monoclonal antibody therapy, true “tumor lysis syndrome” occurs when a patient is treated with an antibody that targets large numbers of proliferating tumor cells in the blood stream, but does not happen when non-replicating cells are removed from the circulation [170, 175, 184]. This is of particular concern in the setting of acute leukemia, lympholeukemias of mantle cell and large B cell lymphoma, and prolymphocyte-predominate chronic lymphocytic leukemia (CLL) in which large numbers of antigen-positive tumor cells are present in the circulation, where the antibody can opsonize large numbers of cells resulting in their lysis in the circulation and in the reticuloendothelial system [79, 179]. Many of the descriptions of so-called “tumor lysis syndrome” in the literature were really predictable antigen– antibody infusion related reactions rather than true tumor lysis syndrome with the complications that result from lysis of large numbers or proliferating cells and release of intracellular products including high levels of nucleic acids that lead to hyperuricemia [79, 787, 798]. Antibody-induced tumor lysis syndrome has only been observed in the setting of large numbers of rapidly proliferating circulating target cells; it has not been observed in the absence of a large leukemic component of disease, probably because of the slower penetration of antibodies into tumor tumor masses [345]. Unless the patient is truly at high risk for tumor lysis syndrome there is no need for prophylactic measures such as prehydration, sodium bicarbonate to alkalinize the urine, and anti-uric acid agents such as allopurinol or rasburicase. For example, in classical CLL, there are primarily mature lymphocytes in the circulation whose excessive number is more the result of being long-lived rather than hyperproliferation, and true tumor lysis syndrome does not occur in that setting, even with the removal of hundreds of thousands of cells in just a few minutes, although, infusion reactions are common, and can be life threatening in a patient who is medically compromised by cardiopulmonary, hepatic, or renal disease. However, a CLL patient with a large number of prolymphocytes is at risk for tumor lysis syndrome.

Acute Hypersensitivity Reactions and Anaphylaxis Because the first Mab were mouse proteins, there was great concern that infusion of these products would be associated with acute anaphylactoid reactions. Fortunately,

Monoclonal antibody therapy such complications were rare, and are even less common in association with chimeric and humanized antibodies. Patients with a known history of allergic reaction to rodents or their by products, were typically excluded from trials of murine antibodies. Acute allergic reactions have included anaphylactic shock, less severe anaphylactoid reactions such as bronchospasm, and generalized pruritus and urticaria [170, 175, 184]. The more severe reactions can be successfully managed with epinephrine. Patients who experience full blown anaphylaxis during or shortly following an antibody infusion should not be retreated with the agent, but it is important that a predictable infusion reaction not be confused with such a severe allergic reaction. Dermatologic effects including pruritis, diffuse or focal maculopapular rashes and urticaria may be seen alone, or as part of a full anaphylactic reaction with laryngeal edema, hypotension, and bronchospasm. Pruritus and urticaria alone typically resolve without treatment, but may be responsive to diphenhydramine or epinephrine.

Side Effects Secondary to Binding to Non-Malignant Tissues Adverse events may also occur as a consequence of direct effects on noncancerous tissue that also express the target antigen. This has been especially true for antibodies that cross-react with adenocarcinomas and cells of the gastrointestinal tract. Such antibodies have been associated with a high frequency of diarrhea, nausea and vomiting, abdominal pain, and even large and/ or small bowel mucosal damage in some patients [109, 240, 593]. Other antibodies that are known to crossreact with antigens on neural tissue have been associated with specific pain syndromes in some patients [109, 205, 651, 652]. Cross-reactivity with normal tissue antigens is particularly a concern when antibodies are conjugated to cytotoxic substances such as radioisotopes, chemotherapy agents, or natural toxins. For instance, the incidence of gastrointestinal toxicity was much greater when a frequently tested adenocarcinoma antibody was given conjugated to a vinca chemotherapy analog, or methotrexate, as compared to administration of the naked antibody [205, 636]. Another adenocarcinoma antibody that cross-reacted with antigens on neural sheaths, produced unacceptable neurotoxicity when conjugated to the A chain of the natural toxin, ricin [541]. Dermatologic toxicities are predictable complications of agents that interfere with EGFR signal transduction, such as cetuximab and panitumumab [343, 551, 650] and the anti-Her2 Mab trastuzumab is associated with cardiac toxicity [374, 658, 678].

Robert O. Dillman Bevacizumab has been associated with hemorrhage, vascular thrombosis, hypertension, and proteinuria, presumably all because of effects on blood vessels. [211, 538, 698]. The complications associated with these three commercially available antibodies are discussed in more detail later in this chapter.

Immune complexes In some instances, Mab are given to patients who are known to have free or soluble circulating antigen. Examples include the circulating idiotype in lymphoma, and carcinoembryonic antigen (CEA), or prostate specific antigen (PSA). Immune complexes may also be formed because of HAMA, HACA, and HAHA. Only rarely have symptoms been noted in the presence of the immune complexes formed by the binding of antibody to circulating antigen, probably because of the small size of such complexes since the monoclonal antibody binds to only one determinant on the circulating antigen. For this reason, the presence of circulating antigen is not a contraindication to antibody treatment, although the binding to soluble antigen greatly alters the pharmacokinetics of the antibody. However, acute arthralgias, myalgias, nerve palsies, fever, and skin rashes have occasionally been seen in this setting and attributed to the acute immune complex formation [170, 175, 184]. During trials of murine Mab, the presence of HAMA predictably decreased the half-life of the antibody, but was rarely associated with complications that could be attributed to immune complexes.

Delayed Allergic Reactions and Serum Sickness Because of the anticipated production of HAMA, there was concern that delayed reactions such as serum sickness would be a significant problem following infusion of murine Mab. Fortunately, immune complex complications related to HAMA have been uncommon, but they can occur [170, 175, 184]. Classic serum sickness has been seen 2 to 3 weeks following exposure to moderate and high doses of murine Mab, and rarely after chimeric Mab. A typical symptom complex includes fever, malaise, arthralgias/arthritis, myalgias, maculopapular erythematous skin rash, and fatigue. Proteinuria has been rarely observed in these few patients and renal insufficiency is extremely rare. Serum sickness can be managed with non-steroidal, anti-inflammatory agents, and corticosteroids in more severe cases.

321

Antibody Issues Immunoglobulin Class and Subclass In lymphoma, murine anti-idiotype Mab of various subclasses produced clinical responses [446, 478, 487]. In melanoma, only murine IgG3 Mab and humanized anti-GD2 or anti-GD3 antibodies produced clear-cut responses [109, 328, 738]. The responses seen with murine anti-CD5 IgG2a Mab in T-cell lymphoma and CLL were limited [38, 177, 179, 180, 227, 228, 485, 486, 825]. Even though it appears that mouse IgG3, and perhaps some mouse IgG2a Mab, have greater potential to effect ADCC than other subclasses of murine Ig [308, 340, 511, 691], the preferred strategy is to use chimeric, humanized, or human Ig because of the advantages associated with the human Fc. Some complement-fixing IgM Mab would be useful, but their size limits tumor penetration in solid tumors, and administration of large doses could lead to hyperviscosity. The issues related to murine Mab are of decreasing importance since most investigators and companies have converted their promising murine Mab into chimeric or humanized Mab. Most of these conversions have been to a human IgG1 subclass because these are predictably associated with efficient CDC and ADCC [660]. Class and subclass switching of antibodies has enabled us to establish more directly the importance of the binding of antibody to Fc receptors on effector cells.

Human Anti-Immunoglobulin Response Because they are foreign proteins, it was not surprising that murine Mab produce human antimouse antibodies (HAMA) responses in immunocompetent patients, especially when large quantities are administered. Trials involving single exposure and multiple exposure to Mab confirmed that this is a substantial obstacle to repetitive therapy, especially in patients with solid tumors, who typically are less immunosuppressed that patients suffering from hematologic malignancies. [131, 164, 185, 382, 640, 664]. Using radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA), investigators detected HAMA in virtually all patients who have received mouse antibodies except for those with CLL, probably because of the immunodeficiency associated with that disorder. It also appears that HAMA is reduced in many B-cell lymphoma patients, and perhaps the anti-B cell antibodies contribute to this. However, recent studies involving radiolabeled anti-CD20 murine Mab as therapy for previously untreated B-cell lymphoma patients resulted in 63% of patients exhibiting

322 HAMA within a median of 3.3 weeks of being treated [362]. This suggests that prior chemotherapy and/or greater mutations of recurrent and progressive disease induce a relative immunodeficiency that lowers the rate of HAMA in more heavily treated patients. Interestingly, many CTCL patients who developed HAMA had previously experienced a clinical response to treatment, perhaps because they are the more immunocompetent patients, or perhaps because their disease was less resistant because of less exposure to chemotherapy. Once HAMA is produced, they effectively neutralize most clinical effects of therapy, although targeting of tumor cells can still be demonstrated. It appears that a small percentage of antimouse antibodies react specifically with the idiotype of the mouse protein rather than only with specific murine Ig isotype determinants [404, 664]. As discussed earlier in this chapter, it has been suggested that this anti-idiotype cascade may contribute to the anti-tumor effect as an indirect vaccine [313, 404]. Efforts to block the antimouse immune response with chemotherapy, radiotherapy, corticosteroids, and cyclosporine, have not been successful. Some investigators suggested that infusions of high doses of antibody would eliminate the antimouse response, but this was not borne out in other trials. When patients receive high doses of antibody, they must be evaluated for a prolonged period of time because, depending on the assay used, the excess of mouse antibody may block any evidence of antimouse antibodies during the early phase of the HAMA, HACA, or HAHA response. Some investigators have suggested that attachment of polyethylene glycol may decrease the immune response [784], but this not been confirmed in humans. In the presence of HAMA, some investigators have combined plasmapheresis with administration of higher doses of antibodies, but this was of limited benefit. Even with modern commercially available antibodies, there is still the potential for human (HAMA) and/or human anti-chimeric antibodies (HACA) following treatment with a chimeric antibody, and even human anti-human antibodies (HAHA) directed against allotypic human antigens on human Mab. Human immune responses that result in the production of HAMA, HACA, and HAHA are readily detected by immunoassays that utilize the therapeutic antibodies in the assays used in the treatment of patients. As expected, most of the antibodies associated with chimeric antibodies are HAMA directed to the residual murine determinants. Most of the antibodies in HACA or HAHA are directed to allotypic epitopes and carbohydrates on the Fc portion. Fortunately the various chimeric and humanized Mab that have entered the clinic have been associated with minimal

Monoclonal antibody therapy HAMA or HACA, or HAHA. Today there is no need to try to obtain approval of a therapeutic mouse monoclonal antibody unless one desires short pharmacokinetics such as in radioimmunotherapy to reduce the amount of total body irradiation [174]. These preparations are a significant improvement over mouse antibodies, and even when HACA or HAHA occur, they tend to appear later and often at lower titers so that repeated intermittent therapy is possible over several months. Based on the large clinical experiences with current chimeric and humanized antibodies, it appears that the risk of HACA or HAMA is only on the order of 1–2% of all patients treated, and this rarely interferes with treatment. There is a clinical caveat associated with HAMA because exposure to such antibodies may sensitize patients who subsequently will have limited therapeutic benefit and/or be at risk for allergic reaction if further antibody doses are given for diagnostic or therapeutic purposes. The HAMA may also confound the interpretation of serum diagnostic tests such as those for prostate specific antigen (PSA) or carcinoma embryonic antigen (CEA) etc. that are measured using momabs. For instance, we have seen a patient develop significant HAMA that resulted in chronic T cell lymphopenia and aberrant PSA assays results secondary to exposure to the OKT3 mouse monoclonal antibody associated with an adoptive cell therapy in which cells were incubated in vitro with the OKT3.

Antigen issues Antigenic Modulation Antigen modulation is a dynamic process in which surface antigen is decreased in the presence of excess antibody. Electron microscopy with autoradiography and 125 I-labeled monoclonal antibody studies showed that modulation is the result of internalization of the antigen and the bound antibody [599, 639, 663]. This is preceded by “capping” of the antibody–antigen complex, a process during which the complex appears to localize to one region of the cell surface. Mab directed against many hematopoietic surface antigens and growth factor receptors on solid tumors induce modulation in vitro and in vivo. Modulation may in reality be an example of antibody acting as a surrogate ligand for a receptor that normally internalizes after binding to a ligand. Modulation occurs within minutes of exposure to Mab, but is reversible once the monoclonal antibody is removed from the system because of the ongoing production or recycling of antigen/receptor. This has been demonstrated both in vitro and in vivo.

Robert O. Dillman Antigenic modulation must be differentiated from immunoselection that might result from the elimination of antigen-positive cells, thereby leaving only cells that express no or only low levels of the target antigen. The conditions for modulation, or antibody-mediated down regulation of a receptor in vivo include presence of immunoreactive Mab in the serum, and persistent existence of tumor cells that exhibit absent or reduced expression of the targeted antigen, but continued expression of another phenotypic marker for the cell in question. For example, the C22 antigen on malignant and normal B cells modulates after binding of antibody, such that CD22 becomes undetectable, but CD19 and CD20 are other markers of B cells that can be measured. In the absence of antibody, the antigen must be re-expressed to prove there is no immunoselection of cells. Thus, if the cells that now appear to be CD22 negative because of modulation are placed in cell culture, re-expression of CD22 proves that only modulation has taken place rather than immunoselection of for a CD22 negative subclone of cells. Modulation is a dynamic process that consists of antigen expression, antibody binding, antibody–antigen internalization, re-expression of surface antigen, additional antibody binding, etc.; such that at any point in time there is always at least some amount of residual surface antigen expression, although this may be difficult to prove depending on the sensitivity of the assay being used. This phenomenon has important implications for passive monoclonal antibody therapy because during modulation, there are insufficient quantities of Mab on the cell surface to effect target cell elimination by complement or effector cells. For circulating cells, there is a threshold of monoclonal antibody-binding that is required before cells are eliminated. In fact, in some cases, cells with a high density of target antigen are rapidly eliminated while cells lower in antigen persist and/ or enter the circulation so that total target cell count is relatively unaffected [177, 179, 180]. Modulation, or down regulation of receptors may be desirable for antibodies that act through a regulatory mechanism, perhaps by altering signal transduction. Continued presence of antibody is needed to sustain this effect, which has important implications for duration of therapy. Rapid internalization greatly limits the therapeutic potential of antibodies that effect complement and/or cell-mediated cytotoxicity, but may be useful or a necessary condition for drug or toxin antibody immunoconjugates. As a general rule, modulation is much more common for hematopoietic cell antigens than solid tumor antigens, but it appears that some of the best targets for treatment of solid tumors are modulating/internalizing antigens that function as growth factor receptors for tumor cells.

323

Free Antigen Many tumor antigens are either shed in small quantities, or may be released during apoptosis. Some tumor antigens are shed in large quantities, constituting potential blocking factors to monoclonal antibody target cell binding. Such immune complexes might also produce damage to normal tissues and organs, as well. Most of the hematopoietic antigens detected by Mab are generally not shed to an extent that interferes with measurement of monoclonal antibody serum levels. However, excess circulating antigen has been a practical problem for anti-idiotype antibodies in the treatment of lymphoma. Immediately following treatment, blood antigen levels decrease precipitously as they complex with antibody, but the remainder of the Mab have access to tumor antigen. Thus, the obstacle of circulating antigen can be overcome with higher monoclonal antibody doses. However, immune complex-mediated disease may occasionally be a complication of this approach.

Antigen Heterogeneity If a given tumor cell does not express the antigen detected by a given monoclonal antibody, there is no basis for monoclonal immune-mediated cytolysis or receptor inhibition for that cell. For many years there was great debate as to whether any single Mab could be clinically efficacious because of the tremendous heterogeneity in human cancer cell phenotypes, especially for solid tumors [220, 307, 522, 637]. Fortunately, in recent years single antibody products have proven effective enough as single agents to gain regulatory approval. With our expanded knowledge of tumor phenotype and tumor cell antigens, in the future it may be possible to employ rationale combinations of Mab to overcome this problem [433, 534]. Ideally, such a combination or “cocktail” would include Mab in quantities directly related to specific antigen expression on an individual patient’s cancer cells rather than fixed proportions being administered to each patient with a given disease. However, just as with chemotherapy, a combination may only be effective if its individual components have some antitumor effects of their own.

Dose, Schedules, and Pharmacokinetics Antibody Dose There is a dose/response relationship at low monoclonal antibody doses (<10 mg) because of the volume of distribution, nonspecific uptake and metabolism, and the

324 importance of number of antibody molecules on cell surfaces for complement, reticuloendothelial, or effector cell-mediated effects. At higher dose levels that provide antibody excess, these dose–response relationships are no longer important, but duration of sustained serum levels (area under the curve) is important for tumors with non-modulating antigen targets. In the case of antigens targeted for a regulatory affect (whether modulating or not), sustained serum levels are needed to continuously block or down regulate receptors that are continually being produced by differentiating cells. The cells may emerge from stem cells or progenitor cells that do not express the target antigen, and therefore not directly affected by antibody therapy. Extrapolation from animal studies, and from studies of biopsies from patients receiving mouse antibodies, suggested that gram quantities of antibodies would be needed for a therapeutic effect because of the importance of tumor penetration and saturation. Several investigators noted that direct in vivo tissue binding (as opposed to binding to circulating cells) could only be demonstrated at doses of 30 to 50 mg or higher [179, 180, 328, 535]. Some radiolabeled monoclonal antibody studies showed that more tumors were imaged with doses of 10 mg or greater [183, 287, 514]. The clinical successes of recently approved Mab have been associated with delivery of high doses repeated at relatively short intervals, such as every 1 to 2 weeks. At these doses and schedules, continually detectable serum levels of antibody are assured in most patients. It seems likely that there are critical thresholds for dose and/or area under the curve of sustained antibody levels to produce an anti-tumor effect. Whether the dose/response relationship is important beyond those threshold levels is unclear, but sustained serum levels is likely to be related to duration of response in some settings.

Infusion Rates/Schedules Rapid infusions of Mab that bind to antigen on circulating blood cells and/or trigger endogenous immune responses leading to cytokine release and cell activation via interactions with Fc receptors, can be life threatening. [79, 170, 175, 178, 179, 184, 435, 787]. When antibodies react with circulating cells, infusion rates greater than 25 μg/ml are typically associated with infusion reactions. Impure preparations that contain microaggregates, pyrogen, or other contaminants may also be more toxic if infused rapidly, but this should not be a problem with products approved for clinical use. Most investigators have been satisfied with 2 to 6-h infusions for delivery of higher doses of antibody, but pure preparations of antibodies which do not react with circulating blood cells can

Monoclonal antibody therapy be given over a few minutes with no untoward effects. Prolonged continuous infusions or repeated bolus infusions are probably necessary for Mab that work via regulatory mechanisms in order to keep receptors down-regulated or blocked. This may also be desirable in order to establish a gradient effect in treating solid tumors because of tumor penetration problems. Bolus infusions are well tolerated by most patients if the monoclonal antibody preparation is free of aggregates and there is no cross-reactivity with circulating cells. Intermittent bolus infusions may be preferred if the down-regulated receptor needs to be re-expressed in order for the antibody/antigen (receptor/ligand) interaction to produce an antitumor effect via CMC or ADCC. In the past, because HAMA was anticipated, some investigators adopted a strategy to deliver the maximum dose of murine Mab within 2 weeks, because HAMA is generally detectable within 2 weeks of initiating treatment. Modern chimeric and humanized antibodies are able to be effectively delivered over many weeks to months because HAMA, HACA, and HAHA have rarely interfered with serum levels. So far there are no studies that have rigorously addressed the correlation between dose, infusion rate, or schedule, even for the antibodies that are now in widespread clinical use.

Serum Levels and Pharmacokinetics Because of known antibody–antigen interactions, it has been easy to devise various enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) to measure serum levels of Mab during and following infusions [25, 34, 205, 443, 665, 706]. Serum Mab levels have been easily detected except during low infusion rates, or in the presence of high levels of circulating antigens, high circulating tumor burden, or in the presence of HAMA. In the leukemias, monoclonal antibody levels tend to fall rapidly following an infusion because of continuing absorption by circulating cells and entry of additional cells into the circulation. However, with some antibodies, with or without the presence of antigenic modulation, serum Mab levels are sustained for many days to weeks depending on the dose given. Using 24- to 48-h infusions, peak Mab concentrations of several μg/ml have persisted for up to 2 weeks. The significance of serum levels at any time point must be viewed in the light of the variables listed above. In addition to tumor burden, other important variables include circulating antigen, antigenic modulation, and the production of antimouse antibodies. With the antiCD20 chimeric antibody rituximab, there is a clear association between the area under the curve for the antibody and tumor response, and return of normal and/or normal B-lymphocytes does not occur until serum levels of ritux-

Robert O. Dillman

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imab are negligible [34]. The dose and schedule for the antibodies in clinical use today are all given at high doses that sustain levels for several weeks to months. Chimeric, humanized, and human Mab consistently demonstrate superior pharmacokinetics, including higher peak levels and prolonged sustained blood levels, compared to their mouse counterparts. This is especially true following repeated administration because of the negative effects of HAMA on mouse antibody pharmacokinetics. Recent studies suggest that recycling of IgG after interaction with Fc gamma receptors may also play a role in sustaining serum levels [357, 422, 442].

Commercially Available Unconjugated Mab for Treatment of Human Malignancy Although many were disheartened by the early observations with murine Mab, the experience with these murine products, and humanization of antibodies through molecular engineering, suggested that Mab would still live up to their potential [161, 163, 166, 167, 172, 173, 428, 533]. “Magic bullets” for cancer therapy became a reality in November 1997 when the anti-CD20 monoclonal antibody rituximab (Rituxan) became the first monoclonal antibody product approved by the US Food and Drug Administration for the treatment of a malignant disease, namely B-cell lymphoma [168]. Ironically, this product was produced by the company IDEC Pharmaceuticals (San Diego, CA), which was originally founded to develop anti-idiotype antibodies, but instead gained success on the basis of a highly effective antibody that reacts with most normal and malignant

B-lymphocytes. In the following decade, eight additional monoclonal antibody products obtained regulatory approval for the treatment of various malignancies. The rest of this chapter will focus on the six unconjugated Mab that have been approved by the United States Food and Drug Administration (US FDA) for the treatment of malignant disease; radiolabeled antibodies and immunotoxins are covered elsewhere in this book.

Rituximab (Rituxan®, Biogen-Idec Pharmaceuticals, San Diego, CA and Genentech, South San Francisco, CA) Rituximab and CD20 The anti-CD20 monoclonal antibody rituximab (Rituxan) became the first commercially available monoclonal antibody for the treatment of a human malignancy when it was approved by the US FDA in November 1997, with a marketing indication for B-cell lymphoma (Table 7) [168]. The anti-CD20 murine Mab that originally had been named 2B8, and later ibritumomab, was converted to a mouse/human chimeric antibody and named rituximab [782]. Rituximab includes murine variable regions from the anti-CD20 murine Mab ibritumomab and human IgG1 kappa constant regions that have been combined using recombinant DNA technology. The genetically engineered chimeric monoclonal antibody is manufactured in CHO cells. In vitro it is cytolytic against CD20positive cells in the presence of human complement and human effector cells [260, 441]. Rituxmab’s binding to CD20 also appears to have a regulatory effect on B cells by promoting apoptosis [473, 661], and it appears to be more effective in this regard than other anti-CD20 antibodies

Table 7. Commercially available monoclonal antibodies for the treatment of human malignancies Generic name

Commercial name Antigen

Antibody type

Fc/Fv Ig type

Cell production

Year FDA approved

Rituximab Trastuzumab Daclizumab Alemtuzumab Bevacizumab Cetuximab Panitumumab

Rituxan Herceptin Zenapax Campath Avastin Erbitux Vectibix

Chimeric Humanized Humanized Humanized Humanized Chimeric Human

IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG2

CHO CHO CHO CHO CHO MP CHO

1997 1998 1998 2001 2004 2004 2006

CD = cluster designation EGFR = epidermal growth factor receptor VEGF = vascular endothelial growth factor CHO = Chinese Hamster Ovary MP = murine plasmacytoma

CD20 EGFR-2 CD25 CD52 VEGF EGFR-1 EGFR-1

326 such as IF5 and anti-B1 [661]. The regulatory consequences of rituximab binding to CD20 may be more important in vivo than the immunologic-based anti-tumor effects. In vitro the presence of rituximab was associated with enhanced sensitivity to chemotherapy which provided a strong rationale for combined modality treatment [156]. Rituximab is used alone or in combination with chemotherapy in indolent B cell malignancies, and is used in combination with chemotherapy in aggressive B cell malignancies. CD20 is a 35 kD antigen with multiple transmembrane domains that is expressed on normal and malignant B cells, but not on non-hematopoietic tissue, B-cell progenitors, plasma cells, T-lymphocytes, monocytes, dendritic cells, or stem cells [782]. CD20 is neither internalized nor shed in significant amounts. No ligand for CD20 has been identified, but its over-expression appears to be important for resistance to apoptosis of B lymphocytes [473, 661].

Clinical Trials with Rituximab Early Clinical Trials with Single-Agent Rituximab Maloney et al. conducted the early trials with C2B8 (rituximab) [459–461]. In a dose escalation phase I trial of single i.v. doses from 10 to 500 mg/m,2 two partial responses and four minor responses were observed among 15 patients [459]. In a subsequent phase II trial, multiple doses of 375–500 mg/m2 were given i.v. weekly for 4 weeks [461]. There were four complete responses and 16 partial responses among 34 evaluable patients for an overall objective response rate of 47%. Most of the partial responders remained in remission for 4–12 months following treatment. The pivotal trial for regulatory approval of rituximab was a multi-institutional study involving 166 patients with low-grade or indolent lymphoma, mostly follicular, who had relapsed after prior chemotherapy [474]. This trial yielded an objective response rate of 50% with a median duration of response of more than a year; 90% of responses lasted more than 6 months. Patients in these trials had already failed a median of four prior chemotherapy regimens. The response rate in patients who had failed high-dose chemotherapy and stem cell transplant was a remarkable 73% (18/23). None of the responding patients relapsed in less than 4 months, and 10% remained in continual remission for 2–5 years. One third of patients had their initial infusion interrupted because of the classical infusion reaction complex of fever and chills and occasional shortness of breath, hypotension, and skin

Monoclonal antibody therapy rash associated with the binding of antibody to circulating B lymphocytes. There was very limited toxicity during subsequent infusions. There was no evidence of hypogammaglobulinemia or infections as a consequence of suppression of humeral immunity. Retreatment with rituximab at a dose of 375 mg/m2 weekly for 4 weeks resulted in a response rate of 41% among 40 patients who previously enjoyed a response to rituximab that had lasted 6 months or longer [149]. The 18-month median duration of response was even longer than the 13 months noted in the pivotal trial, probably because most of the patients had a smaller tumor burden and more favorable pharmacokinetics than the original population of patients treated with rituximab. There was no clear explanation as to why the remaining patients failed to respond to retreatment, but resistance was not due to loss of CD20 expression or HAMA or HACA against rituximab. There was no emergence of a CD20negative subclone as a consequence of a treatment-related immunoselection process. While occasional patients do recur with CD20 negative tumors [145], this has proved to be an exception, and most patients retain CD20 positivity throughout the course of their disease. A study of 13 patients in a similar trial conducted in Japan had a response rate of 38% with rituximab re-treatment but the median duration of response was only 5.1 months in this study [339].

Rituximab in Follicular Lymphoma Rituximab is highly active as a single agent in the treatment of previously treated and previously untreated follicular lymphoma, as shown in Table 8 [15, 56, 97, 125, 147, 231, 251, 277, 460, 461, 474, 562, 790, 791]. In previously treated follicular lymphoma patients, objective response rates have ranged from 46% to 63% [15, 97, 145, 231, 461, 474, 562, 790]. In previously untreated follicular lymphoma patients response rates are generally higher, ranging from 67% to 78% [125, 251, 277, 791]. Most of these trials utilized the standard 4-week course of treatment at 375 mg/m2, but Piro et al. used an 8-week treatment schedule [562], Aviles et al. and Bremer used a 6-week treatment schedule [15, 56], and Hainsworth et al. utilized additional 4 weeks of therapy every 6 months for 2 years [277]. Pooling data from similar trials shows a response rate of 29/37 (78%) for untreated patients who received more than 4 weeks of therapy, 100/143 (70%) for untreated patients who received the standard 4 weeks of therapy, 59/89 (66%) for relapsed patients who received more than 4 weeks of therapy, and 238/458 (52%) for relapsed patients who received the standard 4 weeks of therapy.

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327

Table 8. Rituximab as a single agent in follicular lymphoma Citation [277] [15] [125] [791] [562] [251] [97] [56] [474] [790] [145, 147] [231] [251]

Proportion Clinical setting responding Untreated Relapsed/ refractory Untreated, low tumor burden Untreated Grade 1 Relapsed/ refractory Untreated Relapsed/ refractory Relapsed/ refractory Relapsed/ refractory Relapsed/ refractory Relapsed/ refractory > 10 cm Relapsed/ refractory Relapsed/ refractory

Table 9. Rituximab as a single agent in small lymphocytic lymphoma

% Response Citation

Clinical setting

[277] [231]

Initial therapy Relapsed/ refractory Relapsed/ refractory Relapsed/ refractory

Proportion responding

% Response

15/23 4/29

65% 14%

1/7

14%

4/32

13%

29/37 13/17

78% 76%

36/49

73%

26/37

72%

21/30

69%

38/57 24/38

67% 63%

25/42

59%

75/130

58%

lymphoma

36/70

56%

Citation

Clinical setting

12/22

55%

[127]

32/70

46%

59/128

46%

No prior 21/24 chemotherapy Relapsed/refractory 20/26 gastric Prior 5/11 chemotherapy

[562] [474]

Table 10. Rituximab as a single agent in marginal zone

[469]

Long term follow up of the French trial was reported by Solal-Celigny et al. for 49 untreated follicular lymphoma patients with a low tumor burden who were treated with the standard dose and schedule of rituximab, 375 mg/m2 IV weekly × 4 [683]. Based on the recent Follicular Lymphoma Internal Prognostic Index (FLIPI), 45% of these patients would be considered low risk, 41% intermediate risk, and 14% poor risk. The eventual response rate was actually 80% with 49% achieving a CR. Median PFS was 18 months and 34% had an unmaintained remission that last more than 5 years, and 94% were still living at 5 years.

Rituximab in Small Lymphocytic Lymphoma Clinical results for rituximab in small lymphocytic lymphoma (SLL), another of the indolent lymphomas, are summarized in Table 9. Objective response rates in the setting of relapsed or refractory SLL were much lower than relapsed or refractory follicular lymphoma, but high response rates were observed when rituximab was used as initial therapy for previously untreated patients. These patients are clinically distinguishable from CLL because they had less than 5,000 lymphocytes/μl. The

[127]

Proportion responding

% Response 87% 77% 45%

McLaughlin and Foran trials utilized the standard 4-week course of treatment at 375 mg/m2 [231, 474] while the Piro trial used an 8-week course of rituximab [562], and the Hainsworth trial prescribed the standard therapy followed by planned repeat 4-week courses of therapy every 6 months for 2 years [277]. Patients in the relapsed/refractory setting experienced a relatively brief duration of response, while those who received more than the standard 4 weeks as initial treatment for SLL enjoyed a much longer progression free survival.

Rituximab in Marginal Zone Lymphoma Results of rituximab in mantle zone lymphoma are summarized in Table 10. These indolent lymphomas can occur anywhere, but when they occur in the gastrointestinal tract, they are referred to as mucosa-associated lymphoma tissue (MALT). In the Concani study the primary lymphoma was located in the stomach in 15 patients and was extragastric in 20 [127]. At study entry 12 patients had Ann Arbor stage IE, 3 had stage IIE, and 20 had stage IV disease. The Martinelli trial included 26 evaluable patients presenting with gastric MALT at any stage, relapsed/refractory to initial treatment or not suitable for eradication with antibiotics for anti-Helicobacter pylori therapy [469]. The median survival of 36

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Monoclonal antibody therapy

months reflects the inclusion of previously untreated patients and exclusion of patients with marginal zone lymphoma from other sites. In contrast, in the Conconi trial, the progression free survival was 22 months for those who had not received prior therapy compared to 12 months for patients who had relapsed after previous anti-lymphoma therapy [127].

Rituximab in Lymphoplasmacytic Lymphoma These tumors are characterized by secretion of IgM with a range of clinical presentations that include a very high levels of IgM paraproteinenemia with minimal lymphadenopathy (Waldenströms macroglobulenemia), a chronic lymphoid leukemia, or an indolent lymphoma. These lymphoplasmacytoid lymphomas previously were a small subset of patients in the A group of the Working Group Formulation. Unlike the cells in CLL and SLL and plasma cells, the B-lymphocytes associated with this disease group have a high-expression of CD20. Furthermore, although this entity usually does have increased numbers of circulating B-lymphocytes of the malignant clone, lymphocyte numbers typically are not as high as in CLL. Published reports of rituximab in the treatment of lymphoplasmacytic lymphomas are shown in Table 11. Response rates from 16% to 65% have been reported with progression-free survival rates ranging from 16 to 30 months [187, 231, 249, 726, 728]. In an earlier trial Treon used the standard 4-week dose and schedule of rituximab treatment [726], but subsequently used two 4-week courses of treatment during the first and third months for all patients. Gertz used a single standard 4-week course of treatment at 375 mg/m2 [249] as did Foran [231]. Dimopoulos gave a repeat 4-week

Table 11. Rituximab as a single agent in lymphoplasmacytic lymphoma Citation [728] [726] [187] [187] [249] [249] [231]

course of treatment 3 months later in patients who had not progressed [187]. With standard dosing it often took several months before a response could be declared, and maximum responses were noted more than 12 months after starting treatment. In the trials that utilized repeat dosing, the median time to best response was about 18 months. Pooled data showed that 31/56 (55%) had an objective response when two cycles of treatment were used compared to 34/116 (29%) for patients who received one standard 4-week course of rituximab.

Rituximab in Large B Cell Lymphoma Results of trials for single-agent rituximab in aggressive lymphomas are summarized in Table 12. A French trial of 54 patients with aggressive lymphoma included 30 with large B cell lymphoma [121]. This was designed as a randomized phase II trial in which all patients initially received 8 weeks of rituximab therapy instead of 4, but one half were randomized to receive 375 mg/m2 weekly × 8 weeks, and the other group received 500 mg/m2 on weeks 2–8 after an initial standard dose. There was no difference in response rate between the two groups; so, the response data was grouped together. The overall response rate was 31% with a median response duration of greater than 8 months. The response rate was 37% in large cell. In another European trial a response rate of 25% was observed in 28 patients with relapsed intermediate grade lymphomas who were treated with the standard course of treatment with 375 mg/m2 weekly for 4 weeks [231]. The median duration of response was about 1 year.

Rituximab in Mantle Cell Lymphoma The randomized phase II French trial described above included a 30% response rate for 12 patients with mantle

Table 12. Rituximab in large B-cell lymphoma (LBCL)and mantle cell lymphoma

Clinical setting

Proportion responding

Response rate

Citation

Clinical setting

Untreated and relapsed Relapsed/ refractory Relapsed/ refractory Untreated Untreated Relapsed/ refractory Relapsed/ refractory

19/29

65%

[121]

11/22

50%

[723]

6/12

50%

[231]

6/15 12/34 7/35

40% 35% 20%

[121]

4/25

16%

[231]

Relapsed large B cell Relapsed large B cell Relapsed/ refractory mantle cell Relapsed/ refractory mantle cell Untreated mantle cell

Proportion responding

% Response

11/30

37%

21/57

37%

13/40

33%

3/10

30%

10/34

29%

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329

cell lymphoma who were to receive a planned treatment course of 8 weeks [121]. Other investigators reported a 29% response rate in 34 patients with newly diagnosed mantle cell lymphoma, and a 33% response rate in 40 patients with mantle cell lymphoma that was either refractory to or had progressed after chemotherapy [231]. These patients were treated with the standard 4-week course of treatment at 375 mg/m2. The median duration of response was about 1 year.

Rituximab in Chronic Lymphocytic Leukemia CLL and SLL are morphologically and phenotypically the same with co-expression of CD19/20 and CD5, but clinically CLL is characterized by a peripheral blood lymphocytes count of greater than 5,000/μl. The density of CD20 expression varies greatly among different patients. Theoretically higher or repeated doses would be needed to optimize the efficacy of rituximab in CLL because of the large sink of antigen positive circulating cells that would have to be eliminated before targeting disease in lymph nodes and other tissues. As summarized in Table 13, in patients with relapsed CLL, standard and high doses of rituximab have been associated with responses of 25–45%, but the duration of response only lasted 5 to 10 months with standard dose rituximab [80, 333, 529]. Byrd et al. reported a 45% response rate among 33 patients who received thrice weekly therapy escalating from 100 mg/m2 initially to minimize infusion reactions, up to the standard 375 mg/m2 three times a week [80]. All but one of the responses was partial and the median duration of response was 10 months. O’Brien et al. observed 36% response rate among 40 patients, with a median duration of response of 8 months, in a dose escalation study in which they administered up to 2.25 g/m2 weekly for 4 weeks [529]. Response was correlated with dose: 22% for patients treated at 500 to 825 mg/m2, 43% for Table 13. Rituximab as a single agent in chronic lymphocytic leukemia Citation [716] [277] [278] [80] [529] [342] [333]

Clinical setting

Proportion responding

Response rate

Untreated, low burden Untreated Untreated Relapsed Relapsed Relapsed Relapsed

17/19

90%

18/26 25/43 15/33 14/40 8/23 7/28

70% 58% 45% 36% 35% 25%

those treated at 1.0 to 1.5 g/m2, and 75% for those treated at the highest dose of 2.25 g/m2, but patients who were treated at the higher doses were more often relapsed after, rather than refractory to, fludarabine. Huhn et al. reported a 25% response rate using the standard dose and schedule of four weekly doses of 375 mg/m2, but they restricted the tumor cell load, as measured by the number of circulating lymphocytes and the spleen size, in the first two cohorts of patients included in the study [333]. Median duration of response was only 5 months. A more practical and less expensive strategy in the setting of relapsed CLL is to use Rituximab in combination with chemotherapy or after a response has been achieved with chemotherapy. Those strategies reduce the risk of more intense infusion reactions that are associated with higher numbers of CD20+ circulating cells, and would not necessitate repeated or dose of rituximab. Response rates were much higher in previously untreated CLL who also received more than four weekly doses of rituximab. Thomas et al. treated for 8 weeks [716], while Hainsworth et al. utilized planned retreatment with additional 4-week courses of treatment every 6 months for 2 years [277, 278].

Rituximab in Hairy Cell Leukemia The circulating lymphocytes of hairy cell leukemia (HCL) typically express very high levels of CD20. Rituximab has been used to treat relapsed HCL using both the 4 and 8 week treatment schedules. The 4-week schedule produced responses in 5/10 patients in one study [418], and in 6/24 patients who had all relapsed after cladbribine [819]. The 8-week schedule produced responses in 8/15 relapsed patients [717].

Rituximab in Multiple Myeloma CD20 is rarely expressed on plasma cells, but has been reported to be present on malignant cells from up to 20% of patients with multiple myeloma [727]. Rituximab has rarely produced objective responses in multiple myeloma even in patients whose plasma cells were felt to overexpress CD20. Using the standard dosing of 375 mg/m2, Treon et al. saw objective responses in only 1/19 patients [727], and Hussein et al. reported responses only 2/21 patients prior to starting chemotherapy 5 weeks later [338]. There has been a much greater unpublished experience in the community practice of medicine with anecdotal reports of occasional excellent responses in refractory myeloma patients whose plasma cell overexpressed CD20. Another strategy is to use rituximab as an adjuvant treatment after an initial

330

Monoclonal antibody therapy

response to chemotherapy, in an effort to suppress the B cell clone that leads to the malignant plasma cells. Based on this same rationale, rituximab may be more active against smoldering myeloma than myeloma with a high proliferative index.

Rituximab in Hodgkin’s Disease Hodgkin’s disease is a malignancy of B-lymphocytes [687]. Lymphocyte-predominant Hodgkin disease (LPHD) is characterized by indolent nodal disease that tends to relapse after standard radiotherapy or chemotherapy and the malignant cells of LPHD are CD20+. As shown in Table 14, rituximab alone has been associated with very high response rates in LPHD, but the median progression free survival has usually been no more than a year [203, 586, 803]. Rituximab is also being explored in other Hodgkin’s histologies. In the Younes trial patients had to have received at least two prior treatment regimens, and received 6 rather than 4 weeks of therapy [803].

Extended therapy and Maintenance Rituximab The term “maintenance” has been used to describe the use of rituximab therapy after induction therapy for the treatment of B-cell lymphoma. However, much of this usage actually represents adjunctive, sequential therapy, or consolidation therapy following an initial or induction therapy that that did not include rituximab. Since the mechanism of action of rituximab relates to its Table 14. Rituximab as a single agent in Hodgkin’s disease

Citation [586]

[203] [203] [803]

Median progression-free survival

Clinical setting

Proportion responding

% Response

Relapsed, LPHD > 30% CD20+ Untreated LPHD Relapsed LPHD Relapsed NSHD, 2 prior treatments

12/14

86%

>12 months

12/12

100%

10/10

100%

5/22

22%

10 months 10 months 8 months

LPH = lymphocyte predominate Hodgkin’s disease NSHD = nodular sclerosing Hodgkin’s disease

specific interactions with the CD20 antigen, the term “maintenance” should be reserved for the use of additional doses of rituximab to maintain serum levels of rituximab such that cells that express CD20 do not emerge from pre-CD20-positive B cell clones. Theoretically this should keep CD20+ clones from appearing, but also keeps normal B-cell immunity suppressed since normal B cells also express CD20. No clinical trial has adequately addressed the issue of “maintenance” rituximab in a pure sense. As noted earlier in this section, retreatment with rituximab is often still effective at the time of recurrence in patients who had previously had an objective response to rituximab [148, 339] which suggests that tumor reappeared only because levels of rituximab were insufficient to keep it suppressed. If this is correct, then, continued treatment with rituximab should prolong progressionfree survival, but not necessarily overall survival. The real question is, what strategy is best for preventing becoming rituximab-refractory? True refractoriness would be defined by disease progression during treatment with rituximab. For clinical trials purposes it also has been defined not only as disease progression during therapy, but also as progression of disease within 6 months of treatment in a patient who had stable disease or a response, which has very different implications. The mechanisms of resistance to rituximab appear to relate to (1) loss of cellular dependence on CD20 expression in terms of regulation of cellular resistance to apoptosis (probable) (2) existence or production of CD20 at a rate that rapidly consumes standard doses of rituximab (probable), (3) loss of CD20 expression (uncommon), or (4) induction of neutralizing anti-rituximab antibodies (very rare), (5) lack of complement (unlikely), or (6) insufficient or inadequate effector cells (unlikely). Several trials suggest that patients who have not experienced progressive disease while taking single-agent rituximab benefit from receiving more than the standard 4 weeks of therapy as summarized in the accompanying Table 15. Such approaches have been associated with higher response rates and longer durations of progression free survival than were reported with standard therapy. However, the 95% confidence intervals for response rates overlap with those observed with the 4-week schedule, thus, these results are not clearly better than those seen with 4 weeks of treatment. Randomized trials involving patients with follicular lymphoma show that giving additional dose of rituximab after standard dosing does prolong PFS [251, 280]. In the Swiss trial the addition of one additional 375 mg/m2 dose every 2 months for 4 months was associated with a superior progression free survival of 23 vs. 12 months (p = 0.02) and was even

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Table 15. Rituximab treatment schedules utilizing more than the standard 375 mg/m2 weekly × 4 weeks in patients with indolent or follicular lymphoma (no chemotherapy) Citation

Clinical setting

Schedule after 4 weeks of standard rituximab therapy

Patients

Response rate

PFS

[389] [15] [277] [280] [251] [264] [562] [56]

Relapsed indolent Relapsed follicular Relapsed indolent Relapsed indolent Initial & relapsed follicular Relapsed indolent Relapsed follicular Relapsed indolent

4 doses months 3 if PR or SD 2 more weeks 4 doses months 6,12,18,24 4 doses months 6,12,18,24 1 dose months 3,5,7,9 1 dose monthly if <25 μg/ml 4 more weeks 2 more weeks

25 17 60 35 75 22 37 68

76% 76% 74% 52% 66% 63% 60% 59%

– >34 months 34 months 31 months 23 months >25 months >19 months 14 months

PFS = progression free survival

more striking in patients who had not received chemotherapy before (36 vs. 19 months, p = 0.009) [251]. To date the only trial that has tried to address the issue of time to rituximab-refractoriness is the phase II randomized trial by Hainsworth et al. [280], in which patients with relapsed low-grade lymphoma were treated with the standard 375 mg/m2 weekly × four dosing with rituximab, and then those who did not have progressive disease were randomized to either planned retreatment with rituximab every 6 months for 2 years, using the same dose and schedule, or to delayed treatment until disease progression occurred, then re-instituted rituximab therapy. This design of “maintenance” is not really “rationale” since 6 months is too long to sustain serum levels in some patients, and 4 weeks of therapy is excessive in many patients in terms of rates of cellular catabolism and hepatic metabolism vs. interference with appearance of the CD20 molecule on the cell surface. For these reasons it is not surprising that there was no difference in the time to becoming refractory to rituximab between the retreatment every 6 months compared to retreatment relapse. However, the additional rituximab therapy was associated with a higher response rate 52% vs. 35%, complete response rate (27% vs. 4%). There was a striking difference in the time to disease progression, medians of 31 months vs. 8 months (p = 0.007), but no difference in time to become “rituximab refractory” which was about 30 months in both arms, nor in overall survival at a median follow up of 42 months. However, this was a small trial that was stopped early because it was originally designed for patients with indolent lymphoma who had relapsed after chemotherapy alone, at which time they were treated with rituximab. Once rituximab became a standard part of initial therapy for patients with indolent lymphomas, there were no longer any patients available that met eligibility requirements for the study. There is an ongoing cooperative

group trial (RESORT, ECOG 4402) that also attempts to address this issue in previously untreated low-risk indolent lymphoma patients whose initial therapy is standard-dose rituximab alone. Patients are then randomized to “maintenance” utilizing one 375 mg/m2 dose every 3 months until relapse, instead of four doses every 6 months for 2 years, or are randomized to retreatment with standard-dose rituximab at that time of relapse. The clinical trial definition of refractoriness is the key endpoint, but this trial also attempts to address whether prolonged inhibition of CD20-positive B cells beyond 2 years impairs humoral immunity. In terms of optimizing progression free survival, the optimal way to give “maintenance” rituximab would be based on sustaining serum levels in an effort to prevent the emergence of CD20+ cells. However, a commercial assay for serum CD20 levels is not currently available. The only trial performed to date that utilized serum CD20 levels to guide re-treatment enrolled only 31 patients, and rituximab levels were only sustained for 1 year; so it did not adequately address this issue [264]. In this trial rituximab serum levels were measured monthly, and if the level dropped below 25 μg/ml, an additional 375 mg/m2 dose was administered. For the 22 patients who were monitored as planned, half of the patients required additional dosing by 5 months. The response rate for 29 evaluable patients was 59%, and was 63% for 22 patients with indolent histology. If a commercial assay for measuring serum levels of rituximab became available, it could become the major determinant for optimizing rituximab dosing for individual patients [585]. In practice most physicians have adopted one or more of the published schedules of maintenance therapy for their patients with indolent lymphomas, based on the benefits in terms of response rates and progression free survival, which is a highly valued endpoint by both patients and practicing physicians. However, most

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patients are not initially treated with rituximab alone, but rather with rituximab in combination with chemotherapy. The issue of maintenance rituximab therapy in that setting for both indolent and aggressive lymphomas is discussed latter in this chapter.

Rituximab with Chemotherapy In vitro studies showed additive and/or synergistic benefit from combining rituximab with chemotherapy. [156]. Some studies suggest that the presence of rituximab helps overcome resistance, perhaps because of altered efflux of chemotherapy drugs, or decreased resistance to apoptosis. In the treatment of lymphoma, rituximab has been combined with virtually every chemotherapy regimen in common use including cyclophosphamide, vincristine, and prednisone (CVP) [279, 465], cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) [98, 122, 139, 279, 319, 329, 423, 532, 729, 757], cyclophosphamide, doxorubicin, vincristine, prednisone and etoposide (CHOEP) [555], fludarabine alone [82, 141], fludarabine and cyclophosphamide (FC) [373], fludarabine and mitoxantrone (FM) [476, 813], cyclophosphamide, mitoxantrone, vincristine, and prednisone (CMOP) [197], and fludarabine, chlorambucil, and mitoxantrone (FCM) [233], pentostatin alone [193], pentostatin and

cyclophosphamide (PC) [186, 306, 370, 413], pentostatin and mitoxantrone (PM) [160], cladribine with or without cyclophosphamide [601], chlorambucil [464], chlorambucil, mitoxantrone, and prednisone (CMP) [317], infusional etoposide, platinum, vincristine, cyclophosphamide, and doxorubicin (EPOCH) [350, 786], cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, and cytarabine (hyperCVAD-MA) [605, 718] ifosfamide, cisplatin, etoposide (ICE) [379], irinotecan, mitoxantrone, dexamethasone (IMD) [523]. As a generalization, the addition of rituximab does not increase toxicity in any significant way, although in randomized trials there has been a slightly higher rate of neutropenia, but not febrile neutropenia. There also appear to be no advantages to sequencing chemotherapy and rituximab as opposed to giving the agents together, since they do not have any overlapping toxicities. Several trials have been conducted that confirm the safety and efficacy of administering Rituximab in combination with chemotherapy in various B cell lymphoproliferative disorders.

Indolent and Follicular Lymphoma Combinations of rituximab and a variety of chemotherapies have produced high response rates with durable progression free survival (Table 16). The combinations

Table 16. rials of chemotherapy with concurrent rituximab in patients with indolent lymphoma, mostly patients with follicular lymphoma Citation

Clinical Setting

Chemo Regimen

Proportion Responding

Response Rate

[476] [139,140] [319] [233] [532] [141] [197] [468] [729] [160] [465] [618] [725] [193] [306]

Untreated Relapsed/untreated Untreated follicular Recurrent follicular Untreated Relapsed/Untreated Untreated Untreated/relapsed Untreated/Relapsed LPCL Untreated Untreated indolent Recurrent follicular Recurrent follicular Relapsed Untreated/relapsed LPCL

FMD CHOP CHOP FCM CHOP Fld CMOP Chl CHOP PM CVP FC FC Pento PC

76/76 38/38 214/223 35/37 32/34 35/39 38/42 24/27 11/13 20/24 131/162 37/54 60/75 44/57 10/13

100% 100% 96% 95% 94% 90% 90% 89% 85% 83% 81% 81% 80% 77% 77%

FMD CHOP FCM CMOP PM CVP FC Clb Fld Pento C

= = = = = = = = = = =

fludarabine, mitoxantrone, dexamethasone cyclophosphamide, doxorubicin, vincristine, prednisone fludarabine, chlorambucil, mitoxantrone cyclophosphamide, mitoxantrone, vincristine, prednisone pentostatin, mitoxantrone cyclophosphamide, vincristine, prednisone fludarabine, cyclophosphamide chlorambucil fludarabine pentostatin cyclophosphamide

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have not produced additive toxicity, but because of concerns about the potential for such toxicity, other investigators have utilized rituximab after chemotherapy for patients who were already had a response to therapy. This approach of chemotherapy followed by sequential therapy or consolidation rituximab therapy also has been associated with very high response rates and durable remissions (Table 17). Two randomized phase II trials have compared the concurrent and sequential approaches but were not definitive in showing differences [471, 532]. The larger of the two trials did show a slightly higher response rate and longer progression free survival for the combined approach [471]. Since there has been no evidence of additive toxicity for the combination regimens, and there is no evidence that the sequential approach offers any therapeutic advantages, most physicians use the concurrent approach. Because of the prevalence of follicular lymphoma, more trials have been conducted with chemotherapy and rituximab in this B cell malignancy than any other tumor type. Longer follow up has established that survival for such patients has been improved since the introduction of rituximab in the salvage setting, and as part of initial therapy of patients with follicular lymphoma [141, 327, 619]. In a trial of 38 patients with indolent lymphomas, 80% of whom had not been previously treated, RCHOP was associated with a response rate of 100%, a median progression-free survival of nearly 7 years, and after 9 years of median follow-up, median survival had not been reached [141].

Table 17. Trials of chemotherapy followed by rituximab (sequential therapy) in patients with indolent B cell lymphoma, mostly follicular lymphoma Citation

Clinical setting

Chemo regimen

Proportion responding

Response rate

[813]

Untreated FCL Untreated Untreated FCL Untreated Untreated Untreated FCL Untreated Untreated FCL

CHOP

67/68

98%

CHOP FM

34/35 69/782

97% 96%

FMD FMD FC

69/73 34/36 29/33

95% 95% 88%

FM CHOP

26/30 71/84

87% 84%

[532] [813] [470] [754] [119] [273] [462]

CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone FM = fludarabine, mitoxantrone FMD = fludarabine, mitoxantrone, dexamethasone FC = fludarabine, cyclophosphamide

As summarized in Table 18, randomized trials have confirmed a higher response rate and longer progression free survival when rituximab is added to chemotherapy compared to results for the same chemotherapy alone. Some studies also suggested there may be an improved overall survival as well. This improved survival has been accomplished without any clinically important increase in toxicity. Because of this rituximab with or without chemotherapy has become the treatment of choice for patients with indolent lymphomas who require therapy.

Chemotherapy and Rituximab in Chronic Lymphocytic Leukemia Although rituximab does not have an FDA regulatory marketing indication for CLL, numerous phase II trials have shown that the combination of chemotherapy plus standard doses of rituximab has substantial activity in the disease as shown in Table 19. This has included purine analogs plus rituximab [82, 192, 601, 641], and combinations of alkylating agents, purine analogs, plus rituximab [186, 370, 373, 413, 601, 783]. In one randomized phase II trial, concurrent fludarabine plus rituximab was compared to the sequence of fludarabine followed by rituximab in 104 patients [82]. The response rate was higher for concurrent therapy (90% vs. 77%) as was the complete response rate (47% vs. 28%). A retrospective comparison of these patients to a similar population of patients treated with fludarabine alone at the same dose in an earlier randomized trial suggested that patients treated with the combination of fludarabine plus rituximab had much better outcomes, including a higher response rate (84% vs. 63%), higher complete response rate (38% vs. 20%) better progression free survival after 2 years (67% vs. 45%), and better overall survival at 2 years (93% vs. 81%) [83].

Large B Cell Lymphoma The three large randomized trials summarized in Table 20 have demonstrated the benefits of combining rituximab with chemotherapy in the treatment of large B cell lymphoma. In France Coiffier et al. compared RCHOP to CHOP in 399 previously untreated patients, ages 60–80 with large B-cell lymphoma, using an eightcourse schedule that included antibody and chemotherapy all being given on day one of each course, prednisone, then Rituximab, then the cyclophosphamide, doxorubicin, and vincristine [122]. After 1 year of follow up and 126 events (disease progression or death), the CHOP plus Rituximab arm was superior in response rate (76% vs. 60%, p = 0.004), 1-year event-free

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Monoclonal antibody therapy

Table 18. Randomized trials of chemotherapy ± rituximab in indolent lymphomas Citation

Clinical setting

Treatment Regimen

Proportion Responding

PR Rate

[319]

Untreated follicular

RCHOP CHOP

96% 90%

[745]

Relapsed follicular

RCHOP CHOP

214/223 184/205 p = 0.011 199/234 166/231

CR Rate

PFS

85% 72% p < 0.001 81% 57%

30% 16% p < 0.001 41% 10%

92% 75% p = 0.0009 100% 96% 79% 58% p = 0.01 98%

50% 25% p = 0.004 88% 85% 34% 12%

33 months 20 months p < 0.001 32 months 15 months p < 0.0001 >47 months 29 months p < 0.0001 76% 3-year 60% 3-year – –

[465]

Untreated follicular

RCVP CVP

131/162 91/159

[317]

Untreated follicular

RMCP MCP

166/181 133/177

[476]

Untreated indolent

[233]

Relapsed FCL & Mantle Cell

RFMD FMD RFCM FCM

80 80 52/66 36/62

[75]

Untreated Lympho- plasmacytoid

RCHOP

36

CHOP

36

69% p = 0.01

RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone RCVP = rituximab, cyclophosphamide, vincristine, prednisone CVP = cyclophosphamide, vincristine, prednisone RMCP = rituximab, mitoxantrone, chlorambucil, prednisolone MCP = mitoxantrone, chlorambucil, prednisolone RFMD = rituximab, fludarabine, mitoxantrone, dexamethasone FMD = fludarabine, mitoxantrone, dexamethasone RFCM = rituximab, fludarabine, chlorambucil, mitoxantrone FCM = fludarabine, chlorambucil, mitoxantrone PR = partial response PFS = progression free survival OS = overall survival

Table 19. Phase II trials of chemotherapy plus rituximab in CLL Clinical Citation setting

Median age

% Proportion RespRegimen responding onse

[373] Untreated 58 years FCR 213/224 [370] Untreated 63 years PCR 58/64 [82] Untreated 63 years FR 46/51 [641] Both 59 years FR 27/31 [186] Untreated 72 years PCR 7/9 [783] Relapsed 59 years FCR 138/179 [413] Untreated 62 years PCR 26/34 [601] Relapsed 55 years CCR 14/26 [192] Relapsed 60 years PR 19/59 [186] Relapsed 70 years PCR 2/12 FCR = fludarabine, cyclophosphamide, rituximab FR = fludarabine, rituximab PCR = pentostatin, cyclophosphamide, rituximab CCR = cladribine, cyclophosphamide, rituximab PR = pentostatin, rituximab

95% 91% 90% 87% 78% 77% 75% 54% 33% 17%

survival (69% vs. 49%, p < 0.0005) and overall survival (83% vs. 68%, p < 0.01). Longer follow up at a median of 5-years confirmed the tremendous advantage of RCHOP in terms of PFS and OS, without identification of any long-term toxicities [219]. A similar trial was conducted by Habermann et al. in a U.S. intergroup trial that enrolled 632 patients over the age of 60, except that a different schedule of RCHOP was used, and for the 415 responding patients, there was a secondary randomization to maintenance rituximab vs. observation for 2 years [276]. In this trial the RCHOP regimen was two cycles of rituximab followed by eight cycles of CHOP with three more cycles of rituximab given before the third, fifth, and seventh doses. Patients who had achieved a CR or PR by the end of eight cycles were randomized to either observation or to four 4-week courses of rituximab at 375 mg/m2 per dose, given every

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Table 20. Randomized trials of chemotherapy ± rituximab in large B cell lymphomas Treatment regimen

Patients enrolled

Untreated elderly

RCHOP CHOP

Untreated elderly

RCHOP CHOP

Citation

Clinical setting

[122] [219] [276]

[555]

Untreated low-risk young

RCHOP

PR rate

CR rate

PFS

OS

399

86% 71% p = 0.007

76% 63% p = 0.005

53% 3-year 35% 3-year p = 0.0008

62% 51% p = 0.008

632 or 425

77% 76%

35% 35%

52% 3-year 39% 3-year

67% 58%

81%

p = 0.003 76% 2-year

p = 0.05 94%

60% 2-year p < 0.0001 70% 2-year 57% 2-year p < 0.0001

87% p = 0.001

a

824

CHOP [556]

Elderly

RCHOP-14 CHOP-14

–

67% p = 0.0001

1222

RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone PR = partial response CR = complete response PFS = progression free survival OS = overall survival a Response data are based on all 632 patients, but survival data is limited to 425 patients who did not receive “maintenance” rituximab therapy, four weekly doses every 6 months for 2 years.

6 months for 2 years. For comparability with the French study, an analysis was performed that excluded the 207 patients who received maintenance rituximab, which demonstrated a superiority of RCHOP in terms of PFS and OS, although there was no difference in response rate. Both of the above trials dealt with elderly patients. Pfreundshuh et al. conducted a trial of rituximab and CHOP-like regimens compared to the same chemotherapy alone in 824 patients under the age of 60 years who had was considered low-risk large B cell lymphoma [556]. Low-risk was defined as International Prognostic Index 0–1, stage II–IV disease, or bulky stage I disease. CHOP-like regimens included the addition of etoposide (CHOEP) or addition of methotrexate (MACOP). Any response less than a CR was considered a treatment failure. This trial also confirmed a substantial advantage for patients who received rituximab with the chemotherapy. In elderly patients Pfreundschuh et al. have conducted a four-arm trial (RICOVER-60) comparing six and eight cycles of RCHOP-14 and CHOP-14 [557]. RCHOP-14 was clearly superior to CHOP-14, and six cycles of RCHOP-14 showed a survival advantage over eight cycles of RCHOP-14. What remains to be done is to compare RCHOP-14 to RCHOP-21. Many phase II trials have been conducted with RCHOP and other rituximab plus chemotherapy regimens which also demonstrated excellent response rates as initial treatment or at the time of relapse, as shown

in Table 21. One of the earliest was conducted by Vose et al. in which rituximab was given on day 1 and CHOP on day 3 of each of six treatment cycles to 33 patients with intermediate grade lymphomas, about two third with large B-cell lymphoma (Working Group Formulation G) and the remainder with large cell follicular (Working Group Formulation D) [757, 758]. The trial by Case et al, in which patients received six to eight cycles of RCHOP administered on the same day, followed by 4 cycles of maintenance rituximab for responders, confirmed the activity of concurrent RCHOP therapy in the community setting [98]. The Wohrer trial appears to confirm that RCHOP is highly active in gastric large B cell lymphoma [792]. The encouraging results from the REPOCH regimen have led to randomized trials comparing it to RCHOP [350, 786]. The encouraging results with 14-day RCHOP regimens, which are administered with prophylactic filgastrim or pegfilgastrim, has led to phase III trials using this approach compared to 21-day schedules of RCHOP [62, 283, 596]. Rituximab plus chemotherapy is also active in the salvage setting as evidenced by trials with REPOCH, RICE and RIMD [350, 379, 523].

Mantle Cell lymphoma Table 22 summarizes some of the trials that have been conducted with concurrent rituximab and chemotherapy in patients with mantle cell lymphoma. Howard et al.

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Table 21. Phase II trials of chemotherapy with concurrent rituximab in patients with large B cell lymphoma Citation

Clinical Setting

Chemo Regimen # Patients

[757,758] 76% LBCL CHOP 33 [792] Gastric LBCL CHOP 15 [98] LBCL CHOP 101 [283] LBCL CHOP-14 49 [596] LBCL CHOP-14 26 [62] LBCL CHOP-14 50 [786] Initial, aggressive EPOCH 61 [350] Relapsed or refractory EPOCH 50 [379] Relapsed or refractory ICE 36 [523] Relapsed elderly IMD 30 LBCL = diffuse large B cell lymphoma CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone EPOCH = etoposide, platinum, vincristine, cyclophosphamide, doxorubicin ICE = ifosfamide, cyclophosphamide, etoposide IMD = irinotecan, mitoxantrone, dexamethasone PFS = progression free survival OS = overall survival

Response Rate

PFS

OS

94% 100% 91% >90%? 100% >90% 92% 68% 78% 74%

82% 5-years

88%-5-years

76% 2-years 80% 2-years 70% 2-years 68% 2-years 82% 5-years 30% 2-years

90% 2-years 79% 2-years 72% 2-years 88% 5-years 40% 2-years

37%

45%

Table 22. Clinical trials of rituximab plus chemotherapy in mantle cell lymphoma Citation

Clinical setting

[424]

Untreated

[329] [605]

Untreated Untreated

Treatment regimen

Proportion responding

RCHOP CHOP

58/62 45/60

PR rate

CR rate

PFS

OS

94% 75% p = 0.005 96% 97%

34% 7% p = 0.0002 48% 87%

21 months 14 months p = 0.013 17 months 64% 3-year

NSD

RCHOP 38/40 RHyper94/97 82% CVAD-MA 3-year RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone RHyper-CVAD-MA = rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, cytarabine PR = partial response CR = complete response PFS = progression free survival OS = overall survival

showed that RCHOP was very active in mantle cell lymphoma, but the PFS was not better than what had been seen historically with CHOP alone [329]. However, when Lenz et al. conducted a randomized trial, RCHOP was superior to CHOP in terms of response rate, CR rate, and PFS, but not OS [424]. In a single institution trial, Romaguera et al. found their R-hyperCVAD-MA regimen to be highly active [605]. Both RCHOP and R-hyperCVAD-MA are used as initial therapy in patients with mantle cell lymphoma, and most medically fit patients go on to consolidation with high dose chemotherapy and autologous stem cell rescue.

Burkitt’s Lymphoma and ALL The combination of rituximab and hyper-CVAD with methotrexate and cytarabine (R-hyperCVAD-MA) has

not only been used for mantle cell lymphoma, but also for the treatment of adult acute lymphoblastic leukemia and Burkitt-type lymphomas. Thomas et al. treated 28 evaluable patients with this regimen as initial therapy [718]. Rituximab was given on days 1 and 11 of the hyper-CVAD courses and days 1 and 8 of methotrexate and cytarabine courses. The median age was 46 years, but 29% were over the age of 60 years. The response rate was 27/28 with 86% complete responders. The 3-year progression free and overall survival was 88%.

HIV Associated Lymphoma As summarized in Table 23, the addition of rituximab to chemotherapy has not been shown to improved outcome in HIV-associated, non-Hodgkin’s lymphoma (NHL). These relatively small trials do suggest some survival

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Table 23. Trials of rituximab and chemotherapy in HIV positive patients with B cell lymphoma Citation

Treatment Clinical setting regimen

[823] [53] [365]

Untreated Untreated Untreated

R-CDE R-CHOP R-CHOP CHOP

# Patients 74 52 75 75

CR rate

70% 77% 58% 47% p = 0.147 R-CDE = rituximab, cyclophosphamide, dexamethasone, etoposide RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone CR = complete response PFS = progression free survival OS = overall survival

benefit, but also raise issues regarding an increased risk from infection that was not evident in non-HIV lymphoma patient populations. Spina et al. used 96-h infusion R-CDE (cyclophosphamide 187–200 mg/m2/day, doxorubicin 12.5 mg/m2 per day, and etoposide 60 mg/m2 per day) in 74 patients with HIV-associated NHL, most of whom were receiving concurrent highly active antiretroviral therapy (HAART) [823]. Ten patients were diagnosed with opportunistic infections during or within 3 months of the end of R-CDE, and 17 patients developed non-opportunistic infections for an infection rate of 37%. Two patients died of bacterial sepsis and four of opportunistic infections. In contrast Boue et al. observed a 17% rate of infection, but they did not address the risk of infection in the months after completion of therapy [53]. The one randomized trial that has been conducted showed a trend toward higher response rate and survival with the addition of rituximab, but a higher rate of infectious death in the group that received rituximab [802].

Central Nervous System Lymphoma In the absence of central nervous system (CNS) disease, negligible levels of rituximab are detected following i.v. infusions, presumably because of the blood–brain barrier. However, significant levels can be achieved in the presence of parenchymal disease or other inflammatory processes that alter the blood–brain barrier, or after direct injection into the CSF. Rubenstein et al. treated ten patients with CNS relapse of LBCL with a planned nine injections of rituximab at 10 mg, 25 mg, or 50 mg doses via Ommaya reservoir over 5 weeks [613]. The maximum tolerated dose was determined to be 25 mg based on patients who received the 50 mg dose experiencing dose-limiting hypertension. These patients also had systemic symptoms including abdominal cramps or nausea and vomiting. Responses were observed in all

PFS

OS

59% 2-years

64% 2-years 75% 2-years 32 months 25 months

10.4 months 8.6 months

Infectious death 8% 4% 14% 2% p = 0.035

ten patients including a complete remission in four. Rituximab has also been used in the treatment of primary CNS B-cell lymphoma. Limited benefits were observed in patients with recurrent disease, and rituximab is now being combined with high dose methotrexate as part of initial treatment.

Maintenance Rituximab following Chemotherapy or Rituximab Plus Chemotherapy There has been interest in using maintenance rituximab after an initial response to a combination of rituximab and chemotherapy. As discussed earlier there also have been treatment strategies of chemotherapy followed by rituximab, which is really a consolidation or sequential treatment approach rather than maintenance. Randomized trials have failed to show an advantage for the sequential strategy compared to the combinations of rituximab with the same chemotherapy [276, 476, 532]. Compared to observation, randomized trials have shown that adding rituximab after an initial response to CVP, FCM, or CHOP is associated with longer PFS in indolent lymphoma [233, 322, 745], and adding rituximab after an initial response to CHOP prolongs PFS in large B cell lymphoma [276]. However, chemotherapy alone is no longer considered an appropriate initial option since rituximab plus chemotherapy is consistently superior to chemotherapy alone in terms of response rates and progression free survival for indolent and aggressive lymphomas, and overall survival for large B cell lymphoma. Table 24 shows the results of randomized trials that have evaluated the effect of maintenance rituximab after rituximab plus chemotherapy, but many were trials in which this was a secondary randomization limited to patients who had responded to either chemotherapy alone, or chemotherapy plus rituximab after the primary randomization.

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Table 24. Randomized trials of rituximab + chemotherapy ± “maintenance” rituximab in B cell lymphoproliferative disorders Citation

Clinical setting

[276]

Elderly untreated RCHOP

Induction regimen Maintenance rituximab

[745]

LBCL Relapsed FCL

RCHOP

1 dose q 3 months × 2 years vs. observation 91 98 23 months

[234]

Relapsed

RFCM

4 doses at 3 months & 9 months vs. observation

4 weekly doses q 6 months × 2 years vs. observation

FCL

# randomized PFS

OS

80

77% 2-years

NSD

93

75% 2-years p = 0.81 52 months

NSD

p = 0.004 36 months

NSD

41 40

26 months p = 0.035

LBCL = large B cell lymphoma FCL = follicular lymphoma RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone RFCM = rituximab, fludarabine, chlorambucil, mitoxantrone PFS = progression free survival OS = overall survival

The accompanying table excludes patients who had received only chemotherapy as their induction treatment, since this is not longer considered optimal therapy.

Table 25. Rituximab in combination with biologicals other than antibodies Citation

Rituximab with other Biologics Rituximab with Biologicals other than Monoclonal Antibodies To the extent that rituximab’s clinical benefits are due to CDCC and ADCC, combining rituximab with other biologicals may enhance its clinical benefit. Table 25 summarizes several studies that explored such combinations [8, 9, 148, 239, 259, 368, 380, 475, 609, 617, 743]. Thalidomide was given orally and all of the cytokines were given s.c. Ranges of response were 45–75% for interferon-α, 26–55% for IL-2, 60–83% for GM-CSF, 37–69% for IL-12. The large variance in response rates for specific combinations is almost certainly due to the specific characteristics of the patient populations in each small trial. Only randomized trials can establish the superiority of these responses compared to rituximab alone, which produced response rates from 45% to 85% as a single agent in indolent lymphomas. Whether such trials will be conducted is questionable since rituximab plus chemotherapy is considered standard induction therapy for these malignancies.

Clinical setting

Biological agent

Number of Response patients rate

[617]

Relapsed IFN-α 64 70% indolent [149] Relapsed IFN-α 38 45% indolent [239] Relapsed IL-2 20 55% indolent [259] Relapsed IL-2 34 26% indolent [380] Relapsed IL-2 54 10% indolent [475] Relapsed GM-CSF 12 83% indolent [609] Relapsed GM-CSF 35 60% indolent [8] Relapsed IL-12 43 69% indolent [9] Relapsed IL-12 30 37% indolent [743] Relapsed G-CSF 19 42% indolent [368] Relapsed Thalidomide 16 81% Mantle Cell IFN-α = interferon alpha IL-2 = interleukin-2 GM-CSF = granulocyte macrophage colony stimulating factor IL-12 = interleukin-12 G-CSF = granulocyte colony stimulating factor

Rituximab with other monoclonal antibodies Rituximab has been combined with the anti-CD52 antibody alemtuzumab, and the anti-CD22 antibody epratuzumab as shown in Table 26. Faderl et al. gave rituximab and alemtuzumab at their standard doses [214]. Strauss

et al. and Leonard et al. gave epratuzumab at 360 mg/m2 i.v.ly over 60 min followed by rituximab at 375 mg/m2, weekly for 4 weeks [425, 697].

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Table 26. Rituximab in combination with other monoclonal antibodies Citation [214] [697] [426] [697] [697] [426]

Clinical setting

Biological agent

Number of Response patients rate

Relapsed CLL Relapsed FCL Relapsed FCL Relapsed NHL Relapsed LBCL Relapsed LBCL

Alemtuzumab

25/48

52%

Epratuzumab

21/33

64%

Epratuzumab

10/15

67%

Epratuzumab

2/16

13%

Epratuzumab

7/15

47%

Epratuzumab

4/6

67%

CLL = chronic lymphocytic leukemia FCL = follicular lymphoma NHL = non-Hodgkin’s lymphoma LBCL = diffuse large B cell lymphoma

Toxicity and side Effects The toxicities associated with rituximab were summarized earlier in this chapter in Table 6. Rituximab is generally well tolerated. The most common toxicities observed are the “flu-like” symptom complex associated with i.v. infusion followed by the binding of rituximab to circulating CD20 positive B cells and their subsequent elimination in the reticuloendothelial system [170, 175, 184]. In the pivotal trial for rituximab, 33% of patients had their first infusion interrupted because of side effects, but toxicities were rarely seen with subsequent infusions after circulating B lymphocytes were cleared from the circulation [474]. Because of the predictably of the initial infusion reaction, caution should be taken with patients who have underlying cardiac or respiratory problems who may not tolerate the stress of the cytokines released, and the brief decrease in oxygen saturation associated with the initial congestion of antibody coated cells in the lungs. Care should also be taken when treating patients with high circulating B lymphocyte counts, especially if those cells strongly express CD20, although most patients will tolerate rituximab therapy [79], [363]. When rituximab is administered i.v., antibody molecules immediately contact CD20+ B cells in the circulation. After binding, these are removed in the reticuloendothelial system including the lung, liver, and spleen. Some may also be lysed as part of CDC. Both the binding to CD20 and elimination by CDC and ADCC are associated with the release of cytokines which produce fever, chills, sweats, dyspnea, tachycardia, and sometimes hypotension. Rituximab infusion is rapidly followed by

activation of complement, B-lymphocyte cytolysis, and TNF-alpha release [40, 787]. The severity of the reaction depends on the rate of rituximab infusion, the number of CD20+ cells, and the immune competence of the patient. In patients who have high numbers of rapidly proliferating CD20-positive cells, classic tumor lysis syndrome can occur. The rate of penetration and binding of rituximab to CD20-positive cells located in tumor masses is much slower; therefore in the absence of large numbers of circulating tumor cells, tumor lysis syndrome is only encountered when rituximab is given chemotherapy. Once circulating B cells have been eliminated, rituximab can be infused rapidly with many using an infusion time of 45 min for subsequent weekly infusions [82, 170, 175, 655]. If rituximab therapy is being re-started after being discontinued for several months, and B lymphocytes are again present in the blood stream, then the slow infusion rate should be used. Once circulating target cells are no longer present, subsequent infusions may be safely given at more rapid rates. For instance, the author administers full dose rituximab over 30 min during subsequent weekly treatments once B lymphocytes have been cleared from the circulation. Despite its excellent safety record, rituximab does carry several “Black Box Warnings” from the US FDA. These include fatal infusion reactions, tumor lysis syndrome, severe mucocutaneous reactions, and progressive multifocal leukoencepalopathy. As noted above, depending on the clinical setting and rate of rituximab infusion, patients may experience hypoxia and dyspnea that can be associated with pulmonary infiltrates and acute respiratory distress syndrome, cardiac arrhythmias including ventricular fibrillation and cardiac shock, and myocardial infarction. It is noted that 80% of fatal infusion reactions occurred with the first infusion. Tumor lysis syndrome with renal failure and death can occur with rituximab alone in the presence of high numbers of rapidly proliferating CD20-positive cells in the circulation, or when rituximab is given with chemotherapy in patients who have a high tumor burden of CD20+ cells anywhere in the body. Rare cases of severe mucocutaneous reactions including Stephens-Johnson syndrome and bullous pemphigoid have been reported. Progressive multifocal leukoencephalopathy associated with the Creutzfeld-Jacob virus has been rarely observed.

Summary The approval and resounding success of rituximab opened the doors to other unconjugated or “naked” Mab. In 1998, the year in which the drug was released, it became the most successful cancer drug ever launched, surpassing

340 the achievement of the chemotherapeutic paclitaxel (Taxol), even though the latter had proven useful in multiple solid tumors including lung, breast and ovarian cancer. After introduction as an active therapy for relapsed patients with indolent lymphoma, rituximab quickly become part of the standard treatment of virtually every B-cell malignancy. Rituximab has proven to be an outstanding agent producing high rates of durable responses in a broad range of B cell lymphoproliferative disorders, especially when combined with chemotherapy. Rituximab is appropriate for use either alone or in combination with chemotherapy as the initial treatment of indolent lymphomas. It is appropriate to combine rituximab with chemotherapy for the treatment of newly diagnosed aggressive lymphomas including large B cell and mantle cell. It is appropriate to combine rituximab with a purine analog with or without an alkylating agent in the treatment of newly diagnosed CLL. Because of the phenomenal impact and success of rituximab, there are numerous other anti-CD20 antibody products being developed in an effort to improve on the existing agent. Most of the rationale for such efforts relate to the potential to improved on binding to the CD20 target, or enhancement of immune function because of alterations in the Fc portion of anti-CD20 antibodies based on several reports suggesting that the Fc polymorphisms may predict clinical efficacy [95, 385]. However, others have failed to confirm this observation. [81, 216].

Alemtuzumab (Campath®, Bayer Healthcare, Tarrytown, NY) Alemtuzumab and CD52 The second Mab approved by the U.S. FDA for a hematologic malignancy was alemtuzumab (Campath), an antiCD52 monoclonal antibody that was approved in May 2001 based on data submitted for the treatment of patients with CLL that had recurred or been refractory to the purine analog fludarabine. Development of this product and a humanized version actually started several years before rituximab. The original rat antibody Campath-1G was genetically modified to create Campath1-H, later called alemtuzumab, by joining the hypervariable region of the rodent antibody with a human IgG1 kappa variable framework and constant regions [196]. Rat versions of this antibody showed limited clinical activity [195], but in vitro cytotoxicity in the presence of human complement and/or effector cells was greatly enhanced with the human IgG1 construct, and also showed activity in vivo [284]. Despite some encouraging clinical results with the humanized

Monoclonal antibody therapy construct, Burroughs-Wellcome decided to discontinue trials with this agent [199]. Subsequently, Ilex Oncology obtained the product and elected to proceed with development, and manufactured the humanized derivative in CHO cells. Alemtuzumab reacts with the CD52 antigen, which is present on most lymphomas and chronic leukemias, diseases, and is expressed on many cell types including malignant and normal B and T lymphocytes, natural killer cells, monocytes, macrophages, dendritic cells, some neutrophils [195]. CD52 is also expressed on tissues of the male reproductive system including epididymis, seminal vesicles, and mature sperm. CD52 is not internalized after antibody binding, and it does not appear to be secreted.

Clinical Trials with Alemtuzumab Early clinical trials of with anti-CD52 Mab Initial trials with anti-CD52 were conducted in lymphoma. There were no significant clinical responses among nine patients who received the rat IgG2b monoclonal antibody [195]. In contrast to these results, two of three patients did respond to a rat/human IgG1 chimeric construct [284]. These two patients had lymphadenopathy, splenomegaly, and bone marrow involvement that responded to doses of only 1–20 mg. Tang et al. reported partial responses in three out of seven patients with recurrent indolent lymphoma, who received thrice weekly treatment with 30 mg of the humanized antibody [708]. In early trials, the rat version of the antibody was given to five CLL patients [195, 196]. Three had sustained decreases in circulating leukemic cells, but only one of the three had resolution of marrow infiltrating by leukemic cells. A sixth CLL patient received a rat/human chimeric IgM CAMPATH antibody and had no response. Burroughs-Wellcome sponsored clinical trials of the human IgG1 construct and clinical responses were seen in patients who had failed fludarabine chemotherapy. One report described responses in 6/16 patients who received 30 mg thrice weekly for 16 weeks [347]. However, the company was disappointed in the associated toxicity, and elected to discontinue the trials [199]. Subsequently ILEX Pharmaceuticals obtained the rights to the antibody and proceeded with additional registration trials in CLL patients who had relapsed or been refractory to fludarabine.

Alemtuzumab in CLL Table 27 summarizes the results of various clinical trials of single agent alemtuzumab in patients with CLL [128, 222, 320, 372, 449, 503, 539, 577, 578]. The trials with 30 mg i.v. over 2 h thrice weekly in patients who had relapsed after or progressed during fludarabine treatment were used to support the FDA approval of alemtuzumab

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Table 27. Alemtuzumab as a single agent in chronic lymphocytic leukemia Citation Clinical setting

I.V. dose and schedule

Number of patients Response rate

[449] [320] [503] [128] [577] [539] [372] [578] [222]

30 mg sc thrice weekly for 18 weeks 30 mg iv thrice weekly for 12 weeks 30 mg iv thrice weekly for 12 weeks 10 mg sc thrice weekly for 18 weeks 30 mg iv thrice weekly for 12 weeks 30 mg iv thrice weekly for 12 weeks 30 mg iv thrice weekly for 12 weeks 30 mg iv thrice weekly for 12 weeks 30 mg sc or iv thrice weekly for 12 weeks

38 149 91 16 136 29 93 24 115

Untreated Untreated Relapsed post chemotherapy Relapsed post chemotherapy Relapsed post fludarabine Previously treated Relapsed post fludarabine Relapsed post fludarabine Median 3 prior chemotherapies

for the treatment fludarabine-resistant CLL [372]. These trials demonstrated response rates of about 33%, and durations of response from 7 to 11 months. A high percentage of patients also had stable disease. Because of severe infusion reactions associated with the reactivity of alemtuzumab with B and T lymphocytes, granulocytes, and monocytes, in these trials the dose of alemtuzumab was gradually increased from three to ten and then to 30 mg thrice weekly. A more rationale schedule than escalating boluses is continuous i.v. or subcutaneous (s.c). The s.c. strategy has worked well with a decrease in the severity of infusion reactions with similar efficacy, but local skin reactions occur in almost all patients and can be severe. Based on an analysis of serum samples from 30 patients who were treated with i.v. alemtuzumab, and 20 patients treated by the sc route, blood concentrations were similar and cumulative doses slightly higher using the sc route, but the sc route appeared to be associated with appearance of antialemtuzumab antibodies in some patients [285]. Other investigators have prolonged treatment from 12 weeks to 18 to 30 weeks with some increase in response rates and CR rates with low rates of opportunistic infections in patients who had received extensive prior chemotherapy [128, 449, 714]. In a study of 36 CLL patients who were treated with the standard i.v. dose and schedule, there was no correlation between response to alemtuzumab and the high-affinity, FcgammaR receptor polymorphisms FcgammaR3A and FcgammaR2A [437]. In a European registration trial 297 previously untreated CLL patients were randomized to oral chlorambucil 40 mg/m2 monthly vs. standard i.v. alemtuzumab [320]. The Mab produced a superior response rate, 83% vs. 55% (p < 0.0001) and PFS which has resulted in a marketing indication as initial therapy, even though in the U.S. purine-analog based therapy has been considered the treatment of choice ever since randomized trials showed that fludarabine was superior to oral

87% 83% 55% 50% 40% 38% 33% 33% 23%

chlorambucil 40 mg/m2 monthly for up to 1 year as initial therapy, with response rates of 63% vs. 37% and PFS of 20 vs. 14 months [576]. Alemtuzumab has also been explored in other CD52positive leukemias. In a small study a response rate of 73% was observed among 15 patients with T-cell prolymphocytic leukemia [545]. In a larger trial a response rate of 51% and CR rate of 13% was seen in 75 patients with relapsed T cell prolymphocytic leukemia who were treated with alemtuzumab at a dose of 30 mg i.v. thrice weekly [371]. In a small study that included nine patients with acute myeloid leukemia and six with acute lymphoid leukemia, a 20% response rate was observed, but 87% of patients experienced an infection [722]. Because of the efficacy of purine-analog based chemotherapy regimens with or without rituximab, many recent alemtuzumab trials have focused on its use to eradicate minimal residual disease in responding CLL patients. Thrice weekly doses of 10 to 30 mg have been administered i.v. or s.c. for 4 to 12 weeks as a consolidative therapy [499, 530, 780]. These studies have shown that consolidation with alemtuzumab does increase clinical, phenotypic, and molecular complete response rates, but there is an increased risk of opportunistic infections, even if anti-viral, anti-bacterial, and anti-Pneumocystis prophylaxis is used.

Alemtuzumab in lymphoma Alemtuzumab has activity in both B and T cell lymphoproliferative disorders including lymphomas. As shown in Table 28, response rates were highest in patients in the mycosis fungoides phase of cutaneous T cell lymphoma (CTCL), especially in patients who were previously untreated. Infusional complications from cytokine release were common, especially with the first infusion [37, 377, 383, 448, 450, 736, 814]. Cytopenias were common and associated with high rates of infection, including some deaths, which resulted in early termination of several trials.

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Monoclonal antibody therapy

Table 28. Alemtuzumab as a single agent in lymphoma and other lymphoproliferative disorders other than B cell chronic lymphocytic leukemia Citation

Clinical setting

I.V. dose and schedule

Proportion responding Response rate

Percent infections

[736] [383] [448] [209] [814] [37] [450] [377]

16 indolent, 2 aggressive NHL Relapsed indolent NHL Relapsed Indolent NHL PTCL CTCL & PTCL CTCL Mycosis fungoides CTCL Mycosis fungoides CTCL Mycosis fungoides

30 mg iv thrice weekly 30 mg iv thrice weekly 30 mg iv over thrice weekly 30 mg iv thrice weekly 10 mg iv thrice weekly 10 mg sc thrice weekly 30 mg iv thrice weekly 30 mg iv thrice weekly

8/18 3/18 6/50 5/14 6/10 12/14 12/22 3/8

67% 72% 32% 57% 10% 29% 50% >50%

NHL CTCL MF PTCL

44% 17% 14% 36% 60% 87% 55% 38%

= non-Hodgkin’s lymphoma = cutaneous T cell lymphoma = mycosisi fungoides = peripheral T cell lymphoma

Alemtuzumab with Chemotherapy in CLL

Alemtuzumab has been combined with rituximab in the treatment of patients with CLL. In one trial of 41 CLL patients the response rate for the combination was more than 50% and median PFS was 6 months, but infections occurred in more than 50% of patients [214]. In a second combination trial, patients were divided into three cohorts that received alemtuzumab at doses of 3, 10, or 30 mg during therapy, 1/12 patients had a response [517]. G-CSF was combined with alemtuzumab in the treatment of 14 CLL patients who had been heavily pretreated (median 3.5 chemotherapy regimens, in an effort to reduce infections complications and perhaps increase efficacy [436] G-CSF was administered at a dose of 5 μg/kg daily 5 days before and throughout standard i.v. alemtuzumab therapy. Five patients responded, but nine patients developed neutropenia and six patients had reactivation of cytomegalovirus.

warnings for Alemtuzumab include severe pancytopenias after higher cumulative doses, infusion reactions, and infections, including opportunistic infections. Because it reacts with both B and T cells in the circulation, infusion reactions associated with administration of alemtuzumab are common and often severe. The high percentage of significant infusion reactions limits the ability to deliver high doses i.v. initially, and in trials about 5% of patients had to discontinue treatment because of infusion-associated reactions. For this reason, in the registration trials doses were initially escalated from 3 to 10 mg to 30 mg during the first week, and then administered as 2-h infusions of 30 mg thrice weekly for an additional 11 weeks. Because of the systemic toxicity, approximately one log lower mg doses of alemtuzumab are used compared to rituximab. These low doses are unable to sustain serum levels and promote good penetration into large tumor masses, which is why the best results with this schedule of therapy have been achieved in the blood and bone marrow rather than large lymph nodes. This problem is being overcome by giving s.c. doses and continuing treatment for longer duration, but this also prolongs immunosuppression. A major limitation for clinical use of alemtuzumab is the immunosuppression that accompanies prolonged T-lymphopenia and neutropenia, which is associated with an increased risk of bacterial infections, and opportunistic infections including Herpes virus, cytomegalovirus infections, fungal infections, Pneumocystis carinii, and mycobacterial infections. The risk of opportunistic infections can be reduced, but not eliminated, by administering prophylactic antibiotics and antiviral agents.

Toxicity and Side Effects

Summary

The toxicities associated with Alemtuzumab were summarized earlier in this chapter in Table 6. Black box

Alemtuzumab is active in the treatment of both B and T cell malignancies, and it widely used as a salvage and

Alemtuzumab has been combined with fludarabine in the treatment of CLL. Kennedy et al. reported obtaining complete response in six patients treated with both agents concurrently, who were felt to be refractory to each drug as single agents [377]. Elter et al. reported an 83% response rate among 36 patients who had previously received a mean of 2.6 chemotherapy regimens [207]. Two patients developed fungal pneumonias, one patient died of Escherichia coli sepsis, and two patients had reactivation of subclinical cytomegalovirus.

Alemtuzumab with other Biologicals in CLL

Robert O. Dillman consolidation agent in CLL. It might have had much wider use against B cell lymphoid malignancies had rituximab not been approved and widely adopted before alemtuzumab became available. Thus, even though alemtuzumab has a marketing indication in CLL, and rituximab does not, the latter is more widely used in CLL. Enthusiasm for alemtuzumab has also been muted because of its toxicity, especially immunosuppression, which has greatly limited efforts to combine the agent with chemotherapy.

Trastuzumab (Herceptin®, Genentech, South San Francisco, CA) Trastuzumab and Her2 In September 1998 the U.S. FDA approved trastuzumab (Herceptin), a humanized Mab that reacts with the second component of human epidermal growth factor receptor (EGFR), known as Her2, for the treatment of metastatic breast cancer, making it the first Mab approved for the treatment of a solid tumor [171]. Trastuzumab consists of the idiotopes from the hypervariable region of murine antibody 4D5 united with a human IgG1 kappa antibody that reacts with the p185HER2/neu receptor [93]. The Mab was humanized by inserting the complementary-determining regions (CDRs) of the murine 4D5 into the constant and variable framework of a consensus human IgG1 to produce rHuMAb 4D5, which was more active in ADCC assays than its mouse counterpart. Trastuzumab is produced using recombinant DNA technology with CHO cells serving as the manufacturing factory. Her2 is a 185 kD transmembrane receptor that is a member of the EGFR tyrosine kinase family of receptors and is involved with the autophosphorylation of specific tyrosine residues after being activated by binding of the EGF ligand to the receptor. Early analysis suggested that a subset of about 25% of breast cancer patients had tumors that overexpressed this receptor [673]. Overexpression of the erbB-2 proto-oncogene results in overexpression of the HER-2 receptor on the cell surface and increased cell proliferation [632]. Binding of trastuzumab to the HER2 receptor results in internalization (modulation, down regulation) of the receptor and competitive inhibition to binding of EGF ligands to the receptor [675]. Such inhibition interferes with phosphorylation and the subsequent signal transduction that facilitates cell proliferation. The human IgG1 constant regions on the Fc of the humanized antibody mediate ADCC in vitro; so trastuzumab may also produce anti-tumor effects via the immune system.

343 Indirect support for this comes from a clinical trial in which breast cancer patients, whose tumor cells had high expression of Her2 by immunohistochemistry (IHC), were treated with docetaxel and trastuzumab; tumor samples contained significantly increased numbers of natural killer cells and increased expression of Granzyme B and TiA1 in lymphocytes compared with controls [11]. Based on data submitted to the US FDA, the recommended initial loading dose is 4 mg/kg administered as a 90-min infusion followed by a weekly maintenance dose of 2 mg/kg administered as a 30-min infusion. A 3 week dosing schedule has also been validated [25, 431]. Her2 is overexpressed in only about 25% of patients with breast cancer; so, testing for high expression of the antigen (receptor) by immunohistochemistry (IHC), or for overexpression of the Her2-neu gene by fluorescence in situ hybridization (FISH), is critical for appropriate patient selection. Large trials have confirmed that there is poor reproducibility in performance and interpretation of IHC assays for HER2 in laboratories that do not process and analyze large numbers of samples. Even in the best of hands, there is not complete concordance between IHC and FISH. Most centers who rely on IHC perform FISH for patients who are 2+ by IHC because many trials have demonstrated lower response rates in IHC 2+ compared to 3+, while benefit is almost always seen in patients who are FISH positive. The Dako Herceptest for IHC assay of HER2 was used to determine eligibility in the pivotal trial and is considered the most reliable IHC test available. However, many questions have been raised regarding the reliability and reproducibility of this test, especially on paraffin-fixed rather than fresh tissues [191, 544, 550, 733]. Many samples were felt to be falsely positive for Her2 based on negative FISH tests [733]. Comparative studies confirmed wide variation in interobserver reproducibility, and confirmed that the two extremes, 0 and 3+ were more reproducible than 1+ and 2+, and there was poor discrimination between 2+ and 3+ which resulted in a high rate of false positive tests [544]. Dowsett et al. compared results for 426 breast carcinomas from 37 different hospitals for patients that were being considered for trastuzumab therapy by performing IHC and FISH in three reference centers [191]. Only 2/270 (0.7%) of IHC 0/1+ tumors were FISH positive and only 6/102 (5.9%) of IHC 3+ tumors were FISH negative, which suggested that FISH testing was most valuable for validating that IHC 2+ tumors were Her2 negative. In a large U.S. adjuvant therapy trial, reference laboratories confirmed Her2 positivity for 86% of 2,535

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Monoclonal antibody therapy

registered patients with only 82% concordance between local and high-volume central laboratories for IHC, and 88% for FISH when the same methodologies were used [550]. For the discordant cases, the central and reference laboratories had 94% agreement for IHC 0, 1+, and 2+ with 95% agreement that these were FISH negative. Compared to the central laboratories, rates of discordance were 24% for 75 laboratories that interpreted less than 100 HER2 samples per month and only 3% for 29 laboratories that interpreted 100 or more HER2 samples per month [542].

Clinical Trials with Trastuzumab in Breast Cancer Various clinical trials have confirmed the efficacy of trastuzumab, especially in combination with chemotherapy, for the treatment of Her-2 positive breast cancer in patients with recurrent metastatic breast cancer, and as the initial treatment for metastatic breast cancer, and as adjuvant therapy [331]. At this time most physicians continue trastuzumab maintenance indefinitely or until disease progression since Her2 receptor is continually being produced by some malignant cells.

Trastuzmab Alone Clinical results from various trials with trastuzumab as a single agent in the treatment of metastatic breast cancer are summarized in Table 29. In patients with Her2-positive metastatic breast cancer that has relapsed after chemotherapy, eHer2available data suggests that trastuzumab alone Table 29. Clinical trials of trastuzumab alone in Her2-positive metastatic breast cancer Clinical Citation setting

I.V. dose and schedule

Number of Response patients rate

[68]

Initial therapy

52

33%

[755]

Initial therapy

114

26%

[26]

Initial therapy

105

19%

[118]

Relapsed

222

15%

[26]

Relapsed

8 mg/kg, then 4 mg/kg weekly for 8 weeks 4 or 8 mg/kg, then 2 or 4 mg/kg weekly 8 mg/kg, then 6 mg/kg q 3 weeks 4 mg/kg then 2 mg/kg weekly 250 mg, then 100 mg weekly × 10 weeks

43

12%

produces objective response rates of 12–33% depending on the specific patient population [23, 25, 68, 118, 755]. In a phase II trial in 46 patients with metastatic breast cancer in whom at least 25% of tumor cells expressed Her2 by IHC, a schedule of 250 mg i.v. followed by weekly doses of 100 mg for 9 additional weeks yielded five responses among 43 evaluable patients [23]. Low grade fever was seen in 15% and was believed to be related to immune complexes formed with shed antigen. The largest trial of trastuzumab as a single-agent, which was one of the two pivotal trials that led to regulatory approval of the agent, was conducted in 222 patients with Her2-positive metastatic breast cancer that had recurred after chemotherapy [118]. Trastuzumab alone was given at an initial dose of 4 mg/kg followed by weekly doses of 2 mg/kg. There were eight complete and 26 partial responses with a median duration of response of 13 months. In the 222 patients who had failed chemotherapy, there were four complete and 21 partial responses for an overall objective response rate of 12% by intent to treat analysis. The median duration of response among the 25 responders was 9 months, and their median survival was 13 months, which are comparable to chemotherapy. The patient population included 66% who had received prior adjuvant chemotherapy; 68% had received two or more chemotherapy regimens for metastatic cancer and 55 patients (25%) had relapsed after high-dose chemotherapy and autologous stem cell rescue prior to receiving antibody therapy. The response rate was 14% among patients whose tumors were Her2 2+ or 3+ by IHC, but 20% for patients whose tumors were FISH+ vs. 0% for FISH−. Treatment was generally well tolerated with 40% of patients experiencing fever during the first infusion. Cardiac dysfunction was noted in 5% which was usually reversible when treatment was discontinued. Burris et al. gave trastuzumab at twice the standard doses by the weekly schedule as initial treatment for 8 weeks for patients with Her2-positive metastatic breast cancer who had never received chemotherapy [68]. They reported a response rate of 33% among 52 evaluable patients in a trial that enrolled 61 patients. This response rate is the highest reported for single-agent trastuzumab. However, in a randomized phase II trial conducted by Vogel et al, there was no suggestion that this higher dose produced a higher response rate [755]. In this trial there was no significant difference in results for an 8 mg/kg loading dose and 4 mg/kg per week maintenance vs. the standard 4 mg/kg loading dose and 2 mg/kg per week maintenance. The failure to demonstrate any difference between these doses is not surprising in view of the sustained serum levels of trastuzumab that were achieved at

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the lower dose. In the Vogel trial, trastuzumab alone as initial treatment for metastatic breast cancer resulted in seven complete and 23 partial responses, but the response rates were 35% for patients whose tumors were IHC 3+ compared to 0% for tumors that were IHC 2+, and 34% among patients whose tumors were FISH+ compared to 7% for tumors that were FISH−. The most common adverse events attributed to trastuzumab were chills (25%), asthenia (23%), fever (22%), pain (18%), and nausea (14%) and two patients (2%) with histories of cardiac disease experienced reversible cardiac dysfunction. It would be more convenient for patients if trastuzumab were given at higher doses and less frequently than weekly. Baselga et al. tested an every 3-week schedule of trastuzumab delivery as initial therapy for 105 patients with Her2-positive metastatic breast cancer [25]. They observed response rate was 19%, but was 34% for patients whose tumors were IHC 3+ and/or FISH+ for Her2. As with other trastuzumab schedule, the most common treatment-related adverse events were rigors, fever, headache, nausea, and fatigue.

Trastuzumab Plus Single-Agent Chemotherapy In vitro and animal studies showed that trastuzumab augmented the anti-tumor cytotoxicity of various chemotherapy agents [24], [547]. Table 30 summarizes published phase II clinical trials, grouped by the single-agent chemotherapy that was combined with trastuzumab for treatment of patients with Her2-positive breast cancer [11, 22, 70, 71, 103, 112, 155, 212, 235, 267, 344, 431, 498, 537, 543, 546, 604, 630, 657, 710]. Most of the single agents used in

these trials were the taxanes (paclitaxel or docetaxel), or gemcitabine. The various agents were associated with response rates between 25% and 75% with median PFS of 6 to 12 months. Response rates of 50% or greater were reported in 17 of the 20 trials which appeared to be much higher than observed with chemotherapy alone. Many of these reports noted higher response rates in the subsets of patients who had 3+ IHC Her2 expression by IHC and/ or overexpression by FISH, as opposed to 2+ IHC Her2 or negative FISH. The addition of trastuzumab was not associated with any additive toxicities, other than cardiac disease in association with anthracyclines.

Combination Chemotherapy and Trastusumab Table 31 summarizes published phase II clinical trials that used combination chemotherapy plus trastuzumab for treatment of patients with Her2-positive breast cancer [235, 483, 500, 547, 549, 604, 690, 750]. Most of these trials utilized chemotherapy combinations of taxanes and platinum agents. Combination chemotherapy has consistently produced higher response rates and longer progression free survival in clinical trials in patients with metastatic breast cancer, although survival advantages have been harder to establish. As can be seen in the accompanying table, phase II trials of combination chemotherapy plus trastuzumab appear to produce slightly higher response rates (40–92%) than were seen with single agents plus trastuzumab, and the PFS was somewhat longer (range 6–16 months). In a randomized trial, trastuzumab and paclitaxel with or without carboplatin were compared as first-line therapy

Table 30. Phase II clinical trials of trastuzumab plus concurrent single-agent chemotherapy in metastatic breast cancer Citation

Clinical setting

Chemotherapy

Number of patients

Response rate

[546] [498] [212] [11] [710] [630] [537] [112] [235] [657] [431] [267] [604] [344] [69] [71] [155] [22] [103] [543]

Multiple prior treatments 92% prior chemo 1st or 2nd chemo Neoadjuvant 1st or 2nd chemo Measurable metastatic 95% prior taxane & anthracycline Measurable metastatic No prior therapy 0–3 prior chemo No prior chemo Prior taxane and anthracycline No prior chemo No prior chemo 82% prior chemo Initial chemo 0 or 1 prior chemo for mets No prior chemo No prior chemo Prior chemo

Cisplatin Docetaxel q 3 weeks Docetaxel weekly Docetaxel Docetaxel weekly Docetaxel q 3 weeks Gemcitabine Liposomal Doxorubicin Paclitaxel weekly Paclitaxel weekly Paclitaxel q 3 weeks Paclitaxel weekly Paclitaxel q 3 weeks Vinorelbine Vinorelbine Vinorelbine Vinorelbine Vinorelbine oral Vinorelbine Vinorelbine

37 25 30 23 26 40 61 29 33 88 32 25 88 37 40 54 40 30 62 35

24% 70% 63% 61% 50% 65% 38% 52% 62% 61% 59% 56% 36% 78% 75% 68% 50% 68% 63% 51%

346

Monoclonal antibody therapy

for 196 women with HER-2-overexpressing (2+ or 3+ by IHC) metastatic breast cancer [604]. Treatment consisted of six cycles of trastuzumab 4 mg/kg loading dose and 2 mg/kg weekly thereafter with paclitaxel 175 mg/ m2 every 3 weeks, with or without the addition of carboplatin (AUC = 6) every 3 weeks. Endpoints favored the

Table 31. Combination chemotherapy and trastuzumab for metastatic breast cancer Citation [547] [547] [750] [549] [549] [604] [236] [483] [690]

[500]

Clinical setting

Number of Response Chemotherapy patients rate

15% prior adjuvant taxane No prior taxane Initial therapy Initial therapy

Docetaxel + Carboplatin

Docetaxel + Cisplatin Docetaxel + Epirubicin Paclitaxel + Carboplatin weekly Initial Paclitaxel + therapy Carboplatin q 3 weeks Paclitaxel + No prior Carboplatin chemoq 3 weeks therapy Paclitaxel + No prior Gemcitabine chemotherapy No prior Paclitaxel + chemo Gemcitabine Cisplatin + 60% Gemcitabine taxane, 90% anthracylcine 2nd line Gemcitabine + therapy Vinorelbine

59

58%

62

79%

45

67%

48

81%

43

65%

88

52%

40

52%

13

92%

20

40%

30

50%

addition of carboplatin based on response rates of 52% vs. 36% (p = 0.04), and median PFS of 11 vs. 7 months (hazard ratio 0.66, p = 0.03). In consecutive phase II trials, weekly paclitaxel and carboplatin plus trastuzumab appeared superior to an every 3-week schedule of the same agents with response rates of 81% vs. 65%, median PFS 14 vs. 10 months, and median OS 3.2 vs. 2.3 years, and was associated with less hematologic toxicity [549]. Similar to what has been seen in other trials with anthracyclines, the combination of epirubicin plus carboplatin was associated with a high rate of significant cardiotoxicity (10/45) [750]. The majority of cardiac events occurred late during therapy with trastuzumab alone. Half of the patients were asymptomatic and all cases of CHF were resolved using cardiac therapy.

Randomized Trials of Chemotherapy With or Without Trastuzumab in Breast Cancer Randomized trials comparing chemotherapy plus trastuzumab to chemotherapy alone are shown in Table 32. One of the pivotal trials that led to regulatory approval of trastuzumab compared trastuzumab plus chemotherapy to chemotherapy alone in 469 patients with metastatic disease who had not received prior chemotherapy for metastatic disease [674]. All patients had tumors that overexpressed Her2, which was defined as 2+ or 3+ by IHC on a scale of 0–3. An initial 4 mg/kg trastuzumab dose was followed by 2 mg/kg weekly. The first dose of Mab was infused i.v. over 90 min, but in the absence of significant infusion related toxicity, subsequent doses were infused i.v. over 30 min. Patients who had not received an anthracycline previously were randomized to receive trastuzumab alone or with cyclophosphamide 600 mg/m2 i.v. and doxorubicin 60 mg/m2 or epirubicin (AC) i.v. every 3 weeks for six cycles. Patients who had

Table 32. Randomized trials of chemotherapy ± trastuzumab in breast cancer Citation

Clinical setting

Chemotherapy

Proportion responding Response rate

Median PFS

[674]

Initial therapy metastatic breast, prior dox

Paclitaxel + T Paclitaxel

[674]

Initial therapy metastatic breast, no prior dox

AC + T AC

[470]

Initial chemo

Docetaxel + T

38/92 16/96 p < 0.001 80/143 58/138 p = 0.02 57/93 32/93

7 months 3 months p < 0.0001 8 months 6 months p = 0.002 12 months 6 months p = 0.0001

Docetaxel T = trastuzumab AC = doxorubicin or epirubicin plus cyclophosphamide PFS = progression free survival

41% 17% p < 0.001 56% 42% p = 0.10 61% 34% p = 0.002

Robert O. Dillman

347

received adjuvant chemotherapy that included an anthracycline were randomized to receive paclitaxel 175 mg/m2 i.v. over 3 h alone every 3 weeks, or with trastuzumab. The combination of chemotherapy plus trastuzumab was superior for key end points including: response rate (50% vs. 32%, p < 0.0001), duration of response (9.1 vs. 6.1 months), progression-free survival (median 7.4 vs. 4.6 months, p < 0.001), and overall survival (death at 1 year, 22% vs. 33%, p = 0.008), and median survival 25.1 vs. 20.3 months, p = 0.046) with a 20% risk reduction of death. As shown in Table 32, the differences were most striking for paclitaxel plus trastuzumab vs. paclitaxel alone. The median OS was 22 months for paclitaxel plus trastuzumab vs. 18 months for paclitaxel alone (p = 0.17) and 27 months for AC plus trastuzumab vs. 21 months for AC alone (p = 0.16). This trial clearly demonstrated that cardiotoxicity is associated with trastuzumab. Cardiac dysfunction was noted in 11/91 (12%) patients who received paclitaxel plus trastuzumab alone compared to only 1/95 (1%) who received paclitaxel alone, and in 28/143 (20%) who received AC plus trastuzumab compared to 7/135 (3%) who received AC alone. Even though trastuzumab does enhance the efficacy of anthracycline containing regimens, the increased cardiac toxicity that is seen with such combinations has made this an inappropriate treatment strategy. Another randomized trial compared docetaxel plus trastuzumab to docetaxel alone using in patients with metastatic breast cancer who had received no prior chemotherapy. Docetaxel was given every 3 weeks and trastuzumab was given weekly. This trial confirmed a similar relative advantage for the addition of trastuzumab with roughly a doubling of response rate and PFS, and better OS 31 vs. 23 months (p = 0.032). The

trastuzumab arm was associated with more grade 3 and 4 neutropenia (32% vs. 22%), more febrile neutropenia (23% vs. 17%), and one patient (1%) in the Mabcontaining arm had symptomatic heart failure.

Adjuvant Treatment of Breast Cancer Based on the positive benefits of trastuzumab in measurable metastatic breast cancer, it was widely assumed that a similar benefit would be conveyed in the adjuvant setting against microscopic metastatic disease. Three large randomized trials have confirmed the expected advantage of this approach as shown in Table 33. Because of the cardiotoxicity associated with trastuzumab, patients with active cardiac disease were excluded from these trials. Based on Her2 biology, it is probable that there is more of an advantage to give trastuzumab during, rather than only after chemotherapy, but this has not yet been established in trials. It is also probable that there is an advantage for giving trastuzumab indefinitely, even if it has already been given with chemotherapy, but this also has not been proven. Finally, completed trials have attempted to address whether it is better to give adjuvant trastuzumab for 2 years or 1 year, but it is probable that it is better to give trastuzumab indefinitely if there is reason to believe that the breast cancer has not been completely eliminated. Three large randomized trials have been reported, at least in preliminary form [353, 560, 606, 677]. Two U.S. cooperative group trials were combined for one preliminary analysis [606]. The National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31 compared doxorubicin and cyclophosphamide followed by paclitaxel every 3 weeks with the same regimen plus 52 weeks of trastuzumab beginning with the first dose of paclitaxel. The North Central Cancer Treatment Group

Table 33. Randomized trials of trastuzumab in the adjuvant treatment of high-risk breast cancer Citation

Clinical setting

Treatment arms

Number of patients

DFS

OS

[606]

Node + or high risk node

Trastuzumab no trastuzumab

1,672 1,679

94% 3-years 92%-3 years p = 0.015

[560]

Node + or high risk node

Trastuzumab observation

1,703 1,698

87% 3-years 75% 3-year 0.48 HR p < 0.0001 86% 2-years 78% 2-years 0.54 HR p < 0.0001 91% 3-years 86% 3-years 0.58 HR p = 0.005

[677] [353]

Node + or high risk node

DFS = disease free survival OS = overall survival HR = hazard ratio

Trastuzumab observation

116 116

0.66 HR p < 0.0001 p = 0.15

348

Monoclonal antibody therapy

(NCCTG) trial N9831 compared three regimens: doxorubicin and cyclophosphamide followed by weekly paclitaxel, the same regimen plus 52 weeks of trastuzumab initiated concomitantly with paclitaxel, and the same chemotherapy regimen followed by 52 weeks of trastuzumab after paclitaxel. For an early analysis at a median follow up of 2 years, the two similar arms from each trial were combined and compared: doxorubicin plus cyclophosphamide followed by paclitaxel with or without trastuzumab concurrent with paclitaxel [606]. The international, multicenter trial (HERA) randomized more than 5,000 women with HER2-positive node positive or high-risk node negative breast cancer, compared 1 or 2 years of trastuzumab given every 3 weeks with observation after completion of locoregional therapy and at least four cycles of neoadjuvant or adjuvant chemotherapy [560, 677]. Initial reports have compared the observation and 1-year trastuzumab treatment arms in terms of interim disease free survival [560], and overall survival analyses [677]. In the European trial, 1,010 women with node-positive or high risk node-negative breast cancer were randomized to adjuvant therapy that consisted of three cycles of either docetaxel or vinorelbine, followed by three cycles of fluorouracil, epirubicin, and cyclophosphamide in both groups, with a secondary post-chemotherapy randomization for the 232 patients whose tumors were Her2-positive to either observation or trastuzumab for 9 weeks [353]. Because of the use of anthracyclines in these trial designs, patients have been closely monitored for cardiac toxicity, which has been higher in the trastuzumab arms. In the NSABP B-31 trial 16% of women discontinued trastuzumab therapy due to clinical evidence of myocardial dysfunction or significant decline in left ventricular function. As summarized in Table 33, all three of these trials have shown a benefit for the addition of trastuzumab.

Neo-Adjuvant Treatment of Breast Cancer As summarized in Table 34 trastuzumab is also being combined with chemotherapy as part of neo-adjuvant therapies with pathologic complete response rates ranging from 13% to 65% [70, 77, 78, 130, 334, 495]. After 3 weeks of neoadjuvant treatment with trastuzumab alone, 23% of patients had a PR before going on to receive 12 more weeks of trastuzumab combined with paclitaxel before surgery [495]. One randomized trial, which was designed to accrue 164 patients was closed early because of the marked superiority of the trastuzumab-containing arm [77]. Chemotherapy consisted of four cycles of paclitaxel followed by four cycles of 5FU, epirubicin, and cyclophosphamide, with weekly trastuzumab for all 24 weeks. After 3 years of follow up, there have been no recurrences in the patients who received neoadjuvant therapy, and they have a better PFS (p = 0.041) [78].

Trastuzumab and Hormonal Therapy for Breast Cancer About half of Her2-positive patients are hormone receptor positive which has led to interest in the interaction “cross-talk” between Her2 and hormone receptors which appears to increase resistance to hormonal therapies. There is a recent report of a 26% response rate for 31 evaluable patients who were treated with the aromatase inhibitor letrozole in combination with weekly trastuzumab [464]. Her2 positivity was confirmed for 82% (IHC3+ and/or FISH+) and 82% had previously been treated with tamoxifen. The median PFS was 6 months.

Trastuzumab in Other Tumor Types Her2 is over expressed at a low frequency in many other adenocarcinoma,; so trastuzumab is also being tested in other tumors in the subsets of patients whose tumors overexpress Her2 [2, 171]. However, because of the

Table 34. Trastuzumab plus chemotherapy as neoadjuvant therapy for locally advanced HER2-positive breast cancer Citation

Clinical setting

Neoadjuvant therapy

Number of patients

Response rate

Pathology CR rate

[495] [77, 78]

Locally advanced Locally advanced

Trastuzumab × 3 weeks Chemo alone Trastuzumab + Chemo × 24 weeks

35 19 23

23% – –

[130]

Stage II or III

95%

[70]

Stage II or III

75%

18%

[334]

Locally advanced

Trastuzumab + Docetaxel/Carboplatin × 77 18 weeks Trastuxumab + Paclitaxel q 3 weeks × 40 12 weeks Trastuzumab + Docetaxel + Cisplatin × 30 12 wks

– 26% 65% p = 0.016 35%

–

13%

CR = complete response

Robert O. Dillman variability in IHC methodology for detecting Her2, many of the higher estimates may be fallacious. So far there are no tumor types other than breast cancer for which trastuzumab appears to be efficacious, even in trials restricted to patients with whose tumors are Her2positive by IHC (2+ or 3+). In ovarian cancer 95/837 (11%) were HER2-positive and only 3/45 (7%) responded [48]. In non-small cell lung cancer (NSCLC) 24/209 (11%) patients were Her2-positive tumors by IHC and only 1/24 (4%) had a response [116]. In another NSCLC trial 13/69 had Her2-positive tumors by IHC, and 0/13 responded to trastuzumab [415]. In hormone-refractory prostate cancer 0/18 Her2-positive patients had a response [810]. As summarized in Table 35, several exploratory trials have been conducted in which trastuzumab was combined with chemotherapy in Her2-positive patients with tumor types other than breast cancer [244, 337, 409, 620, 621, 812]. Very few of the patients enrolled had tumors that were Her2 3+ by IHC. For example, less than 5% of patients in two of the NSCLC trials had tumors that were Her2 3+ by IHC [244, 812]. Response rates did not appear to be higher than anticipated for chemotherapy alone, but to determine the true contribution of trastuzumab requires randomized trials. Gatzemeier et al. randomized 101 patients with Her2-positive non-small cell lung cancer to trastuzumab plus gemcitabine and cisplatin or the chemotherapy alone, and found no difference in response rate (36% vs. 41%) or PFS (6.1 vs. 7.0 months), but only six patients had tumors that were 3+ Her2 by IHC, and five of them did have an objective response to treatment [244]. Collectively these studies have been disappointing. This does not appear to be a fruitful area for further investigation given that so few non-breast cancers strongly over express Her2.

Trastuzumab with other Biologicals Even though the efficacy of trastuzumab might be enhanced by combining it with other biological response modifiers, there has been limited exploration of this approach because there are so many efficacious chemotherapy agents with which to combine trastuzumab. Two trials have combined s.c. IL-2 with trastuzumab [225, 589]. In one trial there was a response rate of 9% in 45 patients, but more than 10% of patients experienced grade 3 or 4 pulmonary reactions [225]. In the other trial, one of ten patients had a clinical response [589]. In both trials the use of IL-2 was associated with an increase in natural killer cells, but this did not correlate with toxicity or clinical benefit.

349 Table 35. Trastuzumab plus chemotherapy in tumor types other than breast

Citation [244]

[812]

[409]

[337] [620] [621]

Clinical setting Lung non small cell IIIB & IV Lung non small cell IIIB & IV Lung non small cell IIIB & IV Metastatic urothelial Pancreas With RT primary esophagus

Screened patients who had Her2 + Patients Response tumors treated rate 103/619 (17%)

51

36%

77/360 (21%)

21

38%

28/179 (16%)

64

28%

57/109 (49%)

44

70%

16% –

34 19

38% 50% 2-years OS

OS = overall survival

Toxicity and Side Effects The toxicities associated with trastuzumab in pivotal trials were summarized earlier in this chapter in Table 6. Black box warnings accompanying the label indication include cardiomyopathy and infusion reactions. Trastuzumab infusions can result in serious infusion reactions including dyspnea and hypoxia, which rarely have been fatal. Most of the time transfusion reactions are mild, and are probably associated with binding to circulating white blood cells via either cross reactive antigens or Fc receptors, but much milder than seen with rituximab. Typically, symptoms occurred during or within 24 h of trastuzumab infusion. Infusion should be interrupted for patients experiencing dyspnea or clinically significant hypotension, and patients should be monitored until signs and symptoms completely resolve. The basis for the minor infusion reactions that are sometimes seen with initial infusions is not clear, but may relate to cross reactivity with an antigen expressed on some circulating cells, or a reaction related to binding to Fc on some circulating cells. The major toxicity associated with trastuzumab is cardiac dysfunction or congestive heart failure, that is

350 generally mild and reversible [374, 548, 658, 678, 712, 730]. Trastuzumab treatment can result in left ventricular dysfunction and congestive heart failure; therefore left ventricular function should be evaluated in all patients prior to and during treatment. The frequency and severity of left ventricular cardiac dysfunction and/ or clinical congestive heart failure is highest in patients who receive trastuzuamb concurrently with anthracycline-containing chemotherapy regimens, and is also higher in patients who have recently received anthracyline-containing regimens. If left ventricular dysfunction or clinical heart failure occurs, trastuzumab should be discontinued until cardiac function has improved. Trastuzumab can often be resumed without recurrence of left ventricular dysfunction. As a single agent, trastuzumab alone was associated with a 4–5% frequency of cardiac dysfunction in patients with metastatic disease and in the adjuvant setting [118]. The risk of cardiac toxicity was higher in patients with metastatic breast cancer who received trastuzumab with chemotherapy. Cardiac dysfunction was noted in 11/91 (12%) patients who received paclitaxel plus trastuzumab compared to only 1/95 (1%) who received paclitaxel alone, and in 28/143 (20%) who received AC plus trastuzumab compared to 7/135 (3%) who received AC alone, 27% for combined therapy vs. 8% for anthracycline-based chemotherapy alone, and 13% combined therapy vs. 1% for paclitaxel alone [674]. The higher rate of cardiotoxicity with the doxorubicin plus trastuzumab combination may be due in part to increased drug delivery of doxorubicin to cardiac muscle by loose binding between doxorubicin and the Mab [182], or additive or synergistic cardiotoxic effects of both agents. Other trials have also noted higher rates of cardiotoxicity in patients who have received anthracyclines prior to or concurrent with trastuzumab [376, 548, 674, 707]. The cardiotoxicity of trastuzumab may relate to HER2 expression on cardiac muscle cells that are involved in tissue repair, since absence of the HER2/neu gene in knockout mice is associated with failure to develop an embryonic heart [421]. Results from clinical trials suggest that cardiotoxicity is increased when trastuzumab is administered with or following cardiotoxic agents such as anthracylines. For this reason it is recommended that trastuzumab not be given in combination with an anthracycline or any other agent that is known to damage the myocardium. Based on the adjuvant trials, when trastuzumab is given after an anthracycline, heart failure rates can be expected to be in the range of 4–5% during treatment, but there may be an increased risk that extends beyond treatment.

Monoclonal antibody therapy

Summary The approval of trastuzumab in September 1998 marked the first approval by the US FDA of a therapeutic monoclonal antibody for the treatment of a solid tumor. Because trastuzumab has limited clinical benefit as a single agent, even in patients who overexpress HER2, it is typically administered with chemotherapy agents that have proven benefit in breast cancer, other than anthracyclines, which should be avoided because of the increased risk of cardiotoxicity. A decade after its introduction, trastuzumab with chemotherapy is now standard therapy in the neoadjuvant, adjuvant, and metastatic disease settings for breast cancer patients whose tumors are Her2-positive. It is controversial as to whether trastuzumab should be combined with chemotherapy in patients who have previously progressed during treatment with trastuzumab with or without chemotherapy. Historically overexpression of HER2 was associated with a poorer prognosis. Ironically, trastuzumab is so effective, that the prognosis for HER2-positive breast cancer patients is now better than for those that are HER2-negative.

Bevacizumab (Avastin®, Genentech, South San Francisco, CA) Bevacizumab and Vascular Endothelial Growth Factor The anti-vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab (Avastin) was approved in May 2004 on the strength of randomized trials in which the agent was combined with 5-FU-based chemotherapy for the treatment of metastatic colorectal cancer. In June 2006 it was also approved in combination with FOLFOX4 (5-fluorouracil, leucovorin, and oxaliplatin) for the secondline treatment of metastatic colorectal carcinoma. It also has an indication combined with paclitaxel and carboplatin chemotherapy for the treatment of metastatic adenocarcinoma of the lung. It is also an indicated for first-line treatment of metastatic breast cancer in combination with taxane chemotherapy. Bevacizumab is a recombinant humanized IgG1 Mab that is manufactured in CHO cells [570]. Unlike other commercially available antibodies that target antigens, bevacizumab binds to the VEGF ligand rather than the VEGF receptor. As discussed earlier in this chapter, VEGF is produced by many tumor cells, is the central mediator of tumor angiogenesis, and various lines of evidence suggest that VEGF would be a useful target for anticancer therapy [12, 55, 248, 420, 681].

Robert O. Dillman

351

The binding of bevacizumab to VEGF inhibited its binding to the VEGF receptor in vitro, and had anti-angiogenic and anti-tumor activity in various animal model assay systems [12, 55].

Clinical Trials with Bevacizumab Exploratory trials with bevacizumab Because it blocks VEGF, which is also important in normal angiogenesis, there was concern that the agent might be associated with bleeding. In phase I trials in 25 patients there were no grade III or IV toxicities associated with i.v. doses up to 10 mg/kg [265]. There were no tumor responses, but many patients had stable disease. Bevacizumab was subsequently given in combination with a variety of chemotherapy agents without undo toxicity [466].

Colorectal Cancer Clinical trials of bevacizumab in colorectal cancer are summarized in Table 36. A three-arm randomized phase II trial in metastatic colorectal cancer suggested that bevacizumab augmented 5-FU based chemotherapy [359]. In this trial 144 previously untreated patients were randomized to 5-fluorouracil and leucovorin (FU/LV) + low-dose bevacizumab (5 mg/kg q 2 weeks), or to FU/ LV + high-dose bevacizumab (10 mg/kg q 2 weeks) or to FU (500 mg/m2)/LV (500 mg/m2 alone. FU/LV was given weekly for the first 6 weeks of each 8-week cycle. Better results were seen with the addition of bevacizumab, and there appeared to be an advantage for the lower dose.

In the pivotal trial for regulatory approval, 923 patients with metastatic colorectal cancer were randomized to receive irinotecan, 5-FU and leucovorin (IFL) and bevacizumab (IFL-BV), IFL and placebo (control), or FL with bevacizumab (FL-BV) at a dose of 5 mg/kg every 2 weeks [335, 336]. In the first phase of the trial, 313 patients were randomly assigned to these three arms, then; after a planned initial analysis, the FL-BV arm was discontinued after enrollment of 110 patients, not because of inferior results, but because IFL had become the standard control arm based on other trials in metastatic colorectal cancer. IFL-BV was superior to IFL-placebo for the 813 patients randomized to treatment with one of these arms, in terms of response rate (p = 0.004), PFS (HR 0.54, p < 0.001), and OS (HR 0.66, p < 0.001) [335]. The IFL-BV arm was associated with more grade 3 or 4 hypertension (11% vs. 2%). The FL-BV arm also was superior to the IFL-placebo arm [336]. In a trial of initial therapy for patients with metastatic colorectal cancer who were not considered optimal candidates for first-line irinotecan treatment, 209 patients were randomized to receive FL + becacizumab at 5 mg/kg or FL + placebo [361]. The addition of bevacizumab resulted in a better PFS (HR = 0.50, p < 0.001), higher response rate (p = 0.055), and a trend toward better survival (HR = 0.79, p = 0.16). Hypertension was more frequent with bevacizumab. Because of the emergence of oxaloplatin as an active agent in colorectal cancer, the Eastern Cooperative Oncology Group (ECOG) conducted a three-arm trial in

Table 36. Randomized trials of bevacizumab in the treatment of metastatic colorectal cancer Citation

Clinical setting

Treatment arms

Number of patients Response rate Median PFS

Median OS

[359]

Colorectal metastatic untreated

[335, 336]

Colorectal 1st-line metastatic

[361]

Colorectal 1st-line metastatic

[253]

Colorectal relapsed post IFL

FL + BV-5 FL + BV-10 FL alone IFL + BV-5 IFL + placebo FL + BV-5 FL + BV-5 FL + placebo FOLFOX4 + BV-10 FOLFOX BV-10 BV-10 + gemcitabine gemcitabine

35 33 36 402 411 110 104 105 290

40% 24% 17% 45% 35% 40% 26% 15% 23%

9 months 7 months 5 months 11 months 6 months 9 months 9 months 6 months 7 months

22 months 16 months 14 months 20 months 16 months 18 months 17 months 13 months 13 months

289 243 602

9% 3% 11%

5 months 3 months 6 months

11 months 10 months –

10%

6 months

–

[391]

Pancreas metastatic

FL = 5-flurouracil, leucovorin BV = bevacizumab FOLFOX = folate (leucovorin), 5-fluroruracil, oxaloplatin PFS = progression free survival OS = overall survival

352 which 829 patients whose cancer had recurred after IFL therapy were randomized to receive oxaliplatin, fluorouracil, and leucovorin (FOLFOX4) with bevacizumab or to FOLFOX4 alone, or to bevacizumab alone [253]. In this trial bevacizumab was given at 10 mg/kg every 2 weeks. As shown in Table 36, bevacizumab exhibited limited activity as a single agent, and FOLFOX4 plus bevacizumab was superior to FOLFOX alone for all key endpoints, including OS (HR 0.75, p = 0.001), PFS (HR 0.61, p < 0.0001) and response rate (p < 0.0001). There were higher rates of hypertension and bleeding in the FOLFOX plus bevacizumab arm. A metanalysis was performed using the raw data from three randomized trials that included 5-FU plus leucovorin and bevacizumab (FL-BV) as initial treatment for patients with metastatic colorectal cancer [360]. For the combined data, FL-BV (n = 249) was superior to FL (n = 241) in terms of response rate (34% vs. 24%, p = 0.019), PFS (9 vs. 6 months, HR = 0.63, p < 0.001), and OS (18 vs. 15 months, HR 0.74, p = 0.008). In contrast to these excellent results in previously untreated patients, in an expanded access trial 350 patients with metastatic colorectal cancer, who had relapsed after or progressed during both irinotecan and oxaloplatin based therapy, the response rate was only 4% in the first 100 patients enrolled by investigator analysis, and only 1% based on blinded central review [106]. ECOG conducted a trial with IFL and bevacizumab in 87 patients with untreated advanced colorectal cancer [252]. The response rate among 81 evaluable patients was 49%, but the dose of irinotecan and 5FU both had to be reduced by 20 to 25% because of vomiting, diarrhea and neutropenia. Bleeding occurred in 37 patients (46%) and nine patients (11%) had grade 3 or 4 thromboembolic events. FOLFOX4 and bevacizumab was used to treat 53 patients with previously untreated metastatic colorectal cancer [208]. The response rate was 68%. Hemorrhage was not a problem in this trial, and only one patient had a severe thromboembolic event.

Non-Small Cell Lung Randomized clinical trials involving bevacizumab in tumor types other than colorectal cancer are summarized in Table 37. In a three-arm randomized phase II in non-small cell lung cancer (NSCLC), 99 previously untreated patients were randomized to treatment every 3 weeks with paclitaxel 200 mg/m2 and carboplatin (AUC = 6) (PC) alone or in combination with bevacizumab at 7.5 or 15 mg/kg [354]. The response rate and PFS were higher for the 15 mg/kg bevacizumab arm compared to PC alone with a trend toward better survival. Bleeding

Monoclonal antibody therapy was the most significant complication associated with bevacizumab and included minor mucocutaneous hemorrhage, and major hemoptysis associated with centrally located squamous cell cancers accompanied by tumor necrosis and cavitation. In a large two-arm randomized trial, 878 patients with previously untreated non-squamous NSCLC were randomized to PC + bevacizumab (15 mg/kg) or PC alone on a 3 week schedule [629]. The bevacizumab arm was associated with a higher response rate (p < 0.001), better OS (HR = 0.79, p = 0.003), and PFS (HR 0.66, p < 0.001). Even though patients with squamous cell histology were not enrolled in this trial, there were still 5 hemorrhagic deaths in the bevacizumab arm (1.2%) and significant bleeding was more frequent in the bevacizumab arm (4.4% vs. 0.7%, p < 0.001). In a large European trial 1,043 patients with previously untreated non-squamous NSCLC were randomized to gemcitabine plus cisplatin with bevacizumab at 10 mg/kg or 5 mg/kg or placebo [463]. Results favored the bevacizumab arms, although the benefit was not as impressive as in the U.S. trial with PC. In a phase I/II trial, 40 patients with metastatic nonsquamous NSCLC who had relapsed after previous chemotherapy were treated with bevacizumab 15 mg/kg i.v. every 2 weeks and erlotinib 150 mg orally daily [309]. Eight patients had a partial response.

Breast Miller et al. randomized 462 patients to p.o. capecitabine 2,500 mg/m2 twice daily days 1 through 14 every 3 weeks, with or without i.v. bevacizumab 15 mg/kg on day 1 [484]. There was more grade 3 or 4 hypertension requiring treatment (18% v 0.5%) in patients receiving bevacizumab. The response rate was twice as high for the combination therapy as capecitabine alone, but this did not result in superior survival, which is not surprising since most of these patients had failed at least three chemotherapy regimens prior to entering the trial. In a randomized trial bevacizumab plus paclitaxel was compared to paclitaxel alone as initial therapy for 722 patients with metastatic breast cancer [482]. There was an improvement in response rate and PFS, but not in OS, and there was an increased risk of complications in the bevacizumab arm. The addition of bevacizumab was associated with a longer prolonged progression-free survival (median, 12 vs. 6 months; hazard ratio for progression, 0.60; p < 0.001) and increased the objective response rate (37% vs. 21%, p < 0.001). The overall survival rate, however, was similar in the two groups (median, 27 vs. 25 months; hazard ratio, 0.88; p = 0.16). Grade 3 or 4 hypertension (15% vs. 0%, p < 0.001),

Robert O. Dillman

353

Table 37. Randomized trials of bevacizumab in the treatment of metastatic cancers other than colorectal cancer Citation

Clinical setting

Treatment arms

Number of patients

Response rate

Median PFS

Median OS

[354]

NSCLC IIIB or IV NSCLC

[629]

NSCLC IIIB or IV non-squamous

PC + BV-15 PC + BV-7.5 PC PC + BV-15

35 32 32 434

32% 28% 19% 35%

7 months 4 months 4 months 6 months

18 months 12 months 15 months 12 months

[463]

NSCLC IIIB or IV non-squamous

PC GC + BV-15

444 351

15% 34%

4 months 6.7 months

10 months –

GC + BV-7.5 GC + Placebo Cap + BV-15 Capecitabine

345 347 231 231 368 354 39 37 40 327 322

Gem + BV-10 Gecitabine

301 301

6.5 months 6.1 months 4.9 months 4.2 months NSD 12 months 6 months 4.8 months 3.0 months 2.5 months 10 months 5 months p > 0.001 6 months 6 months

– – 15 months 14 months NSD

Pac + BV-10 Pac BV-10 BV-3 Placebo IFN + BV-10 IFN + Placebo

30% 20% 19% 9% p = 0.001 37% 21% 10% 0% 0% 31% 12% p < 0.001 11% 10%

[484]

Breast metastatic

[482]

Breast metastatic

[799]

Renal cell metastatic

[210]

Renal cell metastatic

[391]

Pancreas metastatic

p = 0.067

NSCLC = non small cell lung cancer BV = bevacizumab, numbers refer to mg/kg PC = paclitaxel, carboplatin GC = gemcitabine, carboplatin IFN = interferon-α Gem = gemcitabine Pac = paclitaxel NSD = no significant difference PFS = progression free survival OS = overall survival NSD = No significant difference

proteinuria (4% vs. 0%, p < 0.001), headache (2% vs. 0%, p = 0.008), cerebrovascular ischemia (2% vs. 0%, p = 0.02), and infection (9% vs. 3%, p < 0.001), were more frequent in patients receiving paclitaxel plus bevacizumab. The combination bevacizumab 10 mg/kg on days 1 and 15 of a 28-day cycle in combination with docetaxel 35 mg/m2 on days 1, 8, and 15 was associated with a 52% response rate in patients with metastatic breast cancer who had not been heavily treated with prior chemotherapy [579].

Renal Cell Hypervascularity has long been noted as a feature of renal cell carcinoma. Most cases of sporadic clear cell carcinoma of the kidney are associated with loss of heterozygosity von Hippel-Lindau (VHL) gene on chromosome 3, and inactivation of the VHL allele. This is

associated with a loss of suppressor gene function and induction of genes that are regulated by hypoxia, one of which is the gene associated with production of VEGF. For this reason, renal cell carcinoma was considered an excellent target for bevacizumab trials. In a randomized double-blind, phase II trial, bevacizumab at 3 and 10 mg/kg every 2 weeks was compared to placebo in patients with metastatic renal cell cancer [799]. After randomization of 116 patients, the trial was stopped early because there was a significant prolongation of progression of free survival in the 10 mg/kg bevacizumab arm compared with placebo (hazard ratio, 2.55; p < 0.001). At the time the study was discontinued, the 5 mg/kg bevacizumab arm also had a slightly longer PFS compared to placebo that did not quite reach statistical significance (hazard ratio, 1.26; p = 0.053). There was no difference in survival, but this analysis was of uncertain significance since cross-over to bevacizumab was permitted in the

354

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placebo group. Hypertension and asymptomatic proteinuria were the main side effects noted. Large randomized phase III trials comparing interferon-alpha (IFN-α) plus bevacizumab with IFN-α alone in patients with metastatic renal cell cancer have shown an advantage for the addition of the antiVEGF antibody. IFN-α was given at a dose of 9 MIU s.c. every other day thrice weekly and bevacizumab 10 mg/kg i.v. every 2 weeks in a large European trial [210]. A similar comparison is ongoing in the US [597]. Hypertension and proteniuria were much more common in the bevacizumab arm. In a phase II trial patients with metastatic clear-cell renal carcinoma were treated with bevacizumab 10 mg/ kg i.v. every 2 weeks and erlotinib 150 mg orally daily [280]. The objective response rate was 20%. The most common adverse events were mild to moderate rash and diarrhea which were attributed to erlotonib, and proteinuria which was attributed to bevacizumab. In dose-finding phase I–II trials, thalidomide, which also has anti-antigenesis properties was combined with bevacizumab in patients with metastatic renal cell cancer whose disease had progressed after receiving placebo in another trial [204]. Sequential cohorts of 10 to 12 patients were treated with bevacizumab 3 mg/kg alone

or bevacizumab plus the maximum tolerated dose of thalidomide per dose escalation. The combination was well-tolerated with more than 50% of patients able to escalate their thalidomide dose to at least 500 mg/day. Toxicities were similar for both treatments and there were no objective responses. Progression free survival was similar between groups, 3.0 months for bevacizumab plus thalidomide and 2.4 months for bevacizumab alone.

Gastric and Gastroesophageal Junction Adenocarcinoma Non-randomized trials involving bevacizumab in various tumor types are summarized in Table 38. Forty-seven patients with metastatic or unresectable adenocarcinoma of the stomach or gastroesophageal junction were treated every 3 weeks with bevacizumab 15 mg/kg on day 1, irinotecan 65 mg/m2, and cisplatin 30 mg/m2 on days 1 and 8 [659]. Possible bevacizumab-related toxicity included grade 3 hypertension (28%), grade 3 to 4 thromboembolic events (25%) grade 3 to 4 thromboembolic events (25%), gastric perforation (6%), myocardial infarction (2%), and significant upper gastrointestinal bleed (2%). The primary tumor was unresected in 40 patients, but only one patient had a significant upper gastrointestinal bleed.

Table 38. Single arm phase II trials of bevacizumab with or without chemotherapy Citation

Clinical setting

I.V. dose and schedule

Number of patients

Response rate

[106]

Colorectal: metastatic refractory to irinotecan & oxaloplatin Colorectal: advanced, untreated Colorectal: chemo-naïve metastatic Breast: metastatic breast 1st or 2nd line Lung: NSCLC non squamous, previously treated, recurrent Gastric and GE: adenocarcinoma

BV-5 q 2 weeks + bolus or continuous FL BV-10 q 2 weeks + IFL BV-5 q 2 weeks + FOLFOX BV-10 q 2 weeks = docetaxel

100

4%

81 53 27

49% 68% 52%

BV-15 q 3 weeks + erlotonib

40

20%

BV-15 q 3 weeks + irinotecan + cisplatin BV-10 q 2 week + GemOx BV 2.5–10 q 2 weeks Capecitabine + RT BV-15 q 3 weeks BV-15 q 3 weeks BV-10 day 1 & 15 + Gemcitabine BV-10 q 2 weeks + erlotonib BV-15 q 3 weeks + Doxorubicin BV-10 q 2 weeks + Irinotecan

34

65%

30 48

20% 20%

62 44 52 59 17 23 9

21% 16% 21% 25% 12% 61% 67%

[252] [208] [579] [310] [659] [809] [132] [66] [87] [390] [281] [143] [759]

Hepatocellular Pancreas: locally advanced, inoperable Ovarian relapsed Ovarian relapsed Pancreas: metastatic Renal cell:clear cell, metastatic Sarcoma: soft tissue Glioblastoma Anaplastic Astrocytoma

BV = bevacizumab IFL = irinotecan, 5-flurouracil, leucovorin FL = 5-flurouracil, leucovorin GemOx = gemcitabine, oxaloplatin RT = radiation therapy

Robert O. Dillman

Hepatocellular Bevacizumab has been given to patients with measurable unresectable or metastatic HCC [809]. For cycle 1 (14 days), bevacizumab 10 mg/kg was administered alone i.v. on day 1. For cycle 2 and beyond (28 days/ cycle), bevacizumab 10 mg/kg was administered on days 1 and 15, gemcitabine 1,000 mg/m2 was administered as a dose rate infusion at 10 mg/m2/min followed by oxaliplatin at 85 mg/m2 on days 2 and 16. The most common treatment-related grade 3 to 4 toxicities included leukopenia/neutropenia, transient elevation of aminotransferases, hypertension, and fatigue.

Pancreas Bevacizumab has been a disappointment in cancer of the pancreas. In one trial, 48 patients with inoperable pancreatic adenocarcinoma received bevacizumab 2 weeks before radiotherapy, then every 2 weeks during radiotherapy, and then after radiotherapy until disease progression [132]. Each cohort included 12 patients at doses of 2.5, 5.0, 7.5, and 10 mg/kg). Capecitabine was administered on days 14 through 52. Four had ulceration and bleeding in the radiation field possibly related to bevacizumab. Three patients had tumor-associated bleeding duodenal ulcers, and one had a duodenal perforation. [132]. In another trial, patients with previously untreated metastatic pancreatic cancer received gemcitabine 1,000 mg/ m2 IV over 30 min on days 1, 8, and 15 every 28 days and bevacizumab, 10 mg/kg IV after gemcitabine on days 1 and 15 [390]. Grade 3 and 4 toxicities included hypertension in 19% of the patients, thrombosis in 13%, visceral perforation in 8%, and bleeding in 2%. Cancer and Leukemia Group B conducted a randomized phase III trial of gemcitabine plus bevacizumab vs. gemcitabine plus placebo [391]. There was no advantage for the addition of bevacizumab in terms of response, rate, PFS or OS.

Ovarian Cancer In contrast to reported results for other tumor types, single-agent bevacizumab is quite active in the treatment of ovarian cancer with response rates of 15–20% in patients who had progressive disease after two or more courses of chemotherapy [66, 87, 292]. The most frequent serious adverse events that were potentially related to bevacizumab and noted by Burger et al. were hypertension (10%) and thromboembolic events (3%) [66]. Cannistra et al. treated reported a 16% response rate in 44 patients, but serious toxicities included proteinuria (16%), GI perforation (11%) hypertension (9%), arterial thromboembolic

355 events (7%), bleeding (2%), and wound-healing complications (2%) [87]. There were three treatment-related deaths (7%). The rate of bowel perforation was unusually high in this trial, but occurred in 24% of patients who had received three prior chemotherapy regimens, compared to 0% in patients who received less than three. It seems likely that the high rate of small bowel perforation is the result of rapid elimination of bowel wall tumor implants, and/or and additional insult to bowel that has been damaged in some manner by chemotherapy [87]. However, a broader experience based on multiple trials suggests that this complication is observed in only about 5% of all ovarian patients [292]. The risk of this complication appears to be increased in more heavily treated patients. A three-arm trial has been started which compares paclitaxel plus carboplatin (PC) with placebo followed placebo, PC with bevacizumab followed by placebo, and PC with bevacizumab followed by bevacizumab for 15 months.

Prostate Bevacizumab was combined with APC8015 (sipuleucelT) in the treatment of 22 patients with hormone-refractory prostate cancer [598]. Sipuleucel-T is a cellular prostate cancer vaccine containing T-lymphocytes and dendritic cells loaded with a recombinant prostatic acid phosphatase and granulocyte-macrophage-colony-stimulating factor fusion protein. Bevacizumab was given at a dose of 10 mg/ kg i.v. on weeks 0, 2, 4, and every 2 weeks thereafter while sipuleucel-T was given i.v. weeks 0, 2, and 4. One patient had a >50% decrease in PSA.

Gliomas In a phase II trial adult patients with recurrent anaplastic astrocytoma or glioblastoma (grade III or IV glioma) received bevacizumab at 10 mg/kg and irinotecan i.v. every 2 weeks of a 6-week cycle [759]. The dose of irinotecan was determined based on whether patients were taking antiepileptic drugs that induced hepatic enzymes that accelerate metabolism of irinotecan. Patients taking enzyme-inducing antiepileptic drugs received irinotecan at 340 mg/m2, while other patients received irinotecan at 125 mg/m2. Although the significance of radiographic changes in treated gliomas is questionable, based on study design the authors alleged an overall response rate of 63% for all patients. In other trials of bevacizumab patients with brain metastases were excluded because of concerns that there might be an increased risk of intracerebral hemorrhage. However, in this trial there were no instances of central nervous system hemorrhage, but four patients (12%) experienced thromboemboic complications.

356

Sarcomas Seventeen patients with metastatic soft tissue sarcoma were treated with doxorubicin at 75 mg/m2 i.v. followed by bevacizumab 15 mg/kg i.v. every 3 weeks [143]. Dexrazoxane was given as a cardio-protectant once the total doxorubicin dose exceeded 300 mg/m2. Six patients developed cardiac toxicity grade 2 or greater despite close monitoring and standard use of dexrazoxane.

Mechanisms of Anti-Tumor Activity Bevacizumab probably promotes anti-tumor effects by a number of mechanisms. The original concept for bevacizumab activity was that it would decrease tumor angiogenesis by blocking VEGF, resulting in a decrease in tumor blood supply which in turn would lead to cancer cell death. However, it appears that bevacizumab treatment is also associated with afferent vascular dilatation and efferent vascular constriction of tumor vessels, which may help concentrate chemotherapy at the tumor site. One study in patients with rectal cancer showed that the bevacizumab decreased tumor perfusion, vascular volume, interstitial fluid pressure, microvascular density and circulating endothelial cells while increasing the proportion of blood vessels with percytes [785]. The decrease in interstial fluid pressure might allow easier penetration for the Mab itself, which might carry some chemotherapy molecules with it. Another study in patients with inflammatory breast cancer demonstrated a decrease of in phosphorylated VEGFR2 in tumor cells, a decrease in vascular permeability, and an increase in tumor cell apoptosis, but no significant changes in microvessel density or VEGF-A expression [771]. In the pivotal trial of IFL and bevacizumab as initial treatment for metastatic colon cancer, levels of epithelial and stromal VEGF, stromal thrombospondin-2, and microvessel density failed to predict clinical benefit associated with the addition of bevacizumab as opposed to placebo [356].

Toxicities and Adverse Events The toxicities associated with bevacizumab are summarized earlier in this chapter in Table 6. The package insert for the product warns of hemorrhage, hypertension, proteinuria, impaired wound healing, GI perforation, and congestive heart failure. The most serious, and sometimes fatal, bevacizumab associated toxicities are gastrointestinal perforation, wound-healing complications, hemorrhage, arterial thromboembolic events, hypertensive crisis, nephrotic syndrome, and congestive heart failure [211]. The impaired wound-healing has led to suggestions of waiting at least 4 to 6 weeks after bevacizumab has been discontinued before taking a patient to surgery,

Monoclonal antibody therapy and waiting at least 2 to 3 weeks after surgery before instituting bevacizumab therapy. Bowel perforation has been a problem in the setting of gastrointestinal and ovarian cancers that are prone to produce tumor implants in the bowel wall, and may be a result of anti-tumor effect. Interestingly, the adverse events have varied somewhat by tumor types, and some may reflect anti-tumor effects rather than non-specific vascular effects. For instance, massive hemoptysis has typically been limited to patients with large centrally located lung cancers, and bowel perforation has been more common in colon cancer and especially ovarian cancer, entities often associated with metastatic implants on bowel surfaces. Although patients with brain metastases have been excluded from many studies, in part because of a fear of intracranial hemorrhage, bleeding has not been a problem in patients with glioblastoma who have received bevacizumab. Hypertension and proteinuria occur in all settings, and may be due to effects on small renal vessels since VEGF is involved in repair of glomeruli endothelial cells. Hypertension is usually readily controlled with a single anti-hypertensive agent. Protenuria can progress to full-blown nepthrotic syndrome. Patients should be monitored for protenuria and treatment discontinued if protenuria exceeds 2 g/24 h. Increased rates of expistaxis have been noted in many trials, but increased rates of vascular thrombosis have also been noted.

Summary Despite some safety concerns, bevacizumab has been a tremendous addition to our therapeutic armamenterarium. Unlike most targeted therapies, it is potentially useful in every type of solid tumor because of the importance of tumor angiogenesis. Bevacizumab seems to enhance chemotherapy in virtually every tumor type in which chemotherapy is active, and is already in widespread use in cancers of the colon, lung, breast, brain, ovary, and kidney. Although its single-agent activity appears limited, bevacizumab alone may be useful in the adjuvant setting, or following complete remission after systemic therapy, because of the inhibition of neoangiogenesis required to develop metastatic tumors.

Cetuximab (Erbitux®), Bristol-Myers Squibb Cetuximab and the Epidermal Growth Factor Receptor The rationale for targeting the epidermal growth factor receptor (EGFR), which is expressed to some extent on all epithelial tumors, was addressed earlier in this chapter. The human epidermal growth factor receptor (EGFR),

Robert O. Dillman

357 The anti-EGFR chimeric Mab cetuximab (Erbitux) was approved in 2004 based on randomized trials in which the agent was combined with the chemotherapy agent irinotecan in the treatment of metastatic colorectal cancer. It is a recombinant chimeric monoclonal antibody that includes a murine Fv and human IgG1 kappa constant regions. Unlike most monoclonal antibody products, cetuximab is manufactured in murine plasmacytoma cells. This cellular factory results in murine glycosylation which may increase the risks of allergic reactions in some patients.

and its ligands, such as transforming growth factor alpha (TGF-α, have long been recognized as a potential targets for antibody-based therapy. EGFR is associated with tyrosine kinase activity, and is frequently overexpressed in cancers of the breast, ovary, bladder, head and neck, prostate, pancreas, and colon. EGFR are expressed at high levels in about one third of all epithelial cancers, and are associated with accelerated growth [480]. Autocrine activation and overexpression of EGFR appears to be crucial for the accelerated growth of many cancers and is associated with increased expression of VEGF. The murine antibody to EGFR-1 called 225 was shown to block receptor function and inhibit cell growth in cultures in nude mouse xenografts [471]. The Mab bound to the extracellular domain of EGFR causing internalization of the receptor and antibody and “downregulation” of the receptor. For this reason most early development with anti-EGFR antibodies focused on immunoconjugates, especially ricin immunotoxins [472]. Cetuximab, the chimeric form of the antibody, was actually more effective in the mouse tumor models than the murine antibody, apparently because of a higher affinity for the EGFR target [571]. Internalization of the receptor is slow enough that cetuximab is cytotoxic in vitro in the presence of human effector cells and complement, but its major anti-tumor mechanism is believed to relate to the receptor blockade preventing signal transduction that is associated with cellular proliferation. In laboratory tests against tumor cells and in animal models with human tumor xenografts, cetuximab enhanced the chemotherapy effects of many different chemotherapy agents including platinums, campothecins, taxanes, fluoropyrimidines,, and gemcitabine.

Clinical Trials with Cetuximab Colorectal cancer Many of the initial trials with cetuximab, as a single agent and in combination with chemotherapy, were conducted in patients with colorectal cancer, as summarized in Table 39. Trial designs included cetuximab alone, and cetuximab in combination with irinotecan in patients with metastatic colorectal cancer that had previously progressed in the face of irinotecan. In these trials cetuximab was given at an initial loading dose of 400 mg/m2 i.v. over 2 h, then 250 mg/m2 i.v. over 1 h weekly. Saltz et al. treated 57 patients whose metstatic cancer had not responded to prior irinotecan alone or as part of combination chemotherapy [628]. Patients were required to have EGFR demonstrated on formalin-fixed paraffinembedded tumor tissue by IHC, but it is now known that quantitative expression of EGFR is not predictive of response to cetuximab treatment. The most common adverse events were an acne-like skin rash, predominantly

Table 39. Trials of cetuximab in patients with metastatic colorectal cancer Citation

Regimen

Clinical setting

# of patients

Response rate

Median time to progression

Median overall survival

[628] [424]

Cetuximab Cetuximab

57 346

9% 12%

1.4 months 1.4 months

6.4 months 6.6 months

[355]

Cetuximab vs. supportive care

Prior irinotecan Prior 5FU, irinotecan, oxaloplatin Prior 5FU, irinotecan, oxaloplatin

287

8%

41% 3 months

6.1 months

285

0%

111 218 60

11% 23% 20%

24% 3 months p = 0.001 1.5 months 4.1 months 3.1

4.6 months p = 0.005 8.6 months 6.9 months 6 months

55

25%

4.7 months

9.8 months

43

72%

12.3 months

30 months

[136] [245]

Cetuximab Cetuximab + irinotecan Cetuximab + irinotecan

[752]

Cetuximab + irinotecan

[703]

Cetuximab + FOLFOX

Irinotecan refractory Prior 5FU, irinotecan, oxaloplatin Refractory to irinotecan or oxaloplatin First-line

5FU = 5-fluorouracil FOLFOX = folate (leucovorin), 5-fluororuracil, oxaloplatin

358 on the face and upper chest (86%) and the constellation of asthenia, fatigue, malaise, or lethargy (56%). Three patients were reported to have had a grade 3 allergic reaction; two were withdrawn. Lenz et al. treated 346 patients whose metastatic cancer was considered refractory to fluoropyrimidines, irinotecan and oxaliplatin [424]. EGFR positivity by IHC was an eligibility requirement, but degree of positivity did not correlate with clinical benefit. The most prevalent toxicity was the acneiform rash which was noted in 83% of patients, which was predictive of clinical benefit. In a randomized trial Jonker et al. randomized 572 patients who had progressed despite 5FU, irinotecan, and oxaloplatin, to standard dose cetuximab or observation [355]. Although the objective response rate was only 8%, OS and PFS were better in the cetuximab arm. Severe adverse events of rash and infection were more common in the cetuximab arm. Hypomagnesemia was also more common with cetuximab. Eleven patients discontinued cetuximab because of infusion reactions. In a regulatory pivotal trial Cunningham et al. randomized 329 patients with colorectal cancer, that had progressed during or within 3 months after discontinuation of irinotecan, to receive irinotecan plus cetuximab or cetuximab alone using a 2: 1 randomization [136]. Protocol prescribed irinotecan was to be the same as used prestudy. The response rate was higher for the combination therapy (p = 0.007), as was PFS (p < 0.001). Gebbia et al. treated 60 patients who had received at least two prior therapies, and whose metastatic cancer was considered refractory to oxaliplatin and irinotecan [245]. Standard cetuximab dosing was combined with irinotecan 120 mg/m2 weekly for 4 out of 6 weeks. Responses were not predicted by EGFR expression. The main grade 3 and 4 toxicities were mostly attributable to the chemotherapy, and included nausea (33%), diarrhea (27%), leukopenia (18%), asthenia (13%), and acneiform skin rash (13%). Vincenzi et al. treated 55 patients whose metastatic cancer was considered refractory to oxaliplatin and irinotecan [752]. Standard cetuximab dosing was combined with irinotecan 90 mg/m2 weekly. Skin toxicity was observed in 89% of patients. The most common grade 3 to 4 adverse events were dermatologic (33%), diarrhea (16%), fatigue (13%) and stomatitis (7%). Fever was noted 25%, typically in association with the first infusion of cetuximab, but no allergic reactions were recorded. More recently cetuximab has been combined with modern combination therapies such as FOLFOX and FOLFIRI as the initial treatment of patients with metastatic colorectal cancer. Tabernero et al. used FOLFOX

Monoclonal antibody therapy and cetuximab as the initial treatment for 43 patients with metastatic colorectal cancer [703]. Cetuximab was given day 1 at a dose of 400 mg/m2 during week 1, and then 250 mg/m2 weekly thereafter. Treatment was welltolerated and the response rate was 72%. In a randomized trial of 337 patients, a preliminary report showed that cetuximab plus FOLFOX was superior to FOLFOX alone in terms of response rate (46% vs. 36%, p = 0.064) [46]. In a randomized trial of 1,198 patients, an initial report showed that cetuximab plus FOLFIRI was superior to FOLFIRI alone in terms of response rate (47% vs. 39%, p = 0.004) and PFS (8.9 vs. 8.0 months, p = 0.048) [741]. Cetuximab is also being explored in rectal cancer. Based on a trial in 40 patients with rectal cancer, preoperative radiotherapy in combination with capecitabine and cetuximab is feasible [452]. Cetuximab in combination with capecitabine, weekly irinotecan, and radiotherapy is being evaluated as neoadjuvant therapy for rectal cancer [324].

Squamous Cell Cancers of the Head and Neck Cetuximab has also been extensively evaluated in squamous cell cancers of the head and neck as shown in Table 40. Vermorken et al. gave single-agent cetuximab at the standard dose and schedule to 109 patients who had recurrent and/or metastatic disease that progressed during platinum chemotherapy [751]. Acneiform skin rash, which occurred in 49%, was the most common toxicity There was one death due to an infusion-related allergic reaction. The response rate was similar to that of single-agent cetuximab in colorectal cancer. Phase I trials suggested that cetuximab could be safely given with radiation therapy and might enhance therapeutic benefit [602]. Bonner et al. conducted a multinational trial comparing cetuximab plus radiotherapy to radiotherapy alone in 424 patients with locoregionally advanced disease, that showed a benefit for the addition of cetuximab including a 26% reduction in death with an increase in survival from 29 to 49 months (p = 0.03) [47]. There was no increase in toxicity other than the acneiform rash. Pfister et al. treated 22 patients locoregionally advanced disease with cisplatin, cetuximab, and 70 Gy of radiotherapy [553]. This trial was closed early because of several adverse events related to infection and cardiac events. Grade 3 or 4 cetuximab-related toxicities included acneiform rash in 10% and hypersensitivity in 5%. With a median follow-up of 52 months, the 3-year overall survival rate is 76%, the 3-year progression-free survival rate is 56%, and the 3-year locoregional control rate is 71%.

Robert O. Dillman

359

Table 40. Trials of cetuximab in patients with squamous cell cancer of the head and neck Citation Treatment regimen

Clinical setting

Number of patients

Response rate

Median PFS

[751]

Cetuximab

103

13%

1.6 months

[47]

Cetuximab or Placebo + 70 Gy RT

Prior platinum, recurrent, metastatic Loco-regional advanced

211 213

– –

[553] [309]

Cetuximab + Cisplatin + RT Cetuximab + cisplatin

22 130

– 13%

[26] [72]

Cetuximab + cisplatin Cisplatin + cetuximab Cisplatin + placebo

Stage III or IV, M0 Refractory to cisplatin + 5FU or paclitaxel refractory to cisplatin Recurrent or metastatic

24 months 15 months p = 0.005 56% 3-year –

96 117 10%

2.8 months 4.2 months

[54]

Cetuximab + 5FU + Cisor carboplatin Carboplatin

Recurrent or metastatic

53

10% 26% 2.7 months p = 0.03 36%

Nasopharynx recurrent or metastatic

59

12%

3.2 months

[102]

5FU = 5-fluoruracil PFS = progression free survival

Herbst et al. treated 130 patients, who did not have an objective response or relapsed within 90 days of completing two cycles of cisplatin/paclitaxel or cisplatin/fluorouracil, with standard cetuximab and cisplatin (75 or 100 mg/m2 i.v.) every 3 weeks [310]. The most common toxicities were anemia, acneiform skin rash, leukopenia, fatigue/malaise, and nausea/vomiting. Seven patients (5%) experienced a grade 3 or 4 hypersensitivity reaction to cetuximab. Baselga et al. treated 96 patients, who were refractory to cisplatin, with standard cetuximab and cisplatin at the same dose and schedule during which progressive disease had occurred [26]. Acneiform rash was the most common toxicity. Burtness et al. randomized 117 patients who had recurrent or metastatic disease to receive cisplatin every 4 weeks with weekly cetuximab or placebo [72]. Response rate was higher with the addition of cetuximab, but PFS and OS were not improved. Survival was better for those patients who developed the skin rash. Bourhis et al. treated 53 patients who had recurrent or metastatic disease with a combination of cetuximab, cisplatin or carboplatin, and escalating doses of 5FU as initial therapy [54]. Dermatologic toxicity was the most common adverse event, but the most common grade 3 or 4 adverse events were leucopenia (38%), asthenia (25%), thrombocytopenia (15%), vomiting (14%). Chan et al. treated 59 patients who had recurrent or metastatic nasopharyngeal cancer that had recurred after cisplatin, with cetuximab and carboplatin [102].

Six patients (10%) experienced serious treatment-related adverse events during cetuximab.

Cancers other than Colorectal and Head and Neck Exploratory trials with cetuximab have been conducted in a variety of other tumor types as shown in Table 41. Pinto et al. treated 34 patients who had advanced gastric or gastroesophageal junction adenocarcinoma with cetuximab and the FOLFIRI regimen as first-line therapy for up to 24 weeks, with cetuximab alone continued for patients whose tumors had not progressed [561]. Median survival was 12 months. Grade 3 to 4 toxicity included neutropenia (42%), acneiform rash (21%), diarrhea (8%), asthenia (5), stomatitis (5), and hypertransaminasemia (5%) with one treatment-related death. In pancreatic cancer, Xiong et al. treated 41 untreated patients who had measurable locally advanced or metastatic pancreatic cancer and whose tumors had EGFR expression by IHC, with cetuximab and gemcitabine [797]. Median survival was 7.1 months and 1-year survival was 32%. The most common grade 3 to 4 adverse events were neutropenia (39.0%), asthenia (22.0%), abdominal pain (22.0%), and thrombocytopenia (17.1%). Despite the encouraging results of this phase II trial, preliminary results of a randomized trial conducted by the Southwest Oncology Group (SWOG) in 735 patients with advanced pancreatic cancer showed no advantage for the combination therapy over gemcitabine alone with a response rate of only 12%, and the same median survival of approximately 6 months that has been

360

Monoclonal antibody therapy

Table 41. Clinical trials of cetuximab in patients with other tumor types other than colorectal or head and neck [561]

Cetuximab FOLFIRI

[797] [559]

Gemcitabine Cetuximab + Gemcitabine Cetuximab alone Paclitaxel + Carboplatin Gemcitabine & Carboplatin Cetuximab

[293] [715] [603] [509]

Number of patients

Response rate

Median PFS

Median OS

Gastric and GE junction untreated Pancreas advanced Pancreas advanced

34

44%

8 months

12 months

41 352

12% 12%

3.8 months 3.5 months

7.1 months 6.5 months

NSCLC previously treated NSCLC untreated metastatic

66 31

5% 26%

2.3 months 5 months

8.9 months 11 months

NSCLC untreated metastatic

35

29%

5.4 months

10.2 months

Renal cell metastatic

55

0%

1 month

49 months 29 months p = 0.03

FOLFIRI = folate (leucovorin), 5-fluorouracil, irinotecan GE = gastroesophogeal NSCLC = non small cell lung cancer PFS = progression free survival OS = overall survival

observed in virtually all randomized trials involving such patients [559]. In NSCLC Hanna et al. observed a response rate of only 5% for cetuximab alone in 66 patients with metastatic disease who had progressed after previous systemic therapy [293]. Grade 3 to 4 toxicities were uncommon, but included acneiform rash (6%), anaphylactic reactions (2%), and diarrhea (2%). Other studies have combined cetuximab with chemotherapy. Thienelt et al. treated 31 previously untreated patients with paclitaxel, carboplatin, and cetuximab [715]. Over 80% of patients experienced dermatologic toxicity, with 13% grade 3 or 4. Robert et al. treated 35 patients with gemcitabine and carboplatin [603]. The most common toxicities attributed to cetuximab were acneiform rash (89%), asthenia (31), fever/chills (20%), and nausea/ vomiting (17.1%). In metastatic renal cell cancer, Motzer et al. observed no responses with single-agent cetuximab in 55 patients [509]. Grade 3 to 4 skin toxicity was noted in 17%. These results were so discouraging that cetuximab has not been evaluated further in kidney cancer. A number of trials are in progress that combine cetuximab with combined modality chemotherapy plus radiation therapy regimens. A trial combining cetuximab with gemcitabine and radiation therapy was being conducted in patients with locally advanced pancreatic cancer [408]. A trial of cetuximab with temozolomide and radiation therapy is being conducted in patients with previously untreated glioblastoma [126]. Cetuximab plus intensity modulated radiation therapy in non small cell lung cancer [349].

Mechanisms of Anti-Tumor Activity and Toxicity Sustained blocking of EFGR is believed to be critical for the anti-tumor effects of cetuximab. Based on randomized trials exploring single doses of 50, 100, 250, 400, or 500 mg/m2, a dose of 250 mg/m2 was predicted to nearly saturate epidermal growth factor receptors [237, 706]. Skin biopsies in 39 patients, before and after i.v. infusions of cetuximab, confirmed a dose-dependent relationship with suppression of EGFR expression [237]. In these trials there was no association between dose and cutaneous rash, but rash predicted for clinical benefit. Pharmacokinetic trials of cetuximab with irinotecan revealed no change in cetuximab clearance or metabolism when combined with irinotecan compared to infusions of cetuximab alone [154, 213]. Because all preclinical testing of cetuximab and its murine precursors were conducted in animals bearing tumors that strongly expressed EGFR, most early trials restricted patient eligibility to those whose tumors had documentable expression of EGFR by IHC. However, in clinical practice it is now generally accepted that IHC testing for EGFR expression is not necessary for determining appropriateness of cetuximab-based therapy, because of inherent limitations in the assays and potential for sampling error, and because responses have been described in patients whose tumors were EGFR-negative by IHC. In eight colorectal cancer patients who were refractory to irinotecan, and whose tumors were negative for EGFR by IHC, four responded to cetuximab plus irinotecan therapy [301]. A small trial of 31 patients

Robert O. Dillman suggested that EGFR gene copy number might predict benefit [504]. However, in a much larger prospective trial, cell surface expression of EGFR, EGFR kinase domain mutations, and EGFR gene amplification did not predict clinical benefit, probably because of the heterogeneity of tumors [355, 424]. A pilot study involving tumors from 39 colorectal cancer patients suggested that cyclin D1 A870G and EGF A61G polymorphisms may predict efficacy of single-agent Cetuximab [806]. The most common side effect associated with cetuximab and other anti-EGFR therapies is a characteristic acneiform rash, which is most prominent on face, chest and upper back [74]. In many trials the prevalence of this side effect was over 80%. The rash usually appears within a week of starting treatment. Serial punch biopsies in patients revealed two main reaction patterns: a superficial dermal inflammatory cell infiltrate, and a superficial folliculitis. The rash can be severe and should be treated to decrease the risk of secondary infection and cellulites. Grade 3 to 4 dermatitis was reported in 5–15% of patients in most trials. Prophylactic topical lubricants are sufficient in many patients, but others require treatment with tetracycline antibiotics or clindamycin. Other common side effects include asthenia and diarrhea. Cetuximab does not appear to increase the toxicity of any of the chemotherapy agents with which it has been combined. Black box warnings for cetuximab include infusion reactions and cardiac arrest. The somewhat high rate of severe infusion related reactions, many of which have been characterized as anaphylactic reactions, is of concern. At this time it is not clear whether this is actually do to cross-reactivity with antigens on circulating cells in some patients, effects of Fc interactions with effector cells, or a true allergic reaction to murine amino acids and/or glycosylation because of the murine plasmacytoma cell line used to produce the product. There was a 2% risk of sudden death in 208 patients with squamous cell cancer of the head and neck who received cetuximab with radiation therapy [47].

Summary Cetuximab is widely used in combination with chemotherapy for patients who have relapsed after primary treatment for colorectal cancer. However, based on the comparative strengths and differences of clinical trials, bevacizumab with combination chemotherapy is usually preferred as the initial treatment. Cetuximab is increasingly being used with radiation therapy in patients who are considered poor candidates to receive combined modality treatment with chemotherapy and radiation therapy, which is currently standard for treatment of

361 locoregionally advanced squamous cell cancers of the head and neck. It will be interesting to see randomized comparisons of chemotherapy plus radiation vs. cetuximab plus radiation, but there are also trial comparing cetuximab plus chemoradiotherapy to chemoradiotherapy alone. There may be a role for cetuximab with chemotherapy in NSCLC, but again bevacizumab with chemotherapy is currently the standard initial therapy.

Panitumumab (Vectibix™) Amgen, Thousand Oaks, California Panitumumab and EGFR The epidermal growth factor receptor (EGFR) monoclonal antibody panitumumab (Vectibix) was approved in 2006 based on randomized trials in which the agent was superior to placebo in patients with relapsed, refractory, metastatic colorectal cancer. Formerly known as ABX-EGF, panitumumab was the first fully human monoclonal antibody to receive regulatory approval and the first totally human Mab approved for cancer therapy [115, 120]. It is a human IgG2. Like the chimeric Mab cetuximab, the human Mab panitumumab binds to the extracellular domain of EGFR causing internalization of the receptor and antibody and “down-regulation” of the receptor with disruption of potential downstream signal transduction that enhances proliferation and resistance to apopotosis in normal and malignant cells. Panitumumab was produced with the hope that its clinical efficacy might be even greater than cetuximab because it is a totally human construct, which may offer advantages in terms of pharmacokinetics, decreased allergenic potential, and possibly enhanced ADCC. The anti-tumor activity of panitumumab was confirmed in vitro and in vivo, against many cancers, including lung, kidney and colorectal. In these models it was well tolerated and clinically active both as monotherapy and in combination chemotherapy agents. Panitumumab was originally made in transgenic mice, but is manufactured in CHO cells.

Clinical Trials with Panitumumab The few clinical trials published for panitumumab are summarized in Table 42. A pivotal trial for regulatory approval compared panitumumab at 6 mg/kg i.v. every 2 weeks to best supportive care in patients with metastatic colorectal cancer whose disease had progressed during or after standard therapy with fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens [254, 255, 741, 742]. All patients had epidermal growth factor receptor (EGFR)-expressing tumors.

362

Monoclonal antibody therapy

Table 42. Trials of panitumumab in patients with metastatic colorectal cancer Citation

Clinical setting

Treatment

# of patients

Response rate

[741]

Progressed after prior chemo

Panitumumab Supportive care

231 232

10% 0%

[302] [35] [35]

Progressed after prior chemo No prior therapy No prior therapy

Panitumumab 148 IFL + panitumumab 19 FOLFIRI + 24 panitumumab

9% 46% 42%

Median time to progression

Median overall survival

8 weeks 7.3 weeks p < 0.0001 14 weeks 5.6 months 10.9 months

– – 9 months 17 months 22 months

IFL = irinotecan, 5-fluorouracil, and leucovorin FOLFIRI = folate (leucovorin), 5-fluorouracil, irinotecan

Panitumumab produced a 10% objective response rate and a longer progression free survival compared to supportive care alone. There was no difference in survival, but approximately 75% of patients in the supportive care alone arm crossed over to receive panitumumab after disease progression. Quality of life was also better in the patients who received panitumumab [668]. For 176 patients who progressed on the supportive care arm, and then subsequently did receive panitumumab, 12% had an objective response and two patients had complete responses [742]. Hecht et al. treated 148 patients whose metastatic colorectal cancer had progressed on chemotherapy that included a fluoropyrimidine and irinotecan or oxaliplatin, or both [302]. Panitumumab was given i.v. at a dose of 2.5 mg/kg weekly for 8 of each 9 weeks until disease progression or excessive toxicity. Skin toxicity occurred in 95% and 5% were grade 3 or 4. Four patients discontinued therapy because of toxicity and one patient had an infusion reaction, but was able to resume treatment. EGFR of at least 1+ by IHC was an eligibility requirement. There was no difference in response for 105 patients who were judged as having high EGFR by IHC compared to 43 patients who were characterized as having a low EGFR. Berlin et al. tested the combination of irinotecan, 5-FU and leucovorin, and panitumumab as initial therapy in patients with metastatic colorectal cancer [35]. This was a two-part, multicenter, phase II study of panitumumab 2.5 mg/kg weekly with bolus 5-FU (IFL) in the first part of the trial, and infusional 5-FU (FOLFIRI) in the second part. Grade 3 to 4 diarrhea occurred in 11 patients (58%) in part 1 and six patients (25%) in part 2. All patients had dermatologic toxicity, but none was grade 4. The authors concluded that panitumumab + FOLFIRI was better tolerated than panitumumab + IFL.

Toxicities The toxicities and adverse events associated with panitumumab are summarized earlier in this chapter in Table 6. Panitumumab black box warnings include dermatologic toxicities and infusion reactions. Panitumumab was developed in the belief that it would be less toxic and more active than cetuximab or other less than completely human antibodies. Panitumumab has been well tolerated whether administered alone or in combination with chemotherapy. However, there was a 1% rate of anaphylaxis in trials submitted for registration of the agent. During eight clinical trials, a very sensitive assay detected anti-panitumumab responses in 25 of 604 (4.1%) subjects, and eight developed neutralizing antibodies [445]. There is a report of at least one patient who had a severe infusion reaction during treatment with cetuximab, who was then successfully treated with panitumumab [332], but because most infusion reactions are seen only with a first infusion, it is not clear that decreased toxicity will be an important benefit, since most infusion reactions are due to antigen–antibody interactions rather than antibody reactions to murine components of the chimeric or humanized Mab. Similar to other agents targeting the epidermal growth factor receptor pathway, skin rash has been the primary toxicity recognized in association with panitumumab therapy, and has occurred in 100% of patients receiving doses of 2.5 mg/kg or higher. Hypomagnesaemia and diarrhea were also most commonly reported. No grade severe or life-threatening reactions were noted in the large randomized trial in patients with colorectal cancer [741, 742]. Other common adverse events noted were paronychia, fatigue, abdominal pain and nausea. The most serious adverse events were severe dermatologic toxicity complicated by infectious sequelae and septic

Robert O. Dillman death, infusion reactions, pulmonary fibrosis, hypomagnesemia, and gastrointestinal problems including abdominal pain, nausea, vomiting, diarrhea, and constipation.

Summary Panitumumab is the first fully human antibody to receive regulatory approval, and joins cetuximab as an anti EGFR Mab. It remains to be seen whether the to totally human construct offers any meaningful advantages of the chimeric construct.

Conclusions “Magic bullets” are now part of the standard anti-cancer therapeutic armamentarium [168]. It has now been more than a decade since rituximab and trastuzumab became the first Mabs approved for the treatment of cancer, and both have become well-established “blockbuster drugs.” Alemtuzumab would be more widely used in the treatment of hematologic malignancies if its target was more specific, and rituximab was not so safe and effective in the treatment of B cell malignancies. While five of these agents are limited in their scope of malignant targets because of their specificity for surface antigens, bevacizumab has the potential to be useful in every cancer setting because of the importance of the VEGF ligand for tumor angiogenesis. Cetuximab and panitumumab are potentially useful in the same patient populations, and it will be interesting to see whether the totally human Ig has any meaningful clinical advantages over the chimeric product. It is interesting that other than rituximab, these agents have rather limited anti-tumor effects, but all dramatically increase the effects of chemotherapy, which is what has led to their rapid and widespread adoption in cancer therapy. The potential for synergistic and additive effects resulting from the use of Mab in combination with other biological response modifiers has not been established, and certainly is not superior to the common chemotherapy plus Mab combinations.

Other Antibodies and Antigens This section is organized by antigenic targets and covers many other Mab that have investigated but are not approved for clinical use as anti-cancer therapy, although some are clinically available based on other indications. Some trials that involved tracer quantities of radiolabeled Mabs and are also included if in fact patients received at least several mg of unmodified antibody. The information described is based on publication in the

363 medical literature and cannot be considered all inconclusive because of the volume of unpublished results known only to certain companies who have pursued commercial development of certain antibodies, and the US FDA.

Anti-Idiotype Antibodies The idiotype of malignant B-cell clones is perhaps the best example of a tumor-specific antigen. More than 15 years before the first approval of a Mab for the treatment of malignancy, the first antibody-mediated CR was reported following administration of a murine antiidiotype antibody [487]. The patient had follicular B cell lymphoma that had progressed after prior treatment with cyclophosphamide–vincristine–prednisone chemotherapy, and interferon-alpha biotherapy. The complete response persisted for over 7 years. Subsequent publications established a 66% response rates among 45 patients with indolent lymphoma who received anti-idiotype antibodies alone, or administered with single-agent chlorambucil, interferon-alpha, or interleukin-2 [146]. Despite the promising early results with anti-idiotype antibodies, commercial development in this area has not evolved because of the need to develop a specific antiidiotype antibody for each patient. Interest for commercial development increased when several investigators suggested that combinations of anti-idiotype antibodies commonly expressed on B-cell lymphomas might be a useful clinical product [16, 394, 489, 614, 692, 702], but enthusiasm dwindled when the frequency of these “shared” or “cross reactive” idiotypes was found to be much lower than originally suggested.. The company IDEC of San Diego, CA, which was originally founded as an anti-idiotype company, subsequently acquired Analytical Biosystems of Sunnyvale, California, established by Levy and Miller of Stanford, which was the first anti-idiotype company to pursue the anti-idiotype approach. IDEC eventually dropped development of a composite anti-idiotype preparation called Specifid® in order to focus its resources on a product called C2B8, a chimeric anti-CD20 antibody that became the blockbuster product rituximab [168]. The largest experience with anti-idiotype antibodies was in lymphoma as initially reported by Meeker et al. [474] and subsequently by others at Stanford who were working with Ron Levy [60, 61, 146, 446, 458]. Among 17 patients receiving individualized anti-idiotype antibodies at doses ranging from 400 mg to over 9 g, there were nine partial responses including the sustained, complete remission in the first patient treated, and others lasting from 1 to 6 months [478, 487]. Favorable responses were

364 associated with increased T-cell infiltration of involved lymph nodes [446]. In some cases, resistance to therapy was shown to result from emergence of idiotype-variant clones [478]. Stanford investigators combined IFN-α with anti-idiotype antibodies based on the rationale that IFN-α might upregulate the idiotype antigen expression, and IFN-α is known to have antiproliferative effects on follicular B-cell lymphoma cells. Of 12 patients treated in this manner, who received total antibody doses ranging from 1.7 to 8.0 g, there were two CRs and seven PRs [61]. Another trial combined chlorambucil with antiidiotype antibodies and one CR and seven PR were described in 13 patients [458]. It was not clear that either of these combination therapies was more effective than anti-idiotype antibodies alone because of changes in patient selection and the known efficacy of both IFN-α or chlorambucil as single agents in the treatment of follicular lymphoma. Other groups have reported on limited trials with anti-idiotype antibodies with limited success [99, 291, 581]. Rankin et al. noted minimal antitumor effects in two lymphoma patients who received murine anti-idiotype Mab at individual doses from 5 to 160 mg and total doses of 3.8 and 5.8 g respectively [581]. Another report relates an initial experience with a mouse/human chimeric anti-idiotype in a patient with B-cell lymphoma who had no tumor response [291]. The brief and limited responses observed in CLL may have been due to the relatively low doses of Mab administered in those trials [99].

Antigens Associated with Hematopoietic Cells Because of the ready availability of clinical material for testing, many of the first murine Mab were developed after immunization with hematopoietic cells. The various antigens identified on blood cells were classified by a cluster designation (CD) numbering system [36]. Many of these antibodies were utilized in early pilot, phase I, and phase I/II trials.

CD3 CD3 is a pan-T cell antigen and an important receptor for activation of T lymphocytes. The first monoclonal antibody approved for any use, was the murine antibody OKT3 which now has the generic name muromonab-CD3 [638], approved to prevent kidney transplant rejection in 1984. Theoretically, CD3 might be a target as a tumor associated antigen in T cell lymphoma, or to activate normal T cells to modulate or induce an indirect antitumor action through other components of the immune system.

Monoclonal antibody therapy Urba et al. treated 36 patients with OKT3 antibody in an effort to activate T cells in the hope of promoting an antitumor effect [737]. Five patients received a 30 ug dose by i.v. bolus and the other 23 received 3-h infusions of 1, 10, 30, or 100 ug. An additional 8 patients received the anti-CD3 daily for 14 days by either bolus 3-h infusion, or 24-h infusion. The dose-limiting toxicity in this study was headache, accompanied by signs and symptoms of meningeal irritation. Eight of the 16 patients tested exhibited HAMA. No tumor responses were observed. Richards et al. treated 13 patients with OKT3. Six patients received 50 ug, and 7 received 100 ug [593]. A partial response was described in one patient with metastatic renal cell carcinoma. Again, neurotoxicity was a significant problem and was observed in 11/13 patients after the first treatment. Headache and confusion were noted. In all patients, neurotoxicity was transient and interestingly, did not recur with re-treatment. In both of these this study and that by Urba et al, a cerebral spinal fluid lymphocytosis was noted in patients who underwent lumbar puncture, and headache was a frequent complaint [593, 737]. Because of the evidence that OKT3 had stimulated an immune response in the central nervous system, Wiseman et al. conducted a trial with OKT3 in patients with gliomas who had failed conventional therapy and evidence of progressive disease [789]. Patients received 25 to 75 ug of OKT3 over 1-h, followed a day later by 300 mg/m2 of cyclophosphamide as a dose intended to reduce effects of suppressor or regulatory T cells. Three of nine patients were reported to have had objective tumor regressions, based on brain studies with magnetic resonance imaging. Anticipated side effects included headache, fever, stupor, nausea, emesis, and transient decreases in T-lymphocyte counts, but no severe or life-threatening toxicity. Several investigators have explored the use of the T-lymphocyte cytokine interleukin-2 (IL-2) in combination with OKT3 because of the potential for synergistic or additive T-cell stimulation. Sosman et al. treated 54 patients with doses of OKT3, ranging between 75 to 600 ug/m2 followed by high-dose bolus IL-2 therapy [684]. The numbers of circulating T cells expressing the IL-2 receptor were not increased, and the tumor response rate was no better than had been observed with IL-2 alone. Buter et al. gave 50 to 400 ug OKT3 with lowdose s.c. IL-2 to eight patients [76]. Neurotoxicity was the limiting toxicity at the highest dose. There was no enhancement of activated lymphocytes and no responses. Another approach involves the production of antibodies that are genetically fused to various cytokines to function as targeted biological response modifiers [388].

Robert O. Dillman These multifunctional products are covered in other parts of this section based on the tumor antigen that is being targeted. A humanized anti-CD3 antibody, initially called HuM291, and now named visilizumab, has been studied in a variety of disorders to suppress activated T lymphocytes, including ulcerative colitis, kidney rejection, and graft vs. host disease following allogeneic stem cell transplants [91, 367, 525, 563, 592]. Visilizumab is a mutated IgG2 isotype whose Fc tail does not bind to Fc gamma-receptors, but can induce apoptosis of activated T lymphocytes. Doses from 0.1 to 15 mg/kg have been infused in various trials, and infusion related toxicities and CNS toxicities seem to be much less than were observed with muromonab. To date there are no reports of its use in cancer patients.

CD4 CD4 is typically associated with helper T cells, and is expressed on many T cell malignancies. Knox et al. treated seven T-cell lymphoma patients with an antiCD4 chimeric Mab [397]. Doses of 10, 20, 40, and 80 mg were given i.v. twice a week for 3 consecutive weeks. Circulating cells were coated with antibody, but there was no significant change in the number of T cells. Some transient benefit was seen in all patients, but these clinical results were not clearly better than those seen with anti-CD5 murine Mab. Zanolimumab is a human IgG1 antibody against CD4 which may exert anti-tumor effects by several mechanisms [594]. Two are regulatory mechanisms that include rapid inhibition of signal transduction mediated by the CD4-associated tyrosine kinase p56lck, and then chronic down-regulation of CD4 molecules. The third is direct Fc-dependent lysis of circulating CD4+ T cells; CD45RO+ cells are more sensitive to ADCC killing than CD45RA+ cells. Zanolimumab is being evaluated in cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphomas (PTCL) Kim et al. reported results of two phase two trials of zanolimumab in 37 patients with refractory CTCL, nine of whom were still in the mycosis fungoides (MF) stage of disease [387]. Patients received up to 4 months of weekly infusions with 280 to 980 mg per dose. The objective response rate was 32%. Adverse events included infusion reactions with the initial depletion of peripheral T cells, and low-grade infections including eczematous dermatitis, especially in MF patients. Based on these encouraging results, additional studies are being conducted in hopes of regulatory approval.

365

CD5 CD5 is a pan T cell antigen that is also co-expressed on the B-lymphocytes of CLL and small B-cell lymphoma (well-differentiated diffuse B-cell lymphoma), and the B-lymphoctyes of mantle cell lymphoma [188]. Normal CD5+ B-cells are prevalent during fetal development, but their number decrease after birth and with age, but increase again in the elderly. In the early years of Mab development, several laboratories produced anti-CD5 antibodies in response to immunization of mice with lymphocytes. CD5 is expressed on most T cells. A number of the early trials with mouse Mabs were conducted in patients with T cell malignancies because of the early availability of antibodies that reacted with the CD-5 antigen on T lymphocytes. Levy and colleagues treated eight T-cell leukemia patients with the anti-CD5 momab Leu1, that induces antigenic modulation, and two anti-leukemia antibodies that did not induce antigenic modulation [429]. Patients received 1 to 92 mg of Leu-1 alone or in combination with the other Mab. Transient reductions in circulating leukemia cell counts were observed, but there were no sustained antitumor effects. There was no obvious advantage to treating with a combination of antibodies in these trials. Miller et al. reported an early success in a patient with CTCL who received 17 infusions of 1 to 20 mg of antiCD5 antibody Leu1 that was administered over a 10-week period [485, 486]. He experienced a partial response including marked shrinkage of lymph nodes, skin lesions, and a neck mass. There was also a transient decrease in circulating T lymphocytes after each treatment. These same investigators treated five additional CTCL patients, and one with a large PTCL [488]. Individual patients received four to seven treatments over 2 to 10 weeks at doses of 250 ug to 100 mg infused over 4–6 h for total doses of 13–761 mg. Four patients had brief tumor regressions lasting from 1 to 4 months. The IgG2a murine Mab T101, which reacts with the CD5 lymphocyte antigen, was one of the first mouse antibodies to be extensively tested in human trials. The CD5 antigen rapidly internalizes after T101 binding, and the antibody was not cytotoxic in in vitro assays of CMC or ADCC with human effector cells or human complement [181]. Dillman et al. treated two CTCL patients with multiple 2-h infusions of the anti-CD5 momab T101 [179], and subsequently treated an additional 10 CTCL patients with T-101 at doses from 10–500 mg, given over 24 h [180]. With the prolonged infusions, brief antitumor responses lasting from days to weeks were seen in four patients. One of the most interesting responses was the

366 dramatic remission of rheumatoid arthritis in a patient with CTCL who was bedridden because of arthritis, and became ambulatory within 24 h of treatment with T101. In other trials with T101, Foon et al. administered the antibody to several CTCL patients. Some patients had minimal improvement in skin lesions [227]. Bertram et al. gave T101 to eight patients with CTCL and to four other patients with various T-cell lymphoproliferative disorders [38]. One patient with CTCL had a partial response of his cutaneous lesions that persisted for 3 months. Another patient with convoluted T-cell lymphoma was alleged to have had a complete remission based on a decrease in bone marrow blasts from 6% to 3% which persisted for 2 months following doses of 1–100 mg T101. Dillman et al. treated 10 CLL patients with T101 [177]. Two patients received 1 to 10 mg over 15 min, two received weekly doses of 10 mg infused over 2 h, and 6 received 24-h infusions of T101 at doses of 10, 50, 100, 150, or 500 mg. Limited unsustained clinical benefit was noted despite confirmation of in vivo binding to CLL cells in blood, lymph nodes, and bone marrow. The same T101 antibody was studied by Foon et al. in 13 patients with CLL [228]. Successive three-patient cohorts received doses of 1, 10, 50, 100 mg and one patient received 140 mg. At all doses there binding to circulating and bone marrow cells could be demonstrated, and a rapid but transient decline in circulating leukemia cell counts. Durable responses were not seen.

CD10 CD10 was originally known as the common acute lymphoblastic leukemia antigen (CALLA). CD10. Ritz and colleagues explored the mouse mab J-5 that reacts with CD 10, in four patients with acute lymphocytic leukemia (ALL) [600]. Doses of 1 to170 mg of J-5 were infused over 15 min to 2 h. In those patients with circulating blasts, treatment was followed by a rapid decrease in circulating CALLA+ blasts, but a large number of CALLAnegative lymphoblasts persisted. However, once J-5 was no longer detectable in the serum, the lymphoblasts reexpressed CALLA, consistent with antigenic modulation or internalization of the surface antigen in the presence of the antibody. There was no decrease in marrow blasts even though J-5 binding was demonstrated. There was no significant toxicity noted in these patients although all three patients who had circulating blast cells had temperature elevations in association with therapy.

CD15 CD15 is a granulocyte-associated antigen. In preliminary trials Ball et al. had treated three AML patients

Monoclonal antibody therapy with one or more of the IgM anti-glycolipid momabs PMN-81, PMN-29, and PM-81 and a fourth antibody, an IgG2b called AML-2-23, which reacts with a protein antigen [19]. None of these antibodies induced antigenic modulation in vitro. Multiple 8 to 12 h infusions of 20 to 70 mg of the various antibodies were associated with rapid, but transient decreases in circulating blasts and elevations of serum lactic dehydrogenase suggesting that leukemia cells had been destroyed, although sustained remission of leukemia did not occur. Ball et al. treated another 16 acute myeloid leukemia (AML) patients with 24-h continuous infusion of anti-CD20 Mab PM81 (also called MDX-11) at doses from 0.5 to 1.5 mg/kg in a dose escalation study [20]. Transient reductions in circulating blasts were noted, but there did not appear to be an effect on marrow blasts at these relatively low doses. Circulating CD15 antigen presented a problem in terms of immune complexes. The toxicities observed in this trial (fever, chills, hypotension, tachycardia) were the same as observed with other Mab that bind to circulating cells.

CD19 CD 19 is a 90 kD glycoprotein differentiation antigen expressed on normal and malignant B cells that might be a useful regulatory target on B cells [753]. The CD19 molecule acts as an internalizing receptor that is physically and functionally associated with certain protooncogene phosphokinases. Hekman et al. treated 6 lymphoma patients with an IgG2a murine Mab against the B-cell CD19 [303]. Total doses from 225 to 1,000 mg were given over 4 h without major toxicity. One patient was felt to have had a partial remission that lasted 8 months following his first treatment, and then 9 months following a second treatment. Because of the internalization (modulation) of this antigen, recent studies have focused more on immunoconjugate approaches.

CD20 This is the target for the chimeric Mab rituximab, which was discussed in detail in this chapter in the section on commercially available antibodies. It is also the target of the commercially available radiolabeleled antibodies I-131 tositumomab (Bexxar) and Y-90 ibritumomab tiuxetan (Zevalin) [174]. The earliest evidence of the therapeutic potential of targeting CD20 was reported by Press et al. [569] who used the anti-CD20 momab 1F5 to treat four patients with refractory B-cell lymphoma. Total treatment was given over 5 to 10 days. Two patients had a 90% reduction in circulating B cells. The total doses delivered were 52 mg in a patient who had

Robert O. Dillman progressive disease, 104 mg in a patient who had stable disease, 1,032 mg in a patient who had a minor response, and 2,380 mg in a patient who had a partial remission including a 90% reduction in lymph nodes that persisted for 6 weeks. This same antibody, which is now called tositumomab, is given as part of I-131 radioimmunotherapy. In one randomized trial that included a control arm in which 36 patients received two 450 mg doses of tositumomab over 1 to 2 weeks, there was a 19% objective response rate [150]. In a similarly designed randomized trial of Y-90 radioimmunotherapy, 4 weeks of standard dose rituximab had an objective response rate of 56% [790]. Because of the tremendous clinical success of rituximab, a number of companies have made new CD20 Mab with variations in Fv and Fc in efforts to enhance therapeutic benefit. It will be interesting to see how many of the products are tested in randomized trials against rituximab as opposed to going for a unique indication in a subset of CD20 positive malignancy.

CD22 CD22 is a modulating antigen widely expressed on B lymphocytes. Epratuzumab, a humanized IgG1 antibody that targets CD22, was originally developed for radioimmunotherapy, although preliminary work with antiCD22 antibodies focused on immunoconjugates because of the internalization of the antigen after antibody binding. Leonard et al. treated 55 patients who had recurrent indolent or large B-cell lymphoma with epratuzumab at doses of 120 to 1,000 mg/m2 over 30 to 60 min weekly for four treatments [425]. There was an 18% response rate among 51 evaluable patients with a median duration of response or more than a year. All nine responses were in patients with follicular lymphoma. One complete response persisted for more than a year in a lymphoma patient who was considered refractory to the anti CD20 ximab rituximab. The treatment with this agent has been well-tolerated with no surprising toxicities described. Subsequently clinical trials were launched in both follicular and large B cell lymphoma using four weekly doses of 360 mg/m2, but these were not completed.

CD23 CD23 has been characterized as a low-affinity IgE receptor that mediates allergic responses, but it is also widely expressed on CLL cells and its expression is used to help differentiated CLL and SLL from mantle cell lymphoma. Lumiliximab is an IgG1 anti-CD23 chimeric Mab that is in clinical trials in CLL [587]. O’Brien

367 et al. conducted a phase I trial involving 46 patients who had relapsed or refractory CLL [84]. Lumiliximab was given i.v. at 125, 250, 375 mg/m2 or 500 mg/m2 weekly for 4 weeks or 500 mg/m2 thrice during week 1 then 500 mg/m2 weekly for the next 3 weeks, or 500 mg/m2 thrice a week for 4 weeks. Little toxicity was reported at any dose level, but there were no objective responses.

CD25 Daclizumab (Zenapax®, Roche Laboratories, Nutley, NJ) The interleukin-2 receptor, CD25, is overexpressed in activated T-cells and many T cell malignancies. It is composed of 3 subunits including an alpha chain (p55), a beta chain (p75), and a gamma chain (p65) which combine noncovalently to bind the important lymphokine interleukin-2 (IL-2) which is not expressed on resting lymphocytes, but is often overexpressed in activated and malignant lymphocytes, especially T-lymphocytes [762]. The anti-CD25 Mab daclizumab (Zenapax™, Roche Laboratories, Nutley, NJ) was approved by the US FDA in 1998 for the marketing indication of kidney transplant rejection [766], but there is a strong rationale for it having clinical activity in certain hematopoietic malignancies [761, 764]. It is a humanized Mab derived by genetic engineering modification of a murine anti-Tac (55 kD subunit of the IL-2 receptor, IL-2Rα) Mab. The humanized form of this antibody was originally called antiTAC-H. Later daclizumab was constructed using a human IgG1 constant framework [358]. The murine version was unable to effect ADCC with human effector cells, but the humanized construct was cytolytic in ADCC assays. As expected, daclizumab was much less immunogenic in monkeys than the mouse Mab. Daclizumab was approved as part of combination prophylaxis regimens for the prevention of renal allograft rejection. These trials utilized five weekly 1 mg/kg doses of daclizumab. Daclizumab was well-tolerated at that dose in kidney transplant patients who were also receiving other immunosuppressive agents (cyclosporine, cyclosporine plus corticosteroids, or those two agents plus azathioprine). Because of its immunosuppressive effects, daclizumab is also being tested in other transplant settings for the treatment of graft vs. host disease (GVHD) [572].

Daclizumab and T Cell Malignancy The expression of CD25 is increased in malignant cells of adult T-cell leukemia which is induced by the human T-cell lymphotrophic virus I (HTLV-I) [272]. Since very few normal cells express IL-2Rα, but abnormal T cells in patients with lymphoid malignancies do, targeting the IL-2Rα with daclizumab might be of therapeutic benefit.

368 Waldmann et al. used the original murine anti-Tac (T-activated cells) Mab (predecessor to the humanized daclizumab to treat 19 patients with adult T-cell leukemia [765]. Patients received 20, 40, 50, 60, or 100 mg doses over 8 to 445 days with variable total doses ranging from 220 mg over 9 days, 490 mg over 51 days, and 220 mg over 445 days. The Mab was given as 3–11 infusions per treatment course. One patient, who had previously failed aggressive combination chemotherapy had a complete response that lasted for 5 months, and then a partial response which lasted 6 months after retreatment. There were two complete and four partial responses observed in this trial as well as one mixed response. The duration of remissions ranged from 2 months to 3 years. Another patient had a reduction in peripheral leukemia cells, but had an increase in malignant lymphadenopathy at the same time. Despite the reactivity with circulating T cells, these patients reportedly had no significant toxicity other than low-grade fever in two patients, and hives in one patient. However, these patients were already markedly immunosuppressed, and they may have been further immunosuppressed by the anti-T-cell antibody. While on study, one patient developed Kaposi’s sarcoma, one developed Pneumocystis carinii pneumonia, and a third developed Staphylococcal-A septicemia. Further evidence of immunosuppression was the observation that only one of the nine patients treated developed HAMA. The CD25 IL-2 receptor is also sometimes expressed in Hodgkin’s disease; so, antibodies such as daclizumab might have activity in selected patients. Few formal studies of daclizumab in T cell malignancies have been published, therefore most hematologists prefer to use other products that target CD25, such as denileukin diftitox (Ontak), the fusion product of IL-2 and diptheria A chain as treatment for CD25 positive malignancies [627]. CD25 is probably a better target for immunoconjugates therapy, such as immunotoxins, because binding leads to internalization of CD25. Theoretically daclizumab therapy may only eliminate circulating cells that express large numbers of CD25 molecules, since CDC and ADCC correlate with the extent of antibody binding. This limitation is supported by the study by Koon et al. who treated 10 CD25+ leukemia patients with daclizumab in a study focused on pharmacokinetics of the antibody and pharmacodynamics of interaction with the CD25 target. [403]. They confirmed that high numbers of CD25+ circulating tumor cells were predictive of accelerated pharmacokinetics, and high levels of CD25 antigen expression correlated with the extent of target cell clearance. Despite clearance of

Monoclonal antibody therapy peripheral leukemic cells, and schedules that sustained daclizumab in the blood, durable responses were not obtained. There are other anti-CD25 Mabs being investigated, including the chimeric Mab basiliximab, that could be investigated in lymphoid malignancy [352].

CD30 CD30 is an excellent target for immunotherapy of Hodgkin’s lymphoma (HL) because it is overexpressed on Hodgkin’s and Reed-Sternberg cells and the lymphocytes of some anaplastic lymphomas, but has limited expression on normal tissue, which makes it a potential target for Mab therapy [402]. Like many targets on hematologic cells, it may not only be a target for CDC and ADCC, but there is also evidence CD30 may be a regulatory target involved in signal transduction. There are a number of different anti-CD30 products under investigation including unmodified Mab, bispecific Mab in which CD30 is one of the targets, and various immunoconjugates. MDX-060 is a human IgG1 kappa anti-CD30 Mab that has exhibited anti-tumor effects in preclinical models. Ansell et al. treated 72 patients who had CD30+ tumors including 63 with Hodgkin’s lymphoma, seven with anaplastic largecell lymphoma, and two with CD30+ T-cell lymphoma [10]. Treatment was well-tolerated during treatment with escalating i.v. doses of 1, 5, 10, and 15 mg/kg, with only 7% of patients experienced grade 3 or 4 treatment-related adverse events. Clinical responses were observed in six patients (8.3%) and more than one third of the patients had a long duration of stable disease. The bifunctional antibody HRS-3/A9 (BiMAb) targets the Fc gamma receptor CD16 that is expressed on various effector cells including NK cells, and CD30 antigen on Hodgkin’s cells. Fifteen patients with refractory Hodgkin’s disease were treated with this antibody in a phase I/II trial [295]. The maximum tolerated dose was 16 mg/m2 and the major side effects were fever, rash and painful lymph nodes. One complete, one partial, one mixed and three minor responses were noted. Nine patients developed HAMA. In a subsequent trial 16 patients with refractory Hodgkins disease were randomized to either 25 mg by continuous i.v. infusion daily for 4 days, or 25 mg i.v. over 1 h every other day for four doses [296]. There was a 25% response rate including one CR. Another bifunctional product consists of F(ab’) fragments derived from the murine anti-CD30 Mab Ki-4 and the humanized CD64-specific Mab H22. Ten patients were treated with escalating doses of 1, 2.5, 5, 10, and 20 mg/m2 per day i.v. on days 1, 3, 5, and 7 [49]. All five

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doses were well tolerated. Mild infusion reactions were observed and four patients had a partial response.

had significant decreases in bone marrow blasts and one was considered a CR.

CD33

CD38

M195 is a murine IgG2a monoclonal antibody that reacts with the CD-33 antigen that is expressed on early myeloid precursors. This receptor is rapidly modulated or down regulated in the presence of M195. Scheinberg et al. treated 10 AML patients using escalating doses of M195 (1, 5, and 10 mg/m2) up to a total dose of 76 mg [633]. Radioisotope tracer studies and bone marrow biopsies demonstrated binding to bone marrow cells, but rapid antigenic modulation took place. Sustained antitumor effects were not seen. Because of the theoretical limitations of HAMA, a CDR-grafted human IgG1 version of M195 (HuM195, lintuzumab) was developed that is able to effect ADCC with human effector cells in vitro [89]. Thirteen AML patients received six i.v. doses over 3 weeks along with a radiolabelled tracer dose. A decrease in marrow blasts was only noted in one patient. Intermittent dosing with 3 mg/m2/day was found to saturate available binding sites [88]. In a subsequent trial 10 patients with refractory AML were given higher doses of HuM195 as daily infusions of 12, 24, and 36 mg/m2 on days 1 to 4 and 15 to 18 [90]. One patient had a complete remission that lasted more than 2.5 years. Mild infusion reactions including fever and rigors were noted. In a successor trial, 50 refractory AML patients were given HuM195 as first salvage therapy in 24 patients and as second or subsequent salvage therapy in 26 patients [217]. Patients were randomized to receive 12 or 36 mg/m2 on days 1 to 4 and 15 to 18. Three responses were recorded, all at the 12 mg/m2 dose. Infusion-related fevers and chills were the predominant toxicities, and were similar for the two doses. Feldman et al. randomized 191 patients with primary refractory or initial relapsed AML to combination chemotherapy consisting of mitoxantrone, etoposide, and cytarabine (MEC) with or without lintuzumab. There was no significant difference in response rate, 36% for MEC plus lintuzumab vs. 28% for MEC alone (p = 0.28) nor in median survival. Mild antibody infusion-related toxicities including fever, chills, and hypotension were common in the lintuzumab arm. HuM195 was preceded by low-dose s.c. IL-2 in 13 patients with relapsed or refractory, followed AML [406]. After 5 weeks of s.c. IL-2 patients received i.v. infusions of HuM195 at a dose of 3 mg/m2 twice a week for 3 weeks. Infusion reactions that included nausea, rigors, and fever were frequently observed. Two patients

CD38 antigen is present on the majority of neoplastic plasma cells and some CLL, and other B cell populations. Stevenson et al. developed a mouse/human chimeric antibody to CD38 that consisted of human IgG1 chemically linked to mouse Fab was able to mediate ADCC in laboratory studies [693] The same group has also developed a CDR-grafted humanized IgG1 from the same murine antibody [206], but noted little difference between the two in various in vitro assays, including down regulation of the receptor. Clinical results with these products have been disappointing [694].

CD40 Interaction with CD40 activates antigen-presenting cells and enhances immune responses, but targeting CD40 that is expressed on solid tumors induces apoptosis. CP-870,893 is a fully human anti-CD40 Mab that was given to 29 patients in a phase I trial [756]. Weekly doses from 0.01 to 0.3 mg/kg were associated with grade 1 to 2 infusion reactions that included fever, chills and rigors as well as brief decreases in lymphocytes, monocytes and platelets, but there was also evidence of B cell activation. Abnormalities in D-dimer and liver function tests were noted 24 to 48 h after infusion, and one patient experienced a deep venous thrombosis. Despite these low doses and the cross reactivity with circulating cells, four metastatic melanoma patients were judged to have had a PR (14%).

CD45 The CD45 antigen is expressed on all hematopoietic cells. Anti-CD45 Mabs cause marrow suppressive effects that range from leucopenia to marrow aplasia in some rodent models, so CD45 may be a useful therapeutic target for hematoproliferative malignancies. Krance et al. gave the rat anti-human CD45 MAbs, YTH25.4 and YTH54.12, which bind to different epitopes on CD45, to 14 patients with hematologic malignancies who were scheduled to undergo stem cell transplantion [407]. Escalating doses were given; the maximum tolerated dose was 400 μg/kg/day for 4 days. Treatment was followed by marked reduction in circulating lymphoid and myeloid cells, but had little impact on normal marrow cells despite reducing the percentage of leukemic blasts in two of three patients who a leukemic marrow.

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CD74 Milatuzumab (hLL1 or IMMU-115) is a humanized anti-CD74 monoclonal antibody [689]. CD74 is a surface membrane protein that functions as a survival receptor in that it enhances cell proliferation and survival [685]. It also functions as an MHC class II chaperone. It is expressed on lymphoid cells, especially malignant B cells, and carcinomas of the kidney, lung, and stomach, and by certain sarcomas, but has limited expression on other normal tissues. In vitro milatuzumab does not induce ADCC or CDC, probably because of the rapid internalization of this receptor molecule. However, despite the rapid internalization of milatuzumab after binding to CD74, preclinical models have suggested anti-tumor activity with unconjugated antibody, in addition to immunoconjugates [688]. Unlike rituximab, CD74 is also highly expressed on the malignant plasma cells of multiple myeloma [73]. CD74 is also expressed on T cells and NK cells, and therapy is associated with depletion of lymphocytes in primate models. The product is being explored in B cell lymphoma, multiple myeloma, and CLL.

CD80 Galiximab reacts with CD80, which is expressed on a variety of lymphoid and myeloid cells. Czuczman et al. treated 37 patients suffering from relapsed or refractory B cell lymphoma with four weekly i.v. infusions of galiximab at doses of 125, 250, 375, or 500 mg/m2 [142]. Four of 35 evaluable patients had an objective response (11%). Treatment was well-tolerated with mild nausea, headache, and fatigue the most common toxicities noted.

CD122 CD122 is the beta-subunit shared by the IL-2 and IL-15 receptors [763]. Both IL-2 and IL-15 activate T cells, but IL-2 also induces apoptosis of self-reactive T cells while IL-15 stimulates their persistence. Mikbeta1 and its humanized version (HuMikbeta1) bind to CD122. In some cell models HuMikbeta1 is more effective in blocking IL-2 stimulation then IL-15 stimulation. The murine Mikbeta 1 was given to 12 cytopenic patients with T cell large granular lymphocyte leukemia (T-LGL) in a phase I dose-escalation trial in which 0.1, 0.5 or 1.5 mg/kg i.v. doses were infused on days 1, 4, 7, and 10 [505]. At these doses there were no significant toxicities and HAMA was not detected, but such patients are already cytopenic and immunosuppressed. The investigators were able to demonstrate saturation and down-

Monoclonal antibody therapy modulation of the CD122 IL-2/IL-15 beta receptor on the surfaces of the TLGL cells, but there was no sustained decrease in leukemia cell numbers. Clinical trials with the huMikbeta1 are planned. Higher doses of the humanized Mab may be associated with more clinical activity, but perhaps also toxicity.

HLA DR LYM-1 is an IgG2a murine Mab that reacts with a polymorphic HLA-Dr antigen on all B cells, but the target antigen is not shed nor does it modulate in the presence of antibody. Ten patients with refractory B cell lymphomas received weekly i.v. infusions of escalating doses of LYM-1 over 4 weeks without any objective tumor responses [330]. Apolizumab (hu1D10) is a humanized Mab against a polymorphic epitope on HLA DRbeta. Rech et al. treated six patients who had relapsed or refractory 1D10-positive non-Hodgkin’s lymphoma with granulocyte colony stimulating factor (GCSF) and apolizumab at doses ranging from 0.15 to 1.5 mg/m2 [582]. There were no clear anti-tumor effects. One patient had skin rash, one thrombocytopenia, and one auto-immune hemolytic anemia.

HM1.24 HM1.24 has been touted as a human plasma cell specific antigen that is over expressed on myeloma cells. Anti-HM1.24 inhibits proliferation of plasma cells that overexpress HM1.24. A humanized IgG1/kappa antiHM1.24 (AHM) has been developed that exhibits strong ADCC activity in vitro with human effector cells against myeloma KPMM2 and ARH77 human myeloma cells in the presence of human PBMCs myeloma cells [540]. It also shows promising activity in animal xenograft models that is diminished by pretreating animals with an anti-Fc gamma receptor II/III antibody [369].

Anti CTLA4 (Cytotoxic T Lymphocyte Antigen 4) CTL-associated antigen 4 (CTLA-4) can inhibit T-cell activation and helps maintain peripheral self-tolerance of antigens expressed on both normal and tumor cells. CTLA-4 is expressed on the surface of activated T-lymphocytes where it suppresses the induction of immune responses that normally follow the interaction between T-cell receptors and HLA molecules on the antigen-presenting cells. Mouse experiments showed that blockade of CTLA4 could yield anti-tumor effects. The two anti-CTLA 4 Mabs, ipilimumab (MDX-010) and tremelimumab (CP-675, 206;

Robert O. Dillman formerly known as ticilimumab), have been extensively tested with promising activity [414]. However, both have been associated with significant immune-related adverse events (IRAE) including dermatitis, inflammatory bowel disease, uveitis, arthritis, hypophysitis, and others. Generally, these IRAE have reversed after cessation of therapy and use of i.v. or topical corticosteroids. However, colectomy has been required in several severe cases of inflammatory bowel disease, and several IRAE-associated deaths have been reported [31]. Despite this toxicity profile, it is likely that one or both of these agents may receive regulatory approval.

Ipilimumab Striking anti-tumor activity has been seen in melanoma, but mostly in conjunction with substantial autoimmune toxicity [133, 531]. Downey et al. reported a 17% response rate among 139 metastatic melanoma patients who were treated with ipilimumab, mostly in conjunction with peptide vaccines [190]. The details of the dose escalations used in these trials had been previously reported [455, 558]. IRAE, including enterocolitis, arthritis and uveitis, were documented in 62% of patients and were highly correlated with a measurable anti-tumor effect. The response rate was 36% among 24 patients who had colitis compared to 11% among 113 patients who did not (p = 0.006) [31]. The three patients who experienced a CR had grade 4 toxicities. Anti-tumor activity has been demonstrated in renal cell cancer. Yang et al. treated 61 metastatic renal cell patients with either 3 mg/kg followed by 1 mg/kg every 3 weeks (n = 21) or 3 mg/kg every 3 weeks [800]. Partial responses were noted for 6/61 patients (10%), five at the higher dose. Grade 3 to 4 toxicity was documented in 33% of patients, most of which appeared to be auto-immune mediated, especially enteritis and endocrine deficiencies. The response rate was 35% among 17 patients who experienced colitis and only 2% among 44 patients who did not (p = 0.002) [31]. A small number of prostate cancer patients have been treated with ipilimumab [676]. Fourteen patients with metastatic hormone-refractory prostate cancer received ipilimumab at a dose of 3 mg/kg i.v. dose. Two patients had a 50% decline in PSA level. Treatment was generally well tolerated and only one patient developed grade 3 dermatititis that required systemic corticosteroids.

Tremelimumab (CP-675,206, Ticilimumab) Striking anti-tumor activity has been seen in melanoma, but mostly in conjunction with substantial autoimmune toxicity [85, 709]. Ribas et al. conducted a phase I dose escalation trial of seven different dose levels of tremeli-

371 mumab in 39 patients, including 34 with melanoma and four with renal cell cancer [591]. Among 29 melanoma patients who had measurable disease, there were two durable CR and two durable PR lasting more than 2 years. Dose-limiting toxicities were IRAE including autoimmune phenomena such as diarrhea, dermatitis, vitiligo, panhypopituitarism and hyperthyroidism. The maximum tolerated dose for a single i.v. infusion was 15 mg/kg. Among 30 melanoma patients who received tremelimumab at a dose of 10 mg/kg monthly (n = 20) or 15 mg/kg every 3 months (n = 10) 4/12 patients with IRAE had an objective tumor responses compared to only 1/18 without IRAE (p = 0.046) [590]. The IRAE and responses were associated with increased production of IL-2 and lack of these was associated with increased production of IL-10. IRAE attributed dermatitis, colitis, hepatitis, and hypophysitis with panhypopituitarism. In a preliminary report on 20 patients, there was uveitis or vision changes noted during extensive opthalamalogic evaluations during and after treatment [696].

Tumor Necrosis Factor-Related ApoptosisInducing Ligand (Trail) Receptor Tumor necrosis factor (TNR)-related apoptosis-inducing ligand (TRAIL) is a TNF family member capable of inducing apoptosis by binding to its two death receptors (DR) DR4 (TRAIL-R1) and DR5 (TRAIL-R2) [64]. There are at least two Mab being investigated that target the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. Both are fully human Mabs. Mapatumumab (HGS-ETR1, TRM-1) targets DR4 (TRAIL-R1) [760]. Lexatumumab (HGS-ETR2), targets DR2 (TRAIL-R2) [467]. TRAIL receptors are upregulated by chemotherapy. DR4 and DR5 may be useful targets for both hematologic malignancies and solid tumors.

Mapatumumab (HGS-ETR1, TRM-1) Tolcher et al. treated 49 solid tumor patients with 158 courses of mapatumumab as i.v. doses ranging from 0.01 to 10 mg/kg infused over 30 to 120 min [724]. Initially mapatumumab was given as a single dose, then every 28 days, and then 10 mg/kg every 14 days. TRAIL-R1 was expressed in 68% of patients whose tumors were assayed. No objective tumor responses were seen. There was minimal toxicity with mild infusion reactions being the most common adverse event reported, including fatigue, fever, and myalgia.

Lexatmumab (HGS-ETR2) Plummer et al. treated 37 solid tumor patients with 120 cycles of lexatumumab at doses ranging from 0.1 to

372 20 mg/kg every 21 days [564]. The maximum tolerated dose was 10 mg/kg based on dose-limiting toxicities that included asymptomatic elevations of serum amylase, transaminases, and bilirubin in three of seven patients treated with 20 mg/kg. There were no tumor responses.

Nuclear Factor-Kappa B Ligand Nuclear factor-kappa B (NF-ΚB) is a family of ubiquitous transcription factors that are believed to control inflammation. NF-KB normally resides in the cytoplasm of cells, but in response to a variety of stimuli, including inflammatory cytokines, growth factors, carcinogens and ionizing radiation, NF-ΚB translocates to the nucleus where it upregulates the expression of over 400 different gene products associated with cell survival, proliferation, invasion, and angiogenesis [5, 194]. Activation of NF-ΚB has been linked to oncogenesis and the biologic behavior of cancer cells, including resistance to chemotherapy and radiation therapy via transcription of anti-apoptotic proteins, leading to increased proliferation of cells and tumour growth. Activation of the NF-ΚB signal pathway follows the interaction of NF-ΚB ligand and receptor (RANKL). Denosumab is a fully human monoclonal antibody to the receptor activator of nuclear factor-kappa B (NF-ΚB) ligand [430]. Lipton et al. gave denosumab to 255 women who had breast cancer-related bone metastases not previously treated with i.v. bisphosphonates [439]. Denosumab was administered s.c. every 4 weeks (30, 120, or 180 mg) or every 12 weeks (60 or 180 mg). Treatment was well tolerated and there was evidence of suppression of bone resorption. Anti-cancer activity was not noted. Body et al. conducted a small randomized, doubleblind trial of single doses of s.c. denosumab (0.1, 0.3, 1.0, or 3.0 mg/kg) or i.v. pamidronate (90 mg), in 29 patients with breast cancer and 25 with multiple myeloma [45]. Patients received a single dose of either agent and a placebo of the other. Treatment was welltolerated. The magnitude of decrease in bone resorption markers was similar to pamidronate, but the effects lasted longer with denosumab.

Integrins Integrins are cell adhesion receptors that mediate intercellular communication. Integrins are implicated in tumor cell survival, angiogenesis, cancer invasiveness, and metastasis, and therefore are a potential target of cancer therapy [520]. The integrin alphaV-beta3 is an adhesion molecule expressed by proliferating endothelial

Monoclonal antibody therapy cells and antibodies blocking this integrin inhibit angiogenesis in preclinical models. Mullamitha et al. treated 24 patients with CNTO 905, a fully human Mab to anti-alphaV integrins that inhibits angiogenesis and tumor growth in preclinical models [512]. CNTO 95 (0.1, 0.3, 1.0, 3.0, and 10.0 mg/kg) was infused on days 0, 28, 35, and 42. CNTO 95 was generally well tolerated with only infusion-related fever being the most common adverse event. At the 10.0.mg dose, a patient with cutaneous angiosarcoma had a 9-month partial response and a patient with an ovarian carcinosarcoma had a lesion that became negative by positron emission tomography, although a stable lesion persisted by computerized axial tomogragphy. McNeel et al. treated 25 solid tumor patients with MEDI-522, a second generation humanized anti-alphaVbeta3 antibody designed for antiangiogenic therapy, in a phase I study in which patients received doses ranging from 2 to 10 mg/kg/wk [477]. Treatment was well tolerated and no dose-limiting toxicities were seen in this dose range. The major adverse events observed were grade 1 and 2 infusion-related reactions (fever, rigors, flushing, and tachycardia), and low-grade constitutional and gastrointestinal symptoms (fatigue, myalgias, and nausea), and asymptomatic hypophosphatemia. There were no objective responses observed, but three patients with metastatic renal cell cancer had prolonged stable disease Serial biopsies of skin showed detected MEDI-522 both in quiescent and in angiogenically active skin blood vessels as well as in the dermal interstitial space [805]. Posey et al. treated metastatic cancer patients with vitaxin, a humanized monoclonal antibody, which has specificity for the integrin alphaV-beta 3 (vitronectin receptor) which inhibits tumor cell mediated angiogenesis in pre-clinical animal models [565]. Vitaxin was given i.v. at doses of 10, 50 or 200 mg in cohorts of three patients. There was no significant toxicity noted in these three dose levels. There were no objective anti-tumor responses. An every 3 week schedule of 200 mg appeared to sustain serum levels.

Human Epidermal Growth Factor Receptor Antigens (EGFR1) Cetuximab and panitumumab, the two FDA-approved products that target EGFR1, were discussed earlier in this chapter. A number of other Mabs have been developed that target this receptor. Mab ch806 is a chimeric antibody that detects a unique epitope on EGFR that is exposed only on mutanted or ligand-activated forms of the receptor on cancer cells in which it is overexpressed [648, 649]. In preclinical studies murine 806 blocked

Robert O. Dillman EGFR-mediated signal transduction resulting in antitumor effects on human tumor xenografts, while in vivo studies in human showed tumor specific targeting of ch806. ABX-EGF is a fully human anti-EGFR Mab that was given to 88 patients with metastatic renal cell cancer at doses of 1.0, 1.5, 2.0, or 2.5 mg/kg weekly [612]. Three patients had an objective response and the majority of patients had stable disease for at least 2 months. A doserelated acneiform rash was the most common toxicity, and occurred 100% of patients who received at least three doses of ABX-EGF at 2.5 mg/kg/wk. The humanized anti-EGFR Mab h-R3 was given to 29 patients with newly diagnosed malignant gliomas including 16 glioblastoma, 12 anaplastic astrocytoma and one anaplastic oligodendroglioma (AO) [580]. Following debulking surgery, patients received six 200 mg infusions of h-R3 weekly in during radiotherapy. Interestingly, no patients developed acneiform rash, and there were no allergic reactions. Median survival was 17months for the glioblastoma patients. Mab h-R3 was given at four dose levels weekly for 6 weeks in combination with radiotherapy, to 24 patients with advanced head and neck cancer [135]. In this trial there wee some infusion reactions, but no dermatologic or allergic toxicities were noted. The overall survival of patients was considered encouraging. HuMax-EGFr is a fully human Mab that was given in escalating doses of up to 8 mg/kg to 28 patients with squamous cell cancer of the head and neck [28]. Most frequently reported adverse event was rash. There were seven responses noted among 11 patients who were treated at the higher doses. Matuzumab is a humanized anti-EGFR Mab formerly known as EMD 72000 that has been explored in patients with a variety of cancers. Nine patients with squamous cell cancer of the head and neck were treated in a phase I trial in which two doses were given before surgery and three doses after surgery [41]. Successive patient groups received a single dose of 100, 200 or 400 followed by four weekly doses of 50, 100, and 200 mg, respectively. Treatment was well-tolerated. The most frequent adverse events attributed to the antibody were fever and a transient elevation of liver enzymes, but there was no significant skin toxicity. In another dose-escalation trial 22 patients with various solid tumors received matuzumab at five different dose levels randing from 400 to 2,000 mg/wk [744]. The maximum-tolerated dose was 1,600 mg/wk because of severe headaches at the 2,000 mg dose. At these dose levels mild acneiform rash was the most common toxicity. Matuzumab was given to 37 women with intra-abdominal platinum-resistant ovarian cancer at doses of 1,600 mg/wk [656]. Therapy was well tolerated,

373 but at this dose dermatologic toxicities were common, including acne, papillary rash, dry skin, and paronychia, but also headache, fatigue, and diarrhea, and case of pancreatitis that was possibly treatment related. There were no objective tumor responses. Matuzumab was given at weekly doses of matuzumab (100, 200, 400 or 800 mg) concurrently with paclitaxel at 175 mg/m2 every 3 weeks in 18 patients with NSCLC [401]. No additive toxicity was noted even at the 800 mg dose. Low grade acneiform skin rash occurred in 14 patients. There were five objective tumor responses. Escalating doses of matuzumab (400 mg weekly, 800 mg biweekly, or 800 mg weekly) were given with gemcitabine to three groups of patients with advanced pancreatic adenocarcinoma [270]. Skin toxicity was the most frequent side effect. There were three partial responses in patients who received 800 mg of Mab weekly. A bispecific antibody called MDX-447 was constructed by cross-linking antigen binding F(ab’) fragments) of a humanized Fab anti-EGFR to a humanized Fab which binds to CD64, the high-affinity immunoglobulin G receptor known as Fc gamma receptor I. Escalating weekly doses from 1 to 15 mg/m2 were administered to 36 patients with various refractory EGFR-positive cancers including cancers of the kidney, bladder, prostate, head and neck, and skin bladder, ovarian, prostate cancer and skin cancer, with and without G-CSF [138]. MDX447 infusions were associated with decreases in monocytes and increases in plasma cytokines in association with fever, chills, hypotension, and myalgias. Fury et al. reported on 41 patients treated with MDX-447 at doses from 1 to 40 mg/m2 and 23 patients who received 1 to 15 mg/m2 of MDX-447 in combination with GCSF [242]. Hypotension was the major dose-limiting toxicity in both cohorts, and accrual to MDX-447 plus GCSF was stopped because of excessive toxicity in association with higher levels of TNF-α and IL-6. The maximum tolerated dose for MDX-447 alone was 30 mg/m2 i.v. weekly. There were no objective responses.

Human Epidermal Growth Factor Receptor Antigens (EGFR2) Trastuzumab, the only FDA-approved product that targets EGFR2, was discussed earlier in this chapter. Other antibodies have been developed that also target HER3. Pertuzumab (rhuMAb 2C4) is a humanized construct of the murine 2C4 Mab that binds to the HER-2 receptor and blocks its ability to dimerize with other HER receptors. In a phase I trial pertuzumab was given at doses of (0.5 to 15 mg/kg) i.v. every 3 weeks to 21 patients with various advanced solid malignancies [3]. Treatment was

374 well-tolerated. Two objective responses were recorded. Patients with relapsed ovarian cancer were treated with pertuzumab, including 61who received a loading dose of 840 mg followed by 420 mg every 3 weeks, and 62 patients who received 1,050 mg every 3 weeks [266]. Pertuzumab was well tolerated, but diarrhea occurred in 69% (11% grade 3, no grade 4). Five patients had asymptomatic left ventricular ejection fraction decreases to less than 50%. There were five PRs. Herbst et al. gave pertuzumab once every 3 weeks to 43 previously treated NSCLC patients [311]. Treatment was well-tolerated, but there were no objective responses. Agus et al. gave pertuzumab as a loading dose of 840 mg followed by 420 mg for subsequent cycles, to 41 patients with hormone refractory prostate cancer who had also experienced disease progression after taxane chemotherapy [4]. Pertuzumab was well tolerated; grade 1 to 3 diarrhea was the most common toxicity. There were no tumor responses. A trial using the same doses of pertuzumab was conducted in 35 patients with hormone refractory prostate cancer who had not been treated with chemotherapy, and another 33 were treated with a fixed dose of 1,050 mg [153]. Treatment was well tolerated, but there were no tumor responses. Attard et al. combined pertuzumab (1,050 mg fixed dose and later the 840 mg loading dose, then 420 mg) with docetaxel chemotherapy with both agents given together every 3 weeks [13]. Based on toxicity they recommended further evaluation with docetaxel 75 mg m2 and 420 mg pertuzumab following a loading dose of 840 mg. Ertumaxomab (2B1) is a bispecific murine monoclonal antibody that binds to the extracellular domains of HER2/ neu and FcgammaRIII. Twenty women with metastatic breast cancer, all but one of whom had received prior chemotherapy were treated in a phase I trial [50]. The initial dose of 2.5 mg/m/day was associated with doselimiting toxicities in three of the first eight; so the remaining 12 patients received 1 mg/m/day. Objective antitumor responses were not seen. A trifunctional version of this antibody which also binds to CD3 on T lymphocytes was tested at doses of 10–200 μg weekly for up to three doses in 17 patients with various malignancies [384]. The maximum tolerated dose was 100 μg because of a systemic inflammatory response syndrome and acute renal failure in a patient treated with 150 μg and severe hypotension and respiratory distress syndrome in one patient and exacerbation of heart failure in two patients who received 200 μg. The most common drug-related adverse events were mild, transient infusion reactions including fever, rigors, headache, nausea, and vomiting, but grade 3 to 4 lymphocytopenia occurred in 76% of patients and elevated hepatic enzymes in 47%. Elevated levels of IL-2,

Monoclonal antibody therapy IL-6, TNF-α, and IFN-γ were seen in association with the infusion reactions. Objective tumor responses were seen in 5 patients. The bispecific antibody MDX-H210 was constructed by cross-linking antigen binding F(ab’) fragments of Mab H22 to CD64, the high-affinity immunoglobulin G receptor FcgammaRI) and mAb 520C9 to HER2 [770]. In a dose escalation trial of single i.v. infusions in patients with breast and ovarian cancer, one partial response was observed in a patient with breast cancer [740]. Elevated plasma levels of TNF-α, IL-6, G-CSF and neopterin were detected at higher doses of the antibody. Seven patients with HER2-positive prostate cancer received 1 to 8 mg/m2 of MDX-H210 as 2-h infusions three times a week [642]. Over this dose range MDXH210 was well tolerated other than mild infusion-related fever, chills, myalgias and malaise, in association with increased plasma levels of TNF-α and IL-6. No doselimiting toxicicty was noted. In another study, 25 patients with HER2+ advanced prostate cancer were treated with s.c. GM-CSF at a dose of 5 μg/m2 for 4 days of each week along with MDX-H210 at 15 μg/m2 weekly for 6 weeks [346]. Toxicities leading to withdrawal from the trial included grade 3 heart failure, dyspnea and allergic reactions, but 35% of patients had at least a 50% decrease in serum PSA levels.

Insulin-Like Growth Factor Receptor The anti insulin-like growth factor-I receptor Mab CP-751,871 was given in doses up to 20 mg every 3 weeks to 24 patients with various malignancies [290]. A maximum tolerated dose was not identified over this dose range. Mild adverse events included hyperglycemia, hyperuricemia, elevated hepatic enzymes, anorexia, nausea, diarrhea, and fatigue. There were no objective responses.

Transferrin Receptor The murine Mab 42/6 reacts with the human transferrin receptor thereby blocking the iron transport protein transferrin, which is crucial for iron transport by cells [732]. In vitro studies showed that 42/6 had inhibitory effects on a various types of cancer cells [704], [705]. In a phase I trial 33 treatments at doses of 2.5 to 300 mg/m2 were given as 24-h infusions to 27 patients [57]. 42/6 was well tolerated, although one patient experienced an allergic-type response associated with a HAMA response during a second course of treatment. Limited mixed tumor responses were observed in three patients with hematological malignancies, but there were no

Robert O. Dillman partial or complete remissions. HAMA was detected 33% of patients.

Vascular Ligands and Receptors Bevacizumab, which reacts with the VEGF ligand, is the only FDA-approved product in this group. Bevacizumab was reviewed in detail earlier in this chapter. Several other Mab products that target VEGF or VEGFR are under development. IMC-1C11 is a chimeric Mab that has anti-angiogenesis properties based on its binding to the VEGF receptor 2 rather than the VEGF ligand. In a dose escalation study, 14 patients with colorectal carcinoma and hepatic metastases received weekly doses at 0.2, 0.6, 2.0, or 4.0 mg/ kg for 4 weeks [566]. There were no serious toxicities, although minor bleeding occurred in four patients treated at the two lowest dose levels. HACA was detected in half the patients and altered pharmacokinetics of IMC-1C11 in two patients. There were no objective tumor responses. HuMV833 is a humanized IgG4 anti-VEGF Mab that was given to 20 patients who had advanced solid tumors by i.v. infusion on days 1, 15, 22, and 29 [348]. Treatment was well-tolerated over the dose range of 0.1 to 10 mg/ kg per dose. One patient with ovarian cancer experienced a partial response. Dose of 1 and 3 mg/kg were selected for further investigation. J591 targets the external domain of prostate-specific membrane antigen (PSMA), but is also expressed on the neovasculature of various solid tumors. J591 was given every 3 weeks in doses of 5, 10, 20, 40, 60, or 100 mg, along with a 2 mg tracer dose of Mab conjugated to 10 mCi of indium-111 to 20 patients with a variety of malignancies [507]. In a second trial 27 patients received doses of 5, 10, 20, 40, or 80 mg and some patients received weekly therapy for up to 6 weeks [490]. Tumor localization was confirmed in both studies and there were no dose limiting adverse events. No tumor responses were observed in either study.

Carcinoembryonic Antigen (CEA) Numerous clinical studies with antibodies directed against carcinoembryonic antigen (CEA) and other antigens of gastrointestinal mucosa have been reported. Several different murine IgG1 anti-CEA antibodies at doses from 1 to 80 mg were infused into 30 colorectal cancer patients utilizing single 1- to 2-h infusions with tracer doses of 111-Indium labeled antibody [183]. With one such Mab three patients had dramatic infusion reactions with fever and rigors which turned out to be due to

375 cross-reactivity with granulocytes, and associated with transient decreases in total leukocyte counts [178]. There were no tumor responses. Other trials utilizing small dose of murine anti-CEA Mab that did not crossreact with granulocytes were associated with a minimum of side effects, and were taken up into tumors, but not associated with tumor responses [451], [30], [288]. In December 1992 the 111-indium conjugate of a murine Mab B-72.3 (OncoScint™) became the first Mab approved specifically for use in cancer patients, albeit as an imaging agent for patients with ovarian and colon cancer rather than a therapeutic agent. approved for clinical use for radioimmunodetection of cancer in patients with ovarian cancer and colorectal cancer in Numerous patients received doses of 10 to 40 mg of antibody along with 1 mg doses of radiolabelled antibody [454]. Tumor regressions were not noted in the trials in patients with known colorectal cancer. However, humanized forms of this antibody appeared promising based on in vitro assays of ADCC. Khazaeli et al. treated 12 patients with 3 to 7 mg of B-72.3 chimeric IgG4 antibody [381]. No toxicities or tumor effects were seen at these low doses. Haisma et al. treated 20 colorectal cancer patients with the human IgM Mab 16.88, labeled with 131iodine for tumor localization [282]. Patients received an 8 mg dose followed 1 week later by 200, 500, or 1,000 mg of antibody. Tumor uptake was seen in at least one tumor site in 80% of the patients, but no tumor regressions were reported.

Epithelial Cell Adhesion Molecule (EpCAM) One of the targets for many murine Mab produced in different laboratories by immunizations with epithelial cancer cells was a surface glycoprotein called 17-1A, GA733-2, or KSA. This molecule was expressed on most malignant epithelial cells and some normal human cells. It was subsequently recognized that this antigen is what is now called epithelial cell adhesion molecule (EpCAM) [820]. Loss of membranous EpCAM is associated with nuclear beta-catenin localization, reduced cell-cell adhesion, and increased migratory potential of cells, which suggests that blocking this molecule could be associated with anti-cancer effects.

10.6.2.27.1 Edrecolomab (17-1A, Panorex®), was the most heavily studied anti-EpCAM Mab [268]. This was actually the first therapeutic Mab approved for the treatment of cancer internationally, but it never received approval in the U.S. Edrecolomab is a murine IgG2a that reacts with a 37–40 kD glycoprotein found on various adenocarci-

376 nomas and on normal epithelial tissues [315]. The EpCAM antigen is not shed into the circulation nor is it internalized. Edrecolomab may produce anti-cancer effects by various mechanisms [404], [316]. It was one of the few murine Mabs that could effect ADCC with human mononuclear cells [312]. Edrecolomab induced a high frequency of HAMA in humans [651], [653]. Some of these HAMA (Ab2) react with the idiotype of the 17-1A mouse antibody (Ab1). As part of the idiotype network control of B-cell proliferation, the Ab2 induces additional antibodies against itself, including antibodies (Ab3) that react with the idiotype of the Ab2. Such an Ab3 is actually an endogenous human antibody with the same reactivity against the tumor antigen as Ab1. This phenomenon has been observed in patients who have received edrecolomab [316], [215]. Single injections of edrecolomab at doses of 15–1,000 mg were well tolerated, but 50% of patients developed HAMA after a single injection [651], [653]. Uptake of 17-1A into tumors was demonstrated by radiolabeled antibodies and by immunohistochemistry [666, 496]. There were no objective responses among 40 patients who received single infusions of 15–1,000 mg of 17-1A during phase I trials in patients with various gastrointestinal adenocarcinomas [651], [653]. In a phase II trial of 20 patients who received 200–850 mg of 17-1A, one response was claimed in a patient with recurrent rectal cancer [240]. In a trial of 25 patients who received one to four doses of 400 mg of 17-1A, one patient was interpreted as having a complete response [443]. Eleven patients experienced mild gastrointestinal toxicity during treatment. Subsequently, this group treated eight patients with a 17-1A chimeric antibody and confirmed the longer serum half-life and reduced rate of HAMA, but there were no responders even though the IgG4 human subclass heavy chain enhanced ADCC [444]. Additional trials with 17-1A were conducted in patients with colorectal carcinoma by Mellstedt and colleagues who reported one objective response among 52 patients who were treated in various ways [240], [821]. There were no responses among 10 patients who received a single 200 to 400 mg dose of 17-1A every 4–6 weeks. There was one minor response among 10 patients who received 200 to 400 mg of 17-1A preceded by 400 mg/m2 of the chemotherapy agent cyclophosphamide, which was given in an effort decrease the frequency of HAMA. There was one CR and two minor responses among 14 patients who received the same dose/schedule of Mab preincubated with autologous peripheral blood mononuclear cells. There were no responses in five patients who received a total dose of 3.6 g of 17-1A given as 400 mg daily on days 1, 3 and 6,

Monoclonal antibody therapy at 3-week intervals for 2 treatment courses. There were no responses among seven patients who received 200 to 400 mg every other day up to a total dose of 4.8 to 7.6 g. There were no responses among six patients who received 500 mg 3 days a week, to a total dose of 12 g. Enthusiasm for edrecolomab as a potentially useful anti-cancer agent peaked after publication of the results of a multicenter randomized German trial of adjuvant therapy that was conducted during 1990–1992 in patients with resected Dukes C colorectal cancer [822]. There were 90 patients randomized to observation, and 99 to a post-operative regimen of edrecolomab 500 mg i.v., then 100 mg i.v. once a month for 4 months. Mild side effects were observed in association with the infusions, including fever, chills, abdominal pain, nausea, and diarrhea, but there were four episodes of anaphylaxis during 371 infusions (1.1%). After a median follow up of 5 years, there was a statistically significant difference in disease-free and overall survival curves with a 30% reduction in death rate and 27% reduction in tumor recurrence in the 17-1A group. The magnitude of improvements in key endpoints were similar to those observed in U.S. trials of 5-fluououracil and levamisole that had led to acceptance of 5-FU based chemotherapy in the adjuvant treatment of colon cancer. After a median follow up of 7 years, the apparent benefits of edrecolomab persisted with a 23% reduction in recurrence and 32% reduction of death in the antibody group [595]. On the basis of this trial, Edrecolomab was approved for the treatment of Dukes C colorectal cancer in Germany in 1995. These promising results were followed by four additional phase III trials in the United States and Europe. Trials in North America compared 5-FU + levamisole, or 5-FU + leucovorin to the same agents plus edrecolomab in Dukes C colon cancer using the same dose and schedule used in the German trial. The European trials involve comparisons among 17-1A antibody alone, 5-FU + leucovorin, and 5-FU + leucovorin + 17-1A. Edrecolomab was also studied in combination with chemotherapy and radiotherapy in Dukes B and C rectal cancer. Results of the large randomized European adjuvant trial did not demonstrate a benefit for the use of edrecolomab as adjuvant therapy [574]. There were 2,761 patients randomized in the three-arm trial. The major toxicities observed in the edrecolomab-alone arm were diarrhea in 32%, although severe or life-threatening diarrhea was noted in only 2%. Various types of hypersensitivity reactions to edrecolomab were documented in 25% of patients. Results in the edrecolomab-alone arm were inferior to the 5FU-containing arms (p < 0.05), and there was no evidence of better results when the antibody was combined with 5FU (p = 0.53).

Robert O. Dillman Three-year survival rates were 76% for 5FU/leuovorin, 75% for the antibody/chemotherapy combination, and 70% for the antibody alone. The differences in 3-year event free survival also revealed inferior results for antibody alone, and no advantage to the chemotherapy plus antibody combination. In another trial 377 patients with stage 2 colon cancer, stratified according by whether the primary tumor was T3 or T4, were randomized post-operatively to treatment with 900 mg edrecolomab or observation [297]. The study was stopped because of discontinuation of drug supply in Germany after negative results from other trials became public. After a median follow-up of 3.5 years there was no difference in overall survival or disease-free survival. Several trials have explored edrecolomab in combination with other biologicals with or without chemotherapy. Edrecolomab was combined with IFN-γ in an effort to increase Fc receptors on effector cells and increase the expression of the antigen. Weiner et al. gave 150 mg of 17-1A on days 2 to 4 in combination with 1.0 MIU/m2 of IFN-γ on days 1 to 4, to 19 colorectal cancer patients, but no antitumor responses were noted [774]. In a second trial 27 patients with colon or pancreatic cancer were given IFN-γ at doses up to 40 MIU/day for 4 days followed by 400 mg 17-1A on day 5 [773]. No objective tumor responses were not seen and the authors concluded that the low dose of IFN-γ was as effective as higher doses.. Saleh et al. treated 15 colorectal cancer patients with 0.1 mg/m2 IFN-γ on days 1 to 15 and 400 mg of 17-1A at a dose of 400 mg on days 5, 7, 9 and 22 [622]. No significant objective tumor responses were described. Weiner et al. conducted a phase II multicenter trial of edrecolomab in patients with unresectable pancreatic carcinoma [776]. A dose of 500 mg was given i.v. thrice weekly for 8 weeks. Because of rapid progression of disease in several patients, only 16/28 patients received the planned total dose of 12 grams. There was one durable PR. Tempero et al. treated 30 patients who had advanced, measurable pancreatic cancer with IFN-γ at a dose of 1 MIU/m2 daily for 4 days and 150 mg of 17-1A admixed with autologous leukocytes on days 2, 3 and 4 [713]. One patient had a CR that persisted for 4 months. The median survival for this group of patients was only 5 months. There was no evidence of increased HLA-DR expression on monocytes or lymphocytes following the administration of IFN-γ. GM-CSF and IL-2 have also been combined with edrecolomab in an effort to enhance the immune response. In a trial of 20 patients who received 17-1A plus GM-CSF to increase effector cells and the expression of Fc receptors, there were two CRs [575]. In a

377 subsequent trial 20 patients with advanced colorectal cancer received edrecolomab 400 mg at day 3 of a 10-day treatment cycle with the simultaneous administration of s.c 250 μg/m2 GM-CSF once daily and 2.4 million U/m2 IL-2 twice daily for 10 days in a planned monthly treatment cycle [321]. Doses of the various drugs had to be decreased because of infusion/allergic type reactions. There was one PR. In a trial of similar design, 12 colorectal cancer patients were treated with 300 μg GM-CSF and 6 million units IL-2 s.c. daily from day 1 to 10 with 400 mg edrecolomab on day 3 of the first cycle, and reduced to150 mg on subsequent cycles up to a maximum of four cycles [221]. There were no tumor responses. Edrecolomab was combined with chemotherapy, GM-CSF, and IFN-α for 27 patients with metastatic colorectal cancer in one trial [434], and with capecitabine alone in 27 patients with various metastatic adenocarcinomas [457]. It was unclear whether the clinical activity seen was any better than what might have been expected with the same chemotherapy alone.

Other anti EpCAM antibodies Elias et al. treated NSCLC patients with the IgG1 momab KS1/4 that reacts EpCAM [205]. Five patients received sequential doses of 1, 10, 60, 100, and 1,000 mg over 2 weeks for a total of 1,661 mg. Minor upper gastrointestinal toxicity was seen in some patients. No antitumor responses were seen. The anti-EpCAM human-engineered Mab ING-1 was given to 25 advanced adenocarcinoma patients at doses of 0.03 mg/kg, 0.10 mg/kg, and 0.30 mg/kg [151]. Two patients experienced reversible, but dose limiting pancreatitis; so, 0.10 mg/kg was felt to be the maximum tolerated dose. There were no responses observed in the first 25 patients, or seven additional patients who were treated with repeated weekly doses of 0.10 mg/kg. Adecatumumab (MT201) is a human recombinant IgG1 Mab that binds EpCAM. Twenty patients with hormone refractory prostate cancer were treated with two adecatumumab infusions on days 0 and 14 in over a dose range of 10 to 262 mg/m2 [528]. Mild infusion reactions including fever and nausea were the most common adverse events. No tumor responses were reported. Catumaxomab is a trifunctional Mab that binds human EpCAM and CD3. Escalating doses of catumaxomab from 2 to 7.5 μg were infused over 8 h in patients with advanced NSCLC who were premedicated with corticosteroids and antihistamines to reduce infusion reactions [654]. Hepatic transaminasemia was the doselimiting toxicity. The MTD was determined to be 40 mg of dexamethasone followed by 5 μg of catumaxomab. No tumor responses were reported.

378 SK-1 is a human Mab generated using hybridoma fusion methods with lymphocytes from regional lymph nodes of colon cancer patients. that recognizes a glycoprotein expressed on the majority of colon cancer tissues that appears to be in the EpCAM family of molecules [398], [801]. In the initial dose escalation study colon cancer patients received 2, 4, or 10 mg given three times. Treatment was well-tolerated; no tumor response were reported [399].

CA125 Oregovomab (B43.13, OvaRex) is a murine Mab that targets CA125 [32]. Because CA125 is shed into the circulation, infusion of oregovomab is associated with immune complexes which probably would complicate a direct therapeutic approach. However, in patients who received low doses of oregovmab, it was possible to demonstrate antibody and T cell responses to CA125 that were not present before injection of the antibody; so it has been hypothesized that these immune complexes are taken up by dendritic cells leading to an anti-CA125 immune response that may be therapeutic [526]. In a phase II pilot study 13 heavily pretreated, platinum-resistant ovarian cancer patients CA125 > 35 U/mL, were given 2 mg of oregovomab i.v. at weeks 0, 2, 4, 8, and 12, followed by additional dose every 3 months for up to 2 years [200]. More than half the patients exhibited enhanced anti-CA125 immune responses, four patients showed decreases in CA125 levels, and three patients had progression-free disease for more than 2 years. In a phase III trial 145 patients with stage 3 or 4 ovarian cancer, who had achieved a complete response following other therapy, were randomized to oregovomab or placebo administered at weeks 0, 4, and 8, and then every 12 weeks for up to 2 years [33]. There was no difference between the two arms in time to recurrence, 13.3 months for oregovomab and 10.3 months for placebo (p = 0.71). Abagovomab (formerly ACA125) is a murine antiidiotypic Mab antibody to a Mab that reacts with CA125 (Ab2). It is being explored as a vaccine in ovarian cancer and appeared promising in phase I trials based on the induction of a human anti-CA125 antibody (Ab3) response [554, 616]. This approach is discussed in more detail in the vaccine chapter of this text. MOv18 is a chimeric Mab that reacts with a folate receptor on ovarian cancer cells. In a phase I trial 15 ovarian cancer patients received single i.v. doses ranging from 5 to 75 mg [497]. At doses of 50 mg and above all patients experienced side effects that included fever, headache and nausea of mild degree. No responses were

Monoclonal antibody therapy reported. A bifunctional momab variant of MOv18 called OC/TR combines the anti-ovarian binding of the MOv18 with anti-CD3 to target T lymphocytes. The product was given as an F(ab’)2 in an effort to avoid systemic toxicity related to removal of T cells in the reticuloendothelial system [721]. However, significant infusion reactions still occurred except at the lowest i.v. doses among five patients, suggesting that the binding to CD3 itself led to the acute release of cytokines that mediated the side effects. This Mab has also been given i.p. as one or two 5-day cycles with IL-2 to 28 patients who had residual limited disease after surgical debulking and chemotherapy [86]. Responses were confirmed in seven patients based on surgical restaging, although one had concurrent progression in retroperitoneal nodes. Most of the responders subsequently progressed outside the peritoneal cavity, but three of the CRs lasted 18 to 24 months. HAMA was detected in 21/25 patients. Elevated plasma levels of TNF-α, IL-6, G-CSF and neopterin were detected following infusions of higher doses of Mov18. A chimeric version of this Mab has been developed and given to small numbers of patients [748]. An IgE construct has also been engineered [366].

Other Anti-Adenocarcinoma Monoclonal Antibodies L6 L6 is a mouse Mab that reacts with a 24 kD epithelial antigen with expression on most adenocarcinomas. It mediates CMC with human complement and ADCC with human NK cells and macrophages. The L6 antigen is now believed to be a distant member of the transmembrane-4 superfamily (TM4SF). Goodman et al. treated five metastatic breast cancer patients with daily doses from 5 to 400 mg/m2 of murine L6 for 7 days to a total dose of 35 to 2,800 mg [262]. One patient with hormone receptor negative cancer, which had progressed on the chest wall despite chemotherapy and radiation therapy, had a CR after receiving a 400 mg dose. The response did not become apparent until about 5 weeks after treatment, and it took about 14 weeks before the CR was attained. Doses from Doses from 5 to 400 mg/m2 were given daily for 7 days to treat nine patients with advanced ovarian cancer [262]. No responses were seen. No evidence of anti-tumor effects in three patients with NSCLC who received L6 [262]. Among five colorectal cancer patients, one patient had a partial response following seven daily 2-h infusions of L6 followed by 1 week of rest, and then 4 days of s.c. IL-2 [811]. A humanized chimeric form of L6 was developed and administered as single infusions ranging from 350 to

Robert O. Dillman 750 mg/m2, but no responses were seen in a phase II trial of 21 patients [263]. Subsequent work with this Mab focused on radioimmunotherapy and a chemotherapy immunoconjugate.

Monoclonal antibody 494/32 Buchler et al. conducted a prospective randomized trial of observation or adjuvant therapy with a 10-day course of 370 mg of the murine IgG1 Mab 494/32 in 61 patients who had undergone a Whipple resection for pancreatic cancer [63]. Analysis after 10 months showed no significant difference in survival between the two treatment groups who had median survivals of 428 days for the treatment group, compared to 386 days for the control group. Ryan et al. administered three different IgM antibreast human Mabs, selected based on their patterns of tissue reactivity, to 10 patients with metastatic breast cancer [615]. One patient each was treated at dose levels of 1, 2, 4, and 11 mg, and then 3 patients were treated at 20 mg, and three other patients received 22 mg in addition to tracer doses of 111-Indium labeled antibody. No tumor regressions were seen in this pilot study or relatively low doses of antibody. Saleh et al. investigated momab D612 in a dose escalation trial of multiple doses of 10 to 180 mg/m2 over 8 days in patients with various gastrointestinal malignancies [625]. The dose limiting toxicity was a secretory diarrhea. HAMA was detected in 18/21 patients. The dose of 40 mg/m2 was selected for phase II trials. No responses were reported. In a subsequent study, 14 patients with metastatic gastrointestinal cancer received the D612, 40 mg/m2, days 4, 7, and 11 along with 80 μg/kg/ day of recombinant human monocyte colony-stimulating factor [626]. Secretory diarrhea occurred in 10 of 14 patients. There were no anti-tumor responses.

Mucin Proteins and Human Milk Fat Globulin MUC1 is a member of a family of mucin transmembrane glycoproteins expressed on most glandular epithelia [672]. Many adenocarcinomas over-express and shed MUC1, and elevated MUC1 serum levels can serve as a marker of tumor burden and progressive disease. Human milk fat globulin (HMFG1) antigen, which is over-expressed in more than 90% of breast cancers and serous ovarian cancers in an underglycosylated form, is the same as MUC1. Mab HMFG1 is a mouse IgG1 that reacts with epitopes of MUC1 on high molecular weight human milk fat globulin glycoprotein antigen. Kosmos et al. treated 15 ovarian cancer patients with intraperitoneal HMFG1 rather than i.v. based on evidence that high circulating levels of antigen result in immune complexes

379 and altered biodistribution [405]. A dose-dependent in vitro T-cell proliferation was observed in 13/15 patients, but no tumor responses were seen. A single-chain Fv (scFv) and Fab fragment from this antibody have been engineered that retain their specificity for MUC1 [552]. The huHMFG-1 (AS1402) antibody is a humanized IgG1 directed against MUC1 and is currently in clinical trials for the treatment of breast carcinoma [502]. Seventeen patients with MUC1-positive cancers were treated with the murine anti-MUC1 BrevaRex mAbAR20.5, which was given i.v. in doses of 1, 2 or 4 mg every 2 to 4 weeks for a total of six doses, with the intent of forming immunogenic complexes as a vaccination approach [152]. MUC1-specific anti-T cell responses were confirmed in five patients.

c30.6

anti-colorectal cancer antibody

The mouse Mab m30.6, which had been used as a diagnostic tool to detect colorectal cancer cells, was engineered to create the chimeric Mab c30.6. Clinical trials were initiated after demonstration of in vitro and in vivo anti-tumor effects in preclinical models. Four patients with metastatic colorectal cancer received 3 mg of 123I-labeled c30.6, and the subsequent 13 patients were treated with single doses up to 50 mg/m2 [769]. The most frequent side effect was infusion-related erythema and a severe burning sensation on the face, neck, ears, chest, palms, soles, and genitalia, which was reduced by premedication with blockade of type I and II histamine receptors. No anti-tumor responses were seen.

A33 The A33 antigen is a cell membrane surface glycoprotein that is sole or predominantly expressed on human gastrointestinal epithelium and on nearly all colorectal cancers [300]. HuA33, is a CDR-grafted humanized Mab against the A33 antigen. In an initial phase I study 11 patients with metastatic, chemotherapy-resistant colorectal cancer were given huA33 at dose of 10, 25, or 50 mg/m2/week every 4 weeks [778]. Significant toxicity occurred in four patients who had developed a strong HAHA response against the Mab. Two patients experienced infusion reactions that included fevers, rigors, facial flushing, and blood pressure changes, while the other two patients had serum-sickness type reactions associated with skin rash, fever, and myalgia. One patient, who was HAHA negative, had a PR. In a second phase I trial 12 colorectal cancer patients were given lower doses of huA33, 0.25, 1.0, 5.0, and 10 mg/m2 along with a tracer dose of radiolabeled Mab prior to surgery [647]. At these doses there was no significant toxicity and tumor localization was confirmed.

380

Lewis Y Antigen Lewis Y antigen is a blood group antigen that is highly expressed on the surface of epithelial tumors. Hu3S193, a humanized Mab that binds to the Le(y) antigen, was given as four weekly doses to eight colorectal cancer patients, six breast cancer patients, and one NSCLC patient, at doses of 5, 10, 20, and 40 mg/m2 [648, 649]. All patients had tumors that were Le (y)-positive. Mild transient nausea and vomiting was observed following the 40 mg/m2 dose. Indium-111 tracer studies confirmed uptake in metastatic sites. No objective tumor responses were recorded. A similar trial with huS193 was carried out in 10 patients with Le (y)-positive small cell lung cancer [410]. Tumor uptake of radiolabeled Mab was confirmed. Toxicities, which again were mild, and only seen at the 40 mg/m2 dose, included two patients with nausea/vomiting, and one who developed urticaria.

G250 (CAIX, MN/CA9) Renal Cell Cancer Antigen The monoclonal antibody G250 (mAbG250) was produced by immunizing against human renal cell carcinoma (RCC). The G250 antigen was isolated and determined to be the same as the MN/CA9 antigen of HeLa cells which has carbonic anhydrase activity. The G250 antigen, which is also referred to as G250MN protein, and carbonic anhydrase IX (CAIX), is found on 95% of clear cell RCC, is rarely expressed on other types of kidney cancer or cancers from other organs [412]. The associated carbonic anhydrase activity has been implicated in regulation of cell proliferation in response to hypoxia. For many years it has been proposed as a good target for Mab-directed therapy [734]. Initial studies were done with the murine Mab G250. In a phase I study, 16 RCC patients received five different doses of 131-I labeled G250 a week prior to nephrectomy [536]. Good tumor localization was confirmed and no significant side effects. The murine IgG2a G250 was converted to a mouse kappa light chain/human IgG1 chimeric Mab, cG250 [700]. In a dose escalation trial, 13 patients received doses of 5, 10, 25, or 50 mg/m2 given weekly i.v. for 6 weeks [144]. There were no serious toxicities and one patient had a CR. In a phase II trial 36 RCC patients, 21 of whom were pre-treated with IL-2 and or IFN-α, were given weekly doses of 50 mg cG250 was i.v. for 12 weeks [43, 749]. There were no serious adverse events. There were no objective responses by the end of treatment, but one of ten patients who continued treatment for an additional 8 weeks eventually developed a CR. Doses of 5, 10, 25, or 50 mg/m2 were given weekly by i.v. infusion for 6 weeks.

Monoclonal antibody therapy

Prostate Cancer Associated Antigens Low doses of IgG1 murine Mabs reactive with prostatic acid phosphatase (PAP) or prostate specific antigen (PSA) were given to 19 patients with metastatic prostate cancer [183, 286]. Tracer doses of 111Indium-labeled antibodies showed uptake in some sites of tumor. There were no significant toxicities associated with treatment, but nearly half of patients developed HAMA. No tumor responses were observed. J591 is a humanized IgG1 Mab that targets the extracellular domain of the transmembrane prostate-specific membrane antigen (PSMA). Fourteen patients with hormone refractory progressive metastatic prostate cancer received 10, 25, 50, and 100 mg of J591 with 2 mg of Indium-111 tracer [506]. There were no significant toxicities and tumor uptake was confirmed. Investigators have reported results for more than 2,000 prostate cancer patients who received 111-In CYT-356 (Capromab, Prostascint™) [298, 680]. Uptake in regional lymph nodes has been confirmed in some patients, but there have not been reports of antitumor effects. but tumor responses could not be measured in this trial design.

Melanoma and Neuroectodermal Associated Antigens Melanoma Glycoprotein Antigens Many of the first Mab developed against solid tumors were to melanoma-associated antigens, including melanotransferrin (p97, p96.5 or gp95 antigen) and high molecular weight chondroitin sulfated proteoglycan core protein (p240, p280). None of the trials utilizing anti-p97 and antip240 murine Mab resulted in objective tumor responses, but this may be due to the limited evidence in vitro for CMC or ADCC with these murine Mabs and/or lack of regulatory significance for these antigens, and the relatively small doses that were delivered in these trials. Dillman and Halpern infused 1 to 50 mg of mouse Mabs into melanoma patients as single 2-h infusions, with or without a 1 mg tracer dose of 111Indium radiolabeled Mab [183, 287]. Twenty-four patients received IgG1 Mabs directed against the p97 antigen [287, 679], and another 28 received IgG2a Mabs directed against the p240 antigen [183, 288]. No definitive tumor responses were seen in patients with measurable disease. Similar experiences were reported by other investigators utilizing relatively low quantities of these same Mabs with radiolabeled tracers [392, 514, 515]. Oldham et al. treated eight patients with 9.2.27, an IgG2a murine Mab that reacts with the p240 glycoprotein antigen [535]. Patients received twice weekly escalating doses of 1, 10, 50, 100, and 200 mg. There was a

Robert O. Dillman relationship between dose and tumor penetration and antigen saturation based on immunohistochemical staining of tumor biopsies which showed in vivo localization in s.c. tumors in 6/8 patients following doses of 50 mg or greater. No tumor responses were seen. Goodman treated four melanoma patients with a combination of two mouse Mabs, an IgG1 anti-P97 and an IgG1 anti-p240, and a fifth patient received anti-p97 alone [261]. Escalating antibody doses were administered as 6-h infusions daily for 10 days to a maximum dose of 50 mg per infusion. No objective tumor regressions were observed.

Melanoma Disialoganglioside Antigens Many early murine Mabs derived from immunization with melanoma cells reacted with disialoganglioside glycolipid antigens, such as GD2 and GD3, which are commonly expressed on neuroectodermal tissues [573].

Anti-GD3 The murine anti-GD3 Mab R24 was given to 21 patients at doses of 1, 10, 30, or 50 mg/m2 every other day for 2 weeks for a totals of 8, 80, 240, or 400 mg/m2 [328, 738]. At higher doses all patients developed urticaria and pruritus that typically occurred within 2 to 4 h following treatment and often appeared around tumor or at sites of previous tumor excision. In patients who received 30 and 50 mg/m2, uptake of Mab in tumor was readily demonstrated using biopsies and immunohistochemical analysis. Significant tumor regressions were seen in four patients: 2/6, 1/6, 1/6, and 0/3 for each successive dose level. Responses were first noted within 2 weeks of completing treatment, but continued to increase for several months. An effort was made to intensify the administration of mouse R24 since treatment was associated with a high rate of urticaria for patients receiving cumulative doses of 400 mg/m2 over 2 weeks [18]. Eight patients received R24 at doses of 800 and 1,200 mg/m2 over 1 week by continuous i.v. infusion along with prophylactic histamine blockers. Severe toxicity was noted. One patient developed anaphylaxis. All patients developed lymphopenia and decreases in serum complement. A cytokine induced vascular leak syndrome occurred in seven patients during the first 2 days of therapy. Serum sickness was observed in six patients within a week, which coincided with the ability to detect HAMA. The maximum tolerated dose was felt to be 800 mg/m2 based on dose-limiting toxicities that included hypertension, chest pain, and vision changes at the 1,200 mg/m2. In another phase I trial in 11 patients with advanced melanoma, R24 was given i.v. daily for 5 days at doses

381 of 10 mg/m2, 30 mg/m2 or 50 mg/m2 [453]. Toxicity was substantial and included hypoalbumenemia in all cases, and a death at the highest dose. There was one mixed tumor response. In another phase I trial with murine R24, 37 patients with advanced melanoma received 1, 10, 20, 40, or 80 mg/m2 with five to six patients at each dose [393]. Urticaria was the most frequent adverse event. A doselimiting toxicity of pulmonary capillary leak syndrome occurred in three of five patients at the highest dose. Hepatic enzyme elevation and chest pain were noted even at lower doses. Two responses were seen at the lowest dose. R24 has been given in combination with a variety of different immunostimulatory cytokines in the hopes of enhancing antitumor effects. Caulfield et al. gave five daily 6-h infusions of escalating dose of R24 in combination with intramuscular with IFN-α2a to 15 melanoma patients [100]. Toxicities were similar to those seen in other trials of R24. No tumor regressions were reported, which was disappointing in view of the apparent activity of R24 and IFN-α as single agents. Several trials of R24 and IL-2 have been conducted. Bajorin et al. evaluated the combination of IL-2 and murine R24 in 20 patients with metastatic melanoma in a phase I trial in which IL-2 was given at a dose of 6 MIU/m2 i.v. over 6 h on days 1–5 and 8–12, while the anti-GD3 antibody R24 was given on days 8–12 at 1, 3, 8, or 12 mg/m2 per day with five patients evaluated at each dose level [17]. Some in vitro T-cell activation was demonstrated and one patient had a PR in soft tissue sites lasting 6 months, and two other patients had minor responses [779]. R24 combined with low-dose continuous infusion IL-2 resulted in one PR and two minor responses in 28 melanoma patients who were treated in a dose escalation trial [682]. At higher doses two patients experienced chest and abdominal discomfort that necessitated dose reductions. In a separate trial, Creekmore et al. gave a higher dose of continuous infusion IL-2 followed by R24 [134]. This sequencing of the agents was associated with responses in 10/28 patients while concurrent administration was associated with responses in only 1/17 patients. However, in the sequential trial, there were also five patients who never received R24 because of the severity of IL-2-related toxicity. R24 has also been given to melanoma patients in combination with GM-CSF, M-CSF, or TNF-α. Chachoua et al. gave s.c. GM-CSF for 21 days at a dose of 150 ug/m2/day, and gave R24 by continuous i.v. infusion on days 8–15 at doses of 0, 10, or 50 mg/m2 [101]. There were no tumor responses observed with GM-CSF alone in five patients, or the lower R24 dose in six patients, but two responses

382 were seen at the 50 mg/m2 dose in nine patients. However, 4/9 were unable to complete this single course of therapy because of toxicity. Minasian et al. treated 19 metastatic melanoma patients with a 14-day continuous infusion of recombinant human M-CSF at a dose of 80 ug/kg/day in combination with R24 which was administered daily by i.v. infusion at doses of 1, 3, 10, 0 and 50 ug/m2/day on days 6 to 10 [492]. There were no objective tumor responses, although three patients did have a mixed response with regression of some lesions. In a pilot study R24 was combined with two different doses of TNF in eight patients with melanoma [491]. One patient had a dramatic tumor lysis syndrome associated with hemorrhagic tumor necrosis in multiple visceral sites of disease. Combining data from 11 R24 trials cited above reveals a cumulative response rate of 6/77 for R24 alone, and 6/127 for R24 in combination with other biologicals.

Anti-GD2 The IgG3 anti-GD2 mouse Mab 3F8 was used by Cheung et al. to treat melanoma patients who received 5, 20, 50, or 100 mg/m2 as 8-h infusions given daily for 2 to 4 days [109]. The study was closed at the 100 mg/m2 dose because all patients treated at that dose level developed hypertension. Treatments were associated with focal pain at tumor sites and pain especially over the abdomen, back, and extremities which was attributed to reactivity with neural tissue. Urticaria, fever, nausea, vomiting and sweats were also noted. Inflammatory actions were observed around tumors, and partial responses were reported for 2/9 patients and two others had a mixed response. Six melanoma patients were given a combination of R24 and 3F8 at doses of only 1–10 mg/m2; but no tumor regressions were seen. Saleh et al. conducted a phase I trial of a different murine anti-GD2 Mab 14G2a, an IgG2a switch variant of the IgG3 anti-GD2 Mab 14.18, in which 12 patients with melanoma received single i.v. infusions of doses from 10 to 120 mg [623]. Treatment was complicated by abdominal and pelvic pain, which necessitated narcotic analgesia for control. All 12 patients developed HAMA. There was one PR. Murray et al. gave Mab 14G2a to 11 patients with metastatic melanoma as part of a phase I trial [516]. There were no objective remissions although two patients exhibited mixed responses to the antibody. Significant adverse events were noted including generalized pain, fever, rash, paresthesias, weakness, hyponatremia and postural hypotension were the significant toxicities observed. The investigators suggested 100 mg/ m2 as the maximum tolerated dose of this Mab. A chimeric version of the anti-GD2 antibody (ch14.18) with a constant region of human IgG1κ has been tested

Monoclonal antibody therapy in a phase I trial in 13 melanoma patient, who received 5 to 100 mg of Mab [624]. As has been seen in other trials of anti-GD2 antibodies in adult patients, the major toxicity associated with this therapy was abdominal/pelvic pain syndrome that necessitated use of narcotic analgesics. No tumor responses were seen in this cohort of patients. KM871 is an IgG1 kappa chimeric Mab to GD3, that was given to 17 metastatic melanoma patients at dose levels of 1, 5, 10, 20, or 40 mg/m2 at 2-week intervals for three doses [645]. Specific targeting of melanoma tissue was demonstrated using indium-111 tracer Mab. No dose-limiting toxicity was observed although three patients experienced pain and/or erythema at tumor sites that was not related to dose. One patient had a partial response that lasted 11 months. KW-2871 is another IgG1 kappa chimeric Mab to GD3 that was given to 17 metastatic melanoma patients who received an initial 10 mg/m2 and then 2 weeks later were stratified into four cohorts to receive four doses of KW-2871 at 2-week intervals at doses of 20, 40, 60, and 80 mg/m2 [232]. The maximum tolerated dose for this schedule was 40 mg/m2 based on grade three dose-limiting toxicities of laryngospasm and chest tightness at doses of 80 and 60 mg/ m2. Urticaria were noted in all 16 patients who were not premedicated with antihistamines. Neuroblastomas also express disialogangliosides. Treatment of eight children with the 3F8 anti-GD2 mouse Mab produced two CRs, one at 5 mg/m2, and the other at 20 mg/m2 [109]). The abdominal/pelvic pain syndrome was not as problematic in these pediatric patients as it had been in adults with melanoma. 3F8 was used to treat 16 neuroblastoma patients who had stage 4 disease [111]. Adverse reactions included pain, fever, urticaria, hypertension, hypotension and anaphylactoid reactions. Three patients survived more than 6 years. In an effort to eradicate minimal residual disease, 3F8 was given after completion of chemotherapy, to 34 neuroblastoma patients, most of whom had bone and/or bone marrow metastases [110]. Only 23 were in CR at the time 3F8 was given. Pain, fever, and urticaria were the most frequent adverse events noted. Fourteen patients were still alive after 3 to 10 years of follow up. Murray et al. reported two PR among five neuroblastoma patients who were treated with the 14.18 Mab as part of a phase I trial [516]. They also noted that pediatric patients tolerated the agent much better than adults. The anti-GD2 Mabs have been combined with various cytokines in an effort to enhance anti-tumor efficace. Yu and colleagues treated 17 neuroblastoma patients, aged 2 to 8 years, with GM-CSF and the chimeric Mab ch14.18, which was engineered with a human IgG1κ modification of the murine 14.18 [804].

Robert O. Dillman Toxicity was minimal in these children as opposed to the experience in adult patients with melanoma. Significant tumor responses were noted in eight patients, including three complete remissions. 3F8 and GM-CSF were given to 19 neuroblastoma patients whose disease was resistant to initial therapy [411]. One cycle consisted of s.c. GM-CSF alone for 5 days, and then GM-CSF i.v. followed hour later by i.v. 3F8 on days 6–10, and 13–17 along with additional s.c. doses of GM-CSF on days 11 and 12. The treatment was given in an outpatient setting. Side effects were mild and included manageable pain and occasional rash. Complete responses were recorded for 12/19 patients. Choi et al. treated 23 melanoma and four sarcoma patients with a combination of ch14.18 and R24 antibodies together with continuous IL-2 [114]. When combined with IL-2, the maximum tolerated dose for both Mab was 5 mg/m2/day because of dose-limiting toxicities including severe allergic reactions and pain with both products at the 7.5 mg/m2/day dose. Two melanoma patients had PRs. L612 HuMAb is a human IgM that binds to ganglioside GM3 and effects CDC with human complement. Nine patients with measurable metastatic melanoma were given a continuous infusion of L612 at a dose of 960 mg, 1,440 mg, or 1,920 mg over 48 h [341]. Mild pruritus and skin rash were noted. No objective responses occurred, but one patient reported pain in s.c. tumor sites.

383 extramedullary myeloma with a murine anti-IL6 Mab [29]. Three patients had an objective antiproliferative effect based on labeling index, and one had a minor regression of a tumor mass. Most patients had some worsening of neutropenia and thrombocytopenia. Moreau et al. have used a combination of a murine antiIL-6 monoclonal antibody with dexamethasone and high dose melphalan in the autologous transplant setting [501]. A major obstacle to treatment with anti-IL-6 is the very high rates of IL-6 production in some patients with myeloma, which would need to be reduced by another therapeutic modality [610]

IL-15 Interleukin-15 (IL-15) is a proinflammatory cytokine that stimulates T lymphocytes and NK cells. HuMax-IL15 is a human IgG1 anti-IL-15 Mab that suppresses T cell proliferation and induces apoptosis in vitro. HuMax-IL15 was given for 12 weeks to patients with rheumatoid arthritis in a double-blinded phase I/II dose-escalation, placebo-controlled trial that enrolled 30 patients [27]. At the doses used in this trial there, HuMax-IL15 was well tolerated and there were no significant effects on subsets of T lymphocytes. This product may be developed mostly for the treatment of auto-immune disease, but could also be considered for certain hematologic malignancies in which IL-15 stimulation may be important.

Gastrin Releasing Peptide (Bombesin)

Other Ligand Targets Interleukin-6 Interleukin-6 (IL-6) is a growth factor for plasma cells that is involved in autocrine/paracrine growth regulation [107]. Soluble IL-6 receptors and L-6 are typically elevated in the advanced stages of myeloma. Klein et al. treated a patient whose primary plasma cell leukemia whose disease was refractory to chemotherapy, with daily i.v. anti-IL-6 Mab [396]. Serial monitoring during the 2 months of treatment demonstrated a decrease in serum calcium, serum paraprotein, C-reactive protein, and the percentage of cells in S-phase in the bone marrow, but an objective tumor response could not be claimed. There were no major toxicities but decreases in both platelet and white blood cell counts were noted. A chimeric anti-IL-6 Mab was given to 12 patients with myeloma at daily doses of 5, 10, 20, 40, and total doses of 140, 280, 560 mg, and 1,120 mg given as two 14-day treatment cycles [747]. There were no responses, and much of the anti-IL-6 antibody was complexed to circulating IL-6. Bataille et al. treated 10 patients with

Peptides such as human gastrin-releasing peptide (GRP) and the peptide bombesin, are sometimes produced by lung cancers. Mulshine et al. infused the anti-GRP murine Mab 2A11 into 12 patients with NSCLC, at dose of 1, 10, or 100 mg/m2 i.v. thrice weekly for 4 weeks [513]. No tumor regressions were noted. In a phase II trial, 12 patients with small cell carcinoma of the lung were given 2A11 at dose and schedule of 250 mg/m2 thrice weekly for 4 weeks [375]. In this study one patient achieved a complete radiographic remission which lasted 5 months, and four patients had stable disease. No significant toxicities were observed. In an accompanying radioimmunodetection study, tumor uptake of 111-Indium conjugated 2A111 was detected in 11/12 patients [105]. The receptor for GRP/bombesin could also be a target for Mab therapy [807].

Fibroblast Activation Protein Sibrotuzumab (BIBH 1) is a humanised version of the murine anti-FAP mAb F19 which reacts with fibroblast activation protein (FAP). FAP is highly expressed by activated fibroblasts of the tumor stroma in many malignancies including breast, colon and lung carcinomas [476].

384

Monoclonal antibody therapy autoimmune diseases and associated with similar risks for infection. The specific marketing indications for adalimumab are rheumatoid and psoriatic arthritis, ankylosing spondylitis, and Chron’s disease [739]. At this time there are no published clinical trials in which adalimumab has been used in the treatment of human malignancy.

In a phase I trial, 26 patients, including 20 with metastatic colorectal cancer and six with NSCLC, were given sibrotuzumab at doses of 5, 10, 25, or 50 mg/m2 weekly for 12 weeks, with trace 131-I labeled Mab during weeks 1, 5, and 9 [646, 323]. There was minimal toxicity during 218 infusions of sibrotuzumab administered during the first 12 weeks and only one dose limiting toxicity was observed. Tumor uptake of the Mab was confirmed. There were no tumor responses. In a phase II trial 25 metastatic colorectal cancer patients were given i.v. weekly 100 mg doses of sibrotuzumab for 12 scheduled weeks [777]. Five patients experienced infusion reactions that included rigors or chills, flushing, nausea, and one episode of bronchospasm. HACA were detected in three patients after four to 12 infusions. There were no tumor responses.

Anti-Idiotype Vaccines

Tumor Necrosis Factor Alpha (TNF-α)

Anti-Idiotype Vaccines in Adenocarcinomas

Infliximab (Remicade®, Centocore, Malvern, PA) Infliximab is a chimeric IgG1 kappa that neutralizes tumor necrois factor alpha (TNF-α) by blocking binding of the ligand to its receptor because of its own high affinity binding to the ligand. It is used in the treatment of a variety of chronic inflammatory autoimmune disorders including Crohn’s disease, ulcerative colitis, rheumatoid and psoriatic arthritis, uveitis, and ankylosing sondylitis [739]. Not surprisingly, sepsis and opportunistic infections are significant complications of TNF-α inhibition. Infliximab was given to 19 patients with cytokineresistant renal cell carcinoma (RCC) at doses of 5 mg/kg at weeks 0, 2, and 6, and then every 8 weeks and then to 18 RCC patients at doses of 10 mg/kg at weeks 0, 2, and 6, and then every 4 weeks [294]. Severe adverse events included a grade 3 hypersensitivity reaction and a death from pulmonary infection with sepsis. Clinical activity was promising with partial responses observed in 3/19 patients using the first dose/schedule and 11/18 using the second dose schedule. In a trial that enrolled patients with a variety of advanced malignancies, 21 patients received infliximab at 5 mg/kg and 20 patients were treated at 10 mg/kg at 0 and 2 weeks and then every 4 weeks [59]. These doses and schedule were not associated with any dose-limiting toxic effects. There were no objective responses in this heterogeneous patient population. In both these trials neutralization of plasma TNF-α was demonstrated shortly after the first infusion.

After clinical trials suggested that murine Mab 17-1A could induce an anti-idiotype cascade, Herlyn et al. tried to induce endogenous human anti-tumor antibodies in patients with colorectal cancer [314]. They gave patients repeated s.c. injections of a goat antibody against the idiotype of Mab 17-1A. The goat antibody induced human antibodies against the same antigen targeted by 17-1A. A significant tumor response was seen in 1/30 patients so treated. Foon et al. treated 32 patients following resection of locoregionally advanced colorectal cancer, with 2 mg s.c. injections of CeaVac, an anti-idiotype antibody to an anti CEA antibody, every other week for four injections and then monthly until tumor recurrence or progression [229]. CeaVac induced a potent anti-CEA humoral and cellular immune response in all 32 patients, including 15 patients who received 5-FU chemotherapy during vaccination. The anti-idiotype Mab, an anti-HMFG antibody, vaccine 11D10 (TriAb), was given to 45 patients with metastatic breast cancer that had responded to standard chemotherapy, before and after high dose chemotherapy and autologous stem cell rescue [583]. Idiotype-specific humoral and T-cell proliferative responses were demonstrated in most patients despite the immunosuppressive effects of high-dose chemotherapy. The 48% 3-year overall survival rate is similar to what has been reported in other series of stem cell transplant in patients with advanced breast cancer. In a multicenter cooperative group trial, 56 patients with colorectal cancer who had undergone curative resection of their liver metastases, received a combination of two anti-idiotype monoclonal antibody vaccines,

Adalimumab (Humira®, Abbott, North Chicago, IL) Adalimumab is a recombinant human IgG1 that blocks the binding of TNF-α to its cell surface receptors. Like infliximab, it is also used in a variety of chronic inflammatory

As discussed earlier in this chapter, the anti-idiotype cascade provides a rationale for developing Ab2 Mab products as a vaccine to induce an endogenous Ab3 response against the target antigen. In the nomenclature that is now in use, the generic names of such products have “ab” at the beginning of their name.

Robert O. Dillman one to CEA(CeaVac) and the other to HMFG (TriAb) [567]. About 40% of patients were progression-free after 2 years, which is similar to what is seen with surgery alone. The anti-idiotypic antibody vaccine ACA125, that is designed to induce endogenous immune responses against tumor antigen CA125, was given to 119 women with advanced ovarian carcinoma [588]. A specific anti-antiidiotypic antibody (Ab3) response was detected in 68% and endogenous CA125 antibodies were detected in 50%. Ab3-positive patients had a much longer survival than Ab3-negative patients (23 vs. 5 month, p < 0.0001). In a randomized phase II trial, prior to surgery 67 patients with colorectal cancer were randomized to receive the human anti-idiotypic Mab 105AD7 with or without BCG or to no treatment [735]. Vaccinations were initiated in 32 patients prior to surgery and were planned to be continued for up to 2 years. Most of the immunized patients exhibited immune responses, but specific immune data and comparative data have not been reported.

Anti-Idiotype Vaccines in Melanoma Mittelman and colleagues gave anti-idiotype antibodies directed Mab that targeted the gp240 glycoprotein antigen, in effort to induce endogenous antibodies against gp240 on melanoma cells. In a phase I trial involving 16 patients, s.c. injections of MAF11-39 from 0.5 mg to 4 mg were well tolerated; so a phase II trial was carried out in which an additional 21 patients were given 2 mg on days 0,7, and 28 [493]. At the 2 mg dose 16 patients exhibited a response to gp240 and one patient had a CR. In another trial the MK-23 anti-idiotype antibody was conjugated to keyhole limpet hemocyanin (KLH) and coadministered with BCG in 25 patients [494]. Three PRs were observed, and 14 of 23 patients developed endogenous human antibodies against gp240. Foon et al. treated 47 melanoma patients with TriGem, an anti-idiotype antibody to a Mab that binds to disialoganglioside GD2, using weekly s.c. injections of 1, 2, 4, or 8 mg in combination with QS-21 as an adjuvant [230]. An anti-anti-idiotype (AB3) response was detected in 40/47 patients. One patient had a complete response.

1E10 Anti-Idiotype Vaccines to Gangliosides A series of trials have been conducted with aluminum hydroxide-precipitated 1E10, which is an anti-idiotype murine Mab (Ab2) specific to an Ab1 Mab that reacts

385 with NeuGc-containing ganglioside that is present on various solid tumors. Up to six i.d. injections of 1 to 2 mg of anti-Id mAb were given every 2 weeks. In 20 patients with advanced melanoma, the treatment was well tolerated and 16 patients exhibited strong Ab3 responses against GM3 [7]. In locally advanced or metastatic breast cancer, eight out of nine patients who received four or more injections exhibited strong Ab3 responses against GM3 [159]. Similar reactions were seen among nine patients with advanced NSCLC who received injections of 1E10 following a good response to chemotherapy and/or radiotherapy [521].

Summary The hybridoma and recombinant DNA technologies, combined with advances in our understanding of immunology, tumor biology, and ligand–receptor–signal transduction cell physiology, laid the foundation for monoclonal antibody therapy of cancer. During1980–1990 exploratory clinical trials confirmed many of the points to consider when developing Mab therapy. During 1990–1995 many murine mab were engineered to produce chimeric and humanized antibodies. During 1997–2007 six unconjugated Mab that have clinical indications specifically for the treatment of human malignancy became available for widespread clinical use. Several of these have revolutionized cancer therapy and have become the highest revenue-generating cancer drugs ever. Hundreds of other Mab are in development and/or clinical trials, including several totally human Mab. Many of these may be in wide-spread clinical use by the time this chapter is published. The next several years will see the introduction of many more Mab into the clinic and continued evolution of their integration into cancer therapy, and perhaps eventually replacement of many current forms of therapy.

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11 Immunotoxins ARTHUR E. FRANKEL, JUNG-HEE WOO, AND DAVID M. NEVILLE

Introduction Immunotoxins are protein molecules composed of a cell surface-directed ligand covalently linked to a peptide cytotoxin. This definition excludes a number of important therapeutic compounds with distinct pharmacologic properties which react with intracellular targets. Such molecules would be unlikely to find and react with their intracellular targets due to the permeability barrier of the plasma membrane. Thus, the ligands must bind cell surface receptors or antigens. Ligands which have been used include monoclonal antibodies and antibody fragments, adhesion molecules, growth factors, and cytokines. The toxophore must be peptide in nature. This excludes radiolabels such as 90Y, 213Bi, or 131I and small molecular weight drugs such as calicheamicin and doxorubicin [1, 2]. These immunoconjugates are the subjects of other chapters in this book. We have further restricted the toxin moieties to cytotoxins. Thus, peptides which modify coagulation [3], complement [4], or immune responses [5] are not considered here. Conjugates with these compounds are important potential therapeutics and are considered separately in chapters discussing anti-angiogenesis and cell-directed therapies. There are three major classes of peptide cytotoxins [6, 7]. Class I toxins are proteins which are intracellular enzymes. They catalytically modify critical intracellular functions. Class II toxins bind to cell surfaces and trigger intracellular signal pathways. Class III toxins are pore-forming peptides which cause leaks in the plasma membrane. Immunotoxins have been prepared with toxins of each class, as listed in Table 1 and discussed below. We will first review the structure and molecular mechanisms of cell intoxication for the peptide cytotoxins used in preparation of immunoconjugates.

Peptide Cytotoxins The DNA sequence, amino acid sequence, and threedimensional structures for most of the toxins used in conjugate synthesis have been defined. In most cases separate domains contribute to different toxin functions.

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

The information on toxin structure has been extremely useful in immunotoxin design.

Type I plant A-B Toxins The type I A-B toxins are produced by plants and bacteria. The plant toxins – ricin, abrin, and viscu-min (mistletoe lectin I) – are encoded by single exons. The sequence from the N-terminus for each includes a signal peptide, a catalytic domain, and an alkaline protease-sensitive disulfide loop, followed by a cell binding domain [40–42]. The linker is cleaved by plant vesicle endoproteases, N-linked glycosyl groups are added, and the signal peptide is removed by a signal peptidase prior to toxin secretion [43]. Thus, the fully processed, native proteins are hetero-dimers of approximately 60,000 Mr, with a 30,000 Mr catalytic subunit (A chain) and a 30,000 Mr cell binding subunit (B chain). The B chains have Ω-loop structures (Fig. 1a) [44–46]. There are three lectin binding sites/subunit which bind (3-galactosyl residues on mammalian cell surface glycoproteins (Fig. 2a) [47, 48]). There are also extensive salt and hydrophobic bonds between the A and B chains which stabilize the heterodimeric structures [44]. After cell binding, the heterodimers internalize into endosomes using both clathrin-dependent and clathrin independent pathways [49], and, via a Rab9independent process, reach the transreticular Golgi [50]. In the Golgi, the toxins bind endoplasmic reticulum shuttle proteins which undergo retrograde transport via COP-I-independent Rab6 regulated vesicles [51]. The toxins are then transported to the endoplasmic reticulum (ER) [52]. In the ER, the toxin is reduced, the A chain unfolds with the help of chaperones, and the A chain translocates to the cytosol using the Sec61p transposon [53]. In the cytosol the A chain refolds. The A chain has an α-helical/β-sheet open sandwich tertiary structure with a large open catalytic cleft. The A chains function as RNA N-glycosidases removing a critical adenine base from the 28S ribosomal RNA (Fig. 3) [54]. This alters the interaction of elongation factor 2 (EF2) with the ribosome and prevents the structural modifications leading the GTP hydrolysis (Fig. 4). Protein elongation is blocked irreversibly (Fig. 5). Cell death follows.

407

408

Immunotoxins Table 1. Peptide cytotoxins used in immunotoxin synthesis Protein

Type

Structure

Order

Source (species)

Reference

Ricin Abrin Viscumin Nigrin b Gelonin Saporin Pokeweed antiviral protein Luffin Bouganin Trichokirin Trichosanthin BRIP Ebulin Bryodin Momordin Momorcochin Moschatin Dianthin 30 Ocymoidine Pyramidatine Colocin 1 Botulinum neurotype type C Diphtheria toxin Pseudomonas exotoxin Anthrax toxins Seminal ribonuclease Bosinophil neurotoxin Angiogenin Pancreatic ribonuclease RibonucleaseA Ranpirnase Barnase a-Sarcin Restrictocin Clavin Mitogillin Bax D-(KLAKLAK)2 Phospholipase C TNF sTRAIL Hemolytic toxin Cecropin/Shiva Pyrularia thionin Proaerolysin Cyt1Aa Toxin

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II II II III III III III III

A-B A-B A-B A-B A A A A A A A A A A A A A A A A A A-B A-B A-B Binary A A A A A A A A A A A A A

Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Bacteria Bacteria Bacteria Bacteria Cow Human Human Human Cow Frog Bacteria Fungi Fungi Fungi Fungi Human Synthetic Bacteria Human Human Sea anemone Insect Plant Bacteria Bacteria

Ricinus communis Abrus precatorias Viscumalbum Sambucus nigra Geloniummulfiflorum Saponaria officinalis Phytolacca americana Luffaa egyptiaca Bougainvillea spectabilis Tiichosantheskifilowfi Tiichosantheskifilowfi Hordeum vulgare Sambucus ebulus Bryonia dimca Momordica charanfia Momorfica coohinchinensis Cucurbita moschata Dianthus caryophyllus Saponaria ocymoides Vaccaria pyramidata Citrullus colocynthis Clostridia botulinum Corynbacterium diphthefiae Pseudomonas aeruginosa Bacillus anthracis Bos taurus Homo sapiens Homo sapiens Homo sapiens BosTaurus Rana pipiens Bacillus amyloliquefaciens Aspergillus giganteus Aspergillus restfictus Aspergillus clavatus Aspergillus fumigatus Homo sapiens

[8] [9] [10] [607] [11] [12] [13] [14] [15] [16] [349] [17] [558] [18, 21] [19] [20] [571] [22] [23] [23] [440] [560] [24] [25] [26] [27] [28] [29] [30] [31] [32] [579] [33] [34] [35] [93] [167] [563] [36] [574] [590] [37] [38] [39] [548] [559]

Type I plant A toxins The type I plant A toxins are 25–31 kDa M, proteins. They have similar primary amino acid sequences [55– 57], and similar three-dimensional structures [55, 56, 58–60], and are all ribosomal RNA N-glycosidases which remove the same adenine base from the 28S ribosomal RNA of the mammalian ribosomes [61]. However, they do not crossreact immunologically. The preproproteins have a signal peptide, the catalytic domain, and a C-terminal extension with a vacuolar

Clostfidium perfringens Homo sapiens Homos sapiens Stoichactis helianthus Hyalophora ceaopia Pyrulafia pubera Aeromonas hydrophila Bacillus thuringiensis

targeting sequence of 25–29 amino acid residues. The signal peptide and C-terminal extension are processed on secretion, thus protecting the cytosolic plant ribosomes from intoxication. The three-dimensional structure contains eight α-helices and a β-sheet composed of six strands. The sequence, structure, and enzyme activity are homologous to the catalytic A subunit of type I plant A-B toxins. There is an open cleft containing a conserved glutamic acid residue, an arginine residue, and a tyrosine and a tryptophan residue. These residues function to bind and stabilize the transition

Arthur E. Frankel et al. state for the C-N bond cleavage [62]. There are three conserved lysyl residues in the C-terminus of the mature proteins which likely are involved in ribosomal substrate recognition [56].

Type I Bacterial A-B Toxins The type I bacterial A-B toxins which have been modified for targeted therapy include diphtheria toxin (DT) and Pseudomonas exotoxin (PE). These toxins will be discussed separately.

409 DT has a 25-residue leader sequence followed by 535 amino acid residues [63, 64]. The mature protein has three domains (Fig. 1b) [65, 66]. There is an N-terminal catalytic domain (amino acid residues 186), also called the A fragment. This domain is followed by a 14 amino acid loop bordered by Cys-186 and Cys-201. The loop is arginine-rich and is a substrate for endosomal furin endoprotease. The second domain is the translocation domain (amino acid residues 187–381) and contains multiple amphipathic helices with two negatively charged amino acid residues (Glu-349 and Asp-352) in a helical hairpin between two of the helices (TH8 and TH9). The translo-

Figure 1. Drawings of peptide toxin a-carbon backbone structures. (a) ricin; (b) DT; (c) PA; (d) RNase; (e) restrictocin; (f) Clostridia perfringens PLC; (g) cecropin (similar to Shiva-1); (h) crambin (similar to PT). Based on coordinates determined from references described in the text. Note that the type I A-B and binary toxins have multiple domains which may subserve distinct functions (see Fig. 2). Cylinders are α-helices and ribbons are β-strands. In the ricin structure (a), red tubes are RTB subunit S2-loops, and yellowblue structures are RTA enzymatic subunit. In the DT structure (b), red is β-sheet binding domain, yellow is translocation domain with amphipathic helices, and green in enzymatic domain. In PA (c), blue is receptor binding β-sheet domain, green is the heptamerforming and membrane-inserting domain, and red is 20 kDa protease-sensitive fragment. EDN (d) has an α + β structure with two α-helices, a β-sheet, a third α-helix and four additional β-sheets separated by loops. There are binding sites for bases and phosphates of RNA. The catalytic histidine residues are at the two ends

410

Immunotoxins

Figure 1. (continued) Restrictocin (e) shows structural homology to Rnases. It has catalytic histidines similar to RNase. There is a three-turn α-helix packed against a five-stranded antiparallel β-sheet. PLC (f) has an N-terminal domain (residues 1–246) composed of six stacked α-helices. It has the phospholipase C active site. There is a flexible linker (residues 247–255) followed by a C-terminal domain (residues 256–370) with an eight β-strand jelly-roll topology with Ca++-dependent phospholipid-binding function. The cecropins (g) are small 36 amino acid residue peptides with a strongly basic N-terminal amphipathic helix linked via a flexible Gly-Pro linker to a neutral C-terminal amphipathic helix. These stack in membranes as shown creating pores. PT toxin has two amphipathic Ochelices connected to two antiparallel β-sheets. The related crambin (h) lacks β-sheets, but the loops serve the same function (Fig. 1 h)

a

c

Ricin

Galactosyl-terminated oligosaccharides

PE Alpa2-macroglobulin

Endosomes KDEL-receptor

Endosome

N-glycosidase reaction on ribosomes

Trans-reticular Golgi

Trans-recticular Golgi 37κD PE fragment

ER ER

Translocation of RTA

EF2 Ribosomes

b

DT

d 20κDa fragment

Heparin-binding EGF PA

Protease

LF 63κDa fragment

EF

Endosome PA receptor EF2

DTA Endosome Ribosomes

MAPKK

cAMP

Figure 2. Steps in peptide toxin intoxication of cells. (a) ricin; (b) DT; (c) PE; (d) anthrax toxins. Note for all toxins there is an initial cell binding event. For the shown type I toxins (a–d), cell binding is followed by internalization. After intracellular processing and transport the type I toxins escape to the cytosol where they catalytically inactivate key cellular processes. For type II and III toxins the membrane perturbations lead to altered cell physiology or cell death

Figure 3. Atomic stick model of the S/R stem loop (red) portion of 28S rRNA (black). In addition to elongation factor binding to the ribosome A site which displaces the peptidyl-tRNA (see Figs. 4 and 5), there is also an rRNA conformation change which occurs spontaneously to drive elongation. This is catalyzed by other EF2 (EFG) domains (II-III) in which the GTPase activity modifies ribosome structure. Ricin and type I plant A toxins remove an adenine base (rRNA N-glycosidase activity) from the large rRNA at a specific location on the S/R conserved stem-loop (S = α-sarcin site and R = ricin site). This stem-loop normally switches between two states which change overall rRNA and ribosome structure in the large subunit. Once the site is chemically modified, EF2 or EFG can no longer bind properly and elongation stops. Note that EF2 or EFG interacts with the ribosome at least two sites in domain IV and II-III in addition to its interaction with the mRNA at the anticodon loop and each of these two sites are affected separately by type I peptide toxins

412

Immunotoxins

c TOXIN

O

His H

N H

C N O H

O OPO AMP O

OH OH

Glu N R

N H

Diphthamide

EF2

Figure 4. (a) Structure of EF-G and EF-Tu-Phe-tRNA-GTP ternary complex. Note that domain IV (the lower tip of EF-G and EF2) resembles the tRNA portion of the EF-Tu-Phe-tRNA-GTP complex. Additional data show that this region is adjacent to the anticodon loop and binds the A site of the ribosome. (b) Model of EF2 α-carbon backbone showing in yellow the location of diphthamide residue at the tip of domain IV. The diphthamide residue is the target for DT and PE ADP-ribosylation. (c) The enzymatic reaction catalyzed by DT and PE. The His and Arg residues of the catalytic site of DT and PE interact with the nicotinamide ring and stabilize the position of the NAD molecule in such a way that the electrophilic carbon atom of the ribose group can interact with the nucleophilic residues of diphthamide on EF2

aa-tRNA-EF-Tu-GTP

50S A-site

EF-Tu-GDP

P-site 30S

EF-G-GDP EF-G-GDP

Figure 5. Protein synthesis steps

cation domain ends in a flexible spacer (amino acid residues 382–390) which is then connected to a β-sheet rich cell-binding domain (amino acid residues 391–535). DT binds to cells via amino acid residues in the cell-binding domain to cell-surface-expressed heparin-binding epidermal growth factor-like growth factor (HB-EGF) precursor [67, 68]. The amino acid residues of the DT cell-binding domain, in particular Lys-516, binds a crevice in the extracellular EGF-like domain of the HB-EGF precursor (amino acid residues 122–148). There is a critical salt bridge interaction between Lys-516 of DT and Glu-141 of HB-EGF precursor. The HB-EGF precursor associates on the plasma membrane with CD9 and heparan sulfate proteoglycan [69, 70]. This complex has a higher affinity for DT. Animals and cell lines with absent or

Arthur E. Frankel et al. low-affinity receptors for DT are insensitive to the toxin [67]. After cell binding the DT-HB-EGF precursor complex undergoes clathrin-dependent endocytosis [71]. Interestingly, the cytoplasmic domain of the HB-EGF precursor lacks an internalization motif (although it has a membrane proximal Tyr-192) [72]. The complex internalizes at 1%/min, and dynamin is required to complete vesicle formation [73]. Once internalized into early endosomes [74], DT u Glu zndergoes furin cleavage at the arginine-rich loop [75], low pH-induced protonation of the helical hairpin aspartate and glutamate [76], insertion of the TH8 and TH9 amphipathic helices of the translocation domain into the vesicle membrane [77], unfolding of the catalytic domain [78], reduction of the disulfide bridge linking the A fragment with the remainder of DT [79, 80], and transfer of the A fragment through the membrane to the cytosol (Fig. 2b) [81]. The DT A fragment then catalytically ADP-ribosylates elongation factor 2 (EF2) on the diphthamide residue in EF2 domain IV [82, 83, 92]. A detailed molecular mechanism for the enzymatic activity has been proposed. First, residues 39–46 of the A fragment ‘active site’ loop bind NAD [84]. The dislodged loop then binds EF2 in combination with the exposed NAD. A fragment Glu-148 then attacks the strained N-glycosidic bond of the NAD. The Glu-148 carboxylate group activates the diphthamide imidazole of EF2 for a nucleophilic attack on Cl’N in an SN2-type displacement reaction. This leads to addition of ADP to the domain IV diphthamide residue. This irreversible modification prevents the elongation factor from displacing the tRNA petidyl complex from the A site to the P site [85, 86]. Protein synthesis is inactivated, and cells die by lysis or programmed cell death [87, 88]. The prolonged action of a single A fragment molecule in the cytosol is sufficient to inhibit protein synthesis and prevent cell growth and division [81, 89]. Cellular resistance to DT can occur due to altered DT receptors [67], lack of internalization [73], failure of arginine-rich loop proteolysis [75], lack of endosomal acidification [90], too great a distance between the DT translocation domain and the vesicle membrane [71], incomplete inter-domain disulfide reduction [79], incomplete A fragment unfolding [78], too rapid cytosolic proteolysis of the A fragment [81], and resistant EF2 [91]. PE is a 66 kDa Mr, protein produced by Pseudomonas aeruginosa. It consists of 638 amino acid residues including a 25 amino acid hydrophobic leader peptide [95]. The molecule has three distinct domains [96]. Domain I is an antiparallel β-structure with 17 β-strands. It includes amino acid residues 1–252 (Ia) and 365–404 (Ib). The first 13 strands form an elongated β-barrel. Domain Ia, is the cell-binding domain. Domain II consists of residues 253–364 and has six consecutive α-helices.

413 Domain II is the translocation domain. Domain III is the carboxylterminal third of the molecule and includes residues 405–613. It has an extended catalytic cleft. Domain III is the enzymatic domain. PE in the blood reacts with plasma carboxypeptidase, and the terminal lysine residue is cleaved [97]. PE612 then binds the α2-macroglobulin receptor/low density lipoprotein receptor-related protein (α2MR/LRP) on the surface of mammalian cells including fibroblasts and hepatocytes [98]. PE612 domain Ia, residue Lys-57 is important in this binding reaction [99]. The PE612, α2 MR/LRP complex undergoes receptor-mediated endocytosis [100]. Once reaching the endosomes the PE612 undergoes furin cleavage between Arg-279 and Gly-280 [101, 102], a pH-dependent conformation change [103] followed by reduction of the disulfide bond between Cys265 and Cys-287 [104]. This creates a 28 kDa N-terminal fragment and a 37 kDa C-terminal fragment. The C-terminal REDL of the 37 kDa fragment then binds to the KDEL receptor and the complex is routed to the endoplasmic reticulum (ER) [105, 106]. In the ER the 37 kDa fragment utilizes residues in domain II to translocate the cytosol [107]. Thirty-four amino acid residues at the N-terminus of the 37 kDa fragment appear critical for translocation, including Trp-281, Leu-284, and Tyr289 [108, 109]. There is an unidentified saturable protein which binds the fragment and facilitates transfer. Most of the translocation domain helices, with the exception of the first and last helix, are essential for the translocation process [110]. The translocon may be part of the machinery for PE transfer to the cytosol, since the translocation occurs in the ER (Fig. 2c). The cytosolic C-terminal fragment then catalyzes the ADP-ribosylation of EF2 similar to DT [111]. Glu-553 is important for NAD binding. The NAD is cleaved and the ADP moiety transferred to the diphthamide residue of bound EF2 [112]. Cell death follows by a process indistinguishable from DT [81, 89]. Cell resistance to PE has been seen secondary to alterations of individual steps in the intoxication process. Cells may lack receptors [113]; cell intracellular trafficking may be disrupted [114–115]; endosomal acidification may not occur [116]; furinmediated cleavage may not occur due to low or abnormal furin [117]; EF2 may be mutated so that the diphthamide residue is absent [91]; finally, cells may be resistant to PE-induced apoptosis [87].

Type I Bacterial Binary Toxin Anthrax toxin produced by Bacillus anthracis is a binary toxin. The catalytic domain and binding domains are on completely separate proteins. Anthrax toxins have two catalytic proteins – edema factor (EF) and lethal factor

414 (LF). Either of these proteins can interact with the cellbinding protein, protective antigen (PA). PA has 764 amino acid residues with a 29 residue signal peptide. Mature PA is a 735 amino acid residue, 83 kDa secreted protein [118]. It has four domains [119]. PA domain 4 (residues 596–735) has an initial hairpin and helix which serves as a flexible linker followed by an immunoglobulin-like fold which functions in receptor binding (Fig. 1c) [120, 121]. The nature of the cell surface receptor is unknown, but there are approximately 30,000 receptors/macrophage [122]. After cell binding, cell surface furin or PACE4 nicks PA in the middle of domain 1 (residues 1–258) at residues 164–167 (Fig. 2d) [123]. This releases the N-terminal 20 kDa Mr fragment, PA20. The remainder of PA, PA63, remains on the cell surface. A large hydrophobic surface on the residual domain 1 of PA63 serves as a site for EF or LF binding. EF and LF are 90 kDa Mr, 770 amino acid residue proteins with a similar N-terminal 250 amino acid domain which mediates binding to PA63 [124, 125]. These proteins have distinct C-terminal domains which have adenylate cyclase and Zn2+-dependent metalloprotease activity, respectively [124–132]. The EF/LF-PA63 and PA63 oligomerize on the cell surface to heptamers facilitated by the new N-terminal domain 1 and domain 2 (residues 259–487) of PA63 [133]. There can be up to seven LF or EF molecules bound per heptamer. Both monomers and heptamers then undergo endocytosis [134]. The low pH of the endosomes triggers protonation of critical histidines and unfolding of a large amphipathic hairpin loop in PA domain 2 which is hypothesized to insert into the membrane and, together with those of other PA molecules in the heptamer, form a 14-strand β-barrel [135]. EF or LF then unfolds and translocates to the cytosol through the barrel pore [136, 137]. Once in the cytosol, EF and LF refold. EF is a calcium-dependent-calmodulin-dependent adenylate cyclase that raises the cytosolic cyclic AMP (cAMP). Elevated cAMP leads to paralysis of several macrophage physiological pathways [138]. LF proteolytically cleaves members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In the case of MEKs I and 2, this cleavage results in their inactivation. Consequently, they cannot activate their substrate, MAPK. Depending on the cell type, this may have a variety of consequences including apoptosis or altered physiology [139].

Type I Vertebrate A Toxins Several members of the vertebrate ribonuclease (RNase) superfamily have been linked to tumor selective ligands. These ribonucleic acid-degrading proteins are small

Immunotoxins (14–16 kDa Mr) proteins with an α + β structure (Fig. 1d) [140]. From the N-terminus there are two α-helices, a β-sheet, a third α-helix, and four additional β-sheets separated by loops. There are binding sites for bases and phosphates of RNA. The catalytic residues include His12, Lys-41, Thr45, and His-119. His-12 and His-119 act as the base and acid, respectively, in the transphosphorylation step that generates the cyclic phosphate intermediate. The roles of His-12 and His-119 are reversed in the subsequent hydrolysis step (Fig. 6) [141]. Lys-41 and Thr-45 bind the substrate phosphate and pyrimidine base, respectively, to stabilize the transition state intermediate. The conjugated vertebrate RNases include bovine seminal RNase (BS-RNase), human eosinophil derived neurotoxin (EDN), human angiogenin (ang), frog ranpirnase, bovine pancreatic RNase A, and human pancreatic ribonuclease I (hRNase 1). They each have distinct properties. BS-RNase is a dimeric protein (32 kDa Mr) with strong and selective cytotoxicity to tumor cells, germ cells, and T cells [142]. EDN is a 15 kDa, 134 amino acid residue, highly basic protein which has selective toxicity to Purkinje cells, parasites, and tumor cells [28, 143]. Ang is a 123 amino acid residue, 14 kDa Mr protein that specifically degrades tRNA. It binds 170 kDa and 42 kDa Mr receptors on endothelial and vascular smooth muscle cell surfaces and induces cell proliferation, cell adhesion, proHis119 R HN

His12

NH

O O

O P N

NH

O O H

B O

His12

His119 H HN

N

H

O O

O HN P

O O

NH

O B

Figure 6. Steps in RNase hydrolysis. This is a two-step process in which initially a cyclic phosphate intermediate is formed. His-12 acts as a general-base catalyst and His-119 acts as a general acid to protonate the leaving group (the RNA fragment designated R). The second step is a hydrolysis step in which His-119 activates the attack of water by general base catalysis and His-12 is the acid catalyst, protonating the leaving group

Arthur E. Frankel et al. duction of cell-associated proteases, and cell migration and invasion. The protein’s ribonucleolytic activity is essential in these activities. The protein is internalized by receptor-mediated endocytosis, translocated to the cytosol and then the nucleus [29, 144]. Ranpirnase is a basic 14 kDa Mr protein which binds tumor cells and vascular endothelium and degrades tRNA selectively [32, 145]. Unlike ang, ranpirnase triggers cell apoptosis. Neither BS-RNase nor ranpirnase is bound by cytosolic ribonuclease inhibitor protein (RI), and, since they are not inactivated in the cell, have potent cell toxicity. RI constitutes >0.01% of cytosolic proteins and inactivates ribonucleases by forming tight complexes that prevent RNA substrates from entering the active site. Human pancreatic RNase 1 and bovine pancreatic RNase A are not toxic to cells, lack cell-binding functions, and are good substrates for RI. Interestingly, a variant of RNasel – des. 1-7hpRNase 1 – is not a substrate for RI and is 100-fold more cytotoxic when delivered to cells by conjugation to epidermal growth factor (EGF) [30]. The nature of the cell-binding and internalization functions of BS-RNase, EDN, ang, and ranpirnase are unknown.

415

Type II Toxins Type II toxins interact with cell surfaces and produce changes leading to secondary intracellular signals and often cell death. Clostridia perfringens α-toxin is a 370 residue, zinc metalloenzyme with phospholipase C activity [149]. It is the key virulence determinant in gas gangrene. It binds to membranes in the presence of calcium. It has two domains (Fig. 1f). The N-terminal domain (residues 1–246) is composed of six stacked a-helices. It has the phospholipase C active site. There is a flexible linker (residues 247–255) followed by a C-terminal domain (residues 256–370) with an eight (three-strand jelly roll topology). The C-terminal domain resembles eukaryotic C2 domains and has Ca Z′-dependent phospholipid binding function. Membrane phospholipid hydrolysis results in the perturbation of cell metabolism and activation of the arachidonic acid cascade and protein kinase C. Cell death and altered physiology such as endothelial cell integrin and cytokine expression and platelet aggregation ensues.

Type III Toxins Type I Fungal A Toxins The fungal cytotoxins include α-sarcin, mitogillin, clavin, and restrictocin. These are 17 kDa Mr, basic proteins with 20 lysines, four arginines, and eight histidines. α-Sarcin, restrictocin, clavin, and mitogillin share 86–99% amino acid homology [94]. They have four cysteines forming two disulfide bridges between residues 6 and 148 and residues 76 and 132. The first disulfide bond is not essential for the catalytic activity. The proteins show structural homology to RNase T1 and have catalytic histidines similar to RNase T1 and other RNases (Fig. 1f). There is a three-turn α-helix packed against a five-stranded antiparallel β-sheet [146, 147]. Large positively charged peripheral loops near the active site construct a platform with a concave surface for RNA binding. A large 39-residue loop L3 may contribute to membrane binding. They have acquired a mechanism to enter cells – interacting with negatively charged phospholipids in membranes and translocating across membranes. Further, in the cytosol, they cleave a single phosphodiester bond (3′ to guanosine residue 4325) in a highly conserved stem-loop structure of 28S ribosomal RNA. This target is two bases removed from the target for depurination by the plant rRNA N-glycosidases [148]. This stemloop rRNA structure is a binding site for elongation factors, and its modification irreversibly blocks protein synthesis (Fig. 3). These toxins share antigenicity with Aspergillus fumigatus allergen I, and produce hypersensitivity reactions in patients. These fungal toxins are cytotoxic to tumor cells [33–35, 93].

Type III toxins produce pores in cell membranes leading to cell death. Sticholysin II from the tentacle nematocysts of the sea anemone Stichodactpla helianthus is a member of the actinoporin family. It is a 175 amino acid, 19 kDa Mr basic protein containing five tryptophans, 12 tyrosines, and no cysteines [150]. This water-soluble protein binds membrane lipid (principally sphingomyelin) and partially unfolds [151]. The protein’s β-sheets facilitate tetramer formation [152]. This is followed by membrane insertion of the N-terminal amphipathic helices. One nanometer pores are produced, which causes colloid osmotic shock and cell death. Cecropins are peptides found in the hemolymph of the giant silk moth Hpalophora cecropia [153]. The synthetic cecropin analog, Shiva-1, is a 36 residue basic peptide devoid of cysteine with a strongly basic N-terminal amphipathic helix linked via a flexible Gly-Pro linker to a neutral C-terminal amphipathic helix (Fig. 1g) [154]. The peptide is toxic to bacteria, parasites, and tumor cells. The peptide binds cell surface acidic lipids and forms channels in the membranes followed by cell lysis. Pyrularia thionin (PT), from the parasitic plant Pyrularia pubera, is a 47 amino acid basic peptide with five lysines, four arginines, and eight cysteines (which form four disulfide bonds) [155]. The molecule consists of two amphipathic α-helices connected to two antiparallel β-sheets (Fig. 1h). PT has a hydrophobic and hydrophilic surface. It binds to cell membrane phos-

416 phatidylserine with its hydrophilic side and interacts with phospholipid hydrocarbon tails through the hydrophobic domain. This destabilizes the membrane bilayer with formation of inverted micelles [156]. The altered membrane organization can lead to cell death. The membrane depolarization also produces an influx of calcium and induction of phospholipaseA2 [157]. Proaerolysin from Aeromonas hydrophila is a 53 kDa Mr protein. It exists as a dimer and binds glycophosphatidylinositol-anchored proteins found on the surface of most mammalian cells. It contains a C-terminal inhibitory domain that is cleaved by membrane proteases such as furin. The N-terminal fragment, aerolysin, oligomerizes and enters plasma membranes to form highly stable pores causing cell death [549].

Toxin Modification and Ligand Conjugation To prepare immunotoxins, three tasks must be completed. The toxin normal tissue binding and toxicity functions must be removed or modified; target cell selective peptide ligands must be identified; and the modified toxin and ligand must be covalently linked in such a manner that there are no alterations to the toxin translocation and enzymatic functions or the ligandbinding or internalization functions. An alternative approach can be used to deliver peptide toxins to cells. Bispecific monoclonal antibodies that have one binding domain for target cell surfaces and the other binding domain to react with peptide toxins have been synthesized. These molecules have shown potent antitumor activity in vitro and in animal models [480]. Because of space limitations we have focused our review only on covalently linked ligandtoxin conjugates.

Toxin Modification The type I A-B, type I binary toxin, type II, and type III toxins may react with normal vital tissues and, consequently, produce serious side-effects. The structure/ function studies described above suggest solutions for the type I and one of the type III toxins. The lectin subunit of the plant type I A-B toxins may be removed either by reduction and purification of the A subunit [8] or production of the A subunit by heterologous expression [158]. A third approach is to use affinity crosslinkers to block the B subunit lectin sites [159]. Genetic engineering has been used to produce B subunit variants with modifications of amino acid residues at the lectin sites [160]. The normal tissue receptor-binding domains of the bacterial type I A-B toxins and the binary toxins have been

Immunotoxins approached in a similar way to the plant toxins. Trypsin cleavage followed by reduction and purification have been used to isolate DT A fragment [161]. DT and PE mutants with altered amino acid residues important in receptor binding have been used [24, 162]. The most common approach for the bacterial A-B-toxins has been to genetically delete the receptor binding domain. This has been accomplished successfully for DT with the generation of DT385, DT388, DAB389, DT390 and DT486 and for PE with synthesis of PE35, PE38, and PE40 [163, 164]. The type I binary toxin, anthrax toxin, has been modified both by removing critical receptor-binding residues of PA domain 4 and by changing the protease cleavage sequence in PA domain 1 so that the toxin is processed only on tumor cells [165, 166]. Further, the anthrax toxin lethal factor has been genetically modified to attach the PE catalytic domain to the lethal factor PA binding domain to produce FP59 [550]. Addition of sphingomyelin to type III actinoporin conjugates, including sticholysin and equinatoxin II, blocks normal tissue binding [151, 234]. The type III toxin, proaerolysin, has been modified to replace the furin site with the prostate cancer specific cleavage protease, PSA, cleavage sequence [548, 549]. The type I A toxins lack normal tissue-binding function and hence do not require structural modification for reducing toxicities.

Ligand Selection The peptide ligand must bind all the target cells and, in the case of type I toxins, undergo internalization. In addition, the ligand should have minimal or no binding to vital normal tissues. The identification of selective ligands for immunotoxin synthesis is one of the most important steps. Table 2 shows the variety of ligandreceptor systems which have been used for immunotoxin targeting. Antibodies, antibody fragments, cytokines, growth factors, antigens, and receptor fragments have been linked to toxins. Only a subset of the synthesized immunotoxins have shown selective cytotoxicity to target cells. Problems with constructs have included steric hindrance or misfolding of the ligand, insufficient internalization for type I toxin conjugates, and unexpected normal tissue reactivities. Ligands which make inactive conjugates with one toxin may be highly efficacious when either (a) combined with another toxin, (b) conjugated or fused with a different linker, or (c) expressed using a different vector or host. These will be discussed in greater detail in the next section. Recently, bispecific immunotoxins have been made by Vallera employing two ligands with one toxin (e.g., EGF and IL3, sFv anti-ErbB2 with sFv anti-EpCAM, IL13 with

Arthur E. Frankel et al.

417

Table 2. Ligands used to prepare immuntoxins Ligand

Type

Receptor/antigen

Type linkage

UCHT1F(ab)’2 BisFv SFv-Cys sFv SPV-T3a UCHT1

Fab’2 fragment Bivalent sFv SFv-Cys sFv MAb MAb

CD3ε CD3ε mCD3ε mCD3ε mCD3 CD3ε

C G G G C C

WT32 MAb 64.1 MAb F(ab’)2 sheep Polyclonal anti-mouse Ig

CD3 CD3 CD3-anti-CD3

C C C

FN18

MAb

rCD3

C

898112-6-15 HB-EGF EGF

MAb Growth factor Growth factor

pCD3 EGFR EGFR

C G G, C

EGF

Growth factor

EGFR, IL13R

G

TGFα

Growth factor

EGFR

G

Mints

MAb

EGFR

C

425.3 425 528 r3

MAb sFv MAb Mab

EGFR EGFR EGFR EGFR

B4G7 14171(dsFv) 14171(Fv) AntiEGFRvIII(Fv) MR1-1 L2

MAb dsFv sFv sFv

Compounds

Disease

Reference

UCHT1F(ab’)2-RTA A-dmDT390-bisFv (DT390-sFv-Cys)2 DT390anti-CD3sFv SPV-T3a-RTA UCHT1-CRM9, UCHT1-DTA, UCHT1-MSPSA, UCHT1-DT, UCHT1-ricin WT32-RTA 64.1-RTA F(ab )2-dianthin, F(ab’)2-saporin, F(ab )2-PAP, F(ab’)2-bryodin, F(ab )2-momordin, F(ab’)2momorcochin, F(ab’)2-trichokirin FN18-CRM9, DT390-C207 diabody pCD3-CRM9 HBEGF-saporin DAB339EGF, DAB486EGF, EGF-DTA, EGF-RTA, EGFhpRNasel, EGF-ang, SAP-EGF DTEGF13

T-cell ALL T-cell ALL T-cell ALL T-cell ALL T-cell ALL T-cell ALL

[401] [168] [169] [170] [411] [171, 172, 312]

T-cell ALL T-cell ALL T-cell ALL

[329] [420] [20]

T-cell ALL

Breast cancer

C G C C

Mint5-ocymoidine, Mint5pyramidatine 425.3-PE 425(scFv)-ETA’ 528-RTA egf/r3Mab-HT

[173, 613] [24] [437] [30, 175, 176, 477, 600] [551, 556] [264, 295] [23]

EGFR EGFRvIII EGFRvIII EGFRvIII

C G G G

B4G7-gelonin sFv(14E1)-ETA dsFv(14E1)-ETA Anti-EGFRvIII(Fv)-PE40

dsFv Growth factor

mEGFRvIII IL2R

G G

Mik-betal(Fv) Bell 0 RFT5 Anti-Tac

sFv MAb MAb MAb

UR IL2R IL2R IL2R

G C C C

Anti-Tac(Fab)

Fab

IL2R

G

Anti-Tac(Fv)

sFv

IL2R

G

RTF5(Fv) GMCSF GMCSF

sFv Growth factor Cytokine

IL2R GMCSFR, uPAR GMCSFR

G G G

mGMCSF IL3

Cytokine Cytokine

mGMCSFR IL3R

G G

T-cell ALL Lung cancer Lung cancer

Prostate cancer, glioma TGFα-PE40, PEα53L/TGFα/KDEL Pancreas cancer

Breast cancer Pancreas cancer Lung cancer Lung adenocarcinoma Lung cancer Gliomas Gliomas Gliomas

[283] [584] [425] [557]

MR1-1(dsFv)PE38KDEL DAB389IL2, DAB486IL2, IL2-PE66GIu4, IL2-Bax, IL2-ricin, IL2-PE40, IL2-PAP, IL2-pancRNase1 Mik-beta1(Fv)-PE40 BB10-saporin RFT5-RTA Anti-Tac-RTA, Anti-CD25-MLA

Gliomas Lymphomas

AntiTac(Fab)-PE40, AntiTac(Fab)-phospholipase C Anti-Tac(Fv)-PE40, AntiTac(Fv)-PE38 RFT5(Fv)-ETA’ DTU2GMCSF DT388GMCSF, DT385-L-GMCSF, GMCSF-PE40, GMCSF-ricin DT390mGMCSF, DT388mGMCSF DT388IL3, DT388K116W

Lymphomas

[608] [160, 162, 167, 177, 178, 413, 476] [253] [447] [382] [314, 413, 10] [36, 260]

Lymphomas Lymphomas Lymphomas Lymphomas

Lymphomas Lymphomas AML AML AML AML

[472] [291] [291] [262]

[260, 279] [285] [555] [179, 180, 181] [182, 183] [184, 558] (continued)

418

Immunotoxins

Table 2. (continued) Type linkage

Ligand

Type

Receptor/antigen

26292 mIL3 Transferrin

sFv CytoKne Growth factor

CD123 mIL3R Transferrin receptor/CD71

G G C

Compounds

Disease

Reference

AML AML Gliomas, T-mellALL

[605] [185, 186] [187, 188, 234, 259, 402, 412, 421, 426] [28, 29, 34, 179, 407]

Gliomas, breast cancer Breast cancer

[188–191]

G G G G G

26292(Fv)-PE38-KDEL DT390mIL3, DT389 L-mIL3 Tf-CRM9, Tf-CRM107, Tf-equinatoxin II, Tf-PE, Tf-Saporin, Tf-gelonin, Tf-RNase, Tf-KFT25-RTA DT388-anti-TfR(Fv),antiTfR(Fv)-PE40, anti-TfR(Fv)-EDN, Anti-TfR(Fv)-pancRNase, Anti-TfR(Fv)-angL2, Antii-TfR(Fv) L1, Anti-TfR(Fv) restrictocin 454A12-CRM107, 454A12-RTA, 454A12-rRTA, 454A12-RNase HB21-PE, HB21-RTA, HB21gelonin, HB21-bryodin, HB21-luffin, HB21-α-sarcin mIL18-PE38 B7-2-L-PE40KDEL DT390-IP-10-Sralpha CCL17-EDN, CCL17-PE38 HB21(Fv)-PE40

anti-TfR(Fv)

sFv

Transferrin receptor/CD71

G

454A12

MAb

C

HB21

MAb

Transferrin receptor/CD71 Transferrin receptor/CD71

mIL18 B7 IP-10 CCL17 HB21 (Fv)

Growth factor Modulator Chemokine Chemokine sFv

51E9

MAb

B2/25

MAb

OKT9

MAb

R17217

MAb

Anti-Ly2.1 IL7 ASF

MAb Cytokine Lectin

IL4

Cytokine

mIL18R CD28 CXCR3 CCR4 Transferrin receptor/CD71 Transferrin receptor/CD71 Transferrin receptor/CD71 Transferrin receptor/CD71 Murine transferrin receptor mTcell antigen IL7R Asialogly coprotein receptor IL4R

C

5E9-gelonin

Breast cancer

[414]

C

B2/25-saporin

Breast cancer

[442]

C

OKT9-gelonin

Breast cancer

[459]

C

R17217-rRTA

Breast cancer

[237]

C G C

Anti-Ly2.1-ricin DAB389IL7 ASF-DTA

T-cell ALL Pre-B cell ALL Hepatocarcinoma

[330] [200] [201]

G

DAB389IL4, IL-4(38-37)PE38KDEL, cpIL-4(13D)PE38KDEL DAB389mIL4, DT390mIL4, mIL4-PE40 IL 13-PE38QQR, DT388IL 13, DTEGF13, DTAT13

Gliomas, KS

[202, 203, 587]

mIL4

Cytokine

mIL4R

G

AML, mastocytosis Renal cell cancer, gliomas

[204–206]

IL13

Cytokine

IL 13R

G

αMSH GRP NT-4 CD4

MSHR GRPR/bb2 TrkB HIVgp120

G G G G

3B3(Fv) Anti-gp120 0.5beta Anti-gp120 CyanovirinN 7B2

Growth factor Hormone Growth factor T cell co-receptor sFv MAb MAb Polyclonal Antiviral protein MAb

Melanoma Adenocarcinoma Neuroblastoma HIV + tumors

G C C C G C

DAB389αMSH, DAB486αMSH DAB389GRP DAB389NT4 DAB389CD4, CD4-PE40, CD4-RTA 3B3(Fv)-PE38 Anti-gp120-RTA 0.5beta-RTA Anti-gp120-RTA FLAG-CV-N-PE38 Anti-gp41-RTA

HIVgp120 HIVgp120 HIVgp120 HIVgp120 HIVgp120 HIVgp41

HRG13

Growth factor

HER4

G

HRG13-PE38KDEL

Breast cancer

C

AML

[14, 33, 191, 360] T cell leukemia [585] GVHD [602] Multiple sclerosis [604] T lymphoma [606] Breast cancer [287]

HIV + tumors HIV + tumors HIV + tumors HIV +tumors HIV +tumors HIV + tumors

[207, 247, 551, 553, 556] [208, 209] [210] [211] [212, 213, 337] [235] [236] [336] [355] [274] [236, 318, 564] [299] (continued)

Arthur E. Frankel et al.

419

Table 2. (continued) Ligand

Type

Receptor/antigen

Type linkage

Compounds

Disease

Reference

HRGbetal HRGbeta2 Betacellulin 48–50 Hrg

Growth factor Growth factor Growth factor

HER4 HER4 HER4

G G G

HRGbeta2-PE38KDEL HRGbetal-PE38KDEL BTC-TX50, BTC-TX48

Breast cancer Breast cancer Breast cancer

[299] [299] [300]

Growth factor

HER4

G

Breast cancer

HERB FRP5 FRP5(SFv) e23(dsFv)2 e23(dsFv) e23(sFv) 520C9 741 F8 454C11 Mgr6 BACH250 Anti-HER2 ConA TRH Ricin sIL 15 D3 GCSF H65

sFv Mab SFv (dsFv)2 dsFv sFv MAb MAb MAb MAb Humanized MAlk MAb Lectin Hormone Lectin Cytokine MAb Cytokine MAb

HER2 HER2 HER2 HER2 HER2 HER2 HER2 HER2 HER2 HER2 HER2 HER2 Mannose TRHR Galactose IL 15R p148 GMCSFR CD5

G G G G G G C C C C C C C C C G C G C

STI T101

MAb MAb

CD5 CD5

C C

[214, 248, 277] [582] [614] [281] [290] [290] [292] [316] [422] [422] [35] [11] [444] [215, 216] [217] [218] [219] [220] [223, 224] [93, 394, 466] [395] [187, 190, 228, 312, 327, 328, 10, 19, 25]

OKT1 SM5-1

MAb sFv

CD5 gp230

C G

DT389hrg, hrg-PE38KDEL, hrg-PE40 hERB-hRNase chFRP5-ZZ-PE38 SFv(FRP5)-ETA e23(dsFv)2-PE38 e23(dsFv)-PE38 e23(Fv)-PE38KDEL 520C9-RTA 741 F8-RTA 454C11-RTA Mgr6-clavin BACH250-gelonin Anti-HER2-saporin ConA-DTA, ConA-RTA TRH-CRM45, TRH-CRM26 Ricin-DTA DAB389IL 15 D3-DTA DAB389GCSF, GCSF-PE40 H65-RTA, H65-mitogillin, H65-gelonin STI-RTA Anti-CD5-CRM9, T101-RTA, T101-RNase, T101-ricin, T101-ricin-125l, anti-CD5-MLA, T101 Fab-RTA, T101 F(ab’)2RTA, anti-CD5-momordin anti-CD5-Pyrularia thionin OKT1-sap or in SMFv-PE38KDELmut1

MEL ZME01 8

sFv MAb

G C

MELsFv-rGel, scFvMEL/TNF ZME018-RTA, ZME018-gelonin

9.2.27

MAb

gp240 Proteoglycan, p250 Proteoglycan, p250

C

NR-ML-05

MAb

C

SV10016

MAb

C

Ep2

MAb

BrE3 Anti-MUC1 Mc5 BM7 C242

MAb sFv MAb MAb MAb

Proteoglycan, p250 Proteoglycan, p250 Proteoglycan, p250 Mucin, MUC1 MUC1 Mucin Mucin Mucin

9.2.27-DTA, 9.2.27-abrin, 9.2.27-PE, 9.2 27-gelonin, 9.2.27-RTA, 9.2.27-PAP NR-ML-05-abrin, NR-ML-05-PE

C242rF(ab’)

Antibody fragment MAb

Mucin Mumn

MBR1

Breast cancer Breast cancer Prostate cancer Lung cancer Lung cancer Prostate cancer Breast cancer Breast cancer Breast cancer Breast cancer Breast cancer Breast cancer Carcinoma Pituitary tumors Carcinoma Lymphoma Hepatocarcinoma AML T-cell ALL T-mellALL T-cell ALL

T-cell ALL Hepatocellular cancer Melanoma Melanoma

[451] [612]

Melanoma

[225, 254, 307, 311]

Melanoma

[254]

SV10016-CRM103

Melanoma

[187]

C

Ep2-saporin

Melanoma

[448]

C G C C C

SCLC Breast cancer Breast cancer Breast cancer Breast cancer

[375] [603] [226] [283] [255, 367]

C

BrE3-RTA sFv(MUC1)-ETA Mc5-DTA BM7-PE C242-PE, C242-NlysPE40, C242-RTA C242F(ab’)-PE38QQR

Colon cancer

[268]

C

MBR1-restrictocin

Breast cancer

[322] (continued)

[569, 574] [353]

420

Immunotoxins

Table 2. (continued) Ligand

Type

Receptor/antigen

Type linkage

Compounds

Disease

Reference

260F9 171A Anti-EpCAM 4D5MoCB A9(Fab)’ Anti-SV40 Cholera toxin B Anti-ConA

MAb MAb sFv sFv Fab fragment Polyclonal Toxin fragment Polyclonal

p55 Ep-CAM EpCAM EpCAM p100 SV40 antigens GM1 ganglioside ConA

C C G G C C C C

260F9-RTA, 260F9-rRTA 171A-DTA, 171A-RTA DTEpCAM23 4D5MoCB-ETA A9(Fab)’-DTA Anti-SV40-DT Cholera B-DTA Anti-ConA-DTA

[158, 191] [227] [552] [568] [229] [230] [231] [232]

RTB

Toxin fragment

Galactose

C

TEClgM Protein A 74124 MT151

MAb Toxin fragment MAb MAb

IgM Fc ImmunoglobulinFc pCD4 CD4

C C C C

OVB3 NR-LU-10

MAb MAb

C C

ATF

Protease fragment

Ovarian antigen Breast cancer antigen Urokinase PA receptor

RTB-DTA, RTB-MLA, RTBmomordin TEClgM-saporin Protein A-RTA 74124-PAP Anti-CD4-PAP, MT151-RTA, MT151-blocked ricin, anti-CD4-saporin OVB3-PE NR-LU-10-PE

Breast cancer Breast cancer Colon cancer Colon cancer A.castellani SV40 + tumors Gliomas ConA-treated tumors Carcinomas

11A8 bFGF

MAh Growth factor

bFGFR bFGFR

C G

aFGF PR1(Fv) MRK16

Growth factor sFv MAb

aFGFR PR1 antigen P-glycoprotein

G G C

PA

Toxin peptide

PA receptor

G

IGF-I Anti-CD8 Lym-1 2G5 HB55 KM231 84.1C Phlp56 B3 B3(Fab) B3(dsFv)

Growth factor MAb MAb MAb MAb MAb MAb Allergan MAb Fab fragment dsFv

IGF-IR CDS HLA-DR HLA-DR HLA-DR Sialyl-Lea-antigen mlgE Ant-P5 Lewisy antigen Lewisy antigen Lewisy antigen

G C C C C C C G G G G

B3(Fv) B1(Fv) B1(dsFv) BR96(sFv)

sFv sFv dsFv) sFv

Lewisy antigen Lewisy antigen Lewisy antigen Lewisy antigen

G G G G

Folate

Vitamin

Folate receptor

C

Anti-FRbeta IL9 55.1(Fv) 55.1 (dsFv)

Mab Cytokine sFv dsFv

Folate receptor IL9R Colon antigen Colon antigen

C G G G

ME20

MAh

p105

C

G, C

ATF-PE38, ATF-PE38KDEL, uPA-SAP, DT388N-termURO, ATF-saporin 11 A8-saporin bFGF-PE40, hFGF-PE66Glu4, hFGF-saporin, DT389bFGF aFGF-PE40, aFGF-PE66Glu4KDEL PR1(Fv)-PE38KDEL MRK16-PE, MRK-RTA, MRK-saporin FP59 + PA, FP59 + PA(MMP) IGF-I-PE40 Anti-CD8-ricin, Anti-CDs-saporin Lym-l-gelonin 2G5-RTA HB55-ricin KM231-RTA 84.1c-RTA P5-ETA’ B3-LysPE38 B3(Fab)-PE38 B3(dsFv)-PE38, dsFvB3-granzyme B B3(Fv)-PE38 B1(Fv)-PE38 B1(dsFv)-PE38 BR96sFv-PE40 (SGN10), BR96sFv-bryodin Folate-momordin, FolateLysPE38, Folate-Cys-PE35 anti-FRbeta-PEA rhIL9-ETA’ 55.1(Fv)PE38, 55.1(Fv)PE38KDEL 55.1(dsFv)-PE38, 55.1(dsFv)PE38KDEL ME20-lysPE40

Myeloma Ig + tumors Transplants HIV + tumors

[233, 387, 417] [455] [238] [431] [239–241, 453]

Ovarian cancer Breast cancer

[242] [243]

AML, gliomas

[244–246, 434]

Breast cancer Breast cancer

[435] [249, 435, 599] [249, 252] [250] [251, 365, 450] [256, 286]

Breast cancer Prostate cancer Renal cancer Gliomas, carcinomas Breast cancer Tcell lymphoma Lymphoma Lymphoma Lymphoma Colon cancer Allergies Allergies Breast cancer Breast cancer Breast cancer

[257] [331, 453] [463] [361] [343] [356] [396] [593] [266] [297] [297, 580]

Breast cancer Breast cancer Breast cancer Breast cancer

[258] [267] [267] [21, 278]

KS

[261]

RA Hodgkin’s disease Colon cancer Colon cancer

[592] [263] [265] [265]

Melanoma

[269] (continued)

Arthur E. Frankel et al.

421

Table 2. (continued) Ligand

Type

Receptor/antigen

Type linkage

G285sFv(VL-VH) GnRH

sFv

CD40

G

Hormone

GnRHR

C

MOC31 WF lectin

MAb Lectin

C C

hPL AntiCD30sFv

Hormone sFv

EGP-2 N-acetylGalactosamine hPLR CD30

Ki4

MAb

CD30

C

Ki4(sFv)

sFv

CD30

G

BerH2

sFv

CD30

G

BerH2

MAb

CD30

C

HRS3

MAb

CD30

C

BerH2-saporin, BerH2-RTA, BerH2-momordin, BerH2dianthin, BerH2-PAP HRS3-RTA

HRS3Fab’

Fab’ fragment

CD30

C

HRS3Fah’-RTA

TP3 TP3(sFv)

Mab sFv

C G

TP3(dsFv)

dsFv

RANTES

Chemokine

p80 Osteosarcoma antigen Osteosarcoma antigen CCR5

HD6 HD39 OM124 RFB4 RFB4(dsFv)

MAb MAb MAb MAb dsFv

LL2 RFB4 AchRα Dsg3ΔNI

Compounds

Disease

Reference

G28-5sFv(VL-VH)-PE40, G28-5sFv(VL-VH)-hryodin GnRH-PE66, GnRH-PE40, GnRH-PAP MOC31-ETA252-61 3 WF-DTA

Lymphoma

[18, 270]

Breast cancer

[271, 293, 13] [272] [221]

hPL-DTA Anti-CD30(sFv)-PE38, AntiCD30(Fv)-PE38KDEL Ki4-RTA

Breast cancer Hodgkin’s disease Hodgkin’s disease Hodgkin’s disease Hodgkin’s disease Hodgkin’s disease

[222] [280]

[338]

TP3-PAP TP-3(sFv)-PE38

Hodgkin’s disease Hodgkin’s disease Osteosarcoma Osteosarcoma

G

TP-3(dsFv)-PE38

Osteosarcoma

[26]

G

RANTES-PE40, DT390-RANTES

[282, 561]

CD22 CD22 CD22 CD22 CD22

C C C C G

MAb sFv Antigen

CD22 CD22 Anti-AchR

C G G

HD6-saporin HD39-saporin OM124-PAP, OM124-saporin RFB4-RTA RFB4(dsFv)PE38, RFB4(dsFv) PE38KDEL, HA22 LL2-onconase DT2219ARL AchRα-gelonin

Rheumatoid arthritis Lymphomas Lymphomas Lymphomas Lymphomas Lymphomas

[32] [554] [594]

Anti-desmoglen3

G

hMN14sFv C110 C19 CB-CEA-1 I-1 C27 E4 E4(Fv) M6 38.13

Antigenic peptide sFv MAb MAb MAb MAb MAb MAb sFv MAb MAb

CEA CEA CEA CEA CEA CEA Prostate antigen Prostate antigen Idiotype Pk antigen

G C C C C C C G C C

Fab’anti-L3T4

Fab’ fragment

C

486P3121

MAb

C

486P-RTA + 486P-RTB

Bladder cancer

[310]

RFT11

MAb

Murine Tcellantigen Bladder cancer antigen CD2

DsgΔN1-PE40KDEL, PE37Dsg3ΔN1-KDEL hMN14(Fv)-PE40 C110-RTA-s°Y C19-RTA CB-CEA-1-hemolytic toxin I-1-blocked ricin C27-ahrin A E4-PE35KDEL E4Fv-PE38KDEL M6-ricin, M6-RTA 38.13-RTA, 38.13-gelonin, 38.13-PAP Fab’anti-L3T4-RTA

Lymphomas B-cell ALL, CLL Myasthenia gravis Pemphigus vulgaris Colon cancer Colon cancer Colon cancer Colon cancer Colon cancer Colon cancer Prostate cancer Prostate cancer Lymphoma Burkitt’s lymphoma Lymphoma

C

RFT11-RTA

T-cell ALL

[326]

C G

Ki(sFv)-ETA’, scFvKit4angiogenin BerH2-scFv-hpRNase

SCLC Carcinoma

[398] [294, 565] [609] [22, 373, 416, 428]

[338] [174] [26]

[452] [452] [348] [332] [288, 562]

[289] [296] [357] [304] [37] [391] [9] [298] [298] [302] [306] [308]

(continued)

422

Immunotoxins

Table 2. (continued) Ligand

Type

Receptor/antigen

Type linkage

35.1 OKT11

MAb MAb

CD2 CD2

C C

452D9 Thyroglobulin

MAh Antigen

Anti-CD64

Mab

p74 Ig antithyroglobulin CD64

H2-D(d)tet

Tetramer

2F8 E9 96.5 HB2 TXU 3A1e(Fv) 3A1f

Compounds

Disease

Reference

T-cell ALL T-cell ALL

[312, 326] [393, 473]

C C

35.1-ricin, 35.1-RTA Anti-CD2-RTA, Anti-CD2-saporin, OKT11-gelonin 452D9-RTA Thyroglobulin-RTA

Ha-ras + tumors Thyroiditis

[313] [305]

C

Anti-CD64-RTA

[598]

MHC

C

Mab sFv MAb MAb MAb Fv-Cys sFv

SR-A/CD163 FCRL1 p97 CD7 CD7 CD7 CD7

C G C C C C G

Ovarian cancer CLL Melanoma T-cell ALL T-cell ALL T-cell ALL T-cell ALL

[610] [615] [424] [434] [390] [408] [588]

Anti-CD7 3A1 e WTI 791T/36 8A IORT6 SN5d Anti-CALLA AntiGE2 AR3 8C

sFv MAb MAb MAb MAb MAb MAb MAb MAb MAb MAb

G C C C C C C C C C C

T-cell lymphoma T-cell ALL T-cell ALL Colon cancer Myeloma T-cell ALL Pre-Bcell ALL Pre-B cell ALL Gliomas Gastric cancer Ovarian cancer

[590] [427] [317] [319] [320, 321] [323] [324] [380] [325] [333, 461] [334]

My9 p67.7 HuM195

MAb MAb Humanized MAlk sFv MAb

CD7 CD7 CD7 p72 Myeloma antigen T-cell antigen CD10 CD10 GE2 CAR-3 Ovarian cancer antigen CD33 CD33 CD33

H2-D(d)tetramer-biotin-avidinsaporin 2F8-Saporin E9(Fv)-PE38 96.5-RTA HB2-saporin TXU-PAP 3A1e(Fv)Cys-RTA Rnase-TRHRQPRGWEQL-antiCD7-sFv scFvCD7:sTRAIL 3A1e-RTA WT1-RTA 791T/36-RTA 8A-momordin, 8A-saporin IORT6-hemolytic toxin SN5d-RTA Anti-CALLA-RTA Anti-GE2-ricin, AntiGE2-RTA AR3-RTA, AR3-ricin, AR3-gelonin 8C-RTA

Rheumatoid arthritis GVHD

C C C

My9-blocked ricin p67.7-RTA HuM195-gelonin

AML AML AML

[346] [352] [460]

CD33 Vasopressin

G C

Anti-CD33(sFv)-ETA Anti-vasopressin-RTA

AML Pituitary tumor

[597] [347]

Hepatoma antigen Cluster 2 antigen-SCLC T-cell antigen Bladder cancer antigen Gastric antigen Oncofetal antigen Laryngeal cancer antigen ICAM CD4 and CD26

C C

Hepama-l-trichosanthin Cluster 2 MAb-RTA

Hepatoma SOLO

[349] [350]

C C

SOKT1-RTA Fih75-RTA, Fih75-a-sarmn, Fih75-gelonin MAh2-RTA 16-RTA, 16-MLA Anti-laryngeal cancer-RTA

T-cell antigen Bladder cancer

[351] [354, 471]

Gastric cancer Lymphoma Laryngeal cancer

[358] [359, 374] [362]

C C

W3-RTA Bispecific (CD4/CD26)-blocked ricin

Myeloma Tissue allografts

[363] [364]

CD24 Erythroblast antigen Glycophorin

C C

SWAII-RTA HAE9-RTA

Lymphoma Eyrtholeukemia

[366] [368]

C

HAE3-RTA

Erythroleukemia

Anti-CD33 Antivasopressin Hepama-1 Cluster 2 MAb

MAb MAb

SOKTI Fib75

MAb MAb

MAb2 16 Anti-laryngeal cancer W3 Bispecific MAb(CD4/ CD26) SWAII HAE9

MAb MAb MAb

HAE3

MAb

MAb Bispecific MAlk MAb MAb

C C C

[601]

[368] (continued)

Arthur E. Frankel et al.

423

Table 2. (continued) Ligand

Type

Receptor/antigen

Type linkage

Compounds

Disease

Reference

Bispecific MAb(CD4/ CD29) CLL2m SEN31

Bispecific MAlk

CD4 and CD29

C

Bispecific (CD4/CD29)-blocked ricin

Tissue allografts

[369]

MAb MAb

C C

CLL2m-RTA SEN31-blocked ricin

CLL SCLC

[370] [371]

317G5 SEN36 SEN7 N901 BDI-1 Anti-mu Anti-CD6 Anti-CRF

MAb MAb MAb MAb MAb MAb MAb MAb

C C C C C C C C

317G5-RTA SEN36-RTA SEN7-PE, SEN7-blocked ricin N901-blocked ricin BDI-1-ricin Anti-mu-RTA Anti-CD6-blocked ricin Anti-CRF-RTA

Breast cancer SCLC SCLC SCLC Bladder cancer Myeloma GVHD Pituitary tumor

[372] [376] [383] [400] [377] [378] [379] [381]

CRF ScFvH17

Hormone sFv

C C

CRF-saporin scFvH17-BSRNase

Pituitary tumor Germ cell tumor

[436] [27, 384]

Anti-la Ng76 E6 A5 7E4B11 IB4 HB7 AntiasialoGM2 Anti-vbeta6

MAb Mab Mab sFv Mab Mab MAb MAb

CLLantigen Cluster 5a SOLO antigen p42 NCAWCD56 NCAWCD56 NCAWCD56 Bladder antigen IgM Fc CD6 Corticotropinreleasing factor CRFreceptor Placental alkaline phosphatase la Melanoma antigen PSMA PSMA RPTPβ CD38 CD38 Asialo-GM2

C C C G C C C C

Anti-la-RTA Ng76-luffin, Ng76-moschatin E6-dgA A5-PE40 7E4B11-saporin IB4-saporin-S6 HB7-blocked ricin AntiasialoGM2-RTA

Carcinoma Melanoma Prostate cancer Prostate cancer Gliomas Myeloma Myeìoma Carcinoma

[385] [571, 572] [583] [595] [589] [596] [386] [388]

C

Antivbeta6-RTA

MAb Lectin

C C

14G2a-RTA Peanut agglutinin-RTA

Myasthenia gravis Neumblastomm Lymphoma

[389]

14G2a Peanut agglutinin BU12 B43 HD37 B4 B-c3 Anti-CD19 Anti-CD19 Fv5191/cys Rituximab

vbeta6 T cell receptor Disialoganglioside D-glucosyl moiety

MAb MAb MAb MAb Mab sFv sFv sFv-Cys Mab

CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD20

C C C C C G G C C

Lymphoma Lymphoma Lymphoma Lymphoma CLL CLL ALL Lymphoma Lymphoma

[441] [432] [399, 452] [415] [563] [611] [554] [406] [581, 586]

ONSM21(Fv)

sFv

C

Medulloblastoma

[419]

ONSM21

MAb

C

ONSM21-RTA

Medulloblastoma

[403]

35

MAb

C

35-ricin

Strabismus

[404]

K42C10 44G4 SN6j 8OG 15A8

MAb Mab MAb MAb MAb

C C C C C

K42C10-RTA 44G4-ebulin, 44G4-nigrin b SN6j-RTA 8OG-gelonin 15A8-gelonin

Breast cancer Breast cancer Breast cancer Hepatoma Breast cancer

[405] [558, 607] [418] [471] [467]

HB5 Anti-Lyt2.2

MAb MAb

Medulloblastoma antigen Medulloblastoma antigen Nicotinic acetylcholine receptor Endoglin/CD105 CD105 Endoglin/CD105 Alphafetoprotein Breast cancer antigen C3d receptor Lyt2.2 antigen

BU12-saporin B43-PAP HD37-RTA, HD37-saporin B4-blocked ricin Anti-CD19:K CD19-ETA’ DT2219ARL Fvs191-Cys-RTA Rituximab/Saporin-S6, Rituximab-allinase ONSM21 (Fv)-RTA

C C

HB5-gelonin Anti-Lyt2.2-gel on in

EBV infection T-cell lymphoma

[474] [475]

MAb

[392] [397]

(continued)

424

Immunotoxins

Table 2. (continued) Type linkage

Ligand

Type

Receptor/antigen

SN7 Anti-VIP

MAb MAb

Anti-CMV

MAb

B cell antigen C Vasoactive C intestinal peptide mCMV antigen C

Anti-CMV

Polyclonal

Anti-d(beta)h

MAb

M24

Compounds

Disease

Reference

SN7-RTA Anti-VIP-RTA

B-cell ALL Pituitary tumor

[409] [410]

CMV infection

[411, 462]

CMV infection

[464]

C C

Antid(beta)h-saporin

Pitirflary tumors

[12]

MAb

Human CMV antigens Dopamine betahydroxlase CD80

Anti-mCMV-RTA, Anti-mCMVgelonin Anti-huCMV-gelonin

C

MAb

CD86

C

LHRH Anti-Pbs21(Fv) Antimelanoma 48127 J3109 0×7 O × 7F(ab’)2

Hormone sFv MAb

LHRHR Pbs21 Melanoma antigen

C G C

Hodkgin’s disease Hodkgin’s disease Breast cancer Malaria Melanoma

[15]

1G10

M24-bouganin, M24-gelonin, M24-saporin IG10-bouganin, IG10-gelonin, IG10-saporin, IG10-gelonin LHRH-bovineRNase Anti-Pbs21(Fv)-Shiva-1 Anti-melanoma-BRIP

gp54 CD72 mCD90 mCD90

C C C C

48127-PAP, 48127-saporin J3109-PAP 0 × 7-DT, 0 × 7-saporin O × 7F(ab’)-saporin

Bladder cancer B-mellALL T-cell ALL T-mellALL

[429] [430] [199, 315] [315]

Anti-Thy1.2

MAb MAb MAb F(ab )2 fragment MAb

mCD90

C

T-mellALL

[16, 458]

Anti-Thy1.2 mFclgE IR162 mSCF K1

Polyclonal Fcfragment MAb Cytokine MAb

C G C G G

T-mellALL Allergies Allergies SCLC, AML Mesothelioma

[342] [273] [303, 309] [275] [284]

K1(Fv)

sFv

G

Anti-mesothelin(Fv)-PE38

Menothelioma

[276]

SS1(dsFv)

dsFv

G

SS1(dsFv)-PE38, D-SS1P

Mesothelioma

[543, 616]

Anti-0X40 MBP(66-88)

C G

Anti-OX40-RTA MBP(66-88)-PE40

Multiple sclerosis [423] Multiple sclerosis [301]

MBPp8799 CD64

MAb Antigenic peptide Peptide Mab

mCD90 mFcεRI receptor rIgEFc receptor mc-kit Mesothelin p40 GPI-anchored Menothelin p40 GPI-anchored Mesothelin p40 GPI-anchored T cell antigen Anti-MBP Ig

Anti-Thy1-trichokirin, Anti-Thy1gelonin Anti-Thy1-RTA Fc(2′-3)-PE40 IR162-RTA, IR162-ricin mSCF-PE40 K1-LysPE38QQR

anti-MBP CD64

G, C C

MBPp87-99-cyt1Aa CD64-riA

[559] [566]

m22 anti-JL1 Campath-1 L6

sFv Mab MAb MAb

G C C C

m22(scFv)-ETA’ Anti-JL1-gelonin Campath-l-saporin L6-ricin

Anti-CTLA4 Anti-CTLA4 8H9 LL1 Anti-mFcD SWA11 Anti-T.cruzi

Mab sFv dsFv Fusion L2-H2 MAb MAb MAb

CD64 JL1 CD52 Lung cancer antigen CTLA-4 CTLA-4 glycoprotein CD74 msIgD SCLC antigen T. muzi antigen

Multiple sclerosis Rheumatoid arthritis AML Leukemias Lymphoma Lung cancer

C G G G C C C

Tolerance Tolerance Osteosarcoma Myeloma Lymphoma SCLC Chagas disease

[575] [576] [577] [591] [340] [341] [344]

Anti-CD45RO BMAC1 OX1

Mab MAb MAb

CD45RO CD45 rCD45

C C C

Anti-CTLA4-saporin scFvCTLA4-perforin 8H9(dsFv)-PE38 2L-Rap-hLL1-gamma4P Anti-mFcD-RTA SWA11-RTA Anti-T. cruziRTA, anti-T. cruzi-abrin A Anti-CD45RO-RTA BMAC1-RTA, MBAC1-MLA OX1-RTA, OX1-MLA

HIV Renal transplant Renal transplant

[578] [345] [345] (continued)

[15, 570] [31] [38] [17]

[573] [567] [335] [339]

Arthur E. Frankel et al.

425

Table 2. (continued) Ligand

Type

Receptor/antigen

Type linkage

M20.4 1921g

MAb MAb

primate NGFR p75 rat NGFR p75

C C

OKT10 Insulin BB2 BB4 Anti-epithelial antigen Anti-Ab1 Anti-Id

MAb Hormone MAb MAb MAb

CD38 Insulin receptors Myeloma antigen Myeloma antigen Epithelial antigen

C C C C C

MAb MAb

C C

Anti-CD37 ML30 Anti-AML 8A 62B1 TTC SP IL6

MAb MAb MAb MAb MAb Toxin fragment Hormone Cytokine

Idiotype anti-DNA Idiotype lymphoma CD37 Heat shock protein AML-M5 antigen Myeloma antigen Myeloma antigen p15 Neurokinin-1 IL6R

C C C C C G G G

VEGF165

Growth factor

flk-1, flk-1/KDR

G, C

VEGF121

Growth factor

flk-1/KDR

G, C

oLH gp330

Hormone Antigen

Ovine LHR Anti-gp330 Ig

Anti-pichinde virus NDA4 14G2a MSN-1

MAb, polyclonal MAb MAb MAb

Pichinde virus antigens NDA4 Tcell antigen GD2 ganglioside Endometrial Ca antigen

Compounds

Disease

Reference

M20.4-saporin 192Ig-saporin, 192Ig-trichosanthin OKT10-saporin Insulin-saporin BB2-saporin BB4-saporin Anti-epithelial antigen-colocin 1 Anti-Ab1-saporin Anti-Id-saporin

Neuromas Neuromas

[437] [433]

Myeloma Breast cancer Myeloma Myeloma Lung cancer

[434] [438] [439] [439] [440]

SLE Lymphoma

[443] [456]

Anti-CD37-saporin ML30-saporin Anti-AML-saporin 8A-saporin 62B1-saporin DAB389TTC DAB389SP DAB389IL6, IL6-PE40, IL6-PE66GIu4 DT390VEGF165, VEGF165-DT385 DT390VEGF121, VEGF121-DT385, VEGF121/rGel

Lymphoma AML AML Myeloma Myeloma Neuromas CML, neuromas Myelomas, KS

[445] [446] [449] [454] [454] [192] [193] [194, 195]

KS

[196, 197]

C C

oLH-gelonin gp330-gelonin

[457] [465]

C

Anti-Pichinde virus-gelonin

Prostate cancer Heyman’s nephritis Pichinde virus

C C C

NDA4-gelonin 14G2a-gelonin MSN-1-gelonin

T-cell ALL Melanoma Endometrial cancer

[469] [470] [547]

KS, breast cancer [196–198]

[468]

RTA, ricin toxin A chain; sFv, single chain antibody; Cys, cysteine; DT, diphtheria toxin; DTA, diphtheria toxin A fragment; CRM9, binding site DT mutant; HBEGF, heparin-binding epidermal growth factor; ETA, Pseudomonas exotoxin; PE, Pseudomonas exotoxin; PE40, 40 kDa PE fragment; GMCSF, granulocyte-macrophage colony-stimulating factor; IL3, interleukin-3; CRM107, binding site DT mutant; rRTA, recombinant RTA; mIL3, mouse IL3; mGMCSF, mouse GMCSF; EGF, epidermal growth factor; TGFα, tumor growth factor-alpha; dsFv, disuffide-stabilized sFv; MAb, monoclonal antibody; R, receptor; G, genetic fusion; C, chemical linkage; RNase, ribonuclease; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CA, cancer; IL7, interleukin-7; IL4, interleukin-4; mIL4, murine IL4; IL13, interleukin-13; aMSH, alpha-melanocyte-stimulating hormone; NT-4, neurotrophin-4; GRP, gastrin-releasing peptide; HRG, heregulin; TRH, thyroid releasing hormone; hPL, human prolactin; Wf, Wistaria floribunda; ConA, concanavalin A; HIV, human immunodeficiency virus; SCLC, small cell lung cancer; IL15, interleukin-15; Ep-CAM, epithelial cell adhesion molecule; RTB, ricin toxin B chain; MLA, mistletoe lectin A chain; KS, Kaposi’s sarcoma; LH, luteinizing hormone; VEGF, vascular endothelial growth factor; CML, chronic myeloid leukemia; IL6, interleukin-6; SLE, systemic lupus erythematosis; Id, idiotype; SP, substance P; TTC, tetanus toxin C-terminal domain fragment; T. cruzi, trypanosoma cruzi; SCF, stem cell factor; Gel, gelonin; PAP, pokeweed antiviral protein; Ig, immunoglobulin; NGF, nerve growth factor.MBP, myelin basic protein; VIP, vasoactive intestinal peptide; CMV, cytomegalovirus; LHRH, luteinizing hormone releasing hormone; CRF, corticotropin releasing factor; GVHD, graft-versus host disease; CLL, chronic ymphocytic leukemia; GnRH, gonadotropin-releasing hormone; IL9, interleukin-9; Tf, transferrin; GCSF, granulocyte colony-stimulating facor; ASF, asialofetuin; IGF, insulin-like growth factor; FGF, fibroblast growth factor; a, acidic; b, basic; o, ovine

426 the N-terminal fragment of urokinase, sFv anti-CD19 with sFv anti-CD22) [551–554]. Dual specific immunotoxins have also been tested by Frankel (urokinase activated DT with GMCSF ligand) [555].

Conjugation of Toxin and Ligand The linkage of the modified toxin with the ligand must be covalent and stable, so that metabolism in the bloodstream or interstitial tissues will not occur. Both amide linkage by genetic engineering and chemical crosslinking using bifunctional reagents such as thiolating compounds – 3-(2-pyridyldithio)proprionicacid N-hydroxysuccinimide ester (SPDP) [8] or 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) [24] – have been used. The coupling must not severely alter the ligand affinity for its receptor or impair the ability of the toxin domains to translocate to the cytosol and enzymatically inactivate cell functions (type I toxins) or interact and alter cell membrane functions (type II and III cytotoxins). Genetic fusions are well defined and controlled, and generally have less effect on toxin functions than chemical conjugation. Further, the fusion can be designed so that the linkage is remote from ligand and toxin domains critical for their normal functions. Chemical conjugation methods yield a heterogeneous product due to the reaction of the derivatizing reagent with multiple amino acid residues in the ligand and/or toxin. Cysteines and lysines are the most frequent substrates for bifunctional reagents. Both chemical conjugates and genetic fusions have been developed and used on patients with beneficial results. Successful clinical development has been done with both chemical conjugates and genetic fusions.

Preclinical Studies with Immunotoxins Over a 1,000 immunotoxins have been synthesized in the past 3 decades, but most have been evaluated with markedly different tissue culture and animal model assays. Thus, comparisons are fraught with potential errors. Nevertheless, in the spirit of a review, we will attempt to draw some general observations. The more potent immunotoxins show higher affinity binding to their cell surface receptors and have more receptors/cell [189]. Immunotoxin efficacy is also influenced by the location of interaction of the ligand and the receptor. Antibodies which bind different epitopes on receptors can vary greatly in conjugate cytotoxicity [326, 422]. The ability of the ligand-receptor complex

Immunotoxins to internalize is also a critical parameter, at least for conjugates with type I toxins [329]. While chemical conjugates represent the vast majority of reported immunotoxins, recombinant toxin conjugates offer advantages for production [164]. The molecules are homogeneous with a single site of linkage between the haptophore and toxophore. Furthermore, the synthesis does not require manipulations of different chemical components. The specificity of the immunotoxin is important, since crossreaction with vital normal tissues can produce severe toxicities (see the next section). Both normal human tissue immunostaining and testing in nonhuman primate models have been used to identify normal tissues likely to be damaged by systemic immunotoxin administration [478, 479]. The size of the immunotoxin appears to influence both efficacy and safety. Larger conjugates have greater difficulty in reaching extravascular sites of disease in most cases [228]. In one report, a larger immunotoxin targeting HER2 was more effective in vivo [614]. The larger molecules have prolonged circulating half-life, leading to increased vascular endothelial injury and vascular leak syndrome (VLS) [8]. However, with conjugates much smaller than 58 kDa Mr, the immunotoxin will be rapidly cleared by the kidneys, leading to very short half-life and insufficient antitumor effect. Further, renal tubular injury is more likely [169]. Immunotoxins made with human ligands and toxins are likely to be far less immunogenic than those made with non-human components [28, 29, 32, 190, 407, 426]. Some toxins perform better for particular classes of tumors or target cells. DT conjugates are more toxic to myeloid malignancies than PE, ricin, or other type I conjugates. This is likely due to the requirement for routing to the endoplasmic reticulum for the type I toxins other than DT and anthrax toxins. Since most myeloid cells rapidly traffic internalized materials to the lysosome for degradation, conjugates requiring transport to the endoplasmic reticulum do not intoxicate these cells well [179, 181]. Consequently, improved efficacy has been observed with DT conjugates for myeloid disorders. Stability in the bloodstream is also important. Disulfide-stabilized single chain immunotoxins or tandem or dimeric single chain immunotoxins appear to be more stable in vivo than single-chain Fv fragment conjugates [163, 168]. These molecules also have a better in vivo therapeutic index. None of the immunotoxins which have been tested in clinical trials to date is perfect. The ligands are generally not tumor-specific. Many of the conjugates are not recombinant. The protein components are frequently immunogenic. The size is often too large. In some cases the target cell may not always endocytose the immunotoxin-receptor

Arthur E. Frankel et al. complex. The patients have often not been optimally selected for the individual agent. However, many of the newly described immunotoxins which have entered clinical trials in the past few years, or will enter clinical trials in the near future, have improved characteristics, and better patient selection is being used. The recent results in the clinic are very exciting and will be described in detail in the next section.

Clinical Experience with Immunotoxins Clinical trials have been conducted with only several dozen immunotoxins over the past 3 decades. The complexities in synthesis and purification of these multidomain polypeptides, and the necessary but extensive safety and regulatory hurdles, have limited the rapid application of this technology. Direct comparisons are difficult due to the different conjugate constructions; the different patient populations; and the different routes, doses, and schedules of drug used in the various trials. Nevertheless, we will address the efficacy, pharmacokinetics, immune responses, and toxicities for each agent and try to present common principles. Excitingly, several of these drugs are showing significant anticancer activity in phase I and II clinical studies in patients with chemotherapy-refractory cancers.

Efficacy Immunotoxins with the highest response rate in clinical studies have been active in tissue culture in the picomolar range, have been targeted to hematologic malignancies or diseases, and have been fully recombinant molecules. The only exceptions in which good response rates were seen in non-hematologic diseases were in cases in which immunotoxins were administered locally by intracavitary or interstitial infusions. BL22 consists of a disulfide-stabilized anti-CD22 sFv fused to PE38. When given at doses of 3–50 μg/kg i.v. over 30 min every other day for three doses every month for up to 14 cycles to patients with chemotherapy refractory B cell malignancies, there were 61% CR and 19% PR in purine-analog refractory hairy cell leukemia (HCL) [481, 482, 617]. The remissions were durable with only 27% responders relapsing after 10–23 months. Retreatment again produced CRs. Responses were dose dependent and the MTD was 40 μg/kg/dose. Neutralizing antibodies developed in 24% of patients. A reversible hemolyticuremic syndrome requiring plasmapheresis was observed. Other side effects were hypoalbuminemia,

427 transaminasemia, fatigue, edema. Thus, BL22 immunotoxin appears to be the current best salvage treatment for relapsed HCL patients. An improved higher affinity variant, HA22, has begun clinical testing in patients with refractory B-cell malignancies [562]. LMB2 is composed of an anti-CD25 sFv fused to PE38. LMB2 has been administered to 35 patients with lymphomas at doses of 2–63 μg/kg i.v. over 30 min q.o.d. × 3 [279]. A complete remission was observed in HCL which was ongoing at 20 months. There were seven partial remissions in patients with HCL (three patients), CTCL (one patient), CLL (one patient), HD (one patient), and ATL (one patient). Responders received at least 60 μg/kg total dose of LMB-2 per cycle. The durations of remissions have not been determined, but exceed 20 and 6 months for two patients and 1 month for the remaining patients. Responding patients had clearance of circulating malignant cells, improvement in skin lesions, and regression of lymph nodes and splenomegaly. DLT was reversible transaminase elevations, diarrhea and cardiomyopathy. MTD was 40 μg/kg/dose. Other side effect was fever. 17% of patients developed neutralizing antibodies after the first cycle. Drug half-life was 4 h. Responders received at least 60 μg/kg total dose of LMB-2 per cycle. LMB-2 was also administered with MART-1/gp-100 vaccine to patients with metastatic melanoma in an effort to augment anti-melanoma immunity via Treg depletion [618]. Denileukin diftitox is a fusion protein composed of the catalytic and translocation domains of DT fused to human IL-2. Among CTCL patients with stage IB to IVA disease refractory to other therapies and treated with 9 or 18 μg/kg/day for 5 days every 3 weeks, there were 10% CRs and 20% PRs lasting a median of 6 months [486, 488, 489]. Denileukin diftitox produced a durable complete remission in a patient with transplantrefractory large cell lymphoma that was ongoing after 5 years [485]. Two patients with low-grade lymphoma obtained partial remissions. Based on the above results, in 1999 the FDA made denileukin diftitox the first approved immunotoxin [539]. Drug half-life was 30 min, and side effects included an acute cytokine reaction (fever, chills, nausea, vomiting, myalgias, chest pain, arthralgias, back pain), transient liver enzyme transaminasemia, and a vascular leak syndrome (hypotension, hypoalbuminemia, dyspnea, edema). Most patients developed an immune response to the agent, but this did not correlate with toxicities or response. In patients with non-CTCL relapsed/refractory T-cell non-Hodgkin’s lymphoma, the agent produced a 48% remission rate with 22% CRs and 26% PRs with median response duration of 6 months [619]. Denileukin diftitox also

428 produced remissions in CLL for a total of 27% overall response (4% CRs and 23% PRs) and median response duration of 6 months [620]. Denileukin diftitox given for patients with relapsed or refractory B cell nonHodgkin’s lymphoma yielded 7% CRs and 18% PRs with median response duration of 7 months [621]. There are case reports of denileukin diftitox-induced remissions in patients with systemic mastocytosis [622], extranodal natural killer/T-cell lymphoma [623], peripheral T-cell lymphoma [490], subcutaneous panniculitislike T cell lymphoma [624], and adult T-cell leukemia [625]. Among non-malignant conditions, denileukin diftitox depletes autoimmune activated T cells yielding a 71% response rate after two to six doses at 9 μg/kg in patients with steroid refractory acute GVHD [626] and yielding remissions in patients with psoriasis [491–493]. Additional studies evaluated the role of expression of the individual IL2R subunits in CTCL response. The alpha subunit expression was measured by immunohistochemistry [627]. Denileukin diftitox responses occurred in both CD25 positive and negative tumors, although patients with strongly positive tumors were more likely to respond. Responses were also more likely at 18 μg/kg versus 9 μg/kg [486]. Rare toxicities included thyrotoxicosis and optic neuritis [628, 629]. Combinations of denileukin diftitox with other agents including bexarotene, rituximab and hyper-CVAD enhanced response rates in CTCL, B-cell non-Hodkgin’s lymphoma, and adult T-cell leukemia, respectively [487, 630, 631]. The effects of denileukin diftitox on IL2R expressing T-regulatory cells has been used to augment immune responses to poxvirus vaccine and melanomas [632, 633]. TransMid is a chemical conjugate of transferrin and the DT-binding site mutant, CRM107. Forty-four patients with recurrent high-grade gliomas had placement of one to two catheters into the tumor beds [483, 484]. Forty milliliters of TransMid (0.66 μg/ml) was delivered by convection over 5 days. There were 12 responders including four complete responsers by magnetic resonance imaging (MRI) with median survival of >71 weeks. Toxicity was brain injury. TransMid is being tested in low grade gliomas and metastatic cancer to brain [634]. NBI-3001, IL4(38-37)PE38KDEL, was administered by one to three stereotactic catheters at 0.2–6 μg/mL in 30–185 mL over 4–8 days to nine patients with recurrent high-grade gliomas [517]. Six of nine patients showed tumor necrosis by MRI and histopathology. There was one unmaintained CR lasting >18 months. NB-3001 given at 6 μg/mL × 40 mL to 9 μg/mL × 100 mL intratumorally via one to three catheters over 4–8 days resulted

Immunotoxins in central nervous system toxicity in 22% of patients at the MTD of 6 μg/mL × 40 mL [635]. MRI showed tumor necrosis following treatment. Median survival was 7 months. NBI-3001 was administered IV at 0.008– 0.27 mg/m2 days × 5 every 28 days to patients with renal cancer and non-small cell lung cancer [636]. DLT was transient transaminasemia. Neutralizing antibodies were detected in 71% of patients after two cycles. No responses were seen. The binding site deleted Pseudomonas exotoxin PE38 fragment used to construct BL22 and LMB-2 was fused to an anti-mesothelin disulfide stabilized Fv to create the recombinant immunotoxin, SS1P. SS1P was given as 30 min IV infusions every other day for three to six doses to patients with advanced mesothelioma, ovarian cancer and pancreatic cancer at 18–45 μg/kg/dose [637]. DLT was pleuritis, and the MTD was 45 μg/kg × 3 doses. There were a few minor responses and over half of patients had stable disease. Phase II clinical studies are underway. Combinations with gemcitabine and paclitaxel are planned [638, 639]. Novel constructs with releasable PEGylation have been tested in animals and they maintain efficacy with reduced immunogenicity and toxicity [616]. PRX302 is a furin cleavage site modified proaerolysin. The new site is selectively cleaved by PSA. It was administered intratumorally to 24 patients with locally recurrent prostate cancer after primary radiotherapy failure [640]. The drug was well tolerated without DLT at doses of 0.03–3 μg/g prostate. Delivery was by a single multi-deposit, transrectal ultrasound-guided transperineal intraprostatic injection using a modified brachytherapy technique. PSA levels decreased in 63% of patients. The percentage of positive prostate biopsy cores post-therapy revealed a decrease in 75% of patients with three patients showing no positive biopsy cores at 30 days post-therapy. A phase IB study has been initiated at multiple institutions to optimize the dose and delivery method. IL13-PEQQR or cintredekin besudotox is composed of interleukin-13 fused to PE38 with a C-terminal QQR. Cintredekin besudotox was administered by convectionenhanced delivery to recurrent glioblastoma multiforme tumors for up to 6 days [641]. Optimal dose was 0.4 mL/h at 0.5 μg/mL over 50 h through two catheters with systemic corticosteroids. Toxicity was tumor necrosis and cerebral edema. 33% of patients had remissions lasting 7–117+ weeks. Response and survival depended upon optimal catheter placement. Unique characteristics of patient brain and tumor growth caused complex fluid distribution patterns after catheter placement and convection delivery. Both 123I-labeled human serum albumin PET imaging and MR diffusion tensor

Arthur E. Frankel et al. imaging predicted patient-specific drug distribution by convection-enhanced delivery and may improve prospective selection of catheter trajectories and targeted toxin efficacy [642]. The catalytic and translocation domains of diphtheria toxin (DT388) were fused via a Met-His linker to human interleukin-3 to make DT388IL3. DT388IL3 was administered IV over 15 min every other day for up to six doses to 45 patients with poor-risk, relapsed or refractory AML or MDS [643]. Half-life was 30 min, and peak levels were dose dependent. An inter-patient dose escalation schema was used with doses from 4 to 12.5 μg/kg/dose. DLT was not observed, but side effects included transient transaminasemia, hypoalbuminemia, fever, chills, nausea and vomiting. Antibodies to drug developed between day 15 and day 30 in most patients. Responses included one CR lasting 8 months. And two PRs of 3 and 4 months. Duration. A phase IB inter-patient dose escalation study in AML and MDS patients with 10–40% marrow blast index (cellularity fraction times percent blasts) with five daily doses is ongoing. A higher affinity variant, DT388K116W, is in final stages of testing and is expected to begin clinical trials in late 2008 [644]. ScFv(FRP5)-ETA was prepared by fusing the scFv of the anti-erbB2 monoclonal antibody FRP5 to domains II and III of Pseudomonas exotoxin. ScFv(FRP5)-ETA was injected intratumorally once daily for 7–10 days with daily doses of 60–900 μg in patients with subcutaneous tumor nodules of metastatic breast cancer, colorectal cancer and malignant melanoma [645]. Adverse reactions were local injection site pain and inflammation. Partial or complete regression of injected nodules was seen in 20% and 40% of patients, respectively. Next, patients with metastatic breast cancer, prostate cancer, head and neck cancer, non-small cell lung cancer and transitional cell carcinoma were treated on an inter-patient dose escalation schedule systemically with 2–20 μg/kg bolus infusion daily for 5 days of each wk for 2 weeks [646]. The DLT was transient transaminasemia. The MTD was 12.5 μg/kg, and the peak drug level was 100 ng/mL. Most patients developed anti-scFv(FRP5)-ETA antibodies after 8 days. There were no responses. Phase II studies are ongoing. Responses have been observed with other immunotoxins including Fab’(antiCD22)-dgA, HerH2-saporin, TP40, DAB486IL2, LMB-1, IgG-HD37-dgA, DA7, DAB389EGF, antiB4-blocked ricin, T101-RTA, H65RTA, 260F9-rA, XMMME001-RTA, N901-blocked ricin, IgRFT5-dgA, SPV-T3a-dgA/WT1-dgA, DT388 GMCSF, B43-PAP, IgRFB4-dgA, 791/T36-RTA, antiTAC-PE, OVB3-PE, 454A12-rA, erb38, and LMB-7 but these molecules have not undergone further

429 development [158, 159, 228, 242, 266, 319, 415, 428, 494–538].

Pharmacokinetics and Tissue Distribution Larger molecules have longer half-lives in the circulation and poorer tissue penetration. The half-life of monoclonal antibody conjugates with RTA, ricin, PAP, and PE ranged from 9 to 24 h [266, 427, 494, 497, 499]. The molecular weights were all around 200,000 Mr. There was likely little clearance of these molecules via renal glomeruli, and penetration into extravascular sites such as nodes, marrow, or skin nodules was poor [158, 228, 536]. In contrast, the smaller recombinant immunotoxins (60,000– 70,000 Mr) have had shorter half-lives of 1–5 h [279, 481, 485, 501, 531] and should have better penetration into extravascular sites of disease. However, there are no reports to date regarding the saturation of extravascular tumor deposits in clinical trials with recombinant immunotoxins. The shorter circulation times and increased vascular permeability of the smaller recombinant immunotoxins may contribute to their greater antitumor efficacy and reduced endothelial toxicity (see below).

Immune Responses Because most individuals in the US are immunized with diphtheria toxoid as children, almost half of adults have anti-DT antibody titers pretreatment with DT conjugates [540]. Further, between 10% and 20% of patients have had previous Pseudomonas infections (often subclinical) yielding pretreatment anti-PE antibody titers [541]. Patients exposed to castor oil may show anti-ricin antibodies. These circulating antibodies reduce the half-life and AUC for immunotoxins and, in some cases, neutralize cell cytotoxicity [266, 486, 542]. Even if patients lack pretreatment immunity, after administration of the foreign protein most patients develop anti-immunotoxin antibodies. The exceptions include CLL patients and some patients with severe immunosuppression. PEGylation or use of human ligands and toxins may reduce the immune response [582, 616].

Toxicities There are two general classes of side-effects due to immunotoxins. In some instances the targeted toxin receptor/antigen is present on normal tissues. This can lead to significant toxicities. LMB-1 and LMB-7 bind the Lewisy antigen which is overexpressed on many carcinomas. The Lewisy antigen is also expressed on normal stomach mucosa. Initial DLTs for these immunotoxins

430 were nausea, vomiting, and diarrhea with endoscopic biopsy evidence of gastritis [266, 530]. After prophylaxis with omeprozole, antiemetics, and loperamide, this side effect was alleviated and dose escalation could proceed. 260F9-rA binds a 55 kDa Mr antigen found on breast carcinomas and normal Schwann cells [158]. The clinical trial was stopped after the occurrence of reversible, but prolonged, peripheral neuropathies with biopsy-confirmed axonal loss. Similarly, clinical trials with OVB3-PE and 454A12-rA were discontinued after CNS toxicity and ligand crossreactivity with normal CNS tissue antigens (an antigen in the pons for OVB3 and transferrin receptors on normal brain capillaries for 454A12) were found [242, 524]. DAB389EGF and erb38 reacted with EGFR and HER-2 receptors present on normal hepatocytes and produced dose-limiting liver injury [527, 532]. DT388GMCSF reacts with normal liver macrophages – Kupffer cells. Subsequent cytokine release triggers dose-limiting hepatocyte damage [531]. Tf-CRM107 produced focal brain injury in the peritumoral normal cortex [483]. Histopathology showed crossreactivity and damage to normal brain capillaries. Toxicities that are independent of ligand have been observed in almost every immunotoxin clinical study. The similarity of the toxicities observed with different immunotoxin trials suggests that the lesions are due to the toxin moieties. The five classes of side effects are: (a) transient constitutional symptoms with fever, chills, nausea, vomiting, myalgias, arthralgias, asthenia and hypotension; (b) transient hepatotoxicity with elevated transaminases and, rarely, other liver enzymes; and (c) transient vascular leak syndrome (VLS) consisting of edema, hypoalbuminemia, weight gain, and, rarely, dyspnea and aphasia; (d) assorted syndromes including rhabdomyolysis, hemolytic uremic syndrome, and renal insufficiency with proteinuria and renal tubular acidosis; and (e) allergic reactions. The toxicities may be due to binding of the toxin moieties to normal tissues. Reaction of immunotoxins with macrophages, lymphocytes, or endothelium may lead to cytokine release with consequent constitutional symptoms and elevated circulating cytokines. Elevations of interleukin-6 and tumor necrosis factor alpha have been reported in some but not all cases [481, 495, 497, 531]. Where studied, corticosteroids or infiiximab + rofecoxib appeared to dampen or eliminate this side-effect [481, 487]. The hepatotoxicity may be directly due to immunotoxin binding affected by the pI of the ligand in the conjugate or secondary release of cytokine from macrophages may damage the liver [543, 544]. In most cases, impairments of hepatic function such as factor VII production or bilirubin clearance were not observed, and the liver

Immunotoxins injury was reversible. VLS is a syndrome observed with interleukin-2, monoclonal antibodies, some chemotherapy drugs such as taxotere, and many immunotoxins. The syndrome usually has onset of 4–6 days after initiating therapy and last 410 days. ‘Leaky’ capillaries throughout the body produce low serum albumin, edema, weight gain, dyspnea, aphasia, rhabdomyolysis, and proteinuria. The abnormal vascular endothelium may be intoxicated by the immunotoxin directly or may be ‘activated’ by elevated circulating cytokines. Evidence from tissue culture and animal models has been reported for a direct binding site of toxins on vascular endothelial cells and for an indirect toxic effect of immunotoxin-triggered inflammatory cytokines on endothelial cells [545, 546]. There do not appear to be effective methods of prophylaxis, although paradoxically active hydration appears to reduce the incidence and severity of VLS. Further, smaller recombinant immunotoxins appear to have a lower incidence, possibly due to their shorter circulating half-life. Excluding patients with prior radiotherapy may also lower the incidence or severity of the VLS syndrome [8]. The rare cases of HUS and rhabdomyolysis may be patient-specific types of vascular injury or due to ligand (particularly anti-CD22 antibodies). Allergic or anaphylactoid reactions occur very rarely, are IgEmediated, and are treated in a similar way to allergic reactions to other foreign proteins such as L-asparaginase. Fluids, cardiac monitoring, oxygen, corticosteroids, diphenhydramine, theophyllines, and rarely epinephrine can be given. Patients with anaphylactoid reactions should not be retreated with the same immunotoxin.

Ongoing and Future Clinical Studies The current and near-future applications of immunotoxins are focused on expanding the use of some of the active, established agents described above, as well as testing novel compounds. Further protocols are being performed with denileukin diftitox, TransMID, NBI-3001, DT388IL3, DT388K116W,LMB-2,Cintredekinbesudotox, AdmDT390bisFv(UCHT1), BL22, HA22, PRX302, scFv(FRP5)-ETA, PrAgU2/FP59, and DT2219ARL. These studies should help define the niche for immunotoxins in the eventual multi-agent armamentarium for therapy of cancer. The past 2 decades are finally showing the original promise for these ‘magic bullets’. Discoveries in the next few years of methods to modulate cell surface receptor number and physiology, as well as methods to ameliorate normal tissue toxicities of these agents,

Arthur E. Frankel et al. should refine the applications of these chimeric proteins in the management of cancer and autoimmune disorders.

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Arthur E. Frankel et al. 628. Ghori F, Polder KD, Pinter-Brown LC et al. Thyrotoxicosis after denileukin diftitox therapy in patients with mycosis fungoides. J Clin Endocrinol Metab 2006; 91: 2205–8. 629. Park M, Liu GT, Piltz-Seymour J et al. Vision loss following denileukin diftitox treatment: a case report of possible posterior ischemic optic neuropathy. Leuk Lymphoma 2007; 48: 808–11. 630. Dang NH, Fayad L, McLaughlin P et al. Phase II trial of the combination of denileukin diftitox and rituximab for relapsed/refractory B-cell non-Hodgkin’s lymphoma. Br J Haematol 2007; 138: 502–5. 631. DiVenuti G, Nawgiri R, Foss F. Denileukin diftitox and hyperCVAD in the treatment of human T-cell lymphotropic virus 1-associated acute T-cell leukemia-lymphoma. Clin Lymphoma 2003; 4: 176–8. 632. Litzinger MT, Fernando R, Curiel TJ et al. IL-2 immunotoxin denileukin diftitox reduces regulatory T cells and enhances vaccine-mediated T-cell immunity. Blood 2007; 110: 3192–201. 633. Mahnke K, Schonfeld K, Fondel S et al. Depletion of CD4+ CD25+ human regulatory T cells in vivo: kinetics of Treg depletion and alterations in immune functions in vivo and in vitro. Int J Cancer 2007; 120: 2723–33. 634. Rainov NG, Soling A. Technology evaluation: TransMID, KS Biomedix/Nycomed/Sosei/PharmaEngine. Curr Opin Mol Ther 2005; 7: 483–92. 635. Weber FW, Floeth F, Asher A et al. Local convection enhanced delivery of IL4-Pseudomonas exotoxin (NBI-3001) for treatment of patients with recurrent malignant glioma. Acta Neurochir Suppl 2003; 88: 93–103. 636. Garland L, Gitlitz B, Ebbinghaus S et al. Phase I trial of intravenous IL-4 pseudomonas exotoxin protein (NBI-3001) in patients with advanced solid tumors that express the IL-4 receptor. J Immunother 2005; 28: 376–81. 637. Hassan R, Bullock S, Premkumar A et al. Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers. Clin Cancer Res 2007; 13: 5144–9.

449 638. Hassan R, Broaddus VC, Wilson S et al. Anti Mesothelin immunotoxin SS1P in combination with gemcitabine results in increased activity against mesothelin-expressing tumor xenografts. Clin Cancer Res 2007; 13: 7166–71. 639. Zhang Y, Xiang L, Hassan R et al. Immunotoxin and Taxol synergy results from a decrease in shed mesothelin levels in the extracellular space of tumors. Proc Natl Acad Sci USA 2007; 104: 17099–104. 640. Coffield S, Boyer A, Culp L et al. Intraprostatic treatment of patients with locally recurrent prostate cancer with the PSAactivated protoxin PRX302. ASCO GU Meeting, Feb 14–16 2008, Orlando, FL. Abstract no. 20172. 641. Kunwar S, Prados MD, Chang SM et al. Direct intracerebral delivery of cintredekin besudotox (IL13-PE38QQR) in recurrent malignant glioma: a report by the Cintredekin Besudotox Intraparenchymal Study Group. J Clin Oncol 2007; 25: 837–44. 642. Sampson JH, Raghavan R, Brady ML et al. Clinical utility of a patient-specific algorithm for simulating intracerebral drug infusions. Neuro Oncol 2007; 9: 343–53. 643. Frankel AE, Liu JS, Rizzieri DA et al. Phase I clinical study of diphtheria toxin-interleukin 3 fusion protein in patients with acute myeloid leukemia and myelodysplasia. Leuk Lymphoma, in press. 644. Liu TF, Urieto JO, Moore JE et al. Diphtheria toxin fused to variant interleukin-3 provides enhanced binding to the interleukin-3 receptor and more potent leukemia cell cytotoxicity. Exp Hematol 2004; 32: 277–81. 645. Azemar M, Djahansouzi S, Jager E et al. Regression of cutaneous tumor lesions in patients intratumorally injected with a recombinant single-chain antibody-toxin targeted to ErbB2/HER2. Breast Cancer Res Treat 2003; 82: 155–64. 646. von Minckwitz G, Harder S, Hovelmann S et al. Phase I clinical study of the recombinant antibody toxin scFv(FRP5)-ETA specific for the ErbB2/HER2 receptor in patients with advanced solid malignancies. Breast Cancer Res 2005; 7:R617–26.

12 Drug Immunoconjugates MALEK SAFA, KENNETH A. FOON, AND ROBERT K. OLDHAM

Monoclonal antibodies and their immunoconjugates represent one of the first practical methods for the selective treatment of cancer [49, 96]. Monoclonal antibody technology now allows for the generation of antibodies or “cocktails” of antibodies that have some selectivity for cancer tissue as compared with the normal tissue of origin. These antibodies can be tested as unconjugated antibody alone or in conjunction with effector cells. The “signal strength” of the antibody may be made more powerful by conjugating antibody to drugs, toxins, biologicals, and radioisotopes with different mechanisms of action and different levels of potency. This chapter will focus on the use of drug immunoconjugates for cancer treatment.

The Problem of Heterogeneity: Antibody-Based Therapeutics as a Solution The problem of tumor-cell heterogeneity (Fig. 1) and the implications for therapy have been reviewed in Chapter 2. Curiously, although tumor-cell heterogeneity has been broadly recognized for decades, clinicians continue to take the simplistic view that treatment need not reflect a specific approach to heterogeneity [90, 113–115]. Thus, treatment is still designed as if there are singular underlying common principles useful in cancer therapy. Single modalities or fixed combinations aimed at eradicating cancer without a strategy designed to approach the problem of tumor-cell heterogeneity still dominate cancer research and treatment [60, 67, 68, 71, 74, 89]. Current data on tumor-cell heterogeneity and the biologic basis of that heterogeneity is covered in Chapter 2 and have been previously reviewed [29]. The basic tenant of these studies is that each primary tumor is composed of a smaller or larger number of clones, each of which has its own genotypic and phenotypic characteristics. Metastasis tends to occur from single cells or clumps of cells from within the primary tumor. By virtue of a series of phenotypic analyses, as well as certain

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

kinds of genotypic inferences, these authors and others have demonstrated beyond doubt that heterogeneity is characteristic of both animal and human tumors (Fig. 1). Recently, there have been references to the implications of this tumor-cell heterogeneity for treatment, and proposals have been made to approach the problem clinically [58, 59, 61–63, 74, 89]. There are two types of tumor-cell heterogeneity [73]. There are differences between patients, with heterogeneity being apparent among tumors of the same histological class. This is macroheterogeneity. Tumors such as breast cancer can be very similar by histological examination, and yet lethality can occur in 7 months or 17 years. Clinical observations have made it clear that what is seen under the microscope bears little relationship to the behavior of the cancer in the patient. This sort of “phenotypic analysis” has led to further investigations, and it is now recognized by most cancer biologists that considerable differences exist among patients with respect to the phenotypic characteristics of the cancer. In addition, there is the problem of heterogeneity within each tumor in each patient; microheterogeneity. Thus, many studies have demonstrated that multiple clones may exist within the primary tumor and that these clones may have different metastatic capabilities, giving rise to heterogeneity even among different deposits in a single patient. Many still believe that even with heterogeneity between individuals and within each patient’s cancer, underlying common features still exist and will allow a general treatment to be developed that might be useful for all cancer, or all of a histological type of cancer. This view supports the “drug development paradigm,” wherein drugs are developed as broadly active agents to be tested in different histological types of cancer (see Chapter 3). Another view is that each cancer and its antigenic phenotype (and behavior) are unique. This view, currently held by a minority of investigators, would suggest the need to individualize treatment for each patient and may even require individual therapeutic manipulations for a single patient over the clinical course of his or her disease based on these differences [65, 68, 71–74]. Acceptance of this hypothesis would dramatically change cancer treatment and would require a laboratory-clinic

451

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Figure 1. Illustration of tumor cell heterogeneity

interface of a type not often used in cancer therapeutics. With the advent of the “new biology” using genetically engineered biologicals and monoclonal antibodies, one has diverse mechanisms for the generation of biological responses, which may match the diversity implicit in tumor cell populations. For the first time, there is hope that one can truly approach the heterogeneity of each patient’s cancer and the other biologic differences of each patient in a rational manner [36, 58, 59, 60, 62, 63, 65, 74].

Rationale for Immunoconjugates Most of the early monoclonal antibody clinical trials focused on the use of unconjugated single antibodies in order to determine toxicity, tolerance, the localization of antibody in solid tumor deposits, and the distribution of antibody in normal and neoplastic tissues [21, 31, 53–55, 81, 83, 93–95, 96]. Although the clinical responses to unconjugated antibody have not been consistent, there is unequivocal evidence that antibody does selectively localize in tumor deposits and on individual cancer cells after

intravenous injection [62, 63, 93]. Biodistribution studies utilizing antibody conjugated to tracer quantities of isotope have demonstrated excellent tumor imaging, but a considerable amount of the antibody is distributed to the liver, spleen, and other organs of the reticuloendothelial system [2, 12, 13, 35, 43, 46, 113]. The infrequency of response to unconjugated antibody and the ability of conjugates to induce regression of bulky cancer in animal models support the concept of immunoconjugate therapy. When considering the development of a conjugate– monoclonal antibody complex for drug targeting, the following factors are of importance: 1. The recognition site for the monoclonal antibody should be located on the surface of the cell. 2. The antibodies should have sufficient tumor tissue specificity. 3. The extent of localization of the antibody at the target site. 4. Biodistribution of the conjugate–antibody conjugate in the body relative to that of the parent antibody. 5. Stability of the conjugate–antibody complex in blood. 6. The toxicity of the conjugate. 7. The conjugate must be non-immunogenic and biodegradable. 8. The conjugate must be released upon interaction between the carrier molecule and the cell and the toxic component must be active at the surface or be internalized into the cell.

Malek Safa et al.

Rationale for Antitumor Cocktails There already exist many different monoclonal antibodies to be assessed in clinical trials in patients with solid tumors [18, 19, 65, 74]. A large number of antibodies for leukemia, lymphoma, melanoma, lung, breast, and colon cancer have been described and characterized. Antibody preparations are available as immunoglobulin M (IgM), IgA, and various subclasses of IgG. There are several monoclonal antibodies which have been engineered to be predominantly human. These are: Rituximab (Rituxan) directed against CD-20 antigen, Campath (Alemtuzumab) against CD52, Trastuzumab (Herceptin) against HER-2/neu receptor, Bevacizumab (Avastin) against VEGF, Cetuximab (Erbitux) and Panitumumab (Vectibix) against EGFR receptor. These antibodies have been approved and are in clinical use for the treatment of non-Hodgkin’s lymphoma, CLL, breast, lung and colon cancer [52, 74, 112]. In addition, the use of murine monoclonal antibody MAb-17–1A has resulted in improved survival of patients with Dukes’ C colorectal cancer [82]. A large variety of new monoclonal antibodies, both murine and human, continue to be discovered and developed. The recent success in the humanization process has further improved the bioavailability and reduced the immunogenicity of these antibodies. Furthermore, the technological evolution from murine-based therapeutic monoclonal antibodies to chimeric (part murine part human protein such as cetuximab), humanized (e.g. trastuzumab) and fully humanized antibodies (bevacizumab, panitumumab) has led to reduction in immune-mediated clearance and hypersensitivity, improved the safety and feasibility of repeated administration and thereby created a viable therapeutic strategy. Thus, it is apparent that the limitation for the use of monoclonal antibody preparations in treatment will not be due to a shortage of antibody preparations [58, 74]. Although the “perfect” antibody for use as an unconjugated antibody or as a targeting agent has not been identified for any human cancer, there are a variety of selective antibodies available for clinical investigation. Methods exist to manufacture high-purity (>99%), homogeneous preparations of monoclonal antibodies. Characterization as to antibody isotype, level of purity, degree of contamination by other substances, stability, and other pertinent physical/chemical characteristics is possible. Thus, no insurmountable obstacles exist with respect to testing a wide variety of monoclonal antibodies in patients (Table 1) [74].

453 Table 1. Optimization of monoclonal antibody therapy ANTIBODY SPECIFICITY Immunoperoxidase Immunofluorescence Radiolocalization ANTIGEN CHARACTERIZATION Biochemical nature Topography and density Epitopes Heterogeneity ANTIBODY–ANTIGEN INTERACTION Turnover of antibody bound to tumor cells Degree of antibody internalization Antigen affinity Antigen levels in serum ANTIBODY DELIVERY Dose Regimen Route Pharmacokinetics Comparison of various cytotoxic agents conjugated to the same antibody

Given a wide choice of antibody preparations and the ability to prepare them for the clinic, the next consideration is, how does one select preparations and patients for clinical trials? It seems unlikely that firm rules can be made on the selection of antibody preparations for all solid tumors. Obviously, the distribution of the tumor, its vascularity, its sensitivity to drugs and radiation, and the quantitative expression of antigens on the tumor cell surfaces are all significant factors. Using conjugates of isotopes, drugs, biologicals, and toxins, the issue may be essentially one of biodistribution within the tumor bed and access of the toxic agent to the tumor cell within each tumor nodule. Thus, it is apparent that while certain principles in the use of monoclonal antibody for human solid tumors may be important, each tumor type may have to be individually evaluated for the optimal use of a monoclonal antibody preparation [73, 74]. Antibodies presumably must reach the tumor bed to be effective [58, 62, 63]. One general principle has been that antibody fragments may more quickly diffuse from the vascular compartment to the tumor bed. Data have indicated that the more antibody one infuses into the vascular compartment, the more antibody one delivers to the tumor cell bed [58, 62, 63]. Although access to the tumor bed is clearly quite important, retention within the tumor bed may be equally or more important. The preparations of antibody fragments may diffuse more quickly into the tumor nodule [2], but larger molecules may be retained for a longer time within that same nodule [58]. Therefore, at the level of these elementary principles, there is much to be learned about the use of

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CB4F10.11+AC4C2 99% CB4F10.11+AC4C3 99% MC6G10+140.72 97% MC6G10+6PL4 93% MC6G10+AC4C2 87% MC6G10+AC4C6 92% MC6G10+AC4C3 74% MC6G10+DC3.24 40% MC6G10+CD4F10.15 96.6% MC6G10+CD4F10.11 49.2%

Figure 2. Antibody preparations to a single patient’s melanoma as seen on flow cytometry

BREAST TUMOR C O U N T

BR3 97% MPC 105

BR1 + BR3 100% MPC 185

BR-1 94% MPC 98

1

200 LOG GREEN FLUORESCENCE (VIABLE)

Figure 3. A two-antibody cocktail for breast cancer

these antibody preparations in clinical trials, and these clinical trials should not yet be subject to hard and fast rules [58, 74]. On the basis of early assessments of the heterogeneity of malignant melanoma [45], including studies of both melanoma cell lines and fresh biopsies of melanoma nodules, it became obvious that considerable antigenic heterogeneity exists. When the data were analyzed in detail with a panel of antimelanoma monoclonal antibodies, each patient’s tumor had its own pattern of reactivity. As an extension of this approach, we have generated antibodies for individual patients, making up

to ten antibodies per patient to analyze heterogeneity [4, 50, 73]. As shown in Fig. 2, the flow cytometry patterns for each antibody may differ considerably when tested against the patient’s cancer. By additive testing of the individual antibodies, a cocktail of antibodies, consisting of two to five components, can be created to cover all of the cells in the tumor as assessed by flow cytometry and immunoperioxidase staining (Fig. 3). Our data support the concept that tumor-cell heterogeneity calls for the generation of antibodies and/or the use of typing panels to prepare antibody cocktails containing

Malek Safa et al. multiple components in an attempt to deliver antibody and its conjugated toxic substance to all the replicating cells in the cancer [73]. This approach should substantially improve the ability to target toxic substances selectively to the cancer of an individual patient. Further analysis is needed with respect to heterogeneity within the individual. Our preliminary data appear to indicate that this microheterogeneity is not striking, and it may be possible to cover it with a sufficiently complex cocktail. However, the possibility of somatic mutation and/or selective pressure in treatment leading to further tumor-cell diversity in patients with metastatic disease is real [23]. In lymphoma, there is evidence to indicate a multiclonality; while it is not completely clear, some of this multiclonality may result from selective pressures induced by treatment with anti-idiotypic antibody [33, 54].

Clinical-Laboratory Integration for Drug Immunoconjugate Trials There are certain important principles in the design and execution of clinical trials using monoclonal antibodies [62–64, 72, 74]. To learn from these early trials it is necessary to have an active and competent laboratory available for in vitro assessments. It is essential to demonstrate that the antibody reaches the tumor nodule. For this, the technique of immunohistology using biopsy specimens subsequent to the infusion of antibody has been quite valuable [36, 62, 92–94]. The distribution of antibody within the tumor nodule in contrast to surrounding normal tissues can be assessed. This technique also gives information on heterogeneity of antigen expression. In vitro overlay with the treatment antibody can assess saturation, and the use of other antibodies can define heterogeneity of antigen expression. Isotope-labeled antibody preparations may also be used to evaluate the biodistribution of antibody [12, 13, 35, 43, 46]. In addition to these techniques using “fixed” tissue, cytofluorometry can be used to visualize individual cells with respect to antigen distribution, antibody localization, and saturation. When the tumor nodule is disaggregated after in vivo treatment, one can determine with great precision the level of antibody localization on individual cells and, by adding exogenous antibody, the degree of saturation on these individual cells [62, 92–94]. Thus, we have techniques to assess the delivery of antibody conjugates to the tumor, as well as the degree of saturation of membrane antigens. In addition, actual determinations of heterogeneity within individual tumor biopsies and between patients can easily be made from these studies [73]. The ability to measure antigen and antigen–antibody complexes before and after treatment, and circulating

455 antibody after injection, is critical to an understanding of the pharmacokinetics in these antibody trials [3, 5, 73]. Further, it is important to measure antiglobulin levels initially and after treatment in conducting these clinical trials, since such antiglobulin responses may ultimately be important in the biodistribution, toxicity and efficacy of an infused antibody [20, 62, 91–94].

Chemotherapeutic Drug Immunoconjugates The rationale for conjugating antibody to chemotherapeutic drugs is fairly straightforward. These drugs have been used for many years in clinical studies. The spectrum of activity and the toxicology of these agents are well understood. In addition, there is considerable evidence to support the belief that dose–response curves exist with these drugs in the killing of tumor cells. Their lack of selectivity for cancer and the major problem of normal tissue toxicity are well known. All of these factors constitute a firm basis for attempting antibody delivery to target the chemotherapeutic agent to the tumor site. Surprisingly, very few clinical studies have been done using antibody-targeted chemotherapy. Perhaps the reason relates to the lower specific activity seen in vitro with these conjugates. Compared with immunotoxins, drug conjugates have been less active in preclinical studies, and this finding has discouraged investigators with respect to the possible in vivo activity of such preparations [23, 33].

Pre-Clinical Studies Preclinical studies using chemotherapeutic agents conjugated to antibody have been reported for a variety of drugs [10, 19, 22, 33, 40, 44, 47, 57, 79, 97, 100, 101, 102]. These studies have indicated that drug conjugates can have in vitro and in vivo activity. The drugs most often used have been vinca alkaloids, methotrexate, daunorubicin, and doxorubicin. In one study methotrexate was coupled to an IgM monoclonal antibody targeted to a variety of tumors, including mouse teratocarcinoma [9]. Deposition of immunoconjugate in viable tumor and lack of binding to antigen negative normal tissue was demonstrated in an in vivo animal model. Also, methotrexate conjugated to monoclonal antibody has been compared with methotrexate-IgG (nonspecific antibody) and free methotrexate against EL4 lymphoma [34]. The methotrexate-antibody conjugate was three and seven times more effective against EL4 lymphoma than methotrexate-IgG and free methotrexate, respectively. Similarly, paclitaxel–antibody conjugates afford selective toxicity

456 and are more cytotoxic in vitro than equimolar concentrations of free paclitaxel or free paclitaxel plus free antibody [37]. In an in vivo model of xenografted tumors, systemic administration of paclitaxel–antibody conjugates prevented tumor growth and prolonged survival of mice better than free drugs [37]. In another study, aminopterin which is a more potent antifolate than methotrexate, was coupled to a monoclonal antibody [84]. These investigators demonstrated that administering leucovorin 48–72 h following a sublethal dose of the aminopterin– antibody conjugate resulted in maintenance of the antitumor efficacy of the immunoconjugate and a significant reduction in toxicity. Vindesine (a vinca alkaloid) has been conjugated with different anti-tumor antibodies, such as anti-CEA, melanoma, osteosarcoma and others [78, 86–88]. Studies with vindesine-anti-CEA conjugates [88] indicate that they increase the therapeutic index of vindesine by decreasing the toxicity and increasing the specificity to tumor. Furthermore, evaluation of the conjugate against non-CEA producing colon carcinoma xenograft and CEA-producing xenograft showed that significant retardation in the growth of tumor was observed only in CEA-producing tumor [101]. The anthracycline family of antitumor antibiotics, most notably doxorubicin (DOX) and daunorubicin, has been used extensively for drug targeting applications (102). BR96-DOX, which is chimeric with human IgG1, binds to Ley-related tumorassociated antigen expressed on most human carcinomas and on normal cells of the gastrointestinal tract of humans, dogs, and rats [107]. BR96-DOX induced cures of human lung, breast and colon carcinomas in athymic mice and rats [38, 107, 108], and syngeneic colon tumors in immunocompetent rats [100, 101]. In another study [116], the monoclonal antibody MSN-1 (IgM), which reacts with endometrial adenocarcinomas, was combined with DOX, and the complex showed significant antitumor activity in athymic mice bearing endometrial carcinoma cell tumors. In one study, IMMU-110, an anti-CD74-DOX immunoconjugate was cytotoxic to non-Burkitt and in Burkitt non-Hodgkin lymphoma cell lines. In addition, IMMU110 was therapeutic in drug-sensitive and drug-resistant non-Hodgkin lymphoma animal models, suggesting that antibody targeting can bypass the multi-drug resistant (MDR) drug efflux system that prevents free doxorubicin from being therapeutic [11]. Anti-CD74-DOX immunoconjugate (IMMU-110) is cytotoxic in non-Hodgkin’s lymphoma models and overcomes MDR [11]. In another study, IMMU-110 showed significant activity against multiple myeloma cell lines and in SCID mouse models of disseminated multiple myeloma [103]. A two-step method has been developed for targeting cytotoxic drugs into tumor cells [14]. It first involves the binding to tumor

Drug Immunoconjugates cells of antibody-phospholipase C (PLC) immunoconjugates. Then, liposomes containing daunorubicin are introduced which are specifically lysed at the tumor site by PLC to release their cytotoxic contents in the vicinity of the tumor cells. For tumor xenografts in mice, the antibody-PLC/liposome approach resulted in an inhibition of tumor growth without eradication of tumors [14]. In another report [85], idarubicin, an analogue of daunorubicin, was coupled to a CD19 antibody. A human pre-B-cell acute lymphoblastic leukemia cell line (NALM-6) was implanted into nude mice and this immunoconjugate was demonstrated to have substantial anti-tumor activity, was non-toxic and was stable in vivo. It was proposed as an excellent immunoconjugate for clinical trials. Another anti-tumor antibiotic agent, DU-257 which is a duocarmycin derivative, was conjugated to a monoclonal antibody (KM231) specifically reactive to GD3 antigen which is highly expressed on the surface of many malignant tumors. The conjugate showed significant growth inhibition of a human colorectal carcinoma cell line (SW1116) [106]. Recently, two new antibody–drug conjugates have been developed as potential therapies for colon cancer and lung cancer. The antibodies were hMN-14 (labetuzumab), a non-internalizing humanized antibody that binds to the carcinoembryonic antigen (CEA) expressed by many solid cancers, and hRS7, a rapidly internalizing humanized antibody targeting epithelial glycoprotein-1 (EGP-1) expressed on high amounts in certain human cancers. The drug selected for conjugation was SN-38, the active metabolite of irinotecan, a chemotherapeutic agent used for the treatment of colorectal, lung, and other cancers. SN-38 cannot be systemically administered to patients with cancer due to its toxicity and poor solubility. In an animal model of colon cancer with lung metastases, therapy with labetuzumab-SN-38 increased median survival time two-fold to 73.5 days compared to non-treated and non-targeting conjugate controls. Similar results were observed in animals bearing human non-small cell lung cancer treated with hRS-SN-38 [28]. In a different approach [17] investigators studied maytansinoids. These drugs are 100–1,000 times more cytotoxic than common anticancer drugs. These investigators felt that the limitation of drug immunoconjugates is that they act stoichiometrically, requiring much higher intracellular concentrations to achieve the comparable cytotoxicity as compared to a protein toxin where one molecule in the cytoplasm leads to cell death. Maytansine was coupled to a monoclonal antibody via disulfide containing linkers that are cleaved intracellularly to release the drug. They demonstrated antigen specific cytotoxicity of cultured human cells and minimal systemic toxicity in mice

Malek Safa et al. with an excellent pharmacokinetic profile. In one study, the immunoconjugate of b-76-8, a monoclonal antibody against HM1.24/BST-2 (CD317) which is a cell surface antigen overexpressed on multiple myeloma cells, with the analog of maytansine was shown to be active in a xenograft model of human multiple myeloma [76]. Similarly, SAR3419, a novel humanized anti-CD19 antibody (huB4) conjugated to a maytansine derivative (DM4) produced 100% cures in two non-Hodgkin lymphoma cell lines [1]. These studies and many others in preclinical models using antibody conjugated to chemotherapeutic agents lend support to the idea that drug-conjugated antibodies may allow better targeting of chemotherapeutic agents to the tumor site [7, 16, 19, 23, 30, 41, 42, 64–68, 77, 105, 109]. Src proto-oncogene family protein tyrosine kinases (PTKs) play a key role in cell function and attempts have been made to develop agents that specifically inhibit Srcfamily PTKs. In one report [110], a general PTK inhibitor, Genistein, that inhibits all members of the Src PTK family was used to target to cancer cells. Genistein is an isoflavone derived from fermentation broth of Pseudomonas. It is also a natural occurring tyrosine kinase inhibitor present in soybeans. Genistein inhibits purified Lck kinase from human lymphoid cells at micromolar concentrations. These investigators conjugated Genistein to an anti-CD19 monoclonal antibody. An antiCD19 antibody was chosen because the CD19 receptor is not shed from cells, it is internalized upon association with antibody and is physically and functionally associated with the Src protooncogene family PTKs including Lck. Human acute lymphoblastic leukemia of the pre-B cell variety was treated in a severe combined immunodeficient mouse model with this immunoconjugate. These investigators demonstrated that the immunoconjugate bound with high affinity to the leukemia cells, inhibited selectively the CD19-associated tyrosine kinases, and triggered rapid apoptotic cell death. Furthermore, at less than one tenth the maximum tolerated dose, greater than 99.99% of the leukemia cells were killed, which led to 100% long-term event-free survival in these animals. In another study [55], LL2, an anti-CD22 monoclonal antibody against B-cell lymphoma, was covalently linked to the amphibian ribonuclease, onconase, a member of the pancreatic Rnase A superfamily. The LL2-onconase conjugate showed significant antitumor activity against disseminated Daudi lymphoma in mice with severe combined immunodeficiency disease. Furthermore, the life span of these mice was increased 135% as compared to controls. Similarly, CMC-544, an IgG4 CD-22-humanized targeted immunoconjugate of the antitumor antibiotic calicheamicin, showed activity against a panel of non-Hodgkin lymphoma cell lines. In the same study, lymphoma-bearing

457 mice treated with rituximab in combination with CMC544 had the longest median survival as compared to control mice [39]. Targeting CD20 and CD22 with rituximab in combination with CMC-544 results in improved antitumor activity against non-Hodgkin’s lymphoma preclinical models. In another study, antibody–drug conjugates to CD79 (anti-CD79-MCCDM1 and antiCD79b-MC-MMAF) showed activity in non-hodgkin lymphoma cell lines [80].

Clinical Trials One of the first reports using drug-labeled antibody used antibody 791T/36 conjugated to methotrexate (MTX) [7]. Initial studies with this conjugate demonstrated that methotrexate conjugation did not alter the pharmacokinetics or tumor localization indices for the antibody labeled with 131 I [25]. In a different study [8], 16 patients with primary colorectal cancer were injected intravenously with I131labeled 791T-36-MTX and imaged using a gamma camera after 48–72 h. Assays of tissue radioactivity showed high tumor uptake of drug-antibody conjugate in 13 of 15 tumor specimens. Also, the monoclonal antibody KS1/4, which targets an antigen expressed on epithelial malignancies and some normal epithelial cells were conjugated to methotrexate [26]. Eleven patients with advanced non-small cell carcinoma of the lung were treated up to a maximum tolerated antibody dose of 1,750 mg/m2 with a cumulative dose of methotrexate of 40 mg/m2. Toxicities in this study included fever, anorexia, nausea, vomiting, diarrhea, abdominal pain, guaiac positive stools and hypoalbuminemia. Two patients had episodes of aseptic meningitis. One patient had a greater than 50% decrease of two lung nodules. Post-treatment tumor biopsies demonstrated binding of the immunoconjugate to tumor epithelial cells. Some of the gastrointestinal toxicities may also have been secondary to binding of the immunoconjugate to a similar antigen on gastrointestinal epithelial cells. Mylotarg (gemtuzumab ozogamicin, previously known as CMA-676) is an FDA approved antibody-targeted chemotherapy agent consisting of the humanized murine CD33 antibody to which the calicheamicin γ1 derivative is attached [111]. It is assumed that binding of Mylotarg to the CD33 antigen results in internalization followed by the release of the potent antitumor antibiotic calicheamicin and subsequent induction of cell death. In one study [111], 122 patients with relapsed acute myelogenous leukemia (AML) were treated with Mylotarg as a single infusion at a dose of 9 mg/m2. Each patient received two Mylotarg doses with at least 14 days between the doses. Within 3–6 h after infusion, near complete satura-

458 tion of CD33 antigen sited by Mylotarg was reached for AML blasts. Furthermore, Mylotarg induced dosedependent apoptosis in myeloid cells in vitro. In a multicenter trial [98], 142 patients with CD-33 positive AML in first relapse were treated with two doses of Mylotarg. Thirty percent of patients achieved remission with a favorable safety profile. Based on these results, Mylotarg was approved for the treatment of relapsed CD-33 positive acute myeloid leukemia. In another study, 48 patients with follicular (FL) or diffuse large B-cell lymphoma (DLBCL) were treated with CMC-544 (antiCD22 conjugated to calicheamicin). The objective response rate was 69% and 33% for patients with FL and DLBCL, respectively [27]. In another dose escalation study of AVE9633, an antiCD33-Maytansinoid immunoconjugate, antileukemia activity was observed in patients with relapsed or refractory CD-33 positive acute myeloid leukemia [48]. In a phase I study, huN901-DM1 (BB-10901) which is a humanized monoclonal antibody–maytansinoid immunoconjugate that binds with high affinity to CD56, was shown to be safe and clinically active in patients with relapsed or refractory CD56 positive multiple myeloma [15]. In addition, in a phase II trial, huN901-DM1 showed activity in patients with CD-56 positive relapsed small cell cancer [51]. Other studies further support the possibility of clinical trials with drug immunoconjugates [18, 19, 25, 30, 32–34, 104]. The rationale for such studies has been previously described [61–67, 72–74] and the dosedelivery curves for tissue penetration and antigen saturation have been fully described [62, 69, 73–75]. Issues of antibody specificity, antigen heterogeneity, optimal linkers, drug selection, and specific activity of the immunoconjugate all remain under active investigation. We have utilized cocktails of antibodies custom tailored to individual patient tumors [68, 71, 73, 75]. These studies have utilized a biopsy of the cancer from each patient and either the generation of new antibodies to that patient’s tumor or the use of existing panels of antibodies to type the patient’s tumor [3, 50]. From these two methods, cocktails of two to six antibodies were generated and conjugated to doxorubicin [56, 68–71] or mitomycin-C [75]. The immunoglobulin-to-doxorubicin ratios were in the range of 1:40 to 1:60. This level of coupling did not alter the antibody’s in vitro immunoreactivity, and excellent in vitro cytotoxicity was observed on antigen-positive cell lines compared to antigen-negative controls. The spectrum of clinical toxicities was strikingly different for doxorubicin conjugated to antibody as compared with free doxorubicin [68, 70]. More than 1 g of doxorubicin on up to 5 g of antibody was

Drug Immunoconjugates administered over a 3-week period without alopecia or severe bone marrow depression. In several of these patients, antibody delivery was confirmed by biopsy of skin and analysis of cytologically positive pleural fluid. Persistence of antibody conjugates for up to 1 week post-treatment was noted with doxorubicin doses of 50 mg borne on approximately 300 mg of antibody. These doxorubicin–monoclonal antibody cocktail immunoconjugates were used in 24 patients. Mild toxic reactions were seen in 17/24 (fever, chills, pruritis, and skin rash) after the administration of these drug immunoconjugates. In several patients there was limited antigenic drift among the sequential biopsies within the same patient over time. Five minor responses were seen with these conjugates. Two patients with breast cancer had isolated improvement in skin ulcerations, and three additional patients with other types of cancers had minor responses. None of these responses were sufficient to reach a partial response (50% reduction in mean tumor diameters) by standard oncological criteria. Toward the end of these studies and at the higher doses of doxorubicin, toxicities were noted in these patients. Urine color turned red, and alopecia with bone marrow suppression was noted. On reexamination of the doxorubicin procured from Adria Laboratories, it was noted that the preparation method for the doxorubicin had changed. A stabilizer (methylparaben) had been added to the preparation (RDF-Adriamycin), which likely changed the stability of the doxorubicin immunoconjugates. This change occurred near the end of the study, and the clinical studies were terminated to determine a better method for manufacturing doxorubicin immunoconjugates in light of the changed chemistry [23, 24, 56, 69, 70, 73]. A subsequent study was carried out using mitomycinC immunoconjugates [75]. Nineteen patients were treated with mitomycin-C conjugated to similar cocktails of monoclonal antibodies. Thrombocytopenia at the 60 mg (mitomycin) dose in this protocol was doselimiting. The anti-mouse antibody titers were lower in the mitomycin-C compared to the doxorubicin-treated patients [5]. No responses were seen with mitomycin-C immunoconjugates, although several patients had less tumor-related pain after treatment [73]. The role of drug resistance in cancer treatment is another issue to be considered. The current widely held opinion is that tumor cells, once resistant, cannot effectively be treated with the same drug. A variety of approaches using mechanisms to overcome membrane resistance have been suggested. However, antibodymediated delivery may be another such mechanism, and

Malek Safa et al. it is unclear whether doxorubicin-resistant tumor cells will be resistant to doxorubicin conjugates if delivered in appropriate quantities. In vitro and animal model data have confirmed sensitivity to doxorubicin conjugate where the cell line was resistant to free drug [19, 22–24]. These and many other issues will be approachable through the use of drug–antibody conjugates. Other studies comparing drug and toxin conjugates are of interest in regard to the issue of specific activity [23, 25]. While the immunotoxins had a higher specific cytolytic capability in vitro, and may be most useful where target cells express low levels of antigens, drug conjugates may be preferable when target cells express a high density of antigens or when large doses of conjugate are needed to penetrate tumors. In these situations, higher-specific-activity conjugates (toxins) may be too toxic for clinical use.

Conclusions Our studies, as well as many similar studies, clearly indicate the potential for the new selective approach to cancer treatment. Selective and effective new anti-cancer approaches are needed in the systemic treatment of human malignancies. Many very toxic molecules are well known to medical science (Table 2). Of key importance is the delivery of these toxic substances to tumor

Table 2. Immunoconjugates with cytotoxic agents PROTEIN TOXINS AND CYTOTOXINS Abrin Ricin Calicheamicin Diphtheria Duocarmycins Gelonin Hematoporphyrin Ribonuclease Purothionine Pseudomonas exotoxin Alpha-amanitin CHEMOTHERAPEUTIC DRUGS Vindesine Methotrexate Daunorubicin Doxorubicin Idarubicin Mitomycin-C Paclitaxel Various new drugs with high toxicity profiles BIOLOGIC AGENTS Interferon Tumor necrosis factor Cobra venom factor

459 cells in ways that will avoid the undesirable side effects on normal tissues. Given these considerations, it is probably no longer necessary to distinguish between drugs and toxins. Perhaps we should speak of strong and weak drugs (chemicals), in that toxin molecules are simply biological chemicals with a higher degree of toxicity than the chemicals we commonly identify as drugs. This may, however, only partially be the case, since most toxins are complex biologicals and most chemotherapeutic drugs are relatively simple chemicals. Such differences in size and structure may profoundly affect in vivo trafficking and immunogenicity. With the advent of monoclonal antibodies and the possibility of delivering toxic substances selectively to cancer cells, many toxic chemicals previously rejected as chemotherapeutic agents may now be recognized for use in association with antibody in an immunoconjugate [99]. Pro-drug strategies and methods of in vivo activation may add further selectivity [6]. Thus, cancer treatment can now enter a new era, since selective delivery offers the hope of increased specific activity against the cancer with less toxicity to the patient. With FDA approval of Mylotarg, clinical proof of principle for drug immunoconjugates was achieved [74]. We are now in an era where costs [58, 68, 73], not technology, will now be the limiting factor in developmental therapeutics [59].

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461 66. Oldham RK. Immunoconjugates: drugs and toxins. In: Oldham RK, ed., Principles of Cancer Biotherapy. New York: Raven Press, 1987:319–335. 67. Oldham RK. Monoclonal antibody therapy. In: Chiao JW, ed., Biological Response Modifiers and Cancer Research, Vol. 40. New York: Marcel Dekker, 1988:3–16. 68. Oldham RK, Lewis M, Orr DW, et al. Individually specified drug immunoconjugates in cancer treatment. Imperial Cancer Research Conference, England, 1990. 69. Oldham RK. Who pays for new drugs? Nature 1988;332(28):795. 70. Oldham RK, Lewis M, Orr DW, et al. Adriamycin custom-tailored immunoconjugates in the treatment of human malignancies. Mol Biother 1988;1(2):103–113. 71. Oldham RK, Lewis M, Orr DW, et al. Individually specified drug immunoconjugates in cancer treatment. In: Ceriani RL, ed., Breast Cancer Immunodiagnosis and Immunotherapy Proceeding. Proceedings of the 12th World Congress of Obstetrics and Gynecology, 1990. 72. Oldham RK. Monoclonal antibodies. In: Nathanson L, ed. Management of Advanced Melanoma. Contemporary Issues in Clinical Oncology. New York: Churchill Livingstone, 1986:195–207. 73. Oldham RK. Custom tailored drug immunoconjugates in cancer therapy. Mol Biother 1991;3(3):148–162. 74. Oldham RD, Dillman RO. Monoclonal antibodies in cancer therapy; twenty-five years of progress, JCO 2008;26(11)1774-1777. 75. Orr DW, Oldham RK, Lewis M, et al. Phase I trial of mitomycin-c immunoconjugate cocktails in human malignancies. Mol Biother 1989;1(4):229–240. 76. Ozaki S.,Tomura T., Matsumoto T. et al. Anti-myeloma activity of the Maytanisoid immunoconjugate of internalizing human monoclonal antibody specific for HM1.24/BST2 (CD317). Blood (ASH Meeting Abstracts), Nov 2005;106:3388. 77. Pavanasasivam G, Pearson JW, Bohn W, et al. Immunotoxins to a human melanoma asociated antigen: comparison of gelonin with ricin and other A-chain conjugates. Cancer Res 1987;47:3169–3173. 78. Philpott GW, Gass EH, Panker CW. Affinity cytotoxicity with an alcohol dehydrogenase-antibody conjugate and allyl alcohol. Cancer Res 1979;39:2084–2087. 79. Pietersz GA, Smyth MJ, Kanellos J. Preclinical and clinical studies with a variety of immunoconjugates. Antibody Immunocon Radiopharm 1988;1:79–103. 80. Polson A., Yu S.F., Ebens A., et al. Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma. Blood, 15 July 2007;110(2):616–623. 81. Raso V, Raso J, Basala M, Schlossman S. Monoclonal antibodyricin A chain conjugate selectivity cytotoxic for cells bearing the common acute lymphoblastic leukemia antigen. Cancer Res 1980;42:457–464. 82. Riethmuller G, Holz E, Schlimok G, Hirche H, et al. Monoclonal antibody therapy for resected Duke’s C colorectal cancer: sevenyear outcome of a multicenter randomized trial. J Clin Oncol 1998,16:1788–1794. 83. Ritz J, Schlossman SF. Utilization of monoclonal antibodies in treatment of leukemia and lymphoma. Blood 1982;59:1–11. 84. Rowland AJ and Pietersz GA. Reduction in the toxicity of aminopterin-monoclonal-antibody conjugates by leucovorin. Cancer Immunol Immunother 1994;39:135–139. 85. Rowland AJ, Pietersz GA and McKenzie IFC. Preclinical investigation of the antitumour effects of anti-CD19-idarubicin immunoconjugates. Cancer Immunol Immunother 1993;37:195–202. 86. Rowland GF, Corvalan JRF, Axton CA, et al. Suppression of growth of human colorectal tumor in nude mice by vindesinemonoclonal antibody CEA conjugates. Protides Biol Fluids 1984;31:783–786.

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Drug Immunoconjugates 101. Smith TW. Antitumor properties of vindesine-monoclonal antibody conjugates. Cancer Immunol Immunother 1985;19:1–7. 102. Stastny JJ and Das Gupta TK. The use of daunomycin-antibody immunoconjugates in managing soft tissue sarcomas: nude mouse xenograft model. Cancer Res 53:5740–5744, 1993. 103. Stein, R. ASCO Annual Meeting Proceedings, 2004;22(14S):6535. 104. Takahashi T, Yamaguchi T, Noguchi A, et al. Clinical trial of monoclonal antibody-drug conjugate, A7-NCS, for 70 patients with colorectal cancer. Antibody Immunocon Radiopharm 1990;3(1):60. 105. Thorpe PE, Ross WCJ. The preparation and cytotoxic properties of antibody-toxin conjugated. Immunol Rev 1982;62:119. 106. Toshiyuki S, Nagamura S, Saito H, Yamasaki M, et al. Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly(ethylene glycol)-dipeptidyl linker capable of tumor specific activation. Bioorgan Medicin Chem 8 (2000);2175–2184. 107. Trail PA, Willner D, Lasch SJ, Hellstrom KE, et al. Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates. Science 1993,261: 212–215. 108. Trail PA, Willner D, Hellstrom KE. Site-directed delivery of anthracyclines for cancer therapy. Drug Dev Res 1995,34:196–209. 109. Tsukada Y, Hurwitz E, Kashi R, et al. Chemotherapy by intravenous administration of conjugates of daunomycin with monoclonal conventional anti-rat-alpha-fetoprotein antibodies. Proc Natl Acad Sci USA 1982;79:7896–7899. 110. Uckun FM, Evans WE, Forsyth CJ, et al. Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases. Science 267:886–891, 1995. 111. van der Velden VHJ, te Marvelde JG, Hoogeveen PG, van Dongen JJM, et al. Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internalization by leukemic and normal myeloid cells. Blood. 2001;97:3197–3204. 112. Vogel C, Cobleigh MA, Tripathy D, Stewart SJ, et al. First-line, single-agent Herceptin (trastuzumab) in metastatic breast cancer: a preliminary report. Eur J Cancer Jan 2001;37 Suppl 1:S 25–29. 113. Vogel C-W, ed. Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer. New York: Oxford University Press, 1987. 114. Von Hoff DD. Implications of tumor cell heterogeneity for in vitro drug sensitivity testing. Semin Oncol 1985;12:327–331. 115. Yarbro JW. Introduction: tumor heterogeniety and the new biology. Semin Oncol 1985;12:201–202. 116. Yoshibumi K, Tsukazaki K, Kubushiro K, Nozawa S, et al. Selective cytotoxicity of adriamycin immunoconjugate antibody MSN-1 to endometrial adenocarcinoma in vitro and in vivo. Oncol Reports 2000;7:1099–1106.

13 Targeted radionuclide therapy of cancer JOHN M. PAGEL, OTTO C. BOERMAN, HAZEL B. BREITZ AND RUBY F. MEREDITH

This chapter is an update of the former Chapter 13 “Radiolabeled Monoclonal Antibodies for Management of Metastatic Cancer”. The emphasis on the update is use of antibody as well as non-antibody radionuclide conjugates in treatment of cancer.

Introduction Radiolabeled monoclonal antibodies and peptide based radiopharmaceuticals have been evaluated as vehicles to selectively target radioactivity directly to tumor cells for more than a decade. Success in achieving significant remissions in patients with hematologic malignancies such as Non-Hodgkin Lymphoma (NHL) and acute myeloid leukemia (AML) has encouraged investigators to continue working to optimize radioimmunotherapy (RIT) approaches. RIT has thus become the third standard modality of treatment in hematologic malignancies, while promising results have been obtained in some solid tumors. Until the past few years, these therapies have focused almost entirely on using beta emitting radionuclides, carried directly by antibodies. In NHL, for example, response rates have varied from 50% to >80%, with best responses seen using non-myeloablative doses of therapeutic radionuclide as first-line treatment in radiosensitive tumors [1–4]. RIT has also been employed with success as a boost to the radiation dose in combination with other treatments, particularly in association with stem cell rescue, as adjuvant therapy, or for treating small volume disease. Despite the enormous potential for targeted therapy, problems were identified soon after the early clinical RIT trials were completed. Initially it was thought that any tumor could be targeted efficiently with monoclonal antibodies. However, the long circulatory half-life of the blood-borne radiolabeled antibody causes prolonged high background activity levels leading to non-specific normal organ exposure to radiation. After the detection of peptide receptor expression by tumors, peptide analogues began to be investigated for specific tumor targeting. Peptides used for tumor targeting show several advantages over antibodies: peptides are small and show rapid diffusion into (target) tissues resulting in rapid pharmacokinetics.

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

Their fast blood clearance could lead to high tumorto-background ratios shortly after administration of the radiopeptide. Considerable expansion has been accomplished with use of pretargeted RIT and other methods to enhance tumor-to-normal organ tissue ratios such as the use of alpha emitting radionuclides. This chapter will focus on these clinical therapeutic aspects of targeted radionuclides for diagnosis and therapy of tumors. The current state of clinical use of radiolabeled antibodies and radiopeptides, as well as the state of development of new compounds and future innovations are reviewed. Discussion of pre-clinical studies will be limited to those that offer insights into future direction for clinical study. The radioisotopes appropriate for diagnosis and therapy and the radiolabeling methods that are currently in use are briefly described.

Radionuclides for Radioimmunotherapy The choice of radionuclide with which to label antibodies or other targeting entities is governed by several considerations [5, 6]. These include the clinical indication as well as physical properties such as mode of decay, energy, and abundance of the emissions and half-life, as well as chemical properties affecting protein attachment and in vivo handling; and finally production aspects including specific activity, availability at needed scale, and cost. Recognition of the advantages and disadvantages of the available choices is important since different strategies are required to maximize their potential. Dose rate is a specific factor modifying therapeutic efficacy, particularly for beta emitting radionuclides. Traditional external beam radiation is given at a much higher rate than internally administered radionuclides. In RIT, radiation is delivered in the range of 10–30 cGy/h and continuously decreasing because of decay. Generally, effectiveness of cell killing goes down as the dose rate lowers because more time is available for repair of sublethal damage [7]. Considering the dose rate effect, some have suggested that 20–30% more dose is needed to sterilize tumors compared to fractionated external beam treatment [7, 8], although a review of RIT studies in animal xenografts by Wessels suggests dose

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464 effects from RIT are comparable to external beam [9]. An inverse effect resulting in enhancement of low-dose rate effects has been observed where cells accumulate in the radiosensitive G2M stage of the cell cycle, which may contribute to efficacy of the low-dose rate radiation of RIT. The type of malignancy may also be a factor as lymphomas have been especially sensitive to inverse dose rate effects [10]. The physical half-life of the radionuclide, as well as the biological half-life for tumor uptake, retention, and elimination from normal tissues of the carrying vehicle must be considered for radionuclide selection. In particular, radionuclide half-life considerations include retention time for antibody or another targeting agent such as a peptide in tumor in order to deliver a dose commensurate with the fraction of injected activity that localizes to target tissue. Thus, with conventionally radiolabeled antibodies or peptides (i.e., injected with radionuclide bound to targeting agent, the half-life must be long enough for tumor uptake as well as tumor irradiation during time of useful tumor to normal tissue ratios). As examples, 131I (8 day t1/2), 186Re (3.7 day t1/2) and 90Y (2.7 day t1/2) are suitable for whole antibody pharmacokinetics where maximum tumor uptake of intact antibodies requires 24–48 h, but tumor retention can persist for several days [11, 12]. Conversely 188Re (17 h t½) would be more compatible with short-lived small molecule targeting. Another general limitation of radiotherapy is that the maximum rate of decay, hence therapeutic efficacy, occurs at the time of injection. For any process that involves slow accumulation in tumor, typical for antibody protein, being a large molecule, much of the radiation decay affects non-tumor tissue before selective tumor to non-tumor ratios are achieved. Therefore, much effort has gone into engineering of various antibody and peptide forms over the last several years to further overcome targeting limitations. Intact IgG antibodies are approximately 150 kD molecular weight and thus are relatively large molecules for their role as targeting vehicles. The whole antibody characteristics of slow disappearance from the blood and the slow tumor uptake kinetics have limited their ability to achieve high tumorto-normal tissue and blood ratios, and have resulted in high radiation exposure of the radiosensitive marrow cells. Early efforts to improve on the pharmacokinetic limitations of whole antibodies included removing the Fc portion to yield F(ab’)2 fragments of 100 kD size. These have been further split into Fab’ (50 kD), or in some cases, Fab fragments of similar size. In general, blood disappearance rates, and tumor-to-normal tissue

Targeted radionuclide therapy of cancer ratios increased with use of these smaller constructs, but absolute tumor uptake and retention was decreased. In the case of Fab and Fab’ fragments, increased kidney localization also occurred because the efficiency of filtration for proteins of this size increases and tubular reabsorption occurs. Improved penetration and retention in tumor has been further explained with the use of a variety of novel molecular antibody forms with different sizes in both solid and hematologic malignancies. These include chimeric and humanized whole antibody forms, CH2 deletion constructs, single-chain forms, diabodies, minibodies, and fusion proteins [6, 13–18]. The CH2 deletion constructs are derived from the constant region of about 125 kD, while single chain Fv of 25 kD are from the variable region. Diabodies are divalent forms of Fvs (scFv dimer) whereas minibodies are scFv-CH3 dimers of 80 kD [13, 14, 19]. Evaluation of these immunotargeting forms has indicated gains in tumor-to-normal tissue ratios, but limitations still exist in kidney and liver uptake as well as tumor retention. Types of emissions considered are beta particles (electrons emitted with a wide range of energies), alpha decay (in which helium + two particles are emitted), and low energy Auger electron emissions, a byproduct of electron capture decay. While the particulate property of the radiation decay mode determines the therapeutic potential, gamma ray emission often associated with the radiation decay, provides the ability to image the biodistribution in vivo, thus indicating tumor localization and non-target uptake and retention. Ideally, gamma radiation should be of low abundance such that contribution to non-target organ irradiation is minimized.

Beta Particle Decay A wide range of beta emitter energies of emission and half-life are available for RIT (Table 1). Adelstein [20], Howell [21], Humm [22], and Wheldon [23] have considered the application of beta emitters with respect to emission energy and penetration, number of cell reversals, and size of tumor targets. Beta emitters have the unique advantage of exerting their cytotoxicity by a crossfire Table 1. Selected beta emitting radionuclides for RIT Beta emitting isotopes

t1/2

Path-length Energy delivered (mm) (MeV)

Iodine-131 Yttrium-90 Rhenium-188 Copper-67 Lutetium-177

8.1 days 64 h 17 h 62 days 6.7 days

0.8 2.7 2.4 0.05–2.1 0.04–1.8

0.6 2.3 2.1 0.6 0.5

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effect as only occasional beta particles will achieve lethal double strand DNA breaks. When a sufficient concentration of emission occurs in a tissue volume, the probability of lethal hits increases, predominantly from sources bound to other cells. This crossfire killing property obviates the need for targeting every cancer cell in contrast to antibody targeted delivery of drug or toxin conjugates. The crossfire effect is efficient for tumor masses larger in diameter than the average beta path length. Relatively few beta emitters have been studied in clinical trials for RIT. These include the low energy emitter 131 I that has a maximum energy of 0.61 MeV and rhenium (186Re) with a moderate energy of 1.07 MeV. The higher energy beta emitter that is currently most widely used radionuclide in clinical trials and clinical practice is 90Y (2.3 MeV). Comparing isotopes, Humm [22] estimated 54% absorption of energy from particles of 131I in a 1 mm cluster but only 10% absorption of 90Y in this small volume. Further analysis by Wheldon and coworkers suggest that moderate or lower energy emitters should be used for clusters of up to [107] cells while an emitter such as 90Y should be used for tumors of [108] cells or greater [23].

Alpha Particle Decay There has been significant interest in targeting alpha radiation for RIT over the past several years (Table 2). With the large He particle emitted, alpha decay results in high linear energy transfer (LET) or energy delivery over a distance of only several cell diameters. This results in the advantages of high potency and lack of oxygen dependence with correspondingly no shoulder on the cell survival curve. The short path length, however, results in the need for much more homogeneous targeting for complete tumor cell kill. Alpha emitters that have been studied for antibody mediated tumor targeting include 212Bi (t1/2 1.06 h), 213Bi (t1/2 0.76 h), 211At (t1/2 7.2 h), and 225Ac (t1/2 10 days). As these are short-lived radionuclides, applications have been mainly in leukemia and lymphoma and nonsystemic administration for other tumors such as intraperitoneal injection. Macklis and coworkers evaluated physical characteristics of 212Bi labeled antibody in lymphoma [24]. Table 2. Selected alpha emitting radionuclides for RIT Alpha emitting isotopes

t1/2

Path-length (μm)

Energy delivered (MeV)

Bismuth-213

46 min

84

6.0

Actinium-225

10 days

50–80

8

Astatine-211

7.2 h

60

6

Only 27 212Bi atoms and 4 alpha-particle tracks (“hits”) of Bi were required for a log of target cell killing. Satisfying the requirement of homogeneous targeting of alpha emitters is more difficult with solid tumors which are often poorly vascularized and may be access-limited by high interstitial pressure due to poor lymphatic drainage [25]. Toxicity to normal tissue via antibody cross-reactivity can be high over a short range due to the potency of alpha radiation and lack of repair potential of double-stranded DNA lesions. As expected, good efficacy has been seen in micrometastatic models, and in established solid tumors little more than a delay in tumor growth has been observed. Alpha particle radiotherapy studies have been reviewed in detail by Hassfjell and Brechbiel [26].

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Emerging Radionuclides for use in RIT Additional pre-clinical investigations have focused on a wider range of beta and alpha emitting radionuclides with different energies of emission and half-lives to improve the efficacy of RIT. Two promising beta emitting radionuclides are 177Lu (0.5 MeV) and 67Cu (0.6 MeV). Lutetium-177 has a moderate beta energy that is similar to 131I and possesses a 6.7 day half-life. An advantage for the use of 177Lu is that this lanthanide has similar chelation properties to 90Y, suggesting that use of standard chelating agents will lead to stable delivery of 177Lu to targeted cells. Moreover, 177Lu can be produced in high specific activities relatively inexpensively, making the use of this agent in clinical trials probable for the near future. Two copper radionuclides have emerged with considerable promise for RIT of hematologic malignancies. Copper-67 has a t1/2 greater than 2 days and emits beta particles with reasonable energy for therapeutic benefit. Copper-64 is also a beta emitting isotope, but in addition 64 Cu emits positrons, making this radionuclide appealing for use with positron emission tomography to allow for accurate estimations of the absorbed doses of therapeutic 67 Cu. Readily accessible quantities of these copper radiometals have been limited, however, and must be more widely available at affordable cost before these agents gain widespread application in RIT protocols.

Antibody-Based Radiopharmaceuticals Extensive work exploiting the safety and efficacy of radiolabeled monoclonal antibodies that target specific surface antigens on malignant cells has been incorporated into a new generation of novel treatment approaches

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for cancer. The characteristics of the antibodies that must be considered for RIT are the same as those for immunotherapy, and are listed in Table 3. Beierwaltes’ successful treatment of metastatic melanoma in a single patient with 131I-labeled polyclonal antibody raised to the patient’s own tumor was the first indication that RIT might be feasible [27]. Order et al. reported initial trials of 131I and 90Y-labeled antiferritin polyclonal antibodies [28–30]. These studies showed that 131 I-labeled antiferritin polyclonal antiserum could produce regressions of bulky hepatomas [29]. Four of 24 patients had partial responses (PR) and one had a complete response (CR). Single doses as high as 150 mCi were given with acceptable hematologic toxicity. Subsequent studies combined cytotoxic agents (5-FU and adriamycin) and external beam radiation with lower doses (50 mCi in divided doses) of antibody-labeled radiation and achieved a response rate of 48% (7% CR; 41% PR). Monoclonal anti-ferritin was found to be inferior to polyclonal antisera because of high localization in the liver, and 90Y-labeled anti-ferritin to be superior to 131I-anti-ferritin. Responses following up to 30 mCi 90Y-anti-ferritin antibodies were also seen in patients with Hodgkin disease when used in conjunction with chemotherapy [30]. A response rate of 62% was reported in 27 patients with advanced Hodgkin lymphoma with 90Y-anti-ferritin. Marrow toxicity at 30 mCi limited further dose escalation, although multiple infusions were administered in some patients. In a separate approach polyclonal radiolabeled anti-ferritin antibody was delivered by dose fractionation. At low doses, this did not improve response rates, yet may Table 3. Considerations for radiolabeled antibody studies Antibody

Antigen – Location, cellular density, modulation, circulating Affinity Specificity Mass Molecular form – intact, fragments Form-murine, chimeric, humanized, human

Radionuclide

Half-life Emissions-type, energy, abundance Chemistry Specific activity

Preparation

Percent protein bound Immunoreactivity Purity

Patient

Pharmacokinetics Images, normal organ biodistribution, tumor uptake Radiation absorbed dose estimates Antiglobulin response Tumor response following RIT

need to be evaluated at doses requiring marrow rescue to achieve optimal results [31]. Bierman et al. reported that 30 mCi 90Y-polyclonal anti-ferritin combined with high-dose chemotherapy in patients with Hodgkin disease undergoing bone marrow transplantation did not cause any additional adverse effects [32]. The marrow irradiation from the 90Y did not cause increased toxicity nor did it interfere with re-engraftment in this study. Although no definite improvement in outcome could be attributed to the RIT, the lack of additional toxicities with the additional lowdose rate irradiation suggested that further similar studies in patients with better performance status were warranted. Overall those early radiolocalization studies performed with polyclonal antibodies, however, suggested that more homogeneous antibodies were required in order to be clinically useful. Hybridoma technology permitted careful selection of monoclonal antibodies directed to particular tumors, such that antibodies with the highest localization and binding potential and with the lowest cross-reactivity with normal tissue are identified. The majority of the clinical trials have utilized radiolabeled murine monoclonal antibodies either with or without other concomitant therapy, combined with 131 90 I, Y and less frequently, 186Re as the radiolabel.

Radioimmunotherapy of Hematologic Tumors Lymphoma The most impressive results of RIT to date have been achieved in NHL. Several monoclonal antibodies conjugated with radioactivity have been evaluated in the treatment of NHL. RIT has proven to be particularly effective in the treatment of patients with NHL because of the sensitivity of lymphocytes to radiation, the large number of target antigens on the surface of lymphocytes and the vascular accessibility of the malignancies. The first RIT trials for B-cell lymphomas were using 131I-labeled Lym-1 antibody. Lym-1 is a novel, murine, IgG2a monoclonal antibody that recognizes a 31–35 kD membrane antigen expressed on malignant B cells characterized as HLA-DR10 [33]. The DeNardo group has generally taken the approach of administering lower doses of fractionated RIT, consistent with standard oncological practice using chemotherapy and external beam therapy, to deliver higher overall dosages with lower toxicity. In a study using low dose, fractionated therapy 30 patients with B-cell malignancies received either 30 or 60 mCi 131I-labeled Lym-1 antibody delivered every 2–6 weeks up to a total dose of

John M. Pagel et al. 300 mCi. Acute toxicity (e.g., fever, rash) was mild and transient. Dose limiting toxicity was thrombocytopenia, particularly in patients with low platelet counts at baseline and in patients with lymphomatous marrow involvement. Eleven of the 30 patients received the intended dose of 300 mCi (thought to be the maximum-tolerated dose (MTD) based on marrow dose estimates). The overall response rate (ORR) was 57% for all patients and 94% of the 18 patients who received at least 180 mCi. Ten percent of patients achieved a CR lasting 10–44 months [34]. A second study was designed to define the MTD and efficacy of at least the first two, of a maximum of four, high doses of 131I-Lym-1 given 4 weeks apart [35]. Dosages studied were 40 to 100 mCi/m2 administered every 4 weeks. Twenty patients with different advanced NHL, histologic subtypes that were resistant to standard therapy were treated. Dose-limiting toxicity was found to be thrombocytopenia at an MTD of 100 mCi/m2. The ORR was 52% with seven patients (33%) achieving a CR, persisting for median duration of 14 months. All three patients that received the MTD (100 mCi/m2 × 2) had a CR. The median duration of survival was 19 months in responding patients and 1.9 months in nonresponders. Considering both studies, responses were observed in 30 of 45 patients including 95% of patients who received more than 200 mCi of 131I Lym-1. Of note, the high dose approach achieved a higher CR rate than the low dose approach. The Lym-1 antibody has also been labeled with 67Cu, in four patients to compare the biodistribution and dosimetry to 131I-Lym-1 [36]. Both the uptake in tumor and the retention time in tumor were found to be higher with the 67Cu-Lym-1 than the 131I-Lym-1. Marrow dose estimates, however, were lower with 67Cu and liver dose estimates were higher. Copper-67-Lym-1 was further administered to 12 patients in a Phase I/II dose escalation trial. Up to four doses of 25 or 50–60 mCi/m2 were administered, the lower dose when marrow involvement was present. Dose limiting toxicity was hematologic and the ORR was 58% [37]. Although the supply of 67 Cu is at present uncertain, this remains an isotope worthy of further study. The majority of RIT investigators have aimed however to deliver the MTD in one injection in order to deliver the highest dose rate possible to the patient. This has been, at least in part, due to the concern of immunoglobulin development toward the therapeutic antibody, which could potentially limit the number of infusions that can be safely administered. Results have been reported by various investigators using single non-myeloablative doses of radiolabeled antibodies directed against a variety of lymphoid

467 differentiation antigens, including HLA class II variant molecules, idiotypic immunoglobulins, the CD 5, 20, 21, 22 and 37 antigens, and the IL2 receptor. Targeting radiation to the CD19, CD20, and CD22 antigens has proven, however, the most effective in clinical NHL trials. These target antigens are expressed in high density with >95% of all B-cell lymphomas expressing CD19 and CD20, and 70% expressing CD22. Two anti-CD20 radiopharmaceuticals (131I-tositumomab and 90Y-ibritumomab tiuxetan) have now been approved for use in patients with relapsed or refractory indolent disease. 131

I-Tositumomab

Kaminski et al. first described the use of low-dose 131 I-labeled anti-CD20 antibody (tositumomab) to treat patients with relapsed indolent NHL [39]. Patients receiving this therapy first received tositumomab labeled with a trace amount of 131I (5 mCi) followed by a therapeutic infusion based on a predicted estimate of the whole body radiation absorbed dose. Each dose of radiolabeled antibody was preceded by an infusion of unlabeled anti-tositumomab antibody to reduce normal, antigen-specific binding. A Phase I dose-escalation trial was subsequently conducted to assess the toxicity and efficacy of non-myeloablative doses of 131I-tositumomab [40] Patients were treated with whole-body radiation doses escalating in 10 cGy increments from 25 to 85 cGy. Twenty-eight of the 34 patients enrolled completed treatment. The overall response rate was 79% and 50% achieved a CR. The median duration of response was 12 months for all patients and 16 months for those patients who reached a CR. Sixteen of 17 patients who achieved a response of 6 months or more in duration remained alive 6 years after treatment. The whole body clearance was found to be variable between patients, most likely dependent on the B-cell load and tumor bulk, thus the tracer study was deemed necessary to administer the MTD to each patient. Hematologic toxicity was dose limiting at a whole-body radiation absorbed dose of 75 cGy. These early studies led to development of a diagnostic dose to determine pharmacokinetics for selecting a therapy dose that will deliver a whole body dose of 75 cGy, the MTD for patients without compromised marrow function (in patients with lower baseline counts, 65 cGy is the target whole body dose). Based on the low toxicity and the clinical results found in the Phase I trials, a trial was designed to evaluate 131I-tositumomab with the primary clinical endpoint being the comparison between the patient’s duration of remission following 131I-tositumomab therapy and the duration of remission on the patient’s last chemotherapy [42]. The study included 60 indolent or transformed

468 NHL patients who were refractory prior to chemotherapy. Seventy-four percent of the patients with indolent NHL experienced a longer duration of response to 131 I-tositumomab compared to 26% who experienced a longer duration of response to prior chemotherapy (p < 0.001). The median duration of remission after 131 I-labeled antibody was 6.5 months, approximately doubling the 3.4-month median duration of remission patients experienced on their last chemotherapy regimen. In addition, a response was observed in only 28% of patients following their last chemotherapy regimen compared to 65% of patients following a regimen of 131 I-tositumomab (p < 0.001). Updated and long-term data on 53 chemotherapyrelapsed/refractory patients, including patients who had received a prior autologous stem cell transplant (ASCT), that were treated with iodine 131I-tositumomab have been provided [43]. Dose-escalations were conducted separately in patients who had or had not undergone a prior ASCT until a non-myeloablative maximally tolerated whole body dose was established (non-ASCT = 75 cGy, post-ASCT = 45 cGy) and then 14 additional non-ASCT patients were treated with 75 cGy. Forty-two of 59 patients (71%) responded and 20 patients (34%) had a CR. Thirty-five of 42 patients (83%) with low-grade or transformed NHL responded versus 7 of 17 (41%) with de novo intermediate-grade NHL (p = 0.005). For all 42 responders, the median progression-free survival was 12 months and 20.3 for those with CR. Seven patients remained in CR 3 to 5.7 years. Sixteen patients were retreated after progression; nine responded and five had a CR. Reversible hematologic toxicity was dose limiting. Only ten patients (17%) had human anti-mouse antibodies detected. Long-term, five patients developed elevated thyroid-stimulating hormone levels, five were diagnosed with myelodysplasia and three with solid tumors. An expanded access study of 359 patients with relapsed or refractory low grade or transformed low grade NHL was carried out. In 273 patients evaluable for response, the ORR was 58% and the CRR was 27%. Follow up at 17 months showed that the median duration of response had not yet been reached [44]. A single, well-tolerated treatment with iodine 131I-tositumomab can, therefore, produce frequent and durable responses in NHL, especially low-grade or transformed NHL. As a first line therapy in follicular lymphoma, 131 I-tositumomab is currently showing promising results. Kaminski et al. reported a 95% overall response rate (75% complete response) in 76 previously untreated follicular lymphoma patients. Molecular remissions were demonstrated in 80% of patients who achieved a complete response [45]. Actuarial 5-year survival was 59%

Targeted radionuclide therapy of cancer with a median PFS of 6.1 years. Analysis according to the Follicular Lymphoma International Prognostic Index risk categories demonstrated that 85% of the patients were intermediate or high risk [46]. Phase II studies of RIT as consolidation following upfront therapy with a variety of chemotherapy regimens including CHOP and fludarabine have resulted in high overall response rates and excellent PFS [2, 3]. In a phase II trial conducted by the Southwest Oncology Group, the complete response/unconfirmed complete response rate improved from 39% after CHOP to 69% following consolidative 131I-tositumomab with an estimated 5-year PFS and overall survival of 67 and 87%, respectively [2]. Based on these promising results, an ongoing phase III trial compares consolidation with 131I-tositumomab following CHOP chemotherapy to observation in untreated follicular lymphoma patients. The current US Intergroup phase III trial randomizes untreated follicular lymphoma patients to either six cycles of CHOP followed by 131 I-tositumomab or to rituximab plus CHOP providing a valuable comparison between two contemporary treatment options for the frontline therapy of follicular lymphoma. Whether upfront cytoreduction with chemotherapy benefits previously untreated patients receiving 131 I-tositumomab is unknown. Clinical outcomes following RIT alone were similar to those achieved with chemotherapy followed by RIT and a randomized trial will be required to address this issue. 90

Y-ibritumomab tiuxetan

Ibritumomab (IDEC-Y2B8, Zevalin®) is a murine IgG1 kappa monoclonal antibody that covalently binds MX-DTPA (tiuxetan), which chelates therapeutic 90Y. A multi-institution Phase I/II study evaluated the safety and efficacy of treatment with 90Y-ibritumomab tiuxetan in 58 patients with low or intermediate grade and mantlecell NHL [47]. The amount of 90Y-labeled antibody was dose-escalated from 0.2 mCi/kg to 0.4 mCi/kg. Prior to 90 Y treatment, biodistribution studies were conducted using a surrogate 111In-labeled antibody conjugate to allow for gamma imaging. Rituximab was pre-administered as the unlabeled blocking antibody (250 mg/m2) to allow for known sites of disease to be more easily visualized and this also decreased the projected dose of radiation to this spleen and marrow. The MTD was 0.4 mCi/ kg (0.3 mCi/kg for patients with baseline platelet counts 100 to 149,000/ml). The only significant toxicity was myelosuppression. The ORR for the intent-to-treat population (n = 51) was 67% (26% CR; 41% PR); for lowgrade disease (n = 34), 82% (26% CR; 56% PR); for intermediate-grade disease (n = 14), 43%. Responses occurred in patients with bulky disease (≥7 cm; 41%)

John M. Pagel et al. and splenomegaly (50%). The median time to progression for patients who responded was 12.7 months. The phase I/II trials were followed by a phase III trial that randomized 143 eligible patients to either rituximab or 90 Y-ibritumomab tiuxetan radioimmunoconjugate to demonstrate that the combination of the 90Y radioisotope to the murine anti-CD20 antibody provided additional efficacy over the unconjugated (“cold”) rituximab alone [48]. A planned interim analysis of the first 90 patients demonstrated an ORR of 80% with 90Y-ibritumomab tiuxetan versus 44% for rituximab (P < 0.05). To provide additional evidence of the benefit of 90 Y radioimmunotherapy over rituximab immunotherapy, patients who were nonresponsive or refractory to rituximab were enrolled in an additional trial and treated with 90Y-ibritumomab tiuxetan 0.4 mCi/kg. An ORR of 46% was achieved in these rituximab-refractory patients. These results provide further evidence of the added value of 90Y [49]. More recently a phase II trial combining six cycles of chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) followed 6–10 weeks later by 90Y-ibritumomab tiuxetan was conducted to evaluate the efficacy and safety in untreated elderly diffuse large B-cell lymphoma (DLBCL) patients. In 20 eligible elderly (age ≥ 60 years) patients with previously untreated DLBCL using this novel regimen the ORR to the entire treatment regimen was 100%, including 95% CRs and 5% PRs. Four (80%) of the five patients who achieved less than a CR with CHOP improved their remission status after 90Y-ibritumomab tiuxetan RIT. With a median follow-up of 15 months, the 2-year PFS was estimated to be 75%, with a 2-year OS of 95%. This study therefore has demonstrated the feasibility and tolerability of this regimen for elderly patients with DLBCL [4].

High-dose Radioimmunotherapy with Stem Cell Support for Lymphoma Data from prior studies showing an inverse relationship of recurrence rates to radiation dosage led investigators to hypothesize that if the radiation dose could be further safely escalated to tumor sites, relapse rates would be reduced without incurring additional toxicity. Press et al. explored the use of myeloablative doses of 131I-labeled monoclonal antibodies with ASCT support in 43 patients with B cell lymphomas who had failed conventional chemotherapy [50]. Two anti-CD20 antibodies (tositumomab and 1F5) and one anti-CD37 antibody (MB-1) were evaluated. Patients were selected for treatment after tracer studies with increasing mass doses of antibody that determined that the absorbed dose to tumor would be greater than that to normal organs. Sixty-four

469 percent of patients screened were eligible for therapeutic doses of radiolabeled antibody. Patients received 58–1,168 mg antibody labeled with 234–777 mCi of 131I. At absorbed doses above 27.25 Gy to lungs, two of four patients experienced cardiopulmonary toxicity. Sixteen of 19 patients achieved a CR and 2 of 19 patients achieved a PR. Mean response duration was in excess of 19 months. A phase II study repeated these results and obtained responses in 15 of 19 patients. At 6 years, the projected overall survival was 78% for patients with indolent lymphoma and 43% for patients with aggressive histologies [51]. Based on the results above, a study to evaluate treatment with the MTD of radioactivity was conducted [52]. Twenty-two of 25 patients evaluated with trace labeled doses achieved biodistributions considered adequate to receive a therapeutic infusion. Twenty-one patients were treated with therapeutic infusions of 131I-tositumomab antibody calculated to deliver not more than 27 Gy to normal organs followed by autologous hematopoietic stem cell reinfusion. Seventeen (81%) achieved CR with a median duration of response of 38 months. The relative long-term efficacy of this high-dose RIT strategy was evaluated via a multivariable cohort analysis of 125 patients with relapsed or refractory follicular lymphoma treated with either myeloablative I-131 tositumomab followed by autologous HCT or conventional high dose therapy followed by autologous HCT. In this study, the estimated 5-year OS for high-dose RIT was 67% and for conventional high-dose therapy was 53% (p = 0.004) [53]. Likewise, 5-year PFS was 48% for the high-dose RIT group and 29% for the conventional transplant group (p = 0.03). Furthermore, 100-day treatment-related mortality was lower in the high-dose RIT group than conventional HCT group (3.7% versus 11.2%) with no evidence of increased MDS/AML at 8 years of follow-up. Based on the exceedingly low nonhematopoietic toxicity of single-agent HD-RIT followed by autologous HCT, this strategy was evaluated in older adults with relapsed B-NHL. Older patients with refractory or relapsed NHL typically have limited therapeutic options largely due to the excessive toxicity associated with potentially curative dose intense regimens and co-morbidities [53]. In this study, 24 patients ≥60 years of age with relapsed or refractory B-NHL received highdose RIT targeted to ≤25–27 Gy to normal organs followed by ASCT. Notable findings from this series included low rates of non-hematopoietic toxicity (<10% grade 4), no treatment-related deaths, and an estimated 51% 3 year PFS. These results, although encouraging, led to relapse in the majority of patients. Therefore, Press et al. combined

470 the MTD of 131I-tositumomab with high-dose etoposide and cyclophosphamide prior to autologous HCT [54]. While the toxicities were significantly greater for the combination regimen than for single-agent therapy, the estimated overall survival at 2 years was 83% and the progression free survival is 68%. Non-randomized controls treated with identical doses of chemotherapy but with total body irradiation had inferior PFS (p < 0.002) and OS (p < 0.004) compared to those who received radiolabeled antibody based regimen. More recently, a subset of 16 patients with refractory mantle cell lymphoma who had been treated with this approach were reported and showed 3-year PFS and OS from transplantation estimated at 61% and 93%, respectively [55]. Extended phase II studies using this combined highdose chemoimmunotherapy regimen in various B-NHL histologies are ongoing to better define the safety and efficacy of this regimen.

Leukemia Abs directed against the CD33, CD45, and CD66 hematopoietic differentiation antigens have been most extensively explored for RIT of leukemia. CD33 is an attractive target for RIT of myeloid leukemias, since CD33 is expressed on most myeloid leukemic blasts, but not on hematopoietic stem cells [56]. Anti-CD33 Abs have been shown to rapidly saturate antigenic targets and the Ab-antigen complexes are rapidly internalized into the cell. Other alternative targets for RIT that do not modulate upon Ab interaction have also been explored. CD45 is stably expressed at a high density on the surface of virtually all hematopoietic cells and does not undergo significant modulation upon Ab binding. Most hematologic malignancies, including 85–90% of acute lymphoid and myeloid leukemias express CD45 [57, 58]. The CD66 antigen is another target that also does not internalize or shed into the circulation. CD66 is expressed at high density on maturing hematopoietic cells but is not found on AML blasts [59]. Therefore, cytotoxic radiation is delivered to bystander leukemic cells by targeting normal CD66 expressing myeloid cells.

Beta emitting Radionuclides for RIT of Leukemia A relatively small number of beta emitters have been studied in clinical trials for RIT of leukemia. Radioiodine was first used for RIT of AML because of its well-established experience as a medical radiotherapeutic. Two groups of investigators demonstrated that 131I-labeled monoclonal antibodies (p67 and M195) reactive with

Targeted radionuclide therapy of cancer the CD33 antigen could be used to treat patients with acute myeloid leukemia (AML) [60, 61]. In about half of the patients radiotherapy was delivered with relative specificity to the marrow. The liver was the only other organ showing localization of the radioimmunoconjugate and was considered to be the dose-limiting normal organ. Success appeared limited by low CD33 expression on target cells and by internalization of the antibody–antigen complex, leading to deiodination and release of 131I. Jurcic et al. studied a humanized anti-CD33 antibody (HuM195) radiolabeled with 90Y. Anti-leukemic activity was seen, but with prolonged myelosuppression, suggesting the utility of this approach as a pretransplant preparative agent [62]. Matthews et al. treated patients with leukemia using BC8, a murine anti-CD45 IgG1 antibody that does not internalize but is reactive with 85–90% of cases of acute leukemia, and the majority of nucleated cells in normal marrow, but not with cells outside the lymphoid or hematopoietic lineages. A phase I trial in 44 patients with AML, or acute lymphoblastic leukemia (ALL) beyond first remission or advanced myelodysplastic syndrome (MDS) was carried out in conjunction with cyclophosphamide, 12 Gy TBI, and stem cell rescue. In this dose escalation trial patients were selected for therapy only when a diagnostic dose indicated that the bone marrow and spleen would receive more radiation than the other normal organs. Eighty-four percent of patients had favorable biodistributions. This improved biodistribution rate was likely due to the characteristics of the anti-CD45 antibody, with longer retention of the radiation at the tumor. Liver dose from a tracer study was used to limit the dose escalation. The MTD was 10.5 Gy to the liver. This trial demonstrated that 131I-labeled antiCD45 antibody could deliver two to five times more radiation to the marrow and spleen compared to normal organs. This approach at the MTD resulted in 24 Gy more radiation delivered to the marrow and approximately 50 Gy to the spleen, without excessive toxicity in a setting of conventional cyclophosphamide/TBI [63]. Seven of 25 patients with AML/MDS were disease free for a median of 26–100 months at the time of the report, and three of nine patients with ALL were disease free for 34–82 months post transplant. The same preparative regimen was used in a later study for patients with AML beyond first remission with relapsed/primary refractory AML, or advanced MDS. Results suggested that a greater radiation absorbed dose delivered to marrow may lead to lower post-transplant relapse rates for patients with high-risk AML/MDS [64]. Half of the patients treated received a dose to marrow <7 cGy/mCi while the remaining half of patients received

John M. Pagel et al. >7 cGy/mCi to marrow. Although the number of patients was limited, only one of nine patients relapsed in the high marrow-absorbed dose group while six of nine patients relapsed in the lower dose cohort. The hazard of relapse in the group that received <7 cGy/mCi to marrow was more than six-times higher than that seen in the group that received the higher marrow dose. Since greater estimated radiation doses could be delivered safely by an 131I-anti-CD45 antibody to marrow and spleen compared to non-target organs, a trial for patients with AML in first remission was conducted [65]. Fortynine patients received high dose targeted irradiation delivered by 131I-anti-CD45 antibody combined with busulfan and cyclophosphamide. The radiolabeled antibody delivered an average of 11 Gy to marrow and almost 30 Gy to spleen. These first remission patients had a low relapse rate (20%) and a 62% estimate of 3-year disease-free survival after transplantation. These data appear promising since at least 30% of patients with AML in first remission would be expected to develop leukemic relapse after being treated with standard busulfan and cyclophosphamide conditioning and stem cell transplant [66]. Nevertheless, carefully controlled randomized trials will be necessary to definitively assess the contribution of the 131I-anti-CD45 antibody to standard transplant regimens. These encouraging results suggested that this approach might be applied to older patients. Given the reduced toxicity of a low-intensity conditioning regimen in high-risk older patients, the anti-leukemic effect of a non-ablative approach might also be improved by the addition of targeted hematopoietic irradiation delivered by a radiolabeled antibody. An allogeneic transplant trial using targeted hematopoietic irradiation delivered by an 131I-anti-CD45 antibody (BC8) combined with fludarabine and 200 cGy TBI has been initiated for a particularly poor risk population of advanced older AML and high-risk MDS patients. This study has demonstrated that at least an average of 27 Gy of targeted radiotherapy can be delivered to bone marrow and an average of 81 Gy to the spleen, in addition to a standard reduced intensity transplant regimen, without a marked increase in day 100 mortality [67]. Whether use of a radiolabeled antibody combined with a nonmyeloablative transplant will reduce post-transplant relapse rates for these older patients with high-risk AML/MDS, however, remains to be determined. Rhenium radioisotopes have also been investigated as part of conditioning regimens prior to HCT. In particular, 188Re labeled anti-CD66 antibody has been used in patients with high-risk AML [68]. At a follow-up of 18 months in 36 relatively younger patients who underwent

471 allogeneic transplantation after 188Re-anti-CD66 conditioning, 45% were disease free; in addition, significantly more patients remaining disease free if they were in remission at the time of transplant, compared to those patients who were not in remission at the time of transplant (67% versus 31%). For older high-risk AML or MDS patients, RIT using an anti-CD66 antibody labeled with either 188Re or 90Y was also combined with a reduced intensity conditioning regimen followed by transplant using T-cell-depleted allografts [69]. Despite the low level of toxicities seen in older patients using this approach, the cumulative incidence of relapse remained high (55% at 30 months post-transplant). The risk of relapse was almost 20% higher for patients not in remission compared to those in either first or second remission. These studies highlight the difficulty of targeting multiple low-energy beta emissions to neighboring leukemic blasts by relying on crossfire from normal targeted CD66 positive granulopoietic cells.

Alpha Emitting Radionuclides for RIT of Leukemia The feasibility of using alpha emitting radionuclides was established in an initial study that employed 213 Bi-HuM195 for patients with refractory or relapsed AML or MDS [70]. In 18 patients with refractory or relapsed AML or MDS, 13 patients had decreases in the percentage of marrow blasts and 10 of 12 evaluable patients had reduced peripheral blood leukemia cells [62]. 213Bi-HuM195 was given in three to six fractions over 2–4 days. Uptake of antibody in marrow, spleen, and liver was seen within 10 min. There were no significant acute toxicities, however, transient elevated liver function abnormalities occurred. Myelosuppression lasted 34–50 days. Since alpha-particle RIT also has promise to be effective in the treatment of minimal leukemia states, a follow-up study has been initiated using chemotherapy to first achieve a significant degree of cytoreduction prior to the delivery of 213Bi-HuM195. Clinical use of 213Bi to date has been otherwise limited, however, since this radionuclide is relatively short lived, which may result in a potentially short marrow residence time of the radionuclide. Recognizing this limitation and that 225Ac has a 10 day half-life, a phase I trial investigating the use of 225Ac-HuM195 for advanced myeloid leukemias has been initiated at Memorial Sloan Kettering Cancer Center [71]. This study holds considerable promise for advancing the field of alpha-particle RIT because of the longer half-life and the generation of short-lived daughter therapeutic alpha-particles (221Fr, 217 At, 213Bi) from 225Ac inside a targeted leukemia cell,

472 which largely accounts for the increased potency of 225 Ac-atomic nanogenerators over 213Bi constructs [72]. CD25 and CD30 have recently been explored as rational targets for RIT of leukemia as these antigens are expressed by a high proportion of disease specific neoplastic cells and not by normal resting cells. Recently, a pre-clinical model of leukemia labeled with the alpha emitter 211At has been studied as a potential radioimmunotherapeutic for patients with CD25 and CD30 positive leukemias [73, 74]. These findings have set the foundation for progression to clinical studies using radiolabeled Abs for the treatment of patients with either CD25 or CD30 expressing leukemias.

RIT of Non-Hematologic Tumors A variety of antibody based proteins have been studied for RIT of solid tumors including whole antibodies and various fragments derived from them, as well as chimeric and humanized modifications to minimize immunogenicity. Fractionated as well as single doses have been administered, marrow rescue has been used for highdose studies, and the targeted radionuclide has been selected from 131I, 90Y, 186Re, or the Auger emitting isotope, 125I. Internalizing and non-internalizing antibodies have also been evaluated. Most of the antibodies react with several tumor types, but we have chosen to categorize these studies according to site of primary disease.

Gastrointestinal Carcinomas Patients with gastrointestinal malignancies have been widely studied in Phase I trials. In general these patients have not been exposed to intensive chemotherapy from myelosuppressive drugs and do not have rapidly progressive disease, and thus they are relatively ideal patients for proof of concept and Phase I studies. A review of several RIT trials for treating gastrointestinal cancer revealed an extremely low response rate [41, 75–89]. Of almost 300 patients studied in these phase I trials, tumor responses included only one CR, 4 PR and although stable disease or minimal responses were not always reported, this may have been present in 10% of patients or more. Marrow toxicity invariably limited dose escalation, even in these patients with often relatively little prior myelosuppressive chemotherapy. Additional strategies will have to be implemented for RIT in solid tumor patients to achieve higher response rates. A potential complicating factor in several Phase I trials for colon cancer has been the use of anti-CEA antibodies that interact with circulating CEA antigen to alter the pharmacokinetics of the therapeutic moiety.

Targeted radionuclide therapy of cancer For example, in both NP-4 and COL-1 studies, complexes were detected within 5 min of injection, indicating that antibodies directed against circulating antigen are subject to variable pharmacokinetics related to the level of circulating antigen [77, 90]. In other anti-CEA antibody studies (e.g., c84.66 antibody), however, tumor burden was noted to alter the pharmacokinetics probably even more so than in patients when antibodies targeting non-shedding antigens were used [83].

Other CEA-Expressing Cancers Phase I studies with anti-CEA antibodies have also been carried out in patients with ovarian cancer and unrespectable or metastatic medullary thyroid cancer (MTC). Juweid et al. reported on a dose escalation trial in which 131 I-MN-14 was administered to 11 patients with advanced ovarian cancer, but a clinical response was seen in only one patient [77] Juweid reported a study with non-myeloablative doses of 131I-labeled NP-4 and MN-14 intact antibodies and their bivalent fragments in 17 patients with MTC [91]. Dosages of 131I ranged from 46 to 268 mCi, depending on whether a fragment was administered. Minor responses were observed in 5 of 11 evaluable patients, mostly with the F(ab’)2 fragments of both antibodies. Juweid also reported on the toxicity and therapeutic potential of high-dose 131I-MN-14 F(ab’)2 combined with ASCT in patients with rapidly progressing metastatic MTC [92]. Twelve patients were entered into the study and dose escalation was based on prescribed radiation doses to critical normal organs. The highest dose level reported was 12 Gy to critical organs and was not dose limiting. One patient had a partial remission for 1 year, another had a minor response for 3 months, and 10 patients had stabilization of disease lasting between 1 and 16 months. The authors of this study suggested that the anti-tumor responses seen in these patients with aggressive, rapidly progressing disease was encouraging and warranted further study.

Prostate Cancer CYT-356, Prostascint™, uses an antibody directed against a prostate specific antigen and has been studied extensively as an imaging agent [93]. This antibody has also been radiolabeled with 90Y for a phase I therapy study in prostate cancer patients. Twelve patients were studied, the MTD was below 12 mCi/m2, limited by marrow toxicity. The authors believed marrow toxicity to be related to extensive bony involvement and prior marrow irradiation to more than half the marrow. No objective responses were seen, but improvement in symptoms were reported.

John M. Pagel et al. KC4, is a pancarcinoma antibody directed against a high molecular weight membrane and cytoplasmic glycoprotein. In a dose escalation study, calcium disodium EDTA was administered for 72 h in conjunction with escalating doses of 90Y-KC4 to patients with refractory prostate cancer [94]. Again here, symptomatic improvement was reported, but at the low dose of 9 mCi/m2 severe marrow toxicity was observed. Meredith et al. has reported on several phase I and II trials using 131I-CC49 in prostate cancer patients [95, 96]. A phase II study with 75 mCi/m2 131I-CC49 showed symptomatic improvement in two thirds of patients but all patients developed human anti-mouse antibody (HAMA) within 4 weeks of therapy [95, 96]. A trial with a low-dose of cyclosporin and the dose of 131I-CC49 fractionated to 38 mCi/m2 at 15-day intervals was unsuccessful because HAMA still developed in the majority of patients. The single-dose approach was adapted, with the addition of alpha interferon and the higher 75 mCi/m2 131I dose. Again, no objective anti-tumor responses were seen, but there was substantial bone pain relief seen in several patients. Marrow toxicity was not related to the extent of bony involvement. In a high-dose therapy study, three patients were treated at the initial dose level of 100 mCi/m2 131I-CC49, followed by 13.2 Gy TBI given over 4 days, beginning 8 days following RIT. All three patients treated at the first dose level achieved objective tumor response [96].

Breast Cancer The radiosensitivity of breast cancer, and the results reported from the breast cancer trials so far suggest that RIT may play a clinical role, particularly with some of the additional strategies to improve response rates that are still under investigation. Although non-myeloablative doses have produced disappointing results, ASCT studies have shown that 100–300 Gy can be delivered to metastatic sites with non-toxic doses to normal organs [97]. L-6 is an antibody directed toward an antigen expressed on ∼50% breast adenocarcinomas, with biological activity that has been evaluated using 131I and [90]Y. Preliminary work by DeNardo et al. demonstrated the importance of administering unlabeled antibody prior to radiolabeled antibody to saturate the antigenic sites on normal tissues, in this case the vascular endothelium [98]. In a phase I study, 200 mg cold blocker was administered with 131I murine L-6. The cold blocker reduced lung activity from 19% to 4%, allowing the radiolabeled antibody to be visualized at the tumor sites. It appeared that a transient inflammatory reaction increased the delivery of subsequently administered radiolabeled antibody to tumor.

473 Three to 15 mg L-6 was administered with 10 mCi 131I per mg antibody and the MTD was 60 mCi/m2. One patient achieved a CR, but 40% of patients also developed a HAMA response [98]. In a multiple-dose protocol, ten heavily pretreated patients were treated with monthly injections of 131I-chimeric L-6. The MTD was 60 mCi/m2 twice limited by myelotoxicity. In five of the ten patients, clinically measurable tumor responses were seen [99]. In this study, serum levels of IL-2 receptors were measured and the increase in serum IL-2 was greatest in the patients who responded. Immunoglobulin developed in eight of ten patients at varying times, and did prevent further therapy doses in four patients. The authors speculated that the reason for success with the L-6 antibody is a combination of the increased vascular permeability from the unlabeled biologically active antibody with these relatively low doses of radiation, and the activated effector cell mechanism. On other multiple dose protocols, patients received G-CSF 7 to 20 days following infusion, or patients underwent immunopheresis using a goat anti-mouse antibody to reduce the non-bound circulating antibody [100]. A high dose study using 150 mCi/ m2 given at eight weekly intervals was initiated. Patients had PBSC reinfused when circulating radioactivity reached an acceptable level. Thrombocytopenia was successfully managed with this regimen. Multiple doses were limited by HAMA for the first two patients. The third patient was given cyclosporin which prevented HAMA formation even after three doses, and she showed evidence of tumor response [101]. A study now underway with 90Y-DOTApeptide-ChL6 appears to provide enhanced therapeutic index [102]. BrE-3 is an IgG1 antibody directed against the peptide epitope of the MUC-1 antigen, present on 95% of breast cancers that have been evaluated for RIT labeled with 90Y. DeNardo reported objective responses in three of six patients treated at single low doses. High tumor doses were estimated, however HAMA developed in five of six patients [103]. Schrier treated nine heavily treated patients with stage IV breast cancer with 90Y-labeled BrE-3 antibody [104]. Harvested autologous marrow or PBSC were reinfused at 15 days in conjunction with G-CSF. Fifteen and 20 mCi/m2 90Y was administered. Pharmacokinetic data indicated that the 90Y biodistribution was not identical to the 111In biodistribution. Six patients developed transient grade 4 marrow toxicity but all recovered and no other toxicities occurred. The 90Y conjugated with MX-DTPA dissociated from the antibody and deposited in the bone, causing the high incidence of marrow toxicity, bone biopsies confirmed this localization of 90Y in the bone. Based on 111In imaging, tumor doses 35–56 cGy/ mCi were reported. These values in terms of cGy per mCi

474 are higher than with other radioimmunoconjugates, but the total dose that could be administered was limited by the marrow toxicity. Four of eight evaluable patients achieved a PR, noted in lymph nodes, skin and marrow lesions, and one achieved a clinical CR. Because of immunogenicity, a human BrE was developed, and nine patients received increasing doses of 90Y-hBRe with peripheral blood stem cell support. Although the initial response rates are less encouraging, this study may define the second organ of toxicity [105]. CC49 is an antibody that targets the TAG-72 antigen, which is expressed in 90% of breast carcinomas. Mulligan studied nine patients with 177Lu-DOTA-CC49 (five of whom were breast cancer patients). Luticium-177 was considered an attractive alternative radionuclide with a lower energy beta emission and longer half life than 90Y [106]. However, 177Lu accumulated in the cells of the reticuloendothelial system and was retained there for a prolonged period. Images demonstrated the tumor uptake as well as activity in the bone marrow. This resulted in myelosuppression at low doses and the MTD was 15 mCi/m2. Reduction in the RES uptake would be required for this to become a useful radioimmunoconjugate. Nine breast cancer patients were also treated with 131 I-CC49 in conjunction with Thiotepa and total body radiation, followed by stem cell rescue. No life threatening toxicities were noted and two patients had a partial response by imaging criteria [107]. The biological response modifier alpha interferon is able to increase the antigenic expression of TAG-72 and increase antibody targeting in tumors [81]. Murray tested this in 15 patients with breast cancer, assessing TAG-72 expression and 131I-CC49 uptake [108]. After 3 days of alpha interferon, 3 million units daily, 10 or 20 mCi 131I-CC49 was administered and 48 h later a biopsy was taken and compared with a pre-study biopsy. The TAG-72 expression was increased by 45% following the alpha interferon (p < 0.05), and there was an apparent increased 131I-CC49 localization in tumor. However, because of intra- and inter-patient variability in percentage of tumor cells in the biopsy specimens as well as heterogeneity of TAG-72 expression, this was difficult to evaluate with certainty. Pharmacokinetics and biodistribution were changed with administration of the alpha interferon. RIT at the MTD with concurrent alpha interferon administration is under investigation and in a preliminary report, 1 of 15 patients had achieved a PR at the expense of increased marrow toxicity, from a more prolonged serum clearance at the higher doses. Wong et al. evaluated the high affinity, anti-CEA chimeric antibody, cT84.66 in 22 breast cancer patients, in a dose escalation trial with 90Y-DTPA. Dose limiting

Targeted radionuclide therapy of cancer marrow toxicity was observed at 22 mCi/m2, and a study with stem cell support is ongoing. Results are preliminary but appear encouraging in the six first patients studied [109].

Glioma The high-grade malignant gliomas (anaplastic astrocytomas and glioblastoma) have a very poor prognosis with current surgery, radiotherapy and chemotherapy approaches. The use of specific monoclonal antibodies labeled with a suitable isotope (131I or 90Y) represents an effective approach to reduce tumor regrowth. Because of the difficulty in interpreting diagnostic imaging scans, and the short survival time in these patients (median 10 months) survival has been used as an endpoint. Radiation, following surgical resection, offers a several month advantage in survival time, but has been limited by normal brain tolerance. Targeted radiation is therefore an attractive approach to increase radiation dose to tumor and potentially contribute to increased survival. Some investigators have injected the antibodies intravenously, or have tried to increase the tumor/background ratio with the avidin/biotin system. In several studies the labeled monoclonal antibodies were injected directly into the tumoral bed after surgery.

Systemic Injection Kalofonos reported a study with 131I-anti-epidermal growth factor receptor antibody (EGFr) [110]. Ten patients with recurrent gliomas received intravenous or intra-arterial administration of the radioimmunoconjugate and six showed clinical improvement. Brady treated 101 patients with high grade gliomas by intravenous or intra-arterial injections of 125I-labeled anti-epidermal growth factor receptor antibody, EGF-425 [111]. Patients with both primary and recurrent astrocytomas and glioblastoma multiforme were treated with an average total dose of 139 mCi 125I, administered divided into three weekly infusions. Brady reported 1 CR and 2 PR of short duration following 125I EGF-425 intravenously. The median survival in both groups was improved with these relatively radioresistent tumors. Fifteen of these patients had recurrent malignant astrocytomas and were treated with 25 to 130 mCi intra-arterially. The median survival was 8 ± 7 months [111]. Twenty-five patients with malignant astrocytomas were then treated in a Phase II adjunctive therapy trial [112]. Four to 6 weeks following surgical debulking (2) or biopsy (13), and external beam radiation, one to three doses of 125I-labeled antibody 425 was administered intravenously

John M. Pagel et al. or intra-arterially at 7–14 day intervals. Cumulative doses ranged from 40 to 224 mCi. HAMA did not develop in any of these patients. The 1 year survival was 60% with a projected median survival of 15.6 months.

Compartmental Injection Malignant gliomas tend to spread by local invasion rather than metastases. Thus intralesional, intra cavitary or intrathecal administration of the therapeutic radiolabeled antibody may be useful to deliver the therapeutic radiation directly to these solid tumors.

Intralesional One approach is to inject the radioimmunoconjugate through an Ommaya reservoir into the surgical cavity following surgical resection, or directly into a cystic tumor when the lesion has a predominantly cystic component. Here the antibody binding to antigen serves to prolong retention of the radiation at the target, while sparing normal brain tissue. Initial studies with 131I antibodies into the surgical cavity demonstrated the feasibility of an intralesional or intracavity approach following surgical resection. Riva et al. [113] reported their experience with intralesional administration of 131 I-labeled antibodies for treatment of both newly diagnosed and recurrent glioma. Anti-tenascin antibodies, BC-2 and BC-4 were radiolabeled with up to 65 mCi of 131 I, and injected into the resection cavity through a catheter. The radioactivity remained at the target site for an effective half time of 60 h and thus could deliver high doses to the tumor, on average 42,000 rad/cycle. Up to four cycles were given to 50 patients. Although HAMA developed in some patients, it did not interfere with tumor targeting in subsequent cycles. In all there were 3 CRs, 6 PRs, 11 tumor stabilizations and 19 tumor progressions recorded. Eleven patients with no radiological evidence of disease at the time of treatment remained disease free. In 26 patients the median time to progression was 3 months, and in the group of newly diagnosed patients who were treated, time to progression was 7 months. The median survival was 20 months; 17 months in patients with bulky disease, and 23 months in patients with minimal or microscopic disease. The median survival of these patients is usually 10 months; thus this study suggests an improved outcome for these patients. In addition, Riva has reported an improved survival in the patients with bulky glioblastoma, 30 months [114]. Another anti-tenascin antibody, 81C6, radiolabeled with up to 100 mCi 131I has been studied in patients with recurrent cystic gliomas and in patients with a surgically

475 created resection cavity from primary or metastatic brain tumor [115] High retention in the cysts with little systemic dissemination resulted in absorbed dose estimates of 127–703 Gy. No hematological or neurotoxicity was observed. In preliminary results, all five patients with recurrent cystic glioma appeared to benefit from the treatment with an increased survival. In the majority of patients with administration into the surgically created cavity, stabilization of disease was observed [116] Chimeric 81C6 carrying the alpha emitter 211At is also under study for injection into the surgical cavity of recurrent gliomas. Dose limiting toxicity has not yet been established [117]. ERIC-1 antibody was radiolabeled with up to 60 mCi 131 I and administered to nine patients with relapsed glioma who had a cyst or cavity following prior resection [118]. In two patients with cystic lesions, the need for aspiration was markedly reduced. The short range of the 131I beta particles delivers a high dose to only a rim of tissue approximately 1 mm thick, around the surgical cavity. In a study with 90Y-labeled ERIC-1, 15 patients were treated with up to 18 mCi and the cerebral edema that developed was managed successfully with dexamethasone [119]. Dose estimates from this treatment ranged from 55 to 351 Gy, depending on whether complete binding on antibody occurred or not. Again in this study, the two patients with cystic tumors required less frequent aspirations. While the overall benefit of the RIT could not be easily determined from this group of patients, median survival was 6 months from treatment.

Intrathecal In neoplastic meningitis, external beam therapy is limited by dose to the normal nervous system. The average survival of patients with neoplastic meningitis is 3 months from diagnosis. Tumor cells are found floating freely in the CSF or in sheets lining the meninges. Thus an intrathecal route of administration bypasses the problems of access to tumor antigen that are present when radioimmunoconjugates are administered systemically. Papanastassiou treated patients with diffuse leptomeningeal deposits with intrathecal radiolabeled antibodies administered through an Ommaya reservoir. Patients included those with carcinomatous meningitis (n = 7), primitive neuroectodermal tumors (n = 18), and CNS leukemia (n = 22), with the antibody depending on the disease [120]. The radioimmunoconjugate leaves the intrathecal space relatively quickly, peak blood flow levels of 6–48% of the injected dose reached the vascular system at 24–56 h following injection. Dosage of 131I ranged from 17 to 90 mCi on 1.7 to 9 mg antibody.

476 Transient aseptic mengitis was common following intrathecal RIT, occurring in approximately 60% of patients. Occasional patients developed seizures. No long term sequelae have been observed. Significant myelosuppression occurred in several patients who received more than 54 mCi. Response was difficult to assess and was assessed by clearance of cells from the CNS and clinical improvement. Papanastassiou reported a 33% overall response rate. This was poorest in the carcinoma patients. The mean time to relapse in 37% of PNET patients was 10 months, In patients with leukemia, marked responses of clearance of leukemic cells from the CSF were observed, but for only 4–8 weeks. Brown et al. reported on patients with leptomeningeal disease and brain tumor resection cavities with subarachnoid communication. Patients with anaplastic gliomas, ependyomas, medullablastoma and anaplastic astrocytoma received a single dose of up to 100 mCi of 131I 81C6 antibody administered intrathecally through an Omaya reservoir [121] No nonhematological toxicity occurred and the MTD was 80 mCi, limited by hematological toxicity. Ten of 24 evaluable patient developed HAMA, but in two patients who were retreated, there was no alteration in biodistribution of radiolabeled antibody. In 31 patients with malignant gliomas, one patient showed a radiographic PR and disease stabilization occurred in 42% of patients, representing an apparent increased survival rate although this was difficult to evaluate. Bigner used the (Fab’)2 fragment of Mel-14, antimelanoma antibody directed against anti-proteoglycan chondroitin sulfate associated protein, to treat a patient with a brain metastasis from melanoma [116]. The patient received Mel-14 F(ab’)2 labeled with 37 mCi 131I injected into the surgical resection cavity, and achieved a complete local response. Eleven patients were treated with up to 80 mCi 131I-Mel-14 (Fab’)2 and 81C6 intrathecally via lumbar puncture. They had melanoma (n = 8), melanosis (n = 1), oligodendroglioma (n = 1) and glioblastoma (n = 1). One patient at 80 mCi had hematological toxicity. After treatment, three patients had complete CSF responses, two had partial responses radiologically and survival ranged from 1 to 11 months, an apparent improvement on the 3 month average survival.

Peptide-Based Radiopharmaceuticals After the detection of peptide receptor expression by tumors, somatostatin analogues became the first radiolabeled regulatory peptides used for specific tumor targeting.

Targeted radionuclide therapy of cancer Peptides used for tumor targeting show several advantages over antibodies: peptides are small and show rapid diffusion into (target) tissues resulting in rapid pharmacokinetics. Their fast blood clearance could lead to high tumor-to-background ratios shortly after administration of the radiopeptide [122–126]. As exemplified by somatostatin, analogs of the peptide that are stable in blood show a better tumor uptake than the natural peptides although both are degraded after internalization [124]. To prevent internalized radionuclides from externalization, a so-called residualizing label consisting of a chelator linked to the peptide and a radiometal such as 111In, 177 Lu or 90Y is used. These radiometal/chelator complexes, are retained in the lysosomes and thus they are trapped inside the cell [126]. To date, the 111In-labeled somatostatin analog octreotide (OctreoScan®) is the most successful radiopeptide for tumor imaging and has been the first to be approved for diagnostic use. It is considered the diagnostic gold-standard in several types of gastrointestinal and other neuroendocrine tumors (NET) and plays a crucial role in the diagnosis and follow-up of patients with these tumors [127–129]. Furthermore, somatostatin receptor scintigraphy (SRS) also contributes significantly to decision making in the management of patients with NET [130–133]. Currently, octreotide-based somatostatin analogs labeled with the ß-emitters 90Y or 177Lu are undergoing clinical trials for peptide receptor radionuclide therapy (PRRT) of NET. Other peptides for tumor targeting in development or undergoing clinical trials are CCK2 receptor-binding peptides, bombesin, substance P, neurotensin, glucagonlike peptide-1, and RGD-peptides for imaging of ανβ3 expression in tumors [126, 133–136].

Regulatory Peptides and their Receptors Regulatory peptides are potent small messenger molecules binding to specific receptors. Usually, they are not larger than 30–40 amino acids and are able to rapidly extravasate and penetrate tissues. Regulatory peptides are synthesized mainly in the brain and gastrointestinal tract. They may also play a role in the peripheral nervous system or in the immune system [125, 126, 137]. Due to their hydrophilicity, they do not cross the blood– brain-barrier in either direction. Therefore, the central nervous system and the periphery (e.g., the gastrointestinal tract) form two independent regulatory systems which thus use the same messenger molecules without any danger of confusing interaction of both. All regulatory peptides bind to and act through transmembrane G protein-coupled receptors. The signal transduction of

John M. Pagel et al. these receptors is triggered by the binding of the respective peptide to the extracellular domain of its receptor which will, in turn, activate the intracellular guanine nucleotide binding proteins (G proteins). An important inactivation pathway is the internalization of the ligand– receptor complex, physiologically leading to either degradation or recycling of the internalized receptor to the cell surface [125]. This inactivation pathway plays a crucial role for the use of radiopeptides in scintigraphy and PRRT. Regulatory peptides influence many aspects of mammalian physiology. Their role as flexible messenger molecules requires rapid degradation in the blood to prevent prolonged action of secreted peptides. Ubiquitously occurring peptidases are responsible for rapid deactivation of regulatory peptides. For radiopeptides, rapid degradation in the circulation or in other tissues would be a major obstacle for reaching the target. Thus, peptides used for tumor targeting should be metabolically stable.

Radiolabeling of Peptides The somatostatin analog octreotide was the first peptide used for the scintigraphic detection of NET. Somatostatin itself has a very short half-life in serum. Thus, octreotide with its higher stability was used for scintigraphic purposes. As the easiest method of radiolabeling peptides is radioiodination using 125I, 123I, or 131I [138] a tyrosyl moiety was introduced into the stabilized peptide in position 3, replacing phenylalanine in the natural sequence. Iodination of the tyrosine residue is relatively easy. Thus, 123 I-Tyr3-octreotide was the first radiopeptide used for imaging of NET in a patient [123]. If an amino-acid is to be replaced by tyrosine for radioiodination, it should not be involved in receptor-binding or activation [126]. However, after internalization, peptides will undergo proteolytic lysosomal degradation. Mono- or di-iodinated tyrosine will be formed and these metabolites will rapidly be excreted from the cells. Mono- or di-iodinated tyrosine may then be deiodinated, preferably in the liver. Excretion of the radioiodine label from the targeted cells is the major drawback of the use of this so-called “noninternalizing” radiolabel in peptide receptor targeting. Thus, the tumor-to-background ratios that can be obtained using this approach are suboptimal and the sensitivity of 131I-octreotide for detecting somatostatin receptor expressing tumors was inferior to that of pentetreotide labeled with the radiometal 111In [124]. In contrast to radioiodinated amino acids, following internationalization by the target cells, radiometal chelator conjugated amino acids are trapped in the lysosomes

477 and thus are retained inside the cells. The most widely used radiometal chelators are DTPA (diethylene-triamin e-pentaacetate) and DOTA (tetra-azacyclododecaneN,N’,N”,N” ’-tetra-acetate). For diagnostic purposes, the DTPA can stably chelate 111In. This chelator is used in the commercially available somatostatin analog OctreoScan®. However, chelation of other radiometals by DTPA used for therapeutic purposes is not sufficiently stable in vivo. In contrast to DTPA, the cyclic chelator DOTA demonstrates sufficient stability for a broad range of other metallic ions, such as 90Y, 177Lu, 153 Sm, 212/213Bi, or 212Pb [139, 140]. Therefore, it is the most frequently used chelator in PRRT. Radiometal chelators such as DTPA or DOTA may be covalently linked to the N-terminus of a given peptide or to the ε-amino group of a lysine moiety. If the N-terminus is essential for receptor-binding or internalization, a lysine moiety within the primary structure has to be used for linking the chelator. Even if lysine is already part of the peptide, it may be crucial for binding or internalization. Thus, introduction of an additional lysine may be applied to conjugate the chelator to the peptide. Thus, the problem of development of stable analogs of (regulatory) peptides not only consists of the labeling alone, but also of changing the amino acid sequence to obtain a site for linking the chelator without decreasing the receptor binding affinity. Furthermore, after modification some peptides will still bind, but will no longer internalize any more. Internalization of the peptide/receptor complex is considered a crucial step in the process of in vivo receptor targeting with peptides [141], although it has recently been shown that noninternalizing antagonists could also lead to efficient receptor targeting [142]. As stated above, peptides for receptor targeting should be metabolically stable. In contrast to other drugs, stabilization by binding to serum proteins is not helpful in this context – due to higher blood retention and liver clearance. Although high serum stability is warranted, high tumor-to-background ratios will only be obtained if there is rapid clearance from the blood. Thus, amino acids may be replaced by D-isomers so that serum peptidases will not affect these modified peptides. Other typical modifications for stabilization are the use of unnatural amino acids, N-terminal acetylation, or C-terminal amidation [143]. Only after internalization, lysosomal degradation will occur. For rapid renal excretion, hydrophilicity is required. Lipophilic peptides will show a higher background activity possibly masking tumors in highly perfused organs such as the lungs or the liver in early images. Furthermore, higher liver extraction will even increase liver activity which is not warranted in abdominal tumors.

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Clinical Experience with Somatostatin Analogs As the physical half-life of 111In is 2.8 days, whole body images should be acquired not only 4h but also 24-h after injection of 200 MBq of the 111In-radiopeptide. Due to continuous clearance from non-target tissues, the tumor-to-background ratio will increase over time so that the 24-h images show a higher diagnostic yield than the 4-h images. Twenty-four-hour scans show a markedly higher sensitivity [144]. However, image acquisition at both time points might help to differentiate specific from non-specific uptake. Although 99mTc is the preferred radionuclide in diagnostic nuclear medicine (ideal γ-energy of 140 keV, high photon flux, relatively low radiation burden, unlimited availability), 111In is also quite suitable. Obviously, gamma cameras should be equipped with medium energy collimators, because of the higher γ-energy of 111In in comparison to 99mTc. The scanning speed should not exceed 5–6 cm per minute to obtain high quality images with high count rates. Additional three-dimensional SPECT images (single photon emission computed tomography) may further increase the sensitivity and should thus be acquired routinely [144, 145]. If bowel activity obscures abdominal tumor manifestations, additional 48-h scanning can be performed, possibly combined with the use of laxatives to accelerate the passage of intestinal radioactivity. Organs with physiologic octreotide-uptake are kidney, liver (later gall bladder and bowel because of biliary excretion), spleen, thyroid, and pituitary [126]. Lymphocytes (and activated leukocytes) and epitheloid cells in granulomas express somatostatin receptors so that scar tissue and granulomas could show up in SRS [146, 147]. Lymphocytes are also responsible for the splenic uptake. After irradiation of the mediastinum, “non-specific” uptake may also be observed. However, this uptake is caused by receptors on accumulated leukocytes and epitheloid cells [148]. Cold somatostatin may also decrease tumor uptake thus possibly leading to false-negative results. If a patient is on medication with somatostatin for inhibition of tumor growth [149], receptors may be blocked by cold somatostatin so that the splenic uptake drops in comparison to the liver uptake. This is an important finding in the evaluation of somatostatin scans [132, 145]. Octreotide, the metabolically stable somatostatin analog, has high affinity for the sst2 and sst5 receptor subtypes, while it has no affinity for subtype 1 and 4 and affinity to subtype 3 is low [125, 150]. Thus, clinical somatostatin receptor scintigraphy with 111In-DTPAoctreotide mainly visualizes sst2 and sst5 receptor-posi-

Targeted radionuclide therapy of cancer tive tumors, but not sst1 and sst4 receptor-positive tumors. A broad variety of tumors express the somatostatin receptor subtypes 2 and 5, which includes most neuroendocrine tumors, lung cancer, breast cancer, differentiated thyroid cancers, but also meningiomas, well-differentiated astrocytomas, pituitary tumors, or malignant lymphomas and several others [124, 151–154]. SRS plays a major role in the diagnosis of neuroendocrine tumors and their metastases. In some gastrointestinal NET, it is considered the diagnostic gold-standard [127–129]. In endocrine tumors of the pancreas, SRS is positive in 50–90% of the cases, dependent on the tumor type. As many insulinomas do not express the subtypes 2 and 5 of the somatostatin receptor, the sensitivity of SRS is only 40–60% [127]. On the other hand, virtually all gastrinomas express somatostatin receptors in a high density and indeed, SRS has a sensitivity of up to 90% in these tumors. Carcinoid tumors of the intestine may also be detected using SRS with sensitivities >80% in comparison to only ∼50% for morphologic imaging modalities. Even if CT, MRI, and ultrasonography are combined, they will not reach the sensitivity of SRS in patients with gastrointestinal NET [127, 145]. Only endoscopic ultrasonography will perform better in the detection of pancreatic NET. Figure 1 shows an example of a SRS in a patient with an ileal carcinoid. The sensitivity of SRS is mainly dependent on the expression of the sstr2 and 5 subtypes of the somatostatin receptor. Thus, insulinomas will be visualized in only a limited number of patients with a sensitivity around 50%. Gastrinomas will be visualized with higher sensitivities of at least 60–90%, dependent on their size [155–157]. It has been shown that the scanning technique has a strong influence on the results of SRS in pancreatic tumors as they may be small and undetectable on planar images [145]. Thus, using SPECT, the sensitivity can be expected to be closer to 90% than to 60%. Endoscopic ultrasonography is the only imaging technique that shows results equivalent to SRS or even better [127]. Active and inactive carcinoid tumors of the gastrointestinal tract may be detected by SRS with high sensitivities exceeding 80% [124, 126, 127, 145] Especially metastatic disease may even be detected by SRS when morphologic imaging modalities fail to show tumors in unexpected locations or outside their anatomical range. Furthermore, SRS can provide information about the somatostatin receptor status of tumors in vivo. This is relevant for the decision whether treatment with cold somatostatin is potentially useful or not. In summary, in patients with carcinoid tumors of the intestine, SRS should be used first in the diagnostic process together with abdominal ultrasound [128, 129].

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Figure 1. SRS (24-h scans) of a patient with a carcinoid of the ileum with metastases mainly to liver and bone. Anterior view on the left, posterior view on the right. Uptake in the liver metastases is high, uptake in the tumor in the left shoulder is comparable to that in normal liver tissue

Other Somatostatin Analogs Besides octreotide, other somatostatin analogs have been approved for clinical use or are under clinical evaluation. Depreotide, a 99mTc-labeled somatostatin analog (Neotect®) has been registered in the US for the characterization of solitary pulmonary nodules. Depreotide is a synthetic peptide with a cyclic, 6 amino-acid sequence [158, 159]. It is a cost-effective alternative to FDG-PET. In comparison to 111In-labeled somatostatin analogs, the shorter physical half-life of 99mTc allows a simplification of the imaging protocol. A higher activity is injected (700–800 MBq) and diagnostic images can already be obtained 90–240 min post injection [158, 160] including SPECT. In comparison to CT-guided biopsy, 99mTc-depreotide shows a comparable sensitivity while the specificity is lower [159], due to uptake into inflammatory tissue, such as granulomas. The specificity of 99mTc-depreotide scintigraphy is clearly higher than that of CT [161]. Another interesting 99mTc-labeled somatostatin analog is 99mTc-HYNIC-D-Phe1-Tyr3-octreotide. It shows favorable results as compared to the commercially

available OctreoScan® (111In-DTPA-D-Phe1-octreotide). Further clinical studies are necessary to finally determine whether this promising compound may be able to replace 111In-labeled somatostatin analogs in clinical routine [162, 163]. Lanreotide is an 111In-labeled somatostatin analog that may also be used for cold somatostatin therapy as an alternative to cold octreotide (Sandostatin®) [149]. Although it was reported that lanreotide has a higher affinity for sstr3 as compared octreotide [164], these data could not be confirmed by others [165]. As lanreotide is more lipophilic than octroetide, tumor-to-background ratios may be lower than with octreotide [166] Lanreotide does not show clear advantages over octreotide.

Other Peptides for Tumor Targeting Gastrin Although SRS is a very potent tool in the diagnosis of NET, it is of only limited value in patients with medullary thyroid carcinoma (MTC) due to limited sstr2

480 expression of these tumors. Given the outstanding sensitivity of the pentagastrin test in the detection of persisting or recurrent MTC, the expression of the corresponding receptor type in human MTC was postulated [167–169]. Indeed, more then 90% of medullary thyroid carcinomas (as well as other tumors) were found to express CCK2 receptors [170, 171]. After initial evaluation of several radioiodinated gastrin-derived compounds, a DTPA-derivatized compound with the residualizing 111 In label was developed [126]. First clinical studies show that 111In-DTPA-D-Glu1-Minigastrin is able to

Targeted radionuclide therapy of cancer demonstrate metastatic MTC with the highest sensitivity in comparison to PET, CT, and SRS [172]. Gastrin receptors are also expressed in many gastrointestinal NET [173]. A study comparing SRS with gastrin receptor scintigaphy (GRS) showed that GRS was positive in about half of the patients in whom octreotide uptake was missing [172]. Furthermore, more than 20% of all patients had higher uptake of gastrin than of octreotide into the tumor lesions. Figure 2 shows a gastrin scan in comparison to a somatostatin scan in a patient with a bronchial carcinoid.

Figure 2. Patient with a bronchial carcinoid with metastases to bone, liver, and mediastinum. Gastrin scan on the left, somatostatin scan on the right. Both anterior view, obtained 24 h after injection

John M. Pagel et al.

Vasoactive Intestinal Peptide (VIP) VIP receptors are expressed by most of human epithelial cancers lacking expression of somatostatin receptors or of the subtypes SSTR 2 or 5 detectable by SRS [125]. Thus, VIP receptor scintigraphy was thought to have great potential for diagnosis and possibly even treatment of a wide range of tumors with high incidence, especially colorectal cancer, gastric cancer, and exocrine pancreatic cancers. In comparison to somatostatin analogs, biodistribution of VIP shows high pulmonary uptake due to high levels of physiologic VIP receptor expression in the lungs. Abdominal uptake is lower in comparison to octreotide [174]. In clinical studies, Virgolini and co-workers showed that primary tumors and metastases of intestinal adenocarcinomas as well as NET could be detected with 123I-labeled VIP [175, 176]. However, in a study of Hessenius et al. no adequate 123IVIP uptake into the tumors was observed [177]. These findings were confirmed by autoradiographic studies of resected tumors demonstrating lower VIP receptor expression in the tumors in comparison to physiologic VIP receptor expression in the surrounding tissues.

Neurotensin Pre-clinical data suggest that 111In-DTPA and 111In-DOTA labeled neurotensin analogs may be of high value in the management of patients with exocrine pancreatic cancer [134]. Buchegger et al. have studied the use of Tc-99 m labeled neurotensin analog NT-XI in patients with ductal pancreatic adenocarcinoma. Tumor was only visualized in one out of four patients [178].

Bombesin Receptors for bombesin are expressed on colon cancer, glioblastoma, prostate cancer, and SCLC [126]. Radiolabeled bombesin potentially could be used to visualize prostate cancer. After the development of radiometal labeled metabolically stable bombesin analogs with high receptor affinity, clinical studies are currently undertaken [179, 180]. A new 99mTc labeled bombesin analog (demobesin 1) showed very favorable results in a human prostate cancer mouse model with high prolonged tumor uptake [181]. Clinical evaluation of this compound will have to shows its value.

Glucagon-like Peptide-1 (GLP-1) GLP-1 is an incretin hormone that induces postprandial insulin secretion from pancreatic ß-cells. Thus, receptors for this peptide are expressed by insulinomas (deriving

481 from pancreatic ß-cells), but also by other NET like gastrointestinal carcinoids [173]. For scintigraphic imaging, GLP-1 and its analogs exendin 3 and exendin 4 seem to have potential [135]. Especially for diagnosis and treatment of insulinoma, GLP-1 might offer new possibilities. Exendin-4 was modified C-terminally and conjugated with DTPA. This Exendin-4 analog labeled with 111 In-had extremely high uptake in insulinoma lesions in mice and even showed therapeutic efficacy when higher activity doses were administered [182, 183]. RGD peptides: RGD peptides are a particularly interesting entity of tumor-targeting agents. RGD-peptides contain the amino acid sequence Arg-Gly-Asp and bind to ανß3 integrin. ανß3 integrin is a heterodimeric transmembrane proteins involved in cell-cell interaction and mediating cellular adhesion to extracellular matrix proteins such as vitronectin, fibronectin, a.o. [184, 185] RGD peptides inhibit neoangiogenesis in growing tumors [186]. Specific tumor targeting of radiolabeled RGD peptides could be demonstrated in several animal models for melanoma, breast cancer, osteosarcoma, pancreatic tumors, and ovarian cancer [136, 187–190]. RGD peptides clearing via kidney instead of hepatobiliary secretion have been developed. These peptides were labeled with 99mTc-HYNIC and 111In-DOTA for tumor imaging and evaluation of biokinetics in a mouse model of a human ovarian cancer xenograft. Target-tobackground ratios as high as 92:1 and 26:1 were achieved using this model [136]. Furthermore, the 90 Y-labeled compound showed inhibition of tumor progression in an in vivo mouse model. It has been pointed out that RGD peptides seem to be an interesting more general approach to the diagnosis and treatment of a variety of tumors as neoangiogenesis is not limited to one specific tumor entity only. However, one has to keep in mind that the most effective targeting with radiolabeled RGD peptides has been demonstrated in mouse/ tumor models in which the tumor cells themselves express the ανß3 receptor on their cell surface.

Peptide Receptor Radionuclide Therapy (PRRT) For PRRT, radionuclides with high cytotoxic potential that can be stably chelated are used. The ß-emitter 90Y has been the first radionuclide to be applied for PRRT in humans. Alternative radionuclides are the ß-emitter 177Lu and the auger-electron emitter 111In (which is usually used at lower activities for diagnostic purposes because of its γ-emission). Other interesting candidates are α-emitters such as 213/212Bi, or 211At. However, no stable and secure labels with α-emitters are available for clinical application in PRRT at the moment [126, 191, 192].

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So far, most experience with PRRT is based on the use of 90Y and 177Lu-labeled somatostatin analogs. Dependent on the inclusion criteria, these studies show that the rate of minor/partial responses is between 10–35%, while the rate of stabilizations of previously progressive disease is higher. Interestingly, some patients show a response several months after treatment [193–202]. There is evidence that application of the same cumulative activity results in a better therapeutic response if the number of therapeutic cycles is lower [198]. In general, PRRT is very well tolerated and only a limited number of patients shows relevant haematotoxicity above grade 3. Nephrotoxicity is one of the major problems of this therapeutic concept [203, 204], but may be reduced by co-infusion of basic amino acids [205, 206]. In comparison to the toxicity associated with chemotherapy regimens for NET [207], PRRT is tolerated extremely well by the patients while the overall response rates seem to be higher. As the therapeutic success with 111In-labeled octreotide is rather low, 90Y was preferred as a radionuclide [208–211]. The data on the use of 177Lu labeled somatostatin analogs are extremely promising as the rate of complete and partial remissions rises to over 50% in previously progressive patients. Thus, 177Lu-labeled somatostatin analogs seem to be a real progress in the treatment of NET [212].

include restricted antigen expression on tumor cells, the small fraction of the cardiac output which reaches the tumor (it is estimated that it takes 400 h for all the blood to pass through a 1 g tumor), and poor vascular permeability because of vascular spasm and high interstitial pressure from decreased lymphatic drainage that results in a high pressure gradient compromising transport towards the center [25]. Therefore several challenges associated with RIT exist that limit the radiation absorbed dose, and thus limit the therapeutic ratio. The dose is inadequate because of poor penetration of the antibody for reasons just mentioned, and the amount of radioactivity that can be injected is limited because of marrow toxicity. Although dose fractionation is used in chemotherapy and with external beam radiation therapy, the development of HAMA precluded multiple doses on antibody, particularly with murine antibody. RIT may be most effective for small tumors which are more vascular, but further work is still required to identify the role of RIT in the management of cancer. In order to increase the radiation absorbed dose to the tumor, one can approach the issue of the therapeutic ratio by either increasing the dose intensity or increasing the radiation sensitivity of the tumor, or alternately reducing the toxicity and thereby allowing higher doses of radioactivity to be administered (Table 4).

Strategies to Improve Outcome of RIT

Table 4. Strategies to improve outcome of RIT

Targeting radiolabeled Abs and peptides to malignant cells has led to the establishment of noteworthy principles and encouraging results, yet obstacles still remain that must be overcome to achieve optimal targeting and elimination of cancer cells by radioimmunoconjugates. For example, the above review indicates clearly that a single dose of conventionally radiolabeled murine monoclonal antibody administered at the MTD will not provide sufficient radiation to tumor to be effective as therapy for patients with solid tumors. In Phase I studies with radiolabeled monoclonal antibodies, bone marrow toxicity has been dose limiting and few complete responses have been seen, even at the MTD. An important reason for the poor results with systemic therapy using radiolabeled antibodies has been the low accretion of antibody in tumors and the fact that antibodies are not ideal carriers of radiation to a tumor target. In general, less than 0.01% of the injected dose localizes to each gram of tumor. Explanations for this low accretion

Increasing dose intensity

Increase radioactivity administered Single high dose Reduce immunogenicity to enable multiple dosing Improve the effectiveness of the administered dose Increase tumor localization Compartmental administration Intraperitoneal Intra-lesional Intrathecal Increasing vascular permeability Hyperthermia External beam radiation Interleukin-2 Integrin antagonists Biological response modifiers Increasing the dose rate Smaller molecules Pretargeting

Increasing radiation sensitivity

Chemotherapy agents Halogenated pyrimidines

Reducing the exposure Dose fractionation to normal tissues Immunoadsorption columns Pretargeting

John M. Pagel et al.

Increasing Dose Intensity Increase Radioactivity Administered The initial approach to increase the dose intensity was to administer a single dose of more radioactivity. The dose limiting toxicity is hematological and can be managed with transfusions, cytokines and autologous marrow or peripheral blood stem cell rescue, with reinfusion when the total body radioactivity is at low levels, usually within 10 days. Blumenthal showed that IL-1 and GM-CSF can allow increased doses of radioactivity to be administered in mice [213]. The addition of hematopoietic growth factors has aided the recovery of patients undergoing these procedures [214]. In one study, 90 mCi/m2 of 186Re was the MTD for a pancarcinoma antibody, and with PBSC rescue, doses up to 300 mCi/m2, were used, which was not yet the MTD [215]. Two of three patients with ovarian cancer treated with peripheral blood HCT achieved a PR, whereas there were no responses at non-myeloabaltive doses [216]. Similarly, 300 mCi/m2 of 131I-CC49 antibody was the MTD with marrow rescue [82], compared with 75 mCi/m2 of 131I for this antibody without marrow rescue. This approach has increased the MTD, however, but has not resulted in significantly increased response rates, and thus additional tactics to achieve tumor regression remain necessary. The ability to administer more than one dose of murine monoclonal antibody has been limited by HAMA [96, 217–219]. Immunosuppressive agents have been investigated to reduce HAMA. Lederman et al. [79] and Lane et al. [78] administered Cyclosporin A to suppress the development of HAMA in studies involving radiolabeled anti-CEA murine monoclonal antibodies with modest success. While this was successful in suppressing HAMA to a F(ab’)2 fragment, this approach has not been universally successful with an intact antibody [220]. Low-dose cyclosporin, as used by Meredith with a highly immunogenic antibody, was unable to significantly reduce HAMA following murine CC49 delivery [95]. Thus, cyclosporin may have some efficacy in reducing immunogenicity of murine antibodies in patients, but does not appear to be sufficient to permit administration of multiple doses in all patients. With modern genetic engineering, chimeric antibodies have been produced to in an attempt to overcome HAMA [219]. The immunogenicity of the chimeric antibodies in generally less than murine antibodies, but has not been sufficiently reduced to uniformly administer multiple dose of RIT [41, 80]. Several humanized antibodies, with only the hypervariable region of the antibody molecule being murine, have now been studied

483 in clinical trials and have established that repetitive doses of human monoclonal antibodies can be administered without evidence of alloimmunization [221]. Thus, it appears that the problem of HAMA may be solved with human antibodies and dose fractionation may be becoming more feasible.

Improving the Effect of the Administered dose Increasing Tumor Localization Because of the physical limitation of accessibility of antibodies to tumors, strategies have been developed to increase tumor localization. Initially these included changing the labeling procedures, changing the radioisotope, increasing the mass amount of antibody and using an intra-arterial administration, none of which had a significant impact. More recently other approaches have been more successful.

Compartmental Administration Intraperitoneal administration of the immunoconjugates for RIT was shown to be feasible, relatively safe, and has demonstrated anti-tumor effects as direct tumor cell exposure results in improved antibody–antigen binding [222]. Several trials have been undertaken in patients with ovarian cancer using intraperitoneal administration. 131 I-, 90Y-, 186Re- and 177Lu-labeled antibodies have been used for this purpose [223–228]. Encouraging tumor responses were observed in patients with Stage III ovarian carcinoma who had tumor nodules less than 2 cm in diameter. Stewart initially reported 5/21 PR following delivery of 131I-OC125 antibodies [228]. However, another study with this radioimmunoconjugate showed no benefit [229]. Following intraperitoneal 186Re-labeled NR-LU-10, 5 of 12 patients with minimal ovarian cancer achieved a PR [226]. The MTD following intraperitoneal infusion of this radioimmunoconjugate, 150 mCi/m2 was higher than following intravenous infusion (90 mCi/m2) because of the reduced blood radioactivity accounting for less marrow exposure [76]. Kavanagh reported antitumor effects in patients treated with 90Y-B72.3, and increased tolerance to 90Y with administration of EDTA [230]. Iodine-131-DOTA-CC49 has also been successful in achieving tumor responses in patients with <1 cm nodules or extended progression-free survival in patients with occult disease or microscopic disease [223]. 131I MOV-18 was similarly shown to improve survival in minimal disease [231]. Hird reported anti-tumor activity following 90Y-HFMG but marrow toxicity limited the administered dose to

484 <30 mCi, even with the addition of intravenous EDTA to chelate the unbound 90Y [225], and in patients with subclinical disease there was a prolongation of survival following one IP dose of 90Y-HFMG [232]. Kosmos reported that immunoglobulin against the chelate precluded more than one treatment using a DOTA chelate to 90Y [233]. In a group of 21 patients who had achieved CR following surgery and conventional chemotherapy, one administration of intraperitoneal HMFG1 monoclonal antibody labeled with 18 mCi/m2 90Y improved survival. The median survival had not been reached with a maximum follow-up of 12 years and survival at greater than 10 years was 78% [224]. Riva reported results in patients with peritoneal gastrointestinal carcinomatosis who received 100 mCi 131 I-FO23C5 via the intraperitoneal and intravenous route [114]. Therapy was administered in conjunction with Cyclosporin A every 3 months with up to four injections. Three complete and six partial responses were observed in 34 patients. Seventeen patients received alpha-interferon to increase the expression of CEA, with an increased the response rate to 59% compared with 29% without alpha-interferon.

Targeted radionuclide therapy of cancer endothelial cells, either directly or via the activated lymphocytes [214]. DeNardo et al. reported that modified IL-2, PEGIL-2, administered before radiolabeled antibody enhanced antibody localization by a factor of two in mice by increasing vascular permeability to the antibody [240]. DeNardo also showed the rIL-2 increased the response rate in xenografts treated with 67Cu-Lym-1 by about 20% [241]. In a pre-clinical study, when IL-2 was conjugated to an anti-CEA antibody, ZCE025, Nakamura found that the cytokine function of IL-2 was destroyed, but that vascular permeability specific to the tumor was increased [242]. Epstein used IL-2 conjugated to TV-1, an antibody to fibronectin that targets the basement membrane and causes increased vascular permeability [243]. Followed by a radioimmunoconjugate directed against a tumor-associated antigen, this showed three- to fourfold increased tumor targeting. Increasing the vascular permeability of newly formed vessels also appears to increase antibody accumulation. DeNardo et al. used RGB pentapeptide, which is a αvβ3 integrin antagonist and showed a transient increase of 45–50% deposition of 111In-ChL6 after 24 h in a xenograft study [244].

Biological Response Modifiers Increasing Vascular Permeability RIT may be most effective for small tumors that are more vascular, but manipulations to improve tumor blood flow to larger tumors may be worthwhile. The influence of vascular permeability on antibody uptake and distribution in tumors has been well documented [234]. Studies to increase vascular flow or permeability at the tumor using hyperthermia have been carried out by Stickney et al. [235]. Hyperthermia reduces interstitial fluid pressure and may improve tumor-associated antigen expression. Local hyperthermia can also exert direct cytotoxic effects, particularly on radioresistant and hypoxic cells which are nutritionally deprived and acutely acidic, and also cells in the S phase of the cell cycle. The degree of cell killing depends on the degree and duration of hyperthermia. The results of the preclinical studies to assess vascular permeability from external beam radiation prior to administering immunoconjugate have varied with tumor types and location [236, 237]. A three-fold increase in antibody uptake was reported when antibody was administered 1 day following low dose external beam therapy in patients with hepatoma, without affecting normal liver uptake [96]. Combined RIT/external beam therapy clinical studies are underway for both hepatic tumors [238] and head and neck cancer [239]. Interleukin-2 alone, modified with polyethylene glycol or conjugated with antibody augments vascular permeability and antibody uptake in tumor by a reaction with

IL-2 and alpha interferon appear to be successful in upregulating the antigen expression of CEA and TAG72. However, definitive results on the work with alpha interferon and with IL-2 in clinical trials have yet to be published. In 1990, Murray et al. showed altered biodistribution and an improved relative tumor uptake of 111 In-anti-melanoma antibody in patients who had received alpha-interferon [108]. In other clinical studies evaluating the effects of alpha interferon, Greiner et al. showed increased serum levels of TAG-72 and CEA antigen [245]. Greiner et al. also demonstrated increased levels of both antigens in ascites cells following intraperitoneal injection of gamma interferon to patients with ovarian cancer and increasing targeting in tumors, but whether this will translate into increased tumor response rate is still under investigation [246]. Increased response rates have been noted in patients with breast cancer receiving alpha interferon in conjunction with 131I-labeled CC49 RIT, and IL-2 have been studied further [81].

Increasing the Dose Rate Increasing the dose rate may be able to be achieved by using radiolabeled antibody fragments or small molecules which penetrate the tumor more rapidly. Early preclinical studies supported the theory that total dose delivered may be of primary importance. Thus, the belief that greater exposure of tumor to radioactivity as determined by area

John M. Pagel et al. under the curve led to the acceptance of intact antibodies as the vehicle to carry the therapeutic radiation. Dose rates effects have been examined by assessing whether radioactivity delivered on an antibody fragment that reaches the target earlier than using an intact antibody. Fragments may provide an advantage compared with the slower localization of intact radiolabeled antibodies, even though the retention of the intact antibody at the tumor is longer. Behr et al. compared response rates of patients with CEA expressing tumors who received the intact antibodies and the F(ab’)2 fragment of NP-4 and MN-14 [75]. The data also showed that more frequent responses occurred in patients receiving the radiolabeled fragments, although only minor responses were reported. Recently studies of 90Y on Octreotide peptide have shown responses in phase I RIT studies for patients with neuroendocrine tumors [105].

Increasing Radiation Sensitivity Radiation sensitizing chemotherapeutic agents such as 5-fluorouracil (5-FU) and cis-platinum have been used in combination with 30 Gy external beam therapy and have resulted in improved response rates in carcinoma of the esophagus, colon, anus, cervix, bladder, and head and neck compared with chemotherapy alone [247]. Radiation enhancement with these drugs has also been seen in vitro with low dose-rate radiation. [248, 249] Drugs to increase radiation sensitivity to RIT have been studied in mouse xenograft models. Paclitaxel and 90YchL6 [250], topotecan and 90Y-huBrE [251], cisplatin with 131I-323/A3 [252] and 131A33 [249], gemcitabine and B72.3 [253] have been studied. In pre-clinical studies, combinations of RIT with paclitaxel have induced cures without increased toxicity [250] and topotecan with RIT has shown an increased survival [251]. Clinical studies with taxol are in progress. The effects of these combination approaches are dependent on the timing and dosing of the chemotherapeutic agents, and will require considerable study in clinical trials. Another approach to increase radiation sensitization is to use halogenated pyrimidines as radiation sensitizers [254]; 5-iododeoxyuridine [255, 256], fluorodeoxyuridine (FUdR) and bromodeoxyuridine (BudR) [257–259] have been most widely studied [255–257, 260]. These act as a thymidine analogue that is incorporated into DNA via the thymidine salvage pathway. This appears to increase DNA susceptibility to radiation and inhibits DNA repair. In vitro studies and xenograft studies of these pyrimidines in conjunction with RIT have shown increased tumor growth delay [256]. Clinical studies using BudR and external beam therapy have shown a

485 survival advantage in patients with anaplastic astrocytoma [260] and responses have been reported in patients with unresectable liver metastases from colon cancer and FudR with external beam hepatic irradiation [261].

Reducing the Exposure to Normal Tissues Several strategies have been proposed to reduce the exposure of the marrow to radiation. Dose fractionation appears to have an impact on reducing marrow toxicity, but repeated fractions are limited by the formation of HAMA with murine antibodies as discussed above [34, 35, 99, 104, 262]. An attempt to reduce toxicity was done by fractionating the intraperitoneal 186Re NR-LU10 dose and administering a second dose of intraperitoneal 186Re NR-LU-10 7 days after the first dose [227, 263]. At the highest dose level of 90 mCi/m2 twice (i.e., 180 mCi/m2) there was severe marrow toxicity in one of three patients. In contrast, similar toxicity was seen in two of three patients in the single dose study at only 150 mCi/m2. Meredith et al. have incorporated this strategy into several clinical trials with B72.3 in patients with colorectal cancer and with CC49 in patients with colon and prostate cancer, and have confirmed a reduction in myelotoxicity but the number of infusions was still limited by HAMA [80, 95] Another approach is to remove the circulating radioactivity that has not localized at the tumor site by a clearing agent. Such a clearing agent could either be administered intravenously or be extracorporeal, i.e., part of an immunoabsorption column [264]. Candidate clearing agents include anti-antibodies directed at the Fc portion of the immunoconjugate or the avidin/biotin system with one component linked to the immunoconjugate and the other to the clearing agent [265]. The extracorporeal approach is being evaluated with external immunoadsorption columns [264]. In this procedure, the patient’s blood is separated into cells and plasma by a cell separator, phereis machine, and the plasma is then circulated through a column which specifically removes the radiolabeled antibody, either by another antibody, e.g., goat anti-mouse antibody or by avidin removing a biotinylated radiolabeled antibody. DeNardo et al. have employed some of these approaches and have reported responses in patients with advanced breast cancer with multiple doses of 131I-labeled chimeric antibody, L6 [264]. Some patients received G-CSF 7–20 days following infusion while others underwent immunophoresis using a goat anti-mouse antibody to reduce the non-bound circulating antibody. Four of nine patients who were able to receive more than one dose achieved a PR [101].

486

Targeted radionuclide therapy of cancer Table 5. Potential advantages and disadvantages of pretargeted RIT systems

Pretargeted RIT Conventionally radiolabeled antibodies or other large molecules remain in the circulation for long periods of time exposing the normal organs, especially the radiosensitive bone marrow, to radiation. The prolonged circulating radiation limits the dose that can be safely administered and ultimately limits the efficacy of the approach. An alternative approach, pretargeted RIT, has been used to administer the antibody and the therapeutic radioisotope separately with some means of bringing them together at the tumor. This has been an attractive approach since the targeting specificity of the antibody is retained, but the whole body radiation dose is substantially decreased because the radioisotope is administered on a small molecule, which is rapidly is removed from the body through the kidneys if it does not bind (Fig. 3). The potential advantages and disadvantages of pretargeted RIT systems are summarized in Table 5. Goodwin et al. pioneered the use of pretargeting, initially studying bispecific antibodies. The first step involved pre-localizing an unlabeled antibody in tumors that recognized both the tumor associated antigen and a hapten, and then administering a radiolabeled hapten that disappeared from the blood stream rapidly [266] With a moderate affinity between the hapten and the antibody, tumor uptake and radiation were sub-optimal and a strategy with a higher affinity system, the affinity enhancement system (AES) was developed [267, 268]. Here a bivalent hapten is used, that can cross-link the bispecific antibody bound to the tumor, thus improving tumor localization of the radioactivity. More recent bispecific agents developed include a “universal” molecule where one arm’s specificity reacts with the chelator used for various radionuclides [269]. The AES system has been evaluated for patients with small cell lung cancer. An anti-CEA x anti-DTPA-indium bispecific antibody was injected to assess feasibility, and this was

1st Step Tumor localization Blood clearance

mAb Streptavidin

Potential advantages • • •

Potential disadvantages

Favorable biodistribution • and blood clearance • Achieves high target-to-non-target organ ratios • Flexibility with respect to isotope and antigen target • • •

Ab constructs may be difficult to manufacture Monovalent (to tumor antigen) Ab fragment constructs may decrease tumor residence time Tetravalent constructs (e.g., SA) may cross-link Ab-antigens and internalize Immunogenicity (e.g., SA) Hapten release from processed Ab may poison tumor-associated conjugate Perceived as complex with respect to dosing and timing

followed by the bivalent hapten, labeled with 40 to 200 mCi 131I. The MTD without stem cell rescue was 100 mCi. For patients receiving >100 mCi, 4 of the 15 patients had grade III/IV thrombocytopenia. At 12 months, there were 3 PR, one stable disease and nine progressions in 13 patients evaluated. The pretargeted approach with bifunctional antibodies and AES was also studied in 26 patients with MTC. Biodistribution was variable among patients, and dose limiting myelotoxicity was reached at 60 mCi/m2 131I. Anti-tumor, minor responses and stablilization of disease were also observed [267], but this preliminary study showed no improvement over the conventionally labeled antibody as regards tumor response or MTD. Another popular approach takes advantage of the ultra-high affinity avidin-biotin system (Kd = 10.15 M). Avidin-biotin has been widely used in in vitro applications but was first translated into in vivo localizing strategies by Goodwin, Meares and McCall [266]. Both avidin and its bacterial counterpart streptavidin have been used in pretargeting applications. Two and three

2nd step Radio-delivery

Radiolabeled Biotin

Figure 3. Pretargeted RIT example to remove unbound Ab constructs from the bloodstream prior to the administration of a therapeutic radionuclide. Divalent Ab-SA conjugate followed by delivery of radiolabeled biotin [278]

John M. Pagel et al. step pretargeting approaches were described with the antibody carrying either the avidin/streptavidin or biotin for imaging and therapy [270]. Imaging studies using bifunctional antibodies and the avidin-biotin approach demonstrated the concept and that improved tumor to background activity ratios could be achieved [270–273]. A variety of tumors and antibodies have been studied using this technique. The procedure has been safe and well tolerated, with improved sensitivity due to the higher tumor-to-background activity ratios in comparison to conventional radioimmunoscintigraphy. The multistep approach, often requiring 72–96 h for completion has not been a practical approach for imaging purposes. Rather, the potential of this approach may be greater for therapy applications. A number of approaches have been under study for therapeutic applications. These include the reverse avidinbiotin approach for targeted radionuclide therapy where the antibody is conjugated to streptavadin rather than biotin, and the biotin is linked to the radionuclide. The preclinical performance of this system has been detailed by Axworthy and coworkers, who reported 80–100% cures of subcutaneous small cell, breast and colon carcinoma xenografts (250 mm3) at 90Y doses of up to 800 mCi/mouse with negligible hematologic toxicity [274]. With conventionally labeled antibodies, 200 uCi was dose limiting because of marrow toxicity. An early clinical study evaluated whether an improved tumor-tored marrow therapeutic ratio could be achieved using pretargeted RIT compared with conventional RIT, and at the same time preserve the efficiency of tumor targeting [275]. Forty-three patients with adenocarcinomas reactive to an anti-EPCAM adenocarcinoma murine monoclonal antibody, NR-LU-10 were studied. The antibody–streptavidin conjugate was injected, followed by a biotin-galactose-human serum albumin clearing agent, followed by 90Y-DOTA-biotin as the final step for therapy. In some patients, the conjugate was radiolabeled with 186Re as an imaging tracer to assess biodistribution of the conjugate and effectiveness of the clearing agent. Indium-111-DOTA-biotin was co-injected with 90 Y-DOTA-biotin to assess the biodistribution, pharmacokinetics and dosimetry of the 90Y-DOTA-biotin. No significant adverse events were initially observed after administration of any of the components. The nontumor-bound antibody-conjugate was efficiently cleared from the circulation by the clearing agent. The mean tumor-to-marrow absorbed dose ratio when using the optimized pretargeting schema was 63:1, compared with a 6:1 ratio reported previously for conventional RIT. This initial study confirmed that the pretargeted RIT approach appeared to be safe and feasible, and

487 achieved a higher therapeutic ratio than that achieved with conventional RIT using the same antibody. Clinical data with both avidin-biotin approaches indicate a substantial improvement in the tumor-to-marrow absorbed dose ratios, with acceptable radiation dose to other normal organs. A significant reduction in marrow toxicity using the pretargeting approach to treat patients with Grades III or IV glioma has been noted by Paganelli and coworkers. They used doses of 150 mCi 90Y-DOTAbiotin, about six to ten times the MTD by the conventional approach, with negligible hematologic toxicity and no acute or delayed side effects [270, 276]. At 2 months, in 45 patients there was a tumor effect in 20%, 55% of patients had disease stabilization and 25% progressed. The tumor responses were maintained in 11% of patients at 12 months. Grade III/IV marrow toxicity occurred in two of five patients treated twice at the highest dose level. Antibody developed to the streptavidin, and only after repeated administrations to the biotinylated antibody.

Pretargeted RIT for Lymphoma and Leukemia Pretargeted RIT was evaluated for treatment of patients with relapsed or refractory NHL. In the first study, the ten patients enrolled had received prior therapy, including high-dose chemotherapy and peripheral stem cell transplant (n = 3); 131I-tositumomab antibody therapy (n = 1); and prior rituximab (n = 6). In this preliminary study, chimeric, anti-CD20 antibody (C2B8, rituximab) was chemically conjugated to streptavidin. Thirty-four hours after the antibody conjugate was administered, a clearing agent (synthetic biotin-N-acetyl-galactosamine) was administered to remove non-localized conjugate from the circulation. A DOTA-biotin ligand, labeled with 111In for imaging and/or 90Y for therapy was administered 18 h later [277]. In three patients, the C2B8/SA conjugate was radiolabeled with a trace amount of 186Re in order to assess pharmacokinetics and biodistribution using gamma camera imaging. Rhenium-186 C2B8/SA images confirmed that the conjugate localized to known tumor sites and that the clearing agent removed >95% of the conjugate from the circulation. The images demonstrated tumor targeting of the DOTA-biotin within 10 min after the injection and identified previously unknown disease in several patients. Localization of radioactivity in normal organs was low and unbound radiobiotin was rapidly excreted from the whole body and normal organs. The median tumor-towhole body dose ratio of 35:1 was higher than previously reported with conventional radiolabeled antibodies. Seven patients received 30 or 50 mCi/m2 90Y-DOTA-biotin.

488 The regimen was safe and well tolerated; only grade I/II non-hematologic toxicity was observed, the most prevalent toxicity was primarily fatigue. Hematologic toxicity was also not severe; five of the seven patients who received 30 or 50 mCi/m2 of 90Y-DOTA-biotin experienced only transient grade III (but no grade IV) toxicity. Although six of ten patients developed humoral immune responses to the streptavidin, these were delayed and transient. Six of seven patients who received 30 or 50 mCi/m2 90Y (51 to 109 mCi total dose) achieved objective tumor regression, including 3 CR and 1 PR. Although these results are preliminary, they suggested that even heavily treated patients with NHL can tolerate high doses of radioactivity with the pretargeted RIT approach without requiring stem cell transplantation. Recent murine studies using human leukemia xenografts have demonstrated that a single treatment of pretargeted 90Y-DOTA-biotin after delivery of an anti-CD45 antibody-strepavidin conjugate results in a minimal level of toxicity, and tumor-to-blood ratios of absorbed radioactivity improved by as much as 30-fold over those seen with a directly radiolabeled Ab [278]. It is estimated that at least twice as much absorbed radiation can be delivered to the marrow and five-times more to the spleen, using pretargeted anti-CD45 RIT as compared to doses estimated to be delivered by a directly labeled antibody. Improved reagents for pretargeting have been developed, including a synthetic clearing agent and fusion proteins that include single-chain Fv fused to streptavidin monomers (scFv)4−SA [14, 279]. The synthetic clearing agent consists of a defined dendrimeric type synthetic molecule with multiple galactosamine units and a biotin for binding circulating streptavidin containing protein and transferring it to the liver via the Ashwell receptors [280]. Advantages of the synthetic clearing agent are defined by reproducible in vivo behavior, lack of biotin release following liver uptake and lack of competition for tumor targeting by radiolabeled DOTA-biotin. The fusion protein improvement that has been validated for several different antibodies also results in expression of a defined protein by E. coli, retained binding for biotin, comparable tumor targeting compared to whole IgG antibodies and economic advantages for protein production. These results have prompted large-scale cGMP production of the anti-CD45 fusion protein for pretargeted RIT trials to be conducted in patients with advanced myeloid leukemias. A preliminary clinical study using 90Y-DOTA-biotin pretargeted with either an anti-CD20 fusion protein (B9E9FP) yielded encouraging results in 12 patients [281]. The ratio of average tumor to whole-body radiation dose was 49:1 in this study and no significant hematologic toxicities related to the pretargeted RIT and with

Targeted radionuclide therapy of cancer objective clinical remissions. With continued research, RIT has the potential to find a place as first-line treatment in radiosensitive tumors, as treatment for small volume disease, or as a radiation boost in combination with other treatments, such as with stem cell rescue or as adjuvant therapy. Investigators are optimistic that these approaches may improve the cure rates of the thousands of patients with acute leukemia who are diagnosed each year.

Summary RIT is a complex, multidisciplinary effort that still faces many challenges. We have come to understand the obstacles and have realized that a single high-dose of a radiolabeled moiety administered systemically is unlikely to be successful in curing most cancers. In most of the RIT trials, radiation absorbed doses higher than 30 Gy have only been infrequently reported, and responses cannot be typically induced in the relatively resistant solid tumors. However, these doses may provide clinical benefit in certain circumstances, in small volume or subclinical disease or with additional manipulations. The tumor regressions observed in pre-clinical studies and in patients with B-cell lymphoma and leukemia have encouraged investigators to pursue antibodies and peptides as vehicles for targeted therapy. In the hematological malignancies, response rates are high enough and of long enough duration, that two radioimmunoconjugates for NHL have been approved for use in the United States. For hematological malignancies, there may well be a place for RIT as first line treatment with current technology. Although it seems that the field of RIT has been moving very slowly, because of the multiple obstacles that had to be overcome, there is still much optimism for targeted therapy as the newer approaches are being investigated. The inadequate delivery of radiation to tumor has been the major problem compromising RIT and increasing the fraction of the radioimmunoconjugate that localizes in the target tumor tissue has been difficult. Pretargeting the antibody prior to injection of the radioisotope may be one strategy that offers promise for lowering marrow exposure to radioactivity and increasing the peak dose rate to the tumor site. The dilemma of the proper positioning of RIT and the design of future clinical trials remains. It appears that RIT may be used to treat small disease or as adjuvant therapy for solid tumors, unless the approaches described above are shown to be successful in markedly improving the amount of radiation deposited at the tumor. Despite the use of new peptides in diagnosis and therapy of tumors and non-malignant

John M. Pagel et al. diseases that have already been described, the most exciting development in the near future may be the combination of different peptides and radionuclides in PRRT. New developments include the development of multivalent peptides that have a higher uptake in the target cell [173], and the development of new peptide analogs with affinity to more somatostatin receptor subtypes [282].

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14 Stem cell/bone marrow transplantation as biotherapy ROBERT K. OLDHAM

Autologous Bone Marrow Transplantation It is clear that bone marrow transplantation has become a major technique in the treatment of metastatic cancer. The use of autologous bone marrow transplantation has allowed for much higher doses of chemotherapy to be given in an attempt to eliminate all of the cancer cells from the patient with the sacrifice of normal bone marrow function in the process. Autologous, cryopreserved bone marrow or peripheral blood stem cells (PBSC) can be reinfused to reconstitute stem cells and bone marrow function. This process is still dose limited by damage to the gastrointestinal tract, liver, lung, heart and other critical organs [25, 42]. Some progress has been made using this technique to allow dose escalation with chemotherapeutic agents. Escalation of doses to the level causing damage to secondary target organs is the current limitation of this technique. The problems of graft versus host disease (GVHD) limits the use of allogeneic bone marrow transplantation (ALBMT), although matching techniques and immunosuppression have improved the results for allogeneic grafts [87, 92]. By contrast, autologous bone marrow transplantation (ABMT) is a technique that does not involve GVHD and allows for significant drug dose escalation. Unfortunately, residual tumor cells can exist in autologous bone marrow. Techniques must be developed to effectively eliminate all replicating tumor cells from these specimens such that the cancer is never reinitiated in the patient after cure by high dose therapy [68]. Considerable progress has been made in leukemias and lymphomas, and there is an ongoing and increasing effort with BMT in solid tumors [59, 72, 82, 86]. Histocompatible donors can be selected leading to successful allogeneic transplants [24, 59, 84]. The perfect transplant only occurs with identical twins (available in less than 1 in 300 transplants), but matching of donors at the HLA-A, -B, -C, and -D loci and selecting for those individuals with negative mixed lymphocyte cultures have improved the results of ALBMT. Engraftment of a

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

stable chimeric state can be demonstrated [84], but GVHD (acute and chronic) results in the recognition of recipient tissues by transplanted donor T lymphocytes and occurs in more than 50% of ALBMT patients. More than half of these individuals will have a fatal outcome [35, 47, 84]. A variety of techniques have been used to attempt to eliminate T cells from these marrow grafts. These have included monoclonal antibodies [63, 72], monoclonal antibody with complement [8, 33, 40, 51, 56, 64, 74, 80, 95], immunotoxins [23, 52, 65, 90], and physical methods [6, 36, 62] to eliminate T cells from the donor graft. Allogeneic BMT is covered later in this chapter, but various techniques to eliminate T cells have been reviewed [37]. Many of the techniques to deplete the T cells for ALBMT are similar to techniques being used to eliminate residual tumor cells in ABMT. Therefore, a review of this literature is critical to the reader who wants to understand all the current techniques available for elimination of subsets of cells from bone marrow prior to transplantation. ABMT is the most popular current method of bone marrow reconstitution and offers the advantage of avoiding GVHD. Bone marrow cells in numbers (1 − 5 × 1010) sufficient to reconstitute the individual’s marrow function, through the stem-cell transplant, can easily be obtained by multiple punctures of the bone with marrow aspiration and then storage of the separated cells in liquid nitrogen. These cells can be thawed and reinfused after high-dose chemotherapy with consistent reconstitution of the bone marrow. In fact, bone marrows can now be separated and divided in such a way as to prepare one to three grafts from a single bone marrow donor. More recent application of this reconstituting technique has involved the use of PBSC harvests through leukapheresis. This technique allows for stem cells to be harvested by high volume leukapheresis and represents a selection method for stem cells that avoids many of the problems of tumor cell infiltration in the bone marrow. Although peripheral blood may contain circulating tumor cells, the evidence thus far indicates that considerable positive selection of stem cells can be effected by a peripheral blood harvest. This may obviate purging of

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498 bone marrow or at least make the purging process easier by virtue of having fewer tumor cells in the stem-cell preparations. The techniques for removing tumor cells from autologous marrow are numerous and have been extensively discussed elsewhere [48, 61, 65, 89, 94]. Two comprehensive reviews can be found in the work of McIntyre [53] and Gross and Gee [37].

The Preparation of Bone Marrow for Transplantation in ABMT Marrow manipulation must be carried out to preserve the pluripotent hematopoietic stem cell in sufficient numbers to insure engraftment. All of the procedures to eliminate tumor cells discussed in this chapter must be done in such a way to preserve adequate stem-cell activity. Clonogenic assays can be used to determine the number and function of stem cells by assaying these preparations for granulocyte/macrophage colony forming cells (GM-CFC), burst forming unit-erythroid (BFU-E), and granulocyte, erythroid, macrophage, and megakaryocyte colony-forming cells (GEMM-CFC). Long-term bone marrow cultures may also be used with the Dexter culture system to measure cell renewal in these systems [2, 22, 28, 32, 54]. Recently, simple flow cytometry assessment of CD-34 lymphocyte numbers has been used to predict stem cell numbers. While these assays are useful to measure progenitor proliferation and differentiation, the critical test is marrow engraftment in the patient treated by the ABMT technique. ABMT hematopoietic recovery is usually seen in the 20- to 40-day period after bone marrow infusion. White blood cell recovery generally precedes platelet recovery; and delayed engraftment can lead to fatal complications. These patients are often isolated from potential surrounding infectious risk and must be carefully monitored for viral, bacterial, and fungal infections during the course of ABMT. Once the white count recovers to the level of 500 granulocytes/mm3 and the platelet count exceeds 30,000/mm3, the risk of infection and bleeding markedly decreases. ABMT impairs immune function but much less so than ALBMT. Immune recovery may take months, and in some patients immune competence is never really fully reconstituted [27, 31, 49, 66, 99]. To prepare marrow aspirates for transplantation, some positive selection of the appropriate reconstituting cells by physical means is commonly done. This may include the simple task of cryopreservation, which eliminates a large number of red blood cells, but may also include separation procedures to obtain mononuclear cells from the marrow [9, 21, 43]. Isopyknic centrifugation of

Stem cell/bone marrow transplantation as biotherapy Ficoll-Hypaque gradients, isopyknic sedimentation on discontinuous Percoll gradients, and the use of centrifugation in blood-cell separators without a gradient are all techniques that have been used to separate mononuclear cells containing the stem-cell fraction [9, 21, 29, 43]. These techniques have eliminated the problems of clumping and cell lysis, yield high numbers of progenitor stem cells, and routinely give marrow engraftment in patients. The technique of removal, manipulation, and preparation for ABMT and the technique of peripheral stem-cell isolation by leukapheresis in an outpatient setting have gained wide clinical acceptance. These techniques are now available and being utilized broadly in the clinical practice of oncology, such that ABMT has become a major treatment technique in patients with previously refractory solid tumors [4, 10, 30, 32, 46, 76, 78, 84].

Techniques to Eliminate Specific Subpopulations of Cells (Tumor Cells and Lymphocytes) from Marrow Specimens There has been a huge number of specific techniques utilized to attempt to eliminate either tumor cells and/or T cells from bone marrow. Elimination of the former is essential in ABMT, and elimination of the latter is desirable in allogeneic BMT. Some of these techniques depend on a biological agent and thus represent a form of ex vivo biotherapy, and others utilize chemotherapy, radiotherapy, or physical techniques to effect marrow purging. Each of these techniques will be reviewed with an emphasis on biotherapeutic techniques for bone marrow purging. Although the techniques of purging tumor cells in ABMT studies will be reviewed, it has been reported that the purging of autologous marrow has no impact on survival in patients with lymphoma. Data from the European Blood and Marrow Transplant Lymphoma Registry (EBMT) demonstrated that the purging of bone marrow for tumor cells did not affect the rate of hematologic engraftment or the risk of procedure-related death. There was no significant difference in survival for patients whose bone marrow was purged vs. case controls left unpurged [83, 96].

Ex vivo Purging with Chemotherapy Various agents have been utilized to exploit the differential toxicity for a specific chemotherapy drug on tumor cells in contrast to the activity on the marrow stem

Robert K. Oldham cells. Preclinical studies have demonstrated the effect of corticosteroids [44], VP-16 and Verapamil [11], 4-hydroperoxycyclophosphamide (4-HC) and platinum [60], 1-β-d-arabinofuranosylcytosine (Ara-C) and deoxycytidine [34], VP-16 and corticosteroids [79], and alkyl lysophospholipids (ALP) [94]. These studies indicate that each of these approaches can eliminate tumor cells and/or T cells (glucocorticoids) from marrows to be reinfused in experimental systems and some in clinical trials [41]. Mafosfamide [69] and 4-HC, both relatives of cyclophosphamide, have been utilized in clinical studies to eliminate residual tumor cells. These cyclophosphamide derivatives, as well as other chemotherapy agents, appear to be promising in the elimination of residual tumor cells from these marrow grafts [37].

Biophysical and Physical Approaches A variety of biophysical methods, including photoradiation [38], laser photoradiation [39], and isotope-mediated purging [50] have been used to purge marrow of unwanted tumor cells. These studies, sometimes with chemotherapy purging, have primarily been used in preclinical studies. Physical separation methods such as elutriation [58] can be used to positively select bone marrow stem cells for ABMT, and this technique can be used as a negative selection technique against tumor cells in ABMT and T cells in ALBMT. Clinical studies of this technique are underway and appear promising. Cell separators can be utilized for the separation of mononuclear cells for marrow [1] or peripheral blood stem cells [97] for transplantation. Various other physical approaches have been used, but the most promising and straightforward approaches involve machines that can carry out such processing in an automated format.

Biotherapeutic Approaches A variety of biotherapeutic approaches have been used to treat bone marrow prior to reinfusion. Lymphokine activated killer (LAK) cells induced by interleukin 2 [18] have been used to purge bone marrow of residual tumor cells with interesting preclinical results. Antibody alone and antibody plus complement have been used by a variety of investigators for the elimination of residual tumor cells [70]. The Campath series of antibodies may have the dual use of purging lymphoma or leukemia cells from the marrow and may also be useful in the in vivo elimination of residual tumor cells after ABMT. More than 520 patients have been treated in various European transplant centers in an approach to deplete T cells and lessen GVHD in allogeneic BMT [16]. These results cer-

499 tainly demonstrate the usefulness of this approach in eliminating T cells. The application of this technique to ABMT for purging malignant lymphocytes carrying the antigens recognized by the Campath antibodies is the next logical step. In multiple myeloma, antibody plus complement [88] can eliminate residual myeloma cells from the marrow. Antibody plus complement has been used in preclinical and clinical studies [55] to purge myelocytic leukemia cells from marrow prior to ABMT. An interesting study of an antibody conjugated to Adriamycin, where the antibody also fixed complement, has been reported, but the preclinical activities demonstrated insufficient selectivity for clinical application [98]. In addition to antibody alone and antibody-fixing complement, there has been a series of studies using antibody conjugated to toxins, usually ricin, to prepare immunotoxins that can be used in bone marrow purging [67, 93]. These immunotoxins have been used in ABMT for T-cell acute lymphoblastic leukemia [91] and to eliminate T cells to decrease GVHD in ALBMT. These ricin immunotoxins have been utilized in clinical studies in marrow purging of solid tumors such as neuroblastoma [13] and breast cancer [77].

Combination Techniques Investigators are pursuing combination techniques with antibody tied to magnetic spheres (biotherapy plus physical separation) to eliminate residual marrow tumor cells. These studies are very well reviewed by Gross and Gee [37] and will not be covered in detail here. Suffice it to say that various physical techniques are being developed where bone marrow is being purged by the dispersion of magnetic beads coated with antibody. These antibody-coated beads attach to tumor cells and can be extracted from bone marrow by passing the preparation over a magnet. By pulling out the residual tumor cells, one can prepare a bone marrow free of residual tumor cells for reinfusion. These studies certainly appear interesting, and further preclinical studies and early clinical applications are being pursued [37].

Stem-cell Selection There is now increasing evidence that biological techniques to select stem cells by positive methods will result in enhanced marrow preparations for ABMT. Antibody to CD-34 can be used to select stem cells from bone marrow or peripheral blood [5, 15]. This technique has mainly been applied to bone marrow, but with the development of PBSC collection techniques [97], the same positive stem-cell selection process could be applied to peripheral blood. High speed clinical cell sorters and antibody-based

500 positive selection systems are both used in clinical trials as methods to select and purify peripheral blood stem cells for ABMT. A technique with far-reaching implications is to culture stem cells continuously in vitro [17, 57]. This approach may allow for the repeated infusion of autologous bone marrow stem cells free of any residual tumor cells and without the presence of any immunologically active T cells [3]. The potential for the broad application of culture stem cells is obvious, even to the point of cryopreserving such stem cells for each interested individual, while living and healthy, in anticipation of their use later in life in the presence of disease. Preserving cord blood or fetal tissues for use later in life as stem cells is also being explored. Cord blood is now routinely used for reconstitution of marrow function after marrow ablative therapy [20]. Such techniques, while interesting, bring up the whole question of how far technology should take us in anticipating disease and dealing with the potential for developing specific diseases. There are various social, political, and economic consequences of techniques that may predict or be useful in the treatment of future diseases. In fact, the whole area of genetic therapy is now coming forth as a potential major form of biotherapy. Not only can stem cells be grown in vitro for eventual use in reconstituting bone marrow, gene therapy with electroporation and other techniques can be used to insert specific genes in normal or defective human cells [45]. Very primitive stem cells may carry few transplantation antigens and after long-term culture may be useful broadly in reconstituting stem cells. It is clear that biotherapy and gene therapy will lead to tremendous advances and opportunities but also bring forth major considerations of “who pays for clinical research” and “who should have access to these new techniques.” (See Chapter 3)

Allogeneic Bone Marrow Transplantation Allogeneic BMT has been successful in treating certain lymphoid and hemopoietic neoplasms. Initially, the total body irradiation plus high dose chemotherapy followed by ALBMT was felt to work primarily through the cytoreductive effects of the chemoradiotherapy. Innovative suggestions by Professor Georges Mathé as early as 1965 and follow-up evidence reviewed by George Santos in 1972 suggested that there was an anti-tumor effect independent of the chemoradiotherapy from ALBMT. This has more recently been clarified as a form of graftversus-tumor (GVT) effect similar to the concept of

Stem cell/bone marrow transplantation as biotherapy GVHD as has been well understood in allogeneic bone marrow transplantation since the early days of this technique [71]. GVHD has been well characterized for many years. It is known to have an acute and more chronic variety, primarily manifest by a clinical syndrome of skin rash, liver and gastrointestinal dysfunction as well as a variety of autoimmune type effects [71]. A variety of strategies, primarily total body irradiation and/or systemic chemotherapy has been used to reduce the number of T cells from the graft responsible for GVHD. These strategies have been more or less effective, usually requiring lifelong treatment of patients who have experienced allogeneic GVHD. Of particular interest has been the concept of autologous GVHD which was initially quite a controversial theory [73]. The autoreactive cells found in autologous GVHD can cause damage to a variety of organs, most of which are less severe than that seen in allogeneic BMT. Histocompatibility differences cannot explain this phenomenon since these autologous transplants cannot have histocompatibility differences. Therefore, immune disregulation is felt to play the major role in autologous GVHD. Treatment is usually not necessary since the syndrome is normally benign and symptomatic care is usually sufficient for patients with manifestations of autologous GVHD [19].

Graft-versus-tumor Effects Early evidence that bone marrow cells of allogeneic BMT producing a graft-versus-leukemia effect has prompted the more general idea of graft-versus-tumor (GVT) effect of both ALBMT and ABMT. Most studies found a correlation between GVH and reduced relapse rate for patients with leukemia treated by ALBMT. Similar observations have been seen, though less striking, in ABMT [73]. The presence of autoreactive T cells related to Ia antigens in many of these syndromes and also noting that Ia antigens are expressed on leukemias and lymphomas with perhaps similar antigens being present on certain solid tumors. The potential for identifying these cells and using them after ex vivo application continues to intrigue investigators in this area. Whether these cells may be purely T cells or be some form of LAK or NK cell is currently under investigation. The ability to grow large numbers of activated T cells opens the possibility of utilizing cells identified by transplantation techniques as useful for their GVT activities [19]. The idea of using ABMT as primary therapy to reduce tumor load and then to exploit the immunosuppression

Robert K. Oldham that follows to administer partially or unmatched donor lymphocyte infusions(DLI) for their further biotherapeutic effect until the host eliminates them is being tested in several European centers and less frequently in the USA. Complete responses of persistent disease post ABMT has caused considerable enthusiasm for this approach in some circles [19]. Recent evidence has provided further credence to the concept that the immune system is active in treating leukemia post allogeneic BMT. This information has come from studies in patients with chronic myelogenous leukemia and includes the following factors. 1. Marrow grafts that have been T-cell depleted have a higher rate of leukemic relapse and less GVHD. 2. An inverse relationship has been seen in HLA identical BMT between the occurrence of GVHD and leukemia relapse. 3. The GVT effect arises from a reduction in leukemic recurrences post allogeneic BMT compared to homozygous twin transplants. 4. Relapsed allogeneic BMT CML patients can be reinduced into complete remission simply by infusing donor leukocytes [85].

Conclusion ABMT and ALBMT offer major opportunities in cancer biotherapy [19]. With ABMT the major question relates to the sensitivity of cancer to a dose escalation strategy of chemotherapy and radiotherapy. If these modalities can eliminate cancer from the patient at doses between the marrow lethal dose and the maximum tolerated dose to the next organ system (gut, liver, lung, heart, etc.), then the technique of ABMT with high-dose therapy may be broadly applicable and curative in certain solid tumors. This technique will always have major, inherent toxicity because of the toxicities of the therapeutic agents. However, short-term toxicity and even some risk of longterm effects would be tolerated by patients with lethal diseases if complete remissions and prolonged remission duration can be engendered. On the other hand, if the dose-response curve for human solid tumors is insufficiently steep to allow for the induction of complete response and improved survival with these techniques, then the broader application of this approach may not be warranted, given the significant toxicities involved. Recent results, summarized above, using allogenic peripheral blood mononuclear cells are particularly encouraging, with clear GVT effects now being seen in CML and other leukemias/lymphomas, confirming the

501 earlier, more intuitive observations of Mathé and Santos [71]. As summarized by Bishop [7], the non-ablative (mini) allogenic bone marrow transplant is being investigated as a potentially less toxic transplant where the donor cells are functioning as a form of adoptive cellular therapy using the GVT effect as biotherapy. Studies of ALBMT comparing myeloablative conditioning regimens with non-myeloablative treatment in lymphoma and chronic lymphocytic leukemia has shown that the outcomes were similar and that the nonablative regimens could be applied to patients with more comorbidities and in older age groups [81], however, the greater power of myeloablative ALBMT has also been demonstrated when used after failure of ABMT in lymphoma. Studies from the International Bone Marrow Transplant Registry have demonstrated complete remission in selected patients who have failed ABMT and then treated with myeloablative ALBMT [26]. Biotherapeutic approaches toward the elimination of residual tumor cells appear to be the most promising with regard to ABMT. Similar approaches to eliminate T cells from allogenic bone marrow may broaden its application. The two most promising techniques appear to rest in the use of antibody to negatively or positively select appropriate cell populations in marrow transplantation and the use of physical separation methods, such as leukapheresis, to positively select for replicating stem cells. Most specifically, the technique of peripheral blood leukapheresis to select for stem cells, perhaps with their eventual long-term culture for repetitive use, does appear to be a highly promising technique to provide stem-cell reconstitution in patients lethally damaged by high-dose chemotherapy and radiotherapy. These studies are at an early stage but they are rapidly evolving, with the technique of ABMT being already widespread in the clinical practice of oncology [10, 75]. The availability of colonystimulating factors (see Chapters 17 and 18) provides a further mechanism for the manipulation of stem-cell numbers and activity. The outpatient use of autologous bone marrow transplantation through leukapheresis to select the stem cells and the use of colony-stimulating factors to support stem cell growth in vitro and in vivo appear to be promising approaches in the treatment of advanced solid tumors [12, 14, 76].

References 1. Areman EM, Cullis H, Sacher RA, et al. Automated isolation of mononuclear cells using the Fenwall CS3000 blood cell separator. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:379–385.

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Robert K. Oldham 39. Gulliya KS, Batagllino M, Matthews JL. Breast cancer and laser photoradiation therapy: an in vitro model for autologous bone marrow purging. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:103–107. 40. Hale G, Bright S, Chumbley G, et al. Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood 1983; 62:873–882. 41. Herve P, Racadot E, Flesch M, et al. Prevention of graft-versus-host disease: Elimination of T-lymphocytes from bone marrow cells by complement-dependent cytolysis with a combination of pan-T monoclonal antibodies (letter). Presse Med 1984; 13(14):886–887. 42. Herzig GP. Autologous marrow transplantation for cancer therapy. In: McCullough J, Sandler SG, eds. Advances in immunobiology, blood cell antigens, and bone marrow transplantation. New York: Alan R. Liss, 1984:319–335. 43. Ho WG, Champlin RE, Feig SA, et al. Transplantation of ABH incompatible bone marrow: gravity sedimentation of donor marrow. Br J Haematol 1984; 57:155–162. 44. Kapoor N, Tutschka PJ, Copelan EA. Bone marrow purging with glucocorticoids. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:39–46. 45. Keating A, Toneguzzo F. Gene transfer by electroporation: a model for gene therapy. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:491–498. 46. Kessinger A, Armitage JO, Smith DM, et al. High-dose therapy and autologous peripheral blood stem cell transplantation for patients with lymphoma. Blood 1989; 74:1260–1265. 47. Korngold R, Sprent J. Lethal GVHD across minor histocompatibility barriers: nature of the effector cells and role of the H-2 complex. Immunol Rev 1983; 71:5–29. 48. Krolick KA, Uhr JW, Vitetta ES. Selective killing of leukaemia cells by antibody-toxin conjugates: implications for autologous bone marrow transplantation. Nature 1982; 295:604–605. 49. Lum LC, Seugneuret MC, Storb RF, et al. In vitro regulation of immunoglobulin synthesis after marrow transplantation. I: T-cell and B-cell deficiencies in patients with and without chronic graftversus-host disease. Blood 1981; 58:431–439. 50. Macklis RM. Radioisotope-mediated purging in bone marrow transplantation. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:109–123. 51. Martin PJ, Hansen JA. Quantitative assays for detection of residual T cells of T-depleted human marrow. Blood 1985; 65:1134–1140. 52. Martin PJ, Hansen JA, Vitetta ES. A ricin A chain-containing immunotoxin that kills human T lymphocytes in vitro. Blood 1985; 66:908–912. 53. McIntyre EA. The use of monoclonal antibodies for purging autologous bone marrow in the lymphoid malignancies. Clin Haematol 1986; 15:249–267. 54. Metcalf D. Detection and analysis of human granulocyte-monocyte precursors using semi-solid cultures. Clin Haemotol 1979; 8:263–285. 55. Mills LE, Ball ED, Howell AL, et al. Efficacy of bone marrow purging in AML using monoclonal antibodies and complement. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:165–170. 56. Mitsuyasu RT, Chaplin RE, Ho WG, et al. Prospective randomized controlled trial of ex vivo treatment of donor bone marrow with monoclonal anti-T cell antibody and complement for prevention of graft-versus-host disease: a preliminary report. Transplant Proc 1985; 17:482–485. 57. Naughton BA, Jacob L, Naughton GK. A three dimensional culture system for the growth of hematopoietic cells. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:435–445.

503 58. Noga SJ, Wagner JE, Rowley SD, et al. Using elutriation to engineer bone marrow allografts. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:345–361. 59. O’Reilly RJ. Allogeneic bone marrow transplantation: current status and future directions. Blood 1983; 62:941–964. 60. Peters RH, Brandon CS, Lobelia AA, et al. Combinations of 4-dydroperoxycyclophosphamide (4-HC) and cisplatin for bone marrow purging in autologous marrow transplantation: an update. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:57–68. 61. Poynton CH, Reading CL. Monoclonal antibodies: the possibilities for cancer therapy. Exp Biol 1984; 43:13–33. 62. Poynton CH, Reading CL, Dicke KA. In: Dicke KA, Spitzer G, Zander AR, eds. Autologous bone marrow transplantation. edited by K.A. Houston: University of Texas M.D. Anderson Hospital and Tumor Institute, 1985:433–437. 63. Prentice HG, Blacklock HA, Janossy G, et al. Use of anti-T-cell monoclonal antibody OKT3 to prevent acute graft-versus-host disease in allogeneic bone marrow transplantation for acute leukaemia. Lancet 1982; 1:700–703. 64. Prentice HG, Blacklock HA, Janossy G, et al. Depletion of T lymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic marrow transplant recipients. Lancet 1984; 1:472–476. 65. Quinones RR, Youle RJ, Kersey JH. Anti-T cell monoclonal antibodies conjugated in ricin as potential reagents for human GVHD prophylaxis: effect on the generation of cytotoxic T cells in both peripheral blood and bone marrow. J Immunol 1984; 132:678–683. 66. Rappeport JM, Dunn MG, Parkman R. T lymphocytes in the peripheral blood of bone marrow transplant recipients. Transplantation 1983; 36:674–680. 67. Raso V, Ritz J, Busala M, et al. Monoclonal antibody-ricin A chain conjugate selectively cytotoxic for cells bearing the common acute lymphoblastic leukemia antigen. Cancer Res. 1982; 42:457–464. 68. Rill DR, Santana VM, Roberts WM, et al. Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 1994; 84(2):380–383. 69. Rizzoli V, Mangoni L. Pharmaceutical-mediated purging with mafosfamide in acute and chronic myeloid leukemias. In: Bone marrow purging and processing. New York: Wiley-Liss, 1990:21–38. 70. Rodt H, Kolb JH, Netzel B, et al. Effect of anti-T-cell globulin on GVHD in leukemic patients treated with BMT. Transplant Proc 1981; 13:257–261. 71. Santos GW. History of bone marrow transplantation. Clin Haematol 1983; 12:611–639. 72. Santos GW. Bone marrow transplantation in leukemia: Current status. Cancer 1984; 54:2732–2740. 73. Santos GW. Syngeneic or autologous graft-versus-host disease. Int J Cell Cloning 1989; 7:92–99. 74. Sharp TG, Sachs DH, Fauci AS, et al. T cell depletion of human bone marrow using monoclonal antibody and complement-mediated lysis. Transplantation 1983; 35:112–120. 75. Sheridan WP, Wolf M, Lusk J, et al. Granulocyte colony-stimulating factor and neutrophil recovery after high-dose chemotherapy and autologous bone marrow transplantation. Lancet 1989; 2:891–895. 76. Sheridan WP, Begley GC, Juttner CA, et al. Effect of peripheralblood progenitor cells mobilized by filgrastim (G-CSF) on platelet recovery after high dose chemotherapy. Lancet 1992; 339:640–644. 77. Shpall EJ, Anderson IC, Bast RC Jr, et al. Immunopharmacologic purging of breast cancer from bone marrow for autologous bone

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15 Cellular immunotherapy (CI), where have we been and where are we going? JOHN R. YANNELLI

Abbreviations used CI, Cellular Immunotherapy; NSCLC, Non-small cell lung cancer; DC, dendritic cell; TIL, tumor infiltrating lymphocyte; LAK, lymphokine activated killer cell; MHC, major histocompatibility complex; IL-2, Interleukin-2; GM-CSF, granulocyte macrophage colony stimulating factor; TCR, T cell receptor; LDTA, lymphocyte defined tumor antigens; MLTC, Mixed lymphocyte tumor cell culture

Overview Previous reviews have detailed the development of cellular therapies using immunologically nonspecific lymphokine activated killer cells (LAK), activated killer monocytes (AKM), and eventually tumor specific tumor infiltrating lymphocytes (TIL). The innovations in large scale cell culture methodologies and the developments in immune assessment approaches have made the therapies more widespread as well as more effective against a variety of tumor histologies. While surgery, radiation therapy, and chemotherapy remain the accepted conventional treatments for cancer; immunotherapy continues to make a case as a therapy to be used in the adjuvant setting. Surgery reduces tumor burden, radiation therapy kills residual tumor cells in the surgical field, and chemotherapy kills residual and micrometastatic disease both systemically and also in the surgical field. However, neither radiation nor chemotherapy provide tumor specificity and both can incapacitate patients due to their effects on normal cells, particularly the bone marrow. Cellular immunotherapy (CI), a potential fourth modality of cancer treatment, can target tumor cells sparing normal cells and tissues. The difficulty that is now faced, in 2008, focuses on the overall effectiveness of CI and justifying its use based on the cost which includes: cell culture reagents, technician time and recombinant cytokines. In the current funding environment, these costs have mounted to levels which make it difficult for researchers at academic NIH supported centers to seriously utilize CI, at least in the way it has been practiced in the past. I have a colleague who scoffs at me when I talk about this R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

subject; he says if the field were successful it would bankrupt America. While being accurate to some degree, this concept has forced a re-evaluation and a forced reduction in the in vitro approaches. A new focus has arisen which is on generating the effector cells in the patients, using vaccine therapies as stimulus. The majority of newer CI protocols to date reflect this approach. Vaccines coupled with pre-treatment conditioning using chemotherapeutic agents is the way of the future, at least for the next few years. The current chapter will serve as a historical review and present some of the new and exciting approaches being utilized in CI which is nearing the 25th anniversary of Steve Rosenberg’s report on LAK cells in the New England Journal of Medicine [1]. The Author’s experience since this time should serve to enlighten and hopefully be comprehensive enough to allow the Reader to consider CI and realize its value to cancer treatment.

A Recap of where the Field has been In the early 1980s, focus of CI was on the use of the innate arm (Natural killer cells, NK cells; Lymphokine activated killer cells, LAK cells; and macrophages) [2] of the cellular immune response to treat primary and metastatic cancer. The idea that immune effector cells such as LAK could be activated to recognize and kill tumor cells across a range of tumor histologies with little consideration for immune specificity drove the laboratory and clinical studies [1, 3, 4]. Interestingly, pre-clinical and murine studies being conducted at this time at the NCI ([5–7], began to lay the framework for the future direction that CI would take. Their studies on lymphocytes that infiltrate tumors (tumor infiltrating lymphocytes, TIL) were fundamental to current CI approaches which now focus on antigen targeted therapies. In some cases, especially in melanoma, TIL were shown to have functional reactivity against tumor cells and were shown to traffic to tumor sites [8–11]. This ability to home to tumor is considered critical for 505

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clinical effectiveness [8]. The discovery of the T cell receptor (TCR) and details regarding the three dimensional structure formed by the interaction of antigenic peptides and self-MHC (either class I or class II; major histocompatibility complex, MHC) furthered our understanding and provided more rationale for their clinical use [12–15]. On a practical level, improved and streamlined cell culture techniques, the bulk production of newly identified growth and/or differentiation inducing cytokines such as IL-2 and the identification of lymphocyte defined tumor antigens (LDTAs) facilitated large scale growth of these tumor reactive T cells (TIL) for clinical trials. TIL populations are comprised of varying numbers of CD4 helper and/or CD8 cytotoxic T cells (CTL). CD4 helper cells recognize self MHC class II and 18–23 length amino acid peptides derived from tumor associated antigens. Specific CD4 cells help to orchestrate immune responses through cytokine production. In some cases, certain populations of CD4+ cells, co-expressing CD25, have been shown to suppress immune responses to tumor [16, 17]. This population will be further discussed below. CD8+ CTL recognize MHC class I antigens and eight to ten amino acid length peptides. Following recognition, CTL are capable of cytokine production and killing of tumor cells by direct granule mediated cytotoxicity as well as delivering death signals mediated by FAS (See Reviews) [18–20]. These two mechanisms may actually be preferentially used in different anatomic sites. Thus, a means to tailor CTL therapies is suggested [21]. Much of what was learned concerning lymphocyte recognition of tumors was gained from studies using TIL derived from melanoma patients. In addition to tumor biopsies, melanoma specific T cells have also been derived from both patient and normal donor peripheral blood [5, 6, 22–42]. Both melanoma specific TIL and specific T cells derived from peripheral blood have been utilized as tools to identify LDTAs using a variety of cellular and molecular techniques (see reviews: [37], [43–45]. As mentioned above, in some cases, CI can target tumor cells and spare normal cells. With our greater understanding of the nature of LDTAs, however, it is now clear that a number of tumor antigens are normal cell proteins with altered expression patterns. For example, in TIL trials it was shown that melanoma specific TIL, following reinfusion, were also capable of killing normal melanocytes [46]. Thus, it has become important to understand T cell specificity for tumors within the context of mechanisms which control peripheral tolerance. Emphasis now is to better understand tolerance, apply principals of tolerance to tumor models and

alter tolerigenic mechanisms to allow the destruction of tumor but spare normal tissue. But, considering expression patterns of some of the LDTAs, the overall benefit of immediate tumor cell destruction and the potential for memory against tumor recurrence probably outweigh some of these concerns. Our expertise and initial successes in immunotherapy were gained in melanoma patients [47]. While the majority of LDTAs have been described in melanoma, a highly immunogenic tumor, LDTAs have also been identified in other solid (breast, colon and lung for example) and liquid tumors (lymphomas and leukemias) (See Reviews [48–51]). This provided the rationale to try CI in these tumors, once considered nonimmunogenic and not candidates for experimental therapies. The goal is to improve CI in melanoma and apply the concepts and improvements to existing therapies for other tumor histologies. All this considered, with the extensive knowledge and experiences gained thus far, investigators are now able to do what was originally intended in the early 1980s. That is to use defined tumor antigens in vitro to generate more homogeneous anti-tumor T cell populations. The relative monetary savings over earlier bulk T cell approaches using these selected T cell populations made the therapy attractive for clinical investigators, laboratory personnel, and regulatory agencies. Extending these advances to the current use of vaccines to educate the immune system with these antigen preparations in vivo against specific tumor antigen targets has become a more logical recent focus. It may be more advantageous to generate anti-tumor immunity in vivo, an active form of immunotherapy, and spare the uncertainties associated with large scale cell culture. Using sound immunologic based principals learned in viral and bacterial systems, these vaccines should be capable of boosting existing anti-tumor immune responses and help to generate new responses against tumor antigens that have escaped recognition. A point to consider, however, is whether immunization strategies will be effective against bulky metastatic disease. It is possible that vaccines may only play a role in the adjuvant setting. Currently, the therapies are structured in the following way. Surgery remains the first line of therapy as a de-bulking procedure. Radiation and/or chemotherapy are used for the removal of residual and micro-metastatic disease. Concurrently, construct vaccines and perform safety testing and deliver the vaccines in prime and boost strategies when the patient’s immune system has recovered. This approach should generate “sentinel” lymphocytes which can seek out and destroy small pockets of metastatic tumor which escape recognition or become resistant to conventional therapies.

John R. Yannelli The inherent nature of specific lymphocytes also predicts the development of memory to protect against future tumor recurrence. Thus, the combination of standard therapies with immunotherapies may provide valuable data and an exciting future in cancer therapies [52–57]. This strategy of combined chemotherapy and immunotherapy is being investigated by our group (Hirschowitz and Yannelli, 2008 unpublished observations). A clinical trial of maintenance chemotherapy in non small cell lung cancer using Pemetrexed + or − allogeneic tumor cell vaccine is being tested in our center. It is early in the study but using stage III and IV NSCLC patients who possess bulky disease; we are monitoring time to progression as well as immunologic responses to the vaccine. Both clinical evaluation and immunologic monitoring taken together will provide more impetus for moving forward with vaccines and a better understanding of much needed improvements. Finally, it also remains to be seen, however, whether vaccines can be used as a defense against the initial development of tumors. These studies are best tested in murine models of spontaneous disease. Once we gain experience with these models and novel vaccines are proven efficacious, populations at risk for certain forms of cancer due to environmental factors or genetic disposition should be considered as potential candidates.

Use of LAK and TIL in the Treatment of Cancer, are they gone Forever? In the early 1990s, frustration arose concerning CI trials such as LAK [1, 3, 4] and TIL [22, 58–60]. These studies had been ongoing since 1984 but examination of the clinical responses made investigators consider more closely the cost to benefit ratio. While LAK was working in some melanoma patients, it was a difficult therapy to deliver. The priming steps with large doses of recombinant IL-2, the repeated leukapheresis procedures, the large-scale cell culture, and the requirement for large numbers (>1011) of LAK cells in multiple cycles made this a challenging therapy. In addition, the large doses of IL-2 required for LAK cell maintenance made this clinically difficult for physicians, support staff and patients. The requirement for hospitalization, many times care in the ICU, made LAK therapy a challenge and doomed its spread to the general practice of Oncology. In addition, complete and durable responses were not the norm. The financial costs associated with this therapy were also daunting for both the clinicians and the patients.

507 Unfortunately, the therapy utilizing a short exposure of leukapheresis products to high concentrations of IL-2 (Pulsed-LAK; [61]) did not engender the enthusiasm which was originally intended. Thus, LAK cell therapy overall was considered too cumbersome a therapy to routinely deliver. Today, with the renewed interest in the innate immune response to cancer, an improved form of this therapy may receive more attention and be utilized in combination with more specific cellular approaches. Whether this approach is successful remains to be seen. In the mid 1980s, the TIL trials began with the hope that more durable clinical successes in a wider range of tumor types could be realized. However, the enthusiasm that greeted the Science report by Rosenberg et al. in 1986 [60] detailing the murine TIL studies was not realized in the early human clinical trials of TIL. In hindsight, the reason for this can be attributed to a number of issues. Immunogenicity of the tumors being studied, clinical use of bulk TIL populations containing nonspecific T cell populations, and the failure to co-deliver effective cytoreductive and/or immunosuppressive chemotherapy were some of the reasons for the disappointing results. Additionally, TIL were being used to treat bulky disease. We now realize the tumor burden contributes to a significant tumor and/or host derived suppressive environment. In retrospect, all of these factors served to dampen the initial enthusiasm. A breakthrough occurred in the early 1990s following a retrospective analysis of the accumulated clinical and laboratory data. A 1991 report by Aebersold et al. [24] discussed characteristics of effector cells that were important for clinical responses. For instance, age of the TIL, doubling time of TIL, and the ability of the TIL to immunologically recognize autologous or MHC matched tumor all proved to be predictors of clinical responses. In two follow-up reports by this group, both Rosenberg et al. [60] and Schwartzentruber et al. [62] extended the observations following an analysis of 5 years of laboratory and clinical data. Interestingly, the tumor most responsive to immunotherapy was melanoma. Other tumors such as breast, colon and lung cancer for instance did not appear to be responsive to TIL therapy. The 30% or so of melanoma patients that showed clinical responses all demonstrated the ability of their TIL to either kill in chromium release assays or specifically secrete cytokines following incubation with autologous or MHC matched melanoma tumor cells. The concept of shared tumor associated antigens presented by common MHC antigens had already been described in reports by Topalian et al. [6], and Muul et al. [5, 39]. These investigators had shown that melanoma TIL could recognize autologous tumor cells and

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in many cases melanoma tumor cell lines expressing shared MHC class I antigens (HLA-A2 for example). These observations were later extended to MHC class II antigens [28, 29, 34]. The relatively simple melanoma-TIL culture conditions and the frequency of clinical responses make melanoma an ideal tumor model to study CI. While culture of enzymatically digested tumor biopsies in high concentrations of IL-2 resulted in tumor specific CD4 and/or CD8 T cells from about 40% of melanoma biopsies tested [35], it is also one of the easier 1tumors from which to derive stable long term tumor cell lines [47, 63]. Stable TC lines make detailed immunologic analysis easier. Interestingly, in the early studies, site of melanoma tumor biopsy appeared to predict whether specific T cells were obtained. That was, cutaneous and visceral lesions were more likely to provide anti-tumor specificity than were TIL derived from tumor involved lymph nodes [35]. A recent report however, showed melanoma reactive TIL could be derived from 39% of tumor involved lymph nodes tested [64]. A recent TIL report reiterates the enormous potential of this therapy, particularly in melanoma. With improvements to both laboratory TIL procedures and pre-treatment of patients with targeted chemotherapy, increased clinical effectiveness of the approach was shown [46, 65–67]. Use of highly selected, antigen specific melanoma reactive TIL following pretreatment of patients with the immuno-depleting drugs fludarabine and cyclophosphamide resulted in 6 of 13 objective clinical responses with 4 additional mixed responses. The infused TIL were shown to proliferate in vivo, traffic to tumor sites, and displayed marked functional reactivity. Compared to the early TIL trials, these results were markedly improved. Thus, in melanoma, TIL remains a very viable treatment option and continues to provide the most interesting clinical results of all immunotherapy’s tested to date. This group continues to lead in the laboratory aspects of CI with a study published online. In the most recent report, evidence is presented that very early TIL cultures are the most efficacious in terms of anti-tumor reactivity. These T cells contained in early culture, while possessing the largest number of antitumor T cell clones, do not suffer from long periods of time in cell culture where important clones are overgrown or critical cell surface receptors and costimulatory molecules are down-regulated [68]. Examples of specific TIL derived from other tumor histologies include: colon cancer [69, 70]; esophageal cancer [71]; breast cancer [72–75]; follicular lymphomas [76]; ovarian cancer [77, 78]; renal cell cancer [79–82]; and non small cell lung cancer [83, 84] to name a few.

The frequency of successes, however, using standard TIL culture conditions was far less than seen in melanoma. In the majority of cases, nonspecific TIL or TIL with no functional reactivity were derived. This result may reflect an inherent inability to lyse fresh tumor cells other than melanoma. But, even when tested against stable MHC matched cell lines, no specific lysis or cytokine release was usually observed. Based on the predictions made by the Investigators at the NCI [24, 60, 62], the infrequent generation of specific T cells from non-melanoma tumor biopsies could explain the overall disappointing clinical results obtained in the early TIL trials. This assessment is strengthened by other published reports [81, 85]. Despite these difficulties, novel cell culture methodologies have been developed since the early TIL trials were begun and improved results in non-melanoma T cell cultures. These studies showed that tumor specific T cells could be derived from the peripheral blood of cancer patients with a variety of cancers. Specific immunologic reagents were then made available for clinical and further immunologic studies. Interestingly, however, the proliferative potential of these T cells is not the same as T cells derived from solid tumor. TIL appear to grow more consistently and to higher levels than specific T cells obtained from peripheral blood. TIL can proliferate in the presence of IL-2 alone and often do not need antigen restimulation. In the TIL studies, over 1011 T cells were often derived from less than 1 × 106 cells [23, 35]. In our own laboratory, we generated over 1011 effector T cells from as few as 100 T cells. It is now evident that the growth and differentiation signals vary for T cells derived from different anatomic sites including peripheral blood, solid and liquid tumors, and tumor involved or tumor free lymph nodes. These differences reflect the maturational state of the T cells that are contained in these sites including differences in the expression of the chemokine receptor CCR7 and the isoforms of CD45 [86–88]. Thus, concerning peripheral blood, factors such as when the blood sample is obtained may be critical. There are likely differences in what proportions of immature versus mature T cells are contained in the sample which is most probably influenced by the overall tumor status and trafficking patterns of T cells. It is then unknown what is required for optimal expansion including: antigenic stimuli, frequency of restimulation, growth factors, and costimulation. With this said, the removal of the T cells from the potential suppressive environment of a tumor bearing host [89–95] for in vitro expansion should be advantageous. Autologous or allogeneic melanoma tumor cells were used as stimulators in mixed lymphocyte tumor cell cultures (MLTCs) and MHC class I restricted specific CTL

John R. Yannelli were sometimes observed [96–99]. Peripheral blood from both normal donors and cancer patients were used in these studies. Celis et al. [100] derived CTL from the peripheral blood of normal donors using antigenic peptides derived from melanoma LDTAs. The culture conditions in these studies relied on low concentrations of IL-2 and in some cases IL-7 provided at multiple intervals during the stimulation and growth phases. More recent methodologies used gene transfer of costimulatory molecules such as CD80 into fresh or cultured tumor cell lines. In these studies, tumor specific T cells against melanoma [38, 101] and other solid tumors were observed including NSCLC [102], and renal cell cancer [103–105]. Interestingly, our studies (Bixby and Yannelli [102] and [84] have shown that NSCLC specific CTL could only be generated following exposure of T cells to CD80 gene modified tumor cells confirming a requirement for costimulation. Finally, gene transfer as a tool to improve T cell reactivity has gained recent interest after a series of early problems [106–110]. Our group at the NCI performed early studies using retroviral mediated gene transfer of TIL with genes encoding the cytokines IL-2 and TNFalpha [111, 112]. The novelty of using IL-2 was associated with the desire of infusing anti-tumor T cells that had an inherent capacity to release IL-2 in order to provide growth stimulus to the T cells in vivo. Of course an issue with IL-2 release is controlling the endogenous IL-2 production thus preventing the growth of T cells with other specificities, particularly self reactive T lymphocytes. TNF-alpha, however, was very intriguing during the early days of gene therapy. While we produced autologous melanoma tumor cell vaccines secreting both IL-2 and TNF-alpha [63], much effort was made to transduce T cells with the same retroviral vectors. In murine models TNF-alpha delivered at the tumor site was observed to be a very potent modulator of tumor response, speculated to be the result of tumor vascular destruction. These results led to limited human trials which were eventually halted by the FDA due to safety issues encountered early on [113]. Coupled with the understanding of lymphocyte defined tumor antigens (LDTAs) and the characterization of tumor reactive T cell receptors {Cole, 1994 #28}{Shilyansky, 1997 #26; Cole, 1997 #25; Cole, 1995 #27; Shilyansky, 1994 #30; Nishimura, 1994 #29}{Roszkowski, 2005 #9}, most recent attempts are proving fruitful in the clinical setting [106–110]. Unfortunately, the laboratory requirements in expertise and molecular probes along with regulatory issues make this approach less applicable to the general academic and oncologic setting. None the less, the knowledge of TCRs and particularly LDTAs is critical

509 to the future improvement of CI. Acknowledging the critical importance of the discovery of LDTAs; (Reviewed in: [43–45, 114]), the remainder of this Chapter will focus on the new approaches to CI utilizing defined LDTAs as targets for therapy.

Lymphocyte Defined Tumor Associated Antigens (LDTAs) Lymphocyte defined tumor antigens (LDTAs) have been identified using a variety of molecular techniques (See Reviews: [37, 43–45, 115]. There have been more than 70 identified at the writing of this Chapter. The techniques used to identify antigens include: (1) cDNA cloning (described below) [26, 116, 117]; (2) a “reverse immunology” approach which generate specific T cells against peptides derived from sequences of serologically defined tumor antigens [118–123]; (3) peptide stripping and HPLC [124–127]; and the newer more advanced molecular techniques including SEREX [128–130], Proteomics [48, 131, 132], and Microarray analysis [133, 134]. The technique initially used to describe a number of antigens in melanoma and other histologies which is still in use is cDNA-cloning [25–27, 30–33, 37, 116, 135–137]. A distinct advantage of this technique is that it is based on the availability of already defined tumor specific T cells. It does not speculate as to their existence as some of the newer techniques do, thus bringing into question the relevance of the LDTA. The reagents required are tumor specific CTL, cDNA generated from a tumor cell line recognized in an MHC restricted fashion by the T cells, and an indicator cell line which can be transfected with both MHC antigens and pools of cDNAs. A cDNA library is prepared, pools of cDNA are transfected into 293 human kidney cells or COS cells which express the restriction element recognized by the T cells. The CTL must be tumor specific as defined by extensive specificity analysis, and should specifically release cytokines which can be measured using standard ELISA assays. In addition, a reasonable number of T cells are required (generally 5 × 108 to 1 × 109). When pools of cDNA are identified which confer recognition of the indicator cells by the CTL, the experiments are repeated by screening dilutions of the cDNA pools until a single cDNA is identified. The cDNA is sequenced, the protein identified and characterized for expression on tumors of the same histology, tumors of different histologies, and normal cells. The antigens discovered thus far by all these techniques are classified into three categories based on histologic

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expression of the genes. The first group identified are termed cancer testes antigens and are expressed in normal testes, melanoma, and other tumor histologies including breast, lung, bladder, and squamous carcinomas. These antigens include the MAGE gene family: MAGE-1 [116], MAGE-2 [138] MAGE-3 [139] and BAGE [140]. Additional cancer testes antigens identified include MAGE-12 [141], GAGE [142], and NY-ESO-1 [130]. The second group includes melanocyte lineage proteins which are expressed on normal melanocytes as well as tumor cells. These antigens include gp100 [26], MART-1/Melan-A [25], tyrosinase [143] tyrosinase related protein-1 (TRP-1) [33], tyrosinase related protein-2 [32] and melanocyte-stimulating hormone receptor (MC1R) [144]. The third group are mutated normal proteins which generate unique epitopes, such as the case with Betacatenin [137] and others including MUM-1 [145], CDK4 [146], caspase-8 [147], and KIA0205 [148]. Certain of these melanoma antigens such as tyrosinase, have also been shown to be recognized by class II restricted T cells, one such antigen is tyrosinase [29, 34]. The questions now which remain unanswered are which antigens are most biologically relevant to the cancer patient and which peptides derived from each protein are the most immunogenic? Once these answers are obtained, it is possible that combinations of peptides, both CD4 and CD8 specific, and/or antigens will be shown to be more efficacious as targets for vaccine strategies. There are also a number of antigens, fewer defined at the moment, which have been identified in other cancers such as MUC-1 [149, 150], CEA [151], p53 [152] Her-2/ neu [153, 154], SART-1 [155], ART-1 [156] and ART-4 [157]. As populations of specific T cells are being identified, so are the antigens they recognize. Our lab, utilizing specific T cells generated against CD80 gene modified NSCLC lines, identified two antigens which include a myoinositol monophosphatase protein (IMPA) [158, 159]and a GTP binding protein termed GNAS [160]. Unfortunately, based on normal tissue distribution, both appear to be minor histocompatibility antigens which limit their eventual clinical use [161]. However, cDNA cloning strategy remains a very powerful tool for LDTA identification.

Cancer Vaccines The major thrust of CI over the past 10 years, has been the development of vaccines for cancer treatment (See Reviews [162–169]. Regardless of what constitutes the vaccine, the desired result is to provide tumor antigen in a stimulatory manner which allows the in vivo generation

of a potent anti-tumor immune response. Vaccines constitute an active form of immunotherapy since they are not dependent on the injection of large numbers of in vitro derived effector cells. Rather, in most cases, the vaccine is delivered in hopes that the patient’s own immune system generates the effector cells in vivo. Thus, patients enrolled in vaccine trials must be immunocompetent. Ideally, a cancer vaccine should: (1) exhibit minimal toxicity, (2) induce the appropriate tumorspecific immune responses against primary tumor and metastatic lesions, (3) induce a memory response to protect against tumor recurrence and (4) be designed so that it is affordable by cancer patients. The overall goal of course is to achieve a measurable therapeutic benefit, most likely derived from both the cellular and humoral arms of the immune system. In a Review, Bodey et al. [170] summarizes the potential pitfalls to the approach. Since vaccines rely on specific B and T cell immunity, many factors must be considered including: (1) loss of MHC expression by tumor cells, (2) low immunogenicity of many of the LDTAs, (3) LDTA down-regulation, (4) secretion of immuno-regulatory molecules by tumor and infiltrating host cells, and (5) impaired antigen presentation by Dendritic cells (DCs). These points taken alone or in combination can predispose many vaccines to failure. However, in light of these difficulties, successes have still been reported. In general, cancer vaccines can be divided into three categories. These include purified LDTAs, whole autologous or allogeneic tumor cell vaccines, and antigen pulsed dendritic cells (DCs). Each has its advantages and disadvantages and will not be discussed in any particular order of importance. The first category is based on recently identified LDTAs and utilizes these molecules as immunizing agents. This includes natural or synthetic proteins, protein derived peptides, RNA, “naked” DNA, or DNA encoded in delivery vehicles such as recombinant viruses, plasmids, or recombinant bacteria (See Reviews above). Liposomes have also been used for the delivery of a variety of immunogens [171]. In the development of this type of vaccine, melanoma again has served as the prototype tumor. Similar advantages have been taken of the established immunogenicity, thus, melanoma vaccine development has proceeded rapidly in the past 10 years. Vaccines have been generated using the defined LDTAs including MART-1, gp100, gp75, MAGE-1, MAGE-3, BAGE, GAGE, tyrosinase, GM2 and GD2 [162]. In addition, as antigens are being discovered in other histologies, vaccine development is also proceeding rapidly. This is evidenced by the use of carcinoembryonic antigen (CEA), MUC-1, Her-2neu and Prostate specific

John R. Yannelli membrane antigen (PSMA) as immunogens in clinical trials [163, 164]. Overall, this approach is promising for many cancer patients since it is based on sound immunologic principals we have learned in bacterial and viral systems. The ability to apply LDTAs as a vaccine strategy is very dependent on the knowledge of the patient’s tumor antigen expression. This can only easily be determined by the availability of autologous tumor. Unfortunately, this is not always possible since some patients are unresectable, or previously resected tumor lesions are unavailable at the time of the trial. In addition, since the vaccine relies on priming the patient against tumor proteins and MHC presented peptide antigens, MHC and LDTA expression by the tumor cells is critical. Decreased expression of these proteins by the tumor will hamper the effectiveness of the vaccine. There is much evidence in the literature for the decreased expression of MHC antigens, particularly HLA-A locus antigens by tumor cells [172–174]. One solution is combining the vaccine with agents designed to maintain or increase MHC expression, such as gamma interferon. This will work assuming that genetic changes have not occurred affecting MHC class I or Beta-2 microglobulin production, assembly or transport to the cell surface. Secondly, the inclusion of multiple antigens in the vaccine, a multivalent approach, increases the expression of at least one of the antigens if the other is lost by mutations or mechanisms of protein down-regulation exercised by selected cells in the tumor. Monitoring expression is also important during the course of treatment to insure that changes do not occur affecting the potential long term success of the vaccine. A second vaccine strategy is a whole cellular vaccine using autologous or allogeneic fresh tumor cells or tumor cell lines. Autologous tumor cell vaccines attempt in a controlled fashion to immunize the patient against all the antigens that are endogenously expressed by their tumor. The use of autologous tumor cells to make individualized vaccines is certainly optimal; however, it does have its drawbacks. To accommodate multiple or even a single vaccination requires a reasonable amount of fresh tumor biopsy. This in itself selects against a number of patients. Some patients have small lesions, the tumor is inaccessible or the patient is inoperable. This is a particular problem in patients with NSCLC. Widespread tumors in the thoracic cavity are many times too difficult to obtain and it becomes an issue of cost versus benefit to the patient. In some cases the tumor cell content of the biopsy obtained is variable or the tumor cells are necrotic. An alternative strategy is the development of an autologous tumor cell line but

511 that can be a challenge depending upon the histology being studied. Some histologies such as melanoma are easier to derive a cell line from than other histologies. The development of a stable tumor cell line requires careful attention to growth medium, serum concentration, growth factors, and substrate making some histologies more difficult but certainly not impossible to derive stable long term lines [63, 175–178] (Yannelli et al., Lung Cancer, submitted 2009 still in review). As a solution, short term tumor lines are often attempted and used for study [179–181]. Sometimes, however, one has to question the integrity of the lines in regard to tumor cell content. The difficulty is that tumor biopsies are not 100% tumor cells. The tumor cell content is variable and biopsies are mixed with fibroblasts and host infiltrating leukocytes. In addition, the viability of tumor cells can be a problem. Often, viable tumor cells exist on the periphery surrounding dead necrotic tissue in the center of the lesion. Following dissection and enzyme digestion the tumor cells often die in the processing. Hence, what appears to be a short term tumor cell line is in many cases normal cells such as fibroblasts and/or macrophages. In the vaccine, what has the patient actually been exposed to, tumor cells or adherent normal cells? Many of these studies fail to fully characterize the short term lines before use. Thus, clinical results can be misleading. It is critical to evaluate the line for tumor antigen expression, MHC expression and certainly cell viability. In our own studies with NSCLC, we observe tumor colonies forming early in the cell culture period. With time, however, these colonies fail to increase in size and are overgrown by fibroblasts (Yannelli et al., Submitted to Lung Cancer, 2008). None the less, clinical studies conducted by Berd et al. [182] utilized autologous whole-cell tumor vaccines in stage IV melanoma patients. Their nonrandomized trial demonstrated a 12.5% response rate, as measured by the development of delayed-type hypersensitivity against autologous melanoma tumor cells. In a subsequent study, they conjugated the hapten dinitrophenyl to the autologous vaccine and observed striking inflammatory responses in metastatic lesions of patients. In addition, an increase in disease-free survival was achieved [183]. Use of autologous tumor cell vaccines has also been attempted in other tumor histologies with some degree of success. Vaccination in renal cell cancer also demonstrated an anti-tumor immune response and a survival benefit in treated patients [184]. More recently, Dillman et al. [181] showed that short term lines could be generated 43% of the time in a study including 125 cancer patients of varying histologies. In this study, patients who developed positive delayed type hypersensitivity

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(DTH) responses to autologous tumor cell vaccines showed improved survival. For the difficulties described using autologous tumor, many investigators have chosen to study stable long term allogeneic tumor cell lines as vaccines due to their availability and the presence of shared tumor antigens. Allogeneic lines are normally standardized in terms of growth conditions and are amenable to manipulations like gene transfer. Use of these lines decreases the variability in vaccine preparations used for multiple immunizations. These lines also reduce the variability between patients. Again, however, it is critical to characterize antigen expression by patient’s tumor and be sure the antigen or antigens are expressed by the allogeneic line chosen for the vaccine. It is also important to match at least one or more MHC class I antigens between the cell line and patient to insure the tumor derived peptides are presented properly. The availability of over 30 NSCLC lines in our lab makes an allogeneic tumor cell vaccine very plausible. We have characterized: (1) the growth characteristics of the lines, (2) tumor antigen expression of the lines, and (3) MHC expression of the lines [185]. These are critical parameters in the development of allogeneic tumor cell vaccines. In a published study, a polyvalent whole cell melanoma vaccine, prepared with three allogeneic cell lines, produced a higher median survival than historical controls and resulted in both cellular and humoral immune responses against melanoma antigens [186, 187]. Evaluation of a vaccine (Melacine) consisting of lysates from two allogeneic cell lines in the adjuvant Detox found it comparable to chemotherapy in prolonging survival while causing much less toxicity [188–190]. However, while the clinical results appear promising in melanoma [191], there should be caution when using allogeneic reagents for immunotherapy. These tumor cells, while potentially expressing shared tumor antigens, also express allogeneic MHC class I and in some cases class II antigens which can give rise to potent allogeneic immune responses. These vaccines may be quickly rejected before the tumor response is allowed to develop (direct response involving T cell recognition of allogeneic MHC antigens on tumor cells). Alternatively, a slower developing allogeneic response (indirect response involving processing and presentation of allogeneic MHC antigens by host DCs), may develop along with the anti-tumor response. The response to allogeneic antigens may provide additional levels of critical cytokines necessary for the overall development of the anti-tumor response (bystander effect). It is important, however, to have the reagents available to decipher the response and determine if it is an anti-tumor, an allogeneic, or a mix of the

two. It is possible to be misled. What appears to be an anti-tumor response may actually have simply been an allo response. The vaccine was rejected with little to no impact on the patient’s tumor. Clinical ineffectiveness may be due to a faulty vaccine. On the other hand, there has been literature over the years suggesting that the generation of a third party allo-immune response can be beneficial to the overall development of an anti-tumor response [192]. An additional approach to tumor cell vaccines has been to genetically modify the autologous or allogeneic tumor cells to express agents designed to enhance the anti-tumor immune response [63, 193–196]. Immune stimulating molecules such as the cytokines TNF-alpha, GM-CSF, gamma interferon or IL-2 for example have been used to aid in the development of immune responses. In some cases the tumor cells have been gene-modified to deliver lymphocyte co-stimulatory molecules such as CD80 [197–199]. Allogeneic melanoma cell lines engineered to secrete IL-2 were administered to melanoma patients in separate studies [200, 201] and each produced clinical responses, albeit weak ones. Haight et al. [202]demonstrated an antibody response to IL-2 expressing allogeneic neuroblastoma cell lines in six patients with neuroblastoma. Additional studies have tested a large number of cytokine genes for efficacy. Using a murine melanoma model, Dranoff et al. [203]compared ten genes for their ability to enhance tumor cell immunogenicity. Of all the genes tested, retroviral transduction with GM-CSF produced potent, long-lasting specific anti-tumor immunity. GM-CSF was first recognized for its ability to stimulate the growth and differentiation of myeloid lineage hematopoietic progenitor cells. GMCSF is produced by T cells, macrophages, endothelial cells and fibroblasts in response to immune stimuli, which acts in a paracrine fashion to recruit neutrophils, monocytes and lymphocytes for host defense [24–26]. Central to its use as an immune adjuvant in vaccines, GMCSF is known to enhance macrophage activity and increase dendritic cell (DC) maturation and function, thereby augmenting antigen processing and presentation [204]. Subsequent studies revealed this immunostimulatory cytokine exerts pleiotropic effects on the immune system. Importantly, it promotes the differentiation and stimulation of dendritic cells (DCs) which are professional antigen presenting cells capable of sensitizing naïve T lymphocytes and eliciting a primary T cell response. Interestingly, early TIL trials in melanoma demonstrated that the specific release of GM-CSF by TIL often correlated with significant clinical responses [62]. One can speculate that the CD14+ cells recruited to the tumor site through inflammatory

John R. Yannelli mechanisms become stimulated by the GM-CSF, undergo differentiation and accomplish tumor antigen presentation to T cells in draining lymph nodes. Thus, GM-CSF may be an especially promising cytokine for gene-modified vaccines. Synergy between antigenic protein and GMCSF has been shown in several clinical studies [205–209]. In NSCLC, Nemunatis et al. evaluated the effects of GM-CSF gene-modified autologous cancer cells with encouraging biologic and clinical results [210, 211]. Similarly, an autologous melanoma vaccine secreting GMCSF reported in 1997 produced anti-tumor immune responses associated with a partial but temporary clinical benefit [197, 212]. A Phase I clinical trial investigating the biologic activity of these vaccines found infiltration of T cells, DCs, macrophages and eosinophils at the immunization sites in all 21 patients evaluated [213, 214]. In addition, autologous tumor-reactive CTL were detected following vaccination. GM-CSFsecreting autologous tumor vaccines have also been utilized in clinical trials for renal cell cancer and prostate cancer [195, 215–217], and similar anti-tumor immune responses were observed. Together, these results demonstrate the feasibility and safety of this vaccination strategy, the low level of toxicity to patients, and confirm the bioactivity of this reagent. Thirdly and the most recent approach is the use of antigen-loaded DCs as immunogens [218]. While all the results are not in at the time of the preparation of this Chapter, we would speculate that in a future review the clinical use of DCs will have been proven in one application or another. We anticipate that DCs will constitute a critical component of vaccines delivered to not only cancer patients but also to many patients having a variety of immunologic disorders. DCs are the most potent antigen-presenting cells in the body. It is speculated in many cases that an inherent problem in tumor immunology is the inadequate presentation of tumor antigens to lymphocyte precursors. This vaccine approach is designed to remove DC precursors from the patient, differentiate them in an environment free of the tumorinduced suppression, and pulse them with relevant tumor antigen before injection. The high expression of costimulatory molecules (CD80, 86, 40) and MHC class I and II molecules; the DCs ability to process and present relevant tumor derived peptides; and their ability to secrete IL-12p70 makes them very attractive as immunogens. Interestingly, DC based vaccines do not require that tumor antigens be fully characterized. This approach can be utilized in the treatment of tumors with primarily undefined antigens. If autologous tumor cell preparations are used to pulse the DCs, the patient is

513 receiving his or her tumor-derived peptides in the context of self-MHC. The primary disadvantage of utilizing DCs as a vaccine, however, is the potential development of immune responses against “self” antigens shared by tumor and normal cells. Ludewig et al. [219]reported on the induction of severe autoimmune disease with dendritic cell (DC) immunotherapy. With that stated, however, DCs may still be the most powerful tool in immunotherapy to date.

Dendritic Cells and Vaccines DCs can be derived from normal donors and patients with cancer including: B cell lymphoma [220, 221]; breast [222]; CML [223]; colon [224]; gliomas [225]; gynecologic malignancies [226]; medullary thyroid carcinoma [227]; melanoma [10, 228–230]; myeloma [231]; NSCLC [232]; prostate [233–235]; renal cell cancer [236, 237]; and various leukemias [238]; (additional references in [218]). Dendritic cells are capable of presenting peptides processed from tumor protein antigens to CD4 helper and CD8 cytotoxic T cells. In addition, through cytokine release DCs can regulate the function of NK and NKT cells. In the laboratory, DCs are routinely generated from CD14+ adherent monocytes obtained from either peripheral blood draws or leukapheresis products. In some cases, CD34+ precursors are used and are obtained following mobilization using G-CSF (granulocyte colony stimulating factor) infusions. The potency of DCs to orchestrate T cell immune responses and the technologies recently developed to generate large numbers has made them an attractive component of immunotherapy protocols. Therefore it is not surprising that researchers have focused on their utilization in the development of novel vaccine strategies for the treatment of cancer [218, 239]. CD34+ DC progenitors differentiate into CD14+/ HLA-DR+/CD11+ intermediates that circulate systemically and come to reside in peripheral tissues [240, 241]. The differentiation of DCs from CD14+ precursors is a complex process which results in a heterogeneous population of cells characterized by phenotypically defined subtypes. In addition, depending upon the mode of cellular and/or cytokine stimulation, these DC subtypes may acquire distinct and opposing functions in regard to the cellular immune response (stimulatory, DC1; inhibitory, DC2; See Reviews [218, 242–244]. This is the topic of much debate when considering which differentiation agents to use in the generation of DCs for clinical trials.

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CD14+ cells removed from peripheral blood are less than stimulatory for cellular immune responses due to the large amounts of IL-10 secreted following stimulation with soluble agents (LPS for instance) (Yannelli et al., unpublished observation). Thus, proper differentiation of CD14+ cells to DCs is critical for T cell stimulation. The differentiation steps most likely begin at the site of inflammation. Interestingly, it is the final maturation of DCs which appears to be critical to the development of a potent Th1 cellular immune response. Immature DCs are considerably less stimulatory and may actually be inhibitory toward the development of the desired cell mediated immune response [245]. These maturation steps can be monitored using appropriate in vitro analysis. CD14+ cells begin the differentiation process to “immature DCs”, that is, cells that are highly phagocytic, express moderate levels of MHC class I and II antigens and relatively low levels of adhesion and T cell costimulatory molecules, including CD40, CD80, and CD86 (See Review [243, 244]). At the site of inflammation (bacterial or viral infection, growing tumor nodule), the immature DCs phagocytize particulate matter derived from damaged tissue including viral infected cells, bacteria, and necrotic or apoptotic tumor cell bodies. At this point, presumably in response to local cytokine signals, the DCs begin the final differentiation to mature DCs. These signals can be replicated in vitro as will be discussed below. In vivo, it is thought that passage across an endothelial cell layer and a basement membrane, serves to further differentiate the DCs. These mature DCs traffic to the paracortical region of draining lymph nodes via afferent lymphatics. “Mature DCs”, having stopped phagocytosis, process the proteins into peptides which are presented in both MHC class I and/ or II molecules at the cell surface. The DCs change their cytokine secretion pattern from IL-10 production characteristic of CD14+ cells to the release of IL-12p70 (Th1 supporting cytokine). At this point, the mature DCs up regulate the proteins necessary for antigen presentation. In the paracortical regions of the lymph nodes, the DCs present peptides derived from tumor antigen, for instance, to both CD4 and CD8 precursor T cells which circulate through the node. The antigen stimulated T cells express IL-2 receptors, proliferate and mature to effector cells. These antigen specific T cells leave the node and circulate to the site of inflammation or the tumor bed to provide effector function. Randolf et al. [246] details what may occur in vivo with the maturation of DCs from CD14+ precursors. In this elegant report the investigators demonstrated that mature DCs do not rely on proliferation since irradiated cells were

capable of the differentiation process. These investigators showed that passage across an endothelial cell layer induced the expression of DC phenotypic characteristics including costimulatory molecule expression, ingestion of particulate matter, and expression of CD83, considered by many to be a marker of DC maturation. DCs can be matured from CD14+ precursors in vitro in order to study DC biology and provide cells for clinical studies. The clinical trials currently on-going use DCs pulsed with antigen in a variety of forms ranging from purified peptides, crude cell extracts, necrotic or apoptotic tumor cells, and RNA or DNA through gene transfer techniques. There are a number of good references detailing the generation of DCs for clinical trials [218, 247–249]. The initial cytokine requirements for in vitro derivation of DCs from CD14+ cells was reported by Romani et al. [250] and Sallusto and Lanzavecchia [247]. These Investigators demonstrated that the culture of adherent CD14+ cells in GM-CSF and IL-4 resulted in cells that lost CD14 expression, were CD1a+ and capable of phagocytosis or antigen uptake. The maturation of DCs in vitro following 5–7 days of culture can be accomplished by exposing the DCs to either: (1) monocyte conditioned medium (MCM) [248]; (2) T cell conditioned medium [251]; (3) CD4s bearing CD40L or anti-CD40 mAb [252]; (4) exposure to combinations of cytokines including IL-1Beta/IL-6/TNF-alpha and PGE-2 [223]; or (5) TNF-alpha/Poly (IC) [253]. In considering which maturation factor to use for immunotherapy trials one must consider (1) the need to recover a high percentage of viable DCs, (2) the secretion of high amounts of IL-12p70 with lesser amounts of IL-10, (3) the expression of high levels of T cell costimulatory molecules, and (4) insure at a minimum that the DCs are stimulatory for T cells in allogeneic mixed DC lymphocyte cultures (MLDCC). More importantly, studies should be conducted to demonstrate that the mature DCs can indeed present tumor antigen to T cell precursors. A number of clinical trials are currently underway studying DCs in a variety of tumor histologies (Reviewed in Steinman and Dhodapkar [218, 254]. One of the first reported DC clinical trials involved the vaccination of autologous antigen pulsed DCs into patients with malignant B cell lymphoma [220]. A total of ten patients were treated and a majority developed T cell mediated antiidiotype proliferative responses. Moreover, two patients experienced complete tumor regression, one had a partial response, and three each stable disease or disease progression. This group subsequently initiated trials in patients with multiple myeloma [231] and prostate cancer [255, 256]. One study examined the effects of different routes of DC administration on the ability to induce immune responses in patients, and antigen-specific

John R. Yannelli T cell responses were observed in all patients regardless of the route utilized [256]. However, the quality of the responses was variable. A second group has been utilizing autologous DCs pulsed with HLA-A2 binding peptides derived from prostate-specific membrane antigen (PSMA). In the initial Phase I study, seven partial responders out of 51 participants had decreased serum PSA levels. In a subsequent Phase II trial, GM-CSF was included as an adjuvant to DC vaccination and 31% of the evaluable patient’s experienced partial or complete responses [232, 257–259]. Another trial reported the immunization of patients with advanced ovarian or breast carcinoma with Her-2/neu or muc-1 peptidepulsed DCs. Peptide-specific CTL responses were detected in five of ten patients, as assessed by intracellular gamma-IFN staining and in microcytotoxicity assays [150]. Kugler et al. [260] reported responses in 7 of 17 patients with metastatic renal cell carcinoma following vaccination with tumor cell-dendritic cell hybrids. There has been a recent study in patients with advanced colorectal cancer [224]. Of 15 patients vaccinated with autologous DCs pulsed with tumor RNA and keyhole limpet hemocyanin (KLH), 11 developed a positive skin test reaction to KLH and a decrease in CEA levels were observed in seven of these patients. DC vaccination approaches have been examined in the setting of melanoma as well. In one study, patients received DCs pulsed with either tumor cell lysate or a cocktail composed of different peptides [228]. Delayedtype hypersensitivity reactions to peptide loaded DCs were observed in 11/16 patients and peptide-specific CTL were identified, indicating the vaccination was successful in generating an antigen-specific immune response. Additionally, 5/16 patients exhibited demonstrable regression of organ metastases yielding two complete and three partial responses. A recent Phase I clinical trial administered intravenously, tyrosinase and gp100 peptide-pulsed DCs derived from PBMC to melanoma patients [261]. In the 16 patients treated, one patient had a complete remission of metastatic disease, two had stable disease and two had mixed responses. In another trial, DCs obtained from CD34+ progenitor cells were pulsed with melanoma peptides derived from Melan-A/MART-1, tyrosinase, MAGE-3 and gp100, as well as control antigens, and administered subcutaneously to 18 patients [262]. An immune response was generated against the control antigens in 16/18 patients. Ten of these patients exhibited responses to more than two of the melanoma antigens, and seven of these had regression of metastatic disease. In our own Center at the University of Kentucky, Markey Cancer Center, we have concluded our clinical trial of antigen pulsed DCs in NSCLC [263, 264].

515 In these studies, autologous DCs were generated from adherent or Miltenyi bead purified CD14+ cells obtained from leukapheresis products. The DCs were pulsed with apoptotic bodies derived from an allogeneic NSCLC line, 1650-TC. Following safety testing, DCs were used in prime and boost doses consisting of 100 million antigen pulsed DCs per indradermal/subcutaneous injection. Our results, focused on immune assessment, showed that an immune responses was observed in 24/35 patients receiving the vaccine (69%). Importantly, while the sample size was small, we showed that immune responses were generated regardless of prior conventional therapy or stage of the disease treated, although the final evaluation of the data indicated that early stage patients responded more favorably. None the less, even stages III and IV benefited. In our Center, attention has now shifted from the DC trial to a new vaccine we have constructed consisting of the apoptotic bodies derived from 1650-TC along with recombinant GMCSF. The simplicity of this approach has allowed the immunization of patients in four clinical centers across Kentucky, strengthening the value of the approach in that it is a transferable technology. An evaluation of the pilot data from ten patients has shown that similar immunologic results are being obtained as determined using gamma interferon ELISPOT assay. Thus, our present focus, combining vaccines with chemotherapy, utilize this approach. All in all, these clinical trials verify: (1) the ability to generate significant numbers of antigen pulsed DCs for clinical trials, (2) the route of administration needed for DC vaccination, and (3) in some cases significant clinical responses. Thus, DCs have already proven their worth as a treatment option for patients with cancer. A major question that remains, similar to the LAK and TIL trials, is whether these cells can be routinely generated in small clinical centers. If these cells are only available at the NCI and large Cancer Centers, many patients will not benefit because they will never gain access to the therapy. As we have demonstrated in our approach, making DCs is a cumbersome expensive process which requires FDA oversite. As discussed above, while our results were fruitful and consistent with others in the literature, focus is shifting to easier tumor cell based vaccines.

Treg Cells in Cancer: Biology and Potential Role in Tumor Immunity A review of the immunologic literature over the past 4 decades has suggested the role of immune cells in controlling the immune response. Early on, the focus was on suppression. It was suggested that the immune

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response to tumors which appear to grow uncontrollably even in the presence of an apparent immune response was greatly affected by their presence and function. In the past decade, the biology of suppressor cells, and their increased role in maintaining immunologic homeostasis, has been uncovered. The control of immune responses, that is limiting hyper-responsiveness and maintaining tolerance, has been shown in part to be controlled by a subset of CD4 helper cells called regulatory T cells or Tregs [265]. Early work by Sakaguchi [266, 267] demonstrated that thymectomized newborn mice developed autoimmunity. The transfer of CD4+CD25+ T cells reversed the effects. In rodent models, Treg depletions lead to autoimmunity and increased allo and tumor reactivity leading to rejection of transplanted tumors [268, 269]. Since tumor antigens are in most cases normal antigens, increased numbers of Tregs during anti-tumor responses could inhibit anti-tumor reactivity. There are different phenotypes reported for human Tregs. A naturally occurring form (nTreg) co-expresses CD4, CD25, CTLA4, the newly described glucocorticoid induced receptor (GITR) and FoxP3 [270, 271]. It is difficult to assess suppressive activity to all CD4+CD25+ cells since CD25 is expressed by activated CD4s. However, FoxP3, a transcriptional regulator, appears to be the more attractive marker since induction of FoxP3 on non-regulatory T cells by gene transfer induces a suppressor phenotype [270, 271]. Mutations in the FoxP3 gene causes the absence of Treg cells in both mice and humans [272]. Most recently, it was reported that Foxp3 acts as both a transcriptional activator and repressor [273] and that FoxP3 binds to the promoters of well characterized regulators of T cell activation and function [274]. Phenotypic analysis of normal donor peripheral blood reveals that 5–10% [275]of all peripheral CD4 cells are Tregs. Cancer patients can have as high as 20–25% [276] Tregs in circulation. O’Garra and Vieira in Nature Medicine [277], described two types of Tregs, the naturally occurring nTregs (CD4+CD25+) and Treg producing IL-10 (IL-10Treg, also secrete TGF-Beta). These two populations can take different names including natural (CD4+CD25+ arising in the thymus) and inducible (CD4+CD25− arising in the periphery, iTregs). A third population has also been described which is CD25− but still expresses FoxP3 [277] Tregs have complex functions which result in both nonspecific or specific immune suppression. Tregs act on naïve T cells as well as activated T cells and function depends on both cell:cell contact or through cytokine release. Tregs can inhibit IL-2 production and may have inhibitory effects on CTL

effector function through surface or secreted TGF-beta [278]. While Tregs act directly on lymphocytes, it is believed their activation may follow interaction with DCs [279] or in some cases tumor cells. Elegant studies by Rong Fu Wang have been the first to document antigen specificity of Tregs in a human TIL system, suggesting that tumor peptides may be directly recognized [280]. It is well documented that the percentage of Tregs is higher in cancer patients tumor tissue, blood, pleural effusions, and in draining lymph nodes [16, 275, 281–283]. Interestingly, CD4+CD25+FOXP3+ cells were found to be selectively drawn to tumor sites (tumor Tregs) by the expression of CCL22 by tumor macrophages and tumor cells [284]. This accumulation was correlated with poor clinical outcome. While murine studies have shown that the depletion of Tregs results in maximal tumor rejection [285] and, depletion of human Tregs enhances CD8 T cell responses following DNA immunization [286], there are limited reports on the effects of tumor antigen vaccines on levels of Treg cells before and after vaccination. We have been examining this question in our DC vaccine study and are summarizing results for publication at this time (Yannelli et al., 2008).

Immunological Monitoring In all the discussion presented, the key to understanding the immune response which is a direct result of the immune intervention is the development of immune assessment assays which are reliable, reproducible, and can be utilized from site to site to insure comparative results. Prior to the immunologic assay, however, is the handling, preparation and cryopreservation of PBMC. These issues will be discussed below. In terms of collecting and handling of PBMC for further immunologic testing, it is standard to subject the peripheral blood product or leukapheresis product to Ficoll Hypaque gradient separation in order to obtain enriched mononuclear cells including monocytes, T cells, B cells and NK cells. Within the T cell population of course are contained the cytolytic cells, the helper cells and the regulatory T cells or Tregs. It is from these cell types that many of the immune effector cells can be routinely generated including CTL, DCs, and Tregs. In most cases, immune assessment studies are not done immediately. It is beneficial to wait until the patients have undergone all of the immunizations and a reasonable follow up time has passed. The issue of cryopreparation and eventual thaw is a critical point that does not always appear to receive the attention it should. Recently the NCI has requested

John R. Yannelli uniformity in this approach and has funded a number of studies to consolidate approaches and provide a meaningful, easy technique for cryopreservation. In our studies, we were struck by the use of human serum in cryopreservation and its cost. We determined that the fluid replacement medium Plasmalyte A could be substituted for either human serum or fetal bovine serum in the freezing of human PBMC [287]. The average cost of a liter of Plasmalyte A is around $7. This provides significant savings over conventional serum. In order to assess immune function following vaccine administration, most investigators examine cytotoxicity, proliferation and cytokine production; all of which monitor antigen specific responsiveness of T cells. Unfortunately, as detailed in Keilholz et al., 2002, shortcomings exist for each of the analyses mentioned [288]. In most cases, one is interested in the increase in specific lymphocyte precursor frequency following immune intervention compared to that found before the vaccine is delivered. An issue of course is using peripheral blood as a source of T cells which may not represent the immune response in lymph nodes and tumor tissue. However, because of its universal availability, peripheral blood remains an accepted anatomic site for study. Cytotoxicity as performed using chromium release is not sensitive and relies on expansion of T cells from the original sample. Proliferation assays are highly variable and cytokine release assays also lack sensitivity. Interestingly, an assay which has received much favor has been the enzyme linked immunospot assay or ELISPOT. Many investigators utilize this approach and if standardization of immune assessment is to be made, it is likely in this assay. Lymphocytes to be analyzed are cultured with antigen presenting cells on membranes containing cytokine capture antibodies in a 96 well format. Captured cytokine is visualized using color reagents and quantitated as spots indicating single cytokine producing cells. The results indicate an increase in “spots” which correlate an increase in the relative number of cytokine producing cells. In our own studies, we have observed increases in gamma interferon producing cells following vaccine therapy, usually at multiple time points correlating with the prime and the boost vaccine [262, 263]. The single cell cytokine assays can also be performed using flow cytometry with considerable sensitivity. This assay can be sued to further delineate T cell subsets secreting cytokine. An issue with this assay is the routine availability of a flow cytometer, although ELISPOT analysis requires a reader system which can also be very expensive to purchase. Finally, tetramer staining is a non-functional, quantitative measure of antigen specific T cell frequency in the

517 peripheral blood. Fluorescent labeled tetramers are constructed to bind a unique MHC-peptide complex to the TCR, the tetramer tagged T cells are analyzed using a flow cytometer. The majority of reagents currently available are for class I peptide complexes although class II complexes are becoming more available with time. Since tetramers are specific for a unique MHC-peptide complex, this assay is most useful for the study of HLA-restricted peptide vaccines, although tetramers are currently not available for all antigens and all HLA types. The coupling of immune assessment assays and clinical responses is critical for an overall understanding of the benefit of CI. While examples exist showing that certain immune assessment assays correlate with tumor response, this has not been a consistent finding. The difficulty at the moment is identifying the best in vitro assay which shows the greatest correlation across a range of studies done at different clinical centers. There is much focus in this area and what are needed are standardization guidelines and consistent controls to allow comparison across both study time points and across different geographic sites. When this is accomplished it is likely that immune assays such as ELISPOT will gain favor as a primary endpoint analysis in clinical immunotherapy studies.

Summary As stated in the beginning of this Review, there has been tremendous progress made in the field of CI since the initial reports using LAK and TIL. Therapies have progressed from using nonspecific killer cells to highly specific T cells over the course of almost 20 years. The techniques used to generate these cells have been improved to the point where it is possible for any laboratory with immunologic expertise to participate in cellular immunotherapy clinical trials. This is true of course as long as the participating lab follows GLP procedures for the generation of therapeutic cells. The techniques exist for safe production and efficient monitoring of cellular products for bacterial and viral contamination. The field has progressed even further with the discovery and molecular characterization of a variety of LDTAs. The knowledge of these antigens has allowed their use in generating more specific populations of T cells for infusion into patients. In addition, these LDTAs have been used as targets for vaccines. Finally, the elucidation of the differentiation steps of DCs and the knowledge of large scale culture requirements has introduced DCs as another major component of CI. Most importantly, these cells have allowed the application of immunotherapy to tumors other than melanoma.

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In closing, however, no matter what is accomplished over the next 10 years we need additional insight into the mechanisms where-by tumors evade immune responses. While tumor and host induced suppression has been a major area of study over the years, it must receive continued and more focused attention in order for these therapies to work either alone or in the adjuvant setting. It is especially important to determine why the immune system is failing to recognize developing tumors in the micro-metastatic stage, that is, before the tumor vasculature is developed. Thus, there needs to be a better understanding of what the exact mechanisms are where-by tumor cells are evading the immune system and its recognition properties. If we understood this in the first place, strategies could be implemented to strengthen the early immune response in patients at risk and correct the deficits so the tumor never takes hold nor has the opportunity to metastasize. It is hoped that at the next revision of this Chapter, a complete understanding of tumor and host induced suppression is obtained and it is coupled with accepted and proven CI approaches to cancer treatment. Acknowledgement This Chapter is dedicated to the many patients who have given themselves for these studies. Additionally, on a local level, this Chapter is dedicated to Dr. Bonnie Sigafus whose tireless dedication to patients with lung cancer has increased funding to scientists in need. Dr. Sigafus recently passed on but her memory will always remain.

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16 Growth and differentiation factors as cancer therapeutics KAPIL MEHTA, ROBERT K. OLDHAM, AND BULENT OZPOLAT

Abbreviations used ADCC, antibody-dependent cytotoxicity; ANLL, acute non-lymphoblastic leukemias; APL, acute promyelocytic leukemia; AML, acute myeloid leukemia; ATRA, all-trans-retinoic acid; CML, chronic myelogenous leukemia; CSF, colony-stimulating factor; DBD, DNA-binding domain; DMSO, dimethylsulfoxide; HMBA, hexamethylenebisacetamide; IL-1, -2, etc., interleukin-1, -2, etc.; LBD, ligand-binding domain; MDS, myelodysplastic syndromes; PKC, protein kinase C; RAR, retinoic recepotr; RXR, retinoid X receptor; TNF, tumor necrosis factor

Differentiation therapy for the treatment of malignant disorders offers an attractive alternative to the conventional cytotoxic chemotherapy. The concept of differentiation therapy is based on the principal of ‘reform’ rather than ‘retaliation’. The perception that malignant cell results from a block in the differentiation pathway has led to a conceptual strategy to remove this block and to re-establish normal homeostasis. Differentiation therapy, in general, is associated with a growth arrest and long-term commitment of the cell to die via apoptosis or senescence. The conventional cytotoxic therapy contrasts from the differentiation therapy in that there is no attempt to restore normal homeostasis in the former case and it is accompanied by immediate cell death. The treatment of acute promyelocytic leukemia (APL) by all-trans-retinoic acid (ATRA) revolutionized the treatment of the cancer and provided the first example and proof of concept of differentiation therapy. Therefore, the differentiation therapy may offer the opportunity for the use of the new, relatively non-toxic agents as well as the use of current chemotherapeutic agents at doses significantly lower than those maximally tolerated. Combining low dose of chemotherapy with one or more differentiation agents may also be of particular interest in the management of disorders resistant to conventional drug therapy. Interest in the therapeutic use of differentiation agents has resurged following the discovery of the dramatic effects of retinoic acid in the treatment of acute promyelocytic leukemia (APL). Treatment with all-trans retinoic acid (ATRA) induces differentiation of APL cells into mature phenotype such that they are no longer capable of

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

further division and are destined to undergo programmed cell death. Another line of evidence suggesting that growth and maturation factors may be operative in some form of cancers is the observation that patients with neuroblastoma and germ cell tumors can show maturational effects in the tumor biopsies post treatment. It is not known whether this relates to the treatment since occasional spontaneous maturation had been seen prior to effective therapy for these cancers. These lines of evidence have led some researchers and clinicians to speculate that cancerous growth remains under some degree of control by differentiation and growth factors and that these processes might be augmented or altered therapeutically. A number of observations suggest that differentiation of human myeloid leukemic cells occurs in vivo. The most obvious example is chronic myeloid leukemia (CML), where glucose-6-phosphate dehydrogenase (G6PD) polymorphism revealed the derivation of mature granulocytes, red cells, and platelets from the leukemic clone. Similar analysis revealed differentiation of acute myeloid leukemia (AML) clones to red blood cells and platelets in some patients [95], and the presence of Auer rods in mature neutrophils of AML patients in remission and persistence of cytogenetic abnormalities in the absence of normal metaphases in patients with AML in clinical remission [26, 89]. DNA recombinant technology has also demonstrated leukemic cell differentiation in AML and preleukemia/ myelodysplastic syndrome (MDS). DNA restriction fragment-length polymorphism in heterozygous individuals has provided strong evidence for in vivo differentiation [95, 417]. HPRT gene analysis, quantitative analysis of chromosome 8 trisomy and chromosome 7 monosomy, and immunoglobin (Ig) gene rearrangement analysis showed that five of six patients with AML had maturation to mature granulocytes in the active phase of the disease even in aneuploid leukemia. Another approach has been to label myeloblasts in vivo with bromodeoxyuridine (BUdR), which allowed the demonstration of BUdR in mature granulocytes of AML patients [326]. Premature chromosome condensation has been used to detect the presence of abnormal cytogenetic clones in mature cells. Fusion of mitotic tissue culture cells with the mature cell in question (e.g., granulocytes) results in immediate chromosome condensation of the chromatin

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528 of interphase nuclei into discrete chromosomes. This permitted karyotype analysis of nondividing cells of 13 patients with CML, and with MDS or AML, after lowand high-dose chemotherapy [159]. Twelve of these cases showed evidence of maturation of cytogenetically abnormal leukemic clones in vivo. Cell lines such as the human myelomonocytic leukemia HL-60 cells are promiscuous in undergoing druginduced differentiation. Indeed, over 80 distinct compounds (not including an even greater number of analogues) have been shown to induce differentiation of the HL-60 cell line (vide infra). Many of these agents may operate via common pathways; nevertheless they belong to distinct categories of compounds and can be divided into the following groups: 1. Vitamins, including retinoids and vitamin D3 [1,(25-(OH)2 D3), and vitamin E. 2. Polar/planar compounds such as dimethylsulfoxide (DMSO), hexamethylene bisacetamide (HMBA), N-methylformamide (NMF), cotylenin A 3. Cytokines and hematopoietic growth factors (e.g., Erythropoietin (Epo), G-CSF, GM-CSF, IL-1, IL-3, IL-6, LIF, TNF, TGFβ, IFNα, IFNβ, IFNγ) 4. Phorbol esters (e.g., phorbol myristate acetate) 5. Chemotherapeutic agents that interfere with DNA synthesis (e.g., cytosine arabinoside, tiazofurin, 6-thioguanine) 6. Chemotherapeutic agents that influence topoisomerase II 7. Chemotherapeutic agents that inhibit DNA methyltransferase (e.g., 5-azacytidine, 5-aza-2′-deoxycytidine) 8. Hormones (steroids-dexamethasone and prednisolone) [429]. 9. Chromatine remodeling: Histone deaceltilase inhibitors (HDAC) (butyrates, valproic acid) 10. Others: adenosine analogs (neplanocin A, deoxycoformycin), arsenic trioxide, peroxisosome proliferator-activated recptor gamma (PPARγ)

Human Leukemic Cell Lines as Models for Differentiation Therapy Much of the history of the development of an effective chemotherapy for leukemia was based on the availability of cell lines, or transplantable leukemias, with sensitivity to cell cycle-specific agents. The development of biological response modification therapy and the recognition of leukemic cell differentiation as an obtainable goal led to

Growth and differentiation factors as cancer therapeutics the use of leukemic cell lines of myeloid lineage capable of terminal maturation. The human myeloid leukemic cell lines HL-60, NB4 and U937 that can undergo myeloid differentiation by all-trans-retinoic acid (ATRA) or other differentiation inducing agents have been studied most extensively. HL-60 was isolated from a patient with acute promyelocytic leukemia (APL), and it retains a promyelocytic morphology but does not express translocation t(15;17) represent M2 type of AML (FAB classification) [110]. NB4 cells was isolated from long-term cultures of leukemia blast cells on bone-marrow stromal fibroblasts of a patient with APL [430]. NB4 cells (M3-AML) are true APL cells harboring t(15;17); thus expressing PMLRARα fusion protein, leading to a differentiation block at the promyelocytic stage. U937 (M4/M5-AML) was established from the pleural fluid of a patient with true macrophage-type diffuse histiocytic lymphoma, and has an immature monoblast-macrophage morphology [365]. These lines are cloned, readily maintained in culture and, while resembling immature cells of their lineage, can be induced to terminal neutrophil, eosinophil, and macrophage (HL-60) or monocyte-macrophage (U937) differentiation. NB4 cells have bileneage potential and undergo phenotypic change along monocytoid/macrophage lines with phorbol ester induction and granulocytic differentiation by retinoic acid [431]. The histochemistry and morphology of these cell lines typify them as immature cells of myelomonocytic lineage. The HL-60 cell line exists in many variants with differing doubling times and extents of spontaneous differentiation [123]. The cells weakly phagocytize latex particles or yeast in the uninduced state and possess low numbers of Fc C3b, insulin, and chemotactic fMLP receptors, whose expression can be greatly enhanced following exposure to differentiation-inducing agents [4, 286]. HL-60 cells are positive to myeloperoxidase, ASD chloracetate esterase, and Sudan black B, but unlike normal promyelocytes they do not stain for alkaline phosphatase, and unlike macrophages they are essentially acid phosphatase-negative [110, 410]. Activated forms of beta-glucuronidase, lysozyme, and G6PD, found at high levels in the granules of normal granulocytes, are present at low concentrations in HL-60 and are minimally involved in degranulation. It has been proposed that these enzymes are already synthesized in HL-60 and stored within the granules as zymogens, but are only converted to active enzymes upon stimulation [204, 422]. Uninduced HL-60 cells have a marginal capacity to produce H2O2 and O2−, have a low level of HMPS activity [284], and have a greatly impaired ability to kill staphylococcus and other microorganisms [110].

Kapil Mehta et al. The U937 cell line as originally reported grew slowly; however, later passages of the line have an accelerated population doubling of 20–48 h. The cells have a monoblastic morphology and histochemical profile (strong ANAE, NASDAE, and beta-glucuronidase positivity and weak acid and alkaline phosphatase) [365, 410]. U937 cells also secrete lysozyme, and neutral protease elastase is present within the cells; these are monocytespecific characteristics [323, 365]. The cells bear few Fc, C3, and chemotactic peptide receptors when compared with normal monocytes, but histamine and insulin receptors are expressed [5, 365]. Only a small percentage of U937 cells are phagocytic, and weakly produce H2O2 and O2− and are incapable of killing microorganisms or tumor cells [204, 324, 355]. Upon induction of differentiation, morphological changes involve significant increases in substratum adherence. The HL-60 cell line changes morphology from promyelocytic to later stages of granulocyte degranule formation when treated with 2% DMSO and retinoic acid [37, 41, 110], whereas phorbol esters transform the cells to monocyte-macrophages with the disappearance of azurophilic granules and appearance of pseudopodia and a cerebriform nucleus [337]. There is evidence that these cells can differentiate to eosinophils spontaneously, and in response to stimulatory activities in human placenta-conditioned media [222]. U937 cells, in response to differentiation stimuli, increase in size and acquire lobated nuclei, and cytoplasmic granules are replaced by vacuoles, duplicating monoblast to monocyte transformation. Various granulocyte lysozyme, acid phosphatase, and beta-glucuronidase activities have been measured in induced cells; when HL-60 is induced with phorbol ester (TPA), the activity of these enzymes is increased up to 20-fold [337]. Similar increases in activity are usually seen after induction with all agents used for differentiation induction. The distinctive monocyte lysosomal enzyme, acid phosphatase, can be detected after exposure of HL-60 cells to phorbol ester for several days. The elaboration of this enzyme into surrounding media is also a monocyteassociated characteristic and accompanies the phorbolinduced maturation of these cells. Myeloperoxidase, an enzyme specific to the myelomonocytic lineage, is constitutively expressed in HL-60. However, activity is lost after induction with phorbol esters, lymphokines, or retinoic acid, but is unaffected when DMSO or cAMPinducing agents are used [338]. Lactoferrin, a metal-binding glycoprotein, is present in normal granulocytes in the secondary (specific) granules and is one of the most specific markers for the neutrophil lineage. It has not been detected in HL-60 cells [284],

529 or in the segmented neutrophils of the majority of patients with leukemia [38]. Under normal conditions, lactoferrin is active as a suppressor molecule, and inhibits the production and/or release of granulocyte-macrophage colony-stimulating factors from monocytes and macrophages at concentrations as low as 10−15 M. In vivo lactoferrin inhibits myelopoiesis in mice at concentrations as low as 10−4 mg/mouse [34]. Therefore, detection of the synthesis of lactoferrin in HL-60 cells, even at low concentrations, would be of importance, especially if the synthesis could be increased by differentiationinducing agents and if the lactoferrin so induced was functionally active. We have detected lactoferrin in HL-60 cells at very low levels, using radioimmunoassay; in biosynthesis studies, using autoradiographic gel analysis (3H) leucine incorporated into material immunoprecipitated with lactoferrin antibody. Following induction of differentiation with DMSO or retinoic acid, a two- to fourfold increase in lactoferrin has been noted. This is still much lower than the 10−12 g per segmented neutrophil reported in normal peripheral blood, indicating a persisting abnormality in the differentiated HL-60 neutrophil, comparable to that noted in neutrophils of patients with leukemia [38]. The isozyme pattern of lactate dehydrogenase in HL-60 cells is reported to change after induction with DMSO; however, the resultant pattern is not characteristic of mature granulocytes or macrophages but rather of more immature stages [303]. Simultaneous expression of other granulocytic and monocytic specific markers may also be the result of incomplete or asynchronous maturation. Of particular interest is the observation that cell division is not required for the induction of many of these characteristics and may account for their uncoordinated expression [37]. Messenger RNA species have been isolated from HL-60 cells translated in vitro and analyzed by twodimensional SDS-PAGE after phorbol ester or DMSO induction. The phorbol-induced protein pattern differed significantly from the DMSO pattern and was characterized as monocytoid rather than granulocytic [61]. Poly ADP-ribose metabolism has been linked to the maturation of granulocyte-macrophage progenitors. ADP-ribosyl transferase is a chromatin-bound enzyme catalyzing the transfer of ADP-ribose to chromatin proteins. The activity of this enzyme increases during the CSF-stimulated differentiation of marrow precursors to monocytes [104]. Using DNA array, suppression-subtractive hybridization, and differential-display PCR techniques, the expression of about 169 genes was shown to be modulated in ATRA-treated human APL (NB4) cells [230]. Of these 32 genes appeared to be the direct targets of the ATRA

530 signaling pathway since their transcriptional regulation was not blocked by cyclohexamide. A similar detailed analysis of genes regulated by ATRA-treatment in mouse APL cells (MPRO) was reported [223]. Both the studies suggested the involvement of several transcription regulatory factors during the process of ATRA-induced granulocytic differentiation. One of the most frequently employed and convenient qualitative measures of reactive oxygen intermediates as important microbicidal products or granulocytes of macrophages is the reduction of NBT to a black formizan precipitate. A variety of factors, both physiologic and pharmacologic, can induce HL-60 production of H2O2 [141, 117, 294]. The microbicidal capacity of granulocytes and HL-60 cells is metabolically linked to an increase in the hexose monophosphate shunt activity and consequent production of superoxide anion activity. Arachidonic acid metabolism has been analyzed after induction of maturation with several agents, and recent results suggest that there is enhanced synthesis and release of prostoglandins of the E and F2-alpha type [142]. Maturation of U937 cells is usually characterized by quantitative rather than qualitative changes, since the cells constitutively produce a variety of enzymes and growth factors. Because U937 cells are of monocytic origin, agents that effect a maturational change may also be potent immunoadjuvants. Blood monocytes and U937 cells are characterized by their content of fluorideinhabitable esterase, alkaline and acid phosphatase, and β-glucuronidase. These enzyme levels are elevated by exposure of normal or neoplastic cells to agents known to influence monocytic activation, such as vitamin D metabolites, phorbol esters, gamma-interferon, and lymphokines [141, 142, 147]. Other enzymatic activities that have been studied in U937 cells include elastase, collagenase, and plasminogen activator. U937 elastase is not released constitutively and is not readily modulated [287, 353]. The production of reactive oxygen species and the percentage of NBT-positive cells are increased in U937 cells by a variety of agents, such as phorbol esters, DMSO, lymphokines, α- and β-interferons, retinoic acid, 1,25-dihydroxy vitamin D3, prostaglandin (PG) E and other cAMP-inducing agents, cytosine arabinoside (ara-C), and protein DIFs [2, 34, 141, 292, 293]. On a more quantitative basis, the activity of the hexose monophosphate shunt has been examined. Notably, db-cAMP and PGE are able to increase activity of the shunt, but cannot induce other characteristics of maturity in U937 [291]. Under certain conditions, U937 cells did not appear to produce prostaglandin E2 in response to lipopolysaccharide (LPS), endotoxin, or concanavalin A (Con A)

Growth and differentiation factors as cancer therapeutics stimulation [200], and differed from peripheral blood monocytes and macrophages in this respect. However, if supplied with exogenous arachidonic acid, the cells can release PGE2 [60]. The failure to release esterified arachidonic acid to the cyclooxygenase pathway was believed to be a property shared by U937 cells and certain macrophage subsets. The production of certain biologically active macromolecules such as, interleukin 1 (IL-1) (endogenous pyrogen, LAF) can be induced by treatment of U937 cells with agents like endotoxin or phorbol esters [302]. IL-2 is probably the factor released by U937 that increases the production of collagenase and PGE2 in cultured synovial cells [9]. U937 cells also secrete hematopoietic growth-stimulating factors that may act as autostimulatory growth factors [14]. The release of such factors can be suppressed, as in normal myelopoiesis, by agents such as lactoferrin [38]. U937 also produces the second component of complement, and the release of C2 can be augmented sevenfold by inducing maturation of U937 with phorbol ester or a lymphokine [302, 313]. Similarly, treatment of U937 cells with Con A has been shown to induce the synthesis of a 65 kDa heat resistant lymphocyte proliferation inhibitor that is distinct from lymphotoxin or interferons [405]. The qualitative and quantitative changes in the expression of certain plasma membrane constituents in HL-60 and U937 have been associated with differentiation. Induction of HL-60 with DMSO or phorbol ester, for example, modifies the expression of “fast-eluting” glycopeptides [387]. Comparison of the elution profiles of fucosyl-labeled glycopeptides with normal granulocytes and macrophages revealed that HL-60 glycopeptides were larger and more complex than those on a normal band of polymorphonuclear neutrophils. DMSO or phorbol treatment of HL-60 did not induce a normalization of the elution profile to a mature conformation [385]. Induction of cell-surface glycoproteins on HL-60, particularly as seen in the time of appearance of gp130 following DMSO treatment, was concomitant with development of chemotactic ability [109]. In this context, patients with impaired granulocyte chemotaxis have greatly reduced amounts of GP130 in their granulocytes [342]. Cytoskeletal proteins of HL-60 have also been studied. During induced maturation, there is increased synthesis of vimentin and other structural proteins. As may be expected, the profiles of cytoskeletal proteins induced by DMSO resemble those of granulocytes and, following phorbol ester induction, resemble those of macrophages [27]. Modulation of expression of several cell surface antigens has been characterized by the use of monoclonal

Kapil Mehta et al. antibodies. In one study, two monoclonal antibodies, B9.8.1 (specific for monocytes and metamyelocytes), were used to characterize maturational changes [310]. Treatment of HL-60 with granulocyte- or macrophageinducing agents resulted in enhanced antigen expression. In a similar study, reactivity with the monoclonal antibodies Mo1 and Mo2, recognizing determinants specific to the myelomonocytic lineage, could be induced with phorbol esters, as well as protein inducers of differentiation [69, 379]. HL-60 constitutively expresses HLA-A and -B antigens, and induction of HLA-DR determinants has been reported [69]. Normal myelomonocytic precursors express the DR antigens twice: transiently during early stages (CFU-GM) [98], and later as monocytes. HLA-DR determinants of HL-60 have been detected following treatment with inducers of monocyte macrophage differentiation, but not with agents that induce granulocyte maturation. The expression of Fc and C3 receptors has been studied extensively in U937. The receptor for IgG1 can be induced by a variety of agents, both physiologic and pharmacologic [141, 200]. The receptor for IgE is also present, but its modulation has not been studied. The C3B receptor (as recognized by EAC or Mac-1 monoclonal antibody) is inducible in a similar manner, as are Mac-1 and macrophage-restricted Mac-3 cell-surface antigens [325]. The chemotactic receptor for the potent chemotactic peptide fMLP is weakly expressed in U937 (7,505 sites/ cell). Within 3 days of exposure to phorbol esters, dexamethasone, or protein inducers of differentiation (excluding native or recombinant γ-interferon), up to fivefold increase in fMLP receptors is seen [142, 313]. U937 cells are generally considered devoid of any Ia antigen on their surface; however, small numbers of cells react with antibodies to HLA-DR and HLA-DS/ CD molecules. It has proved possible to clone such Ia-positive cells and develop constitutively Ia antigenpositive variant lines of U937 [121]. Induction of HLA-DR determinants in response to treatment with gamma-interferon and PG has been reported [311]. Beta2-microglobulin expression has also been shown to increase in response to protein inducers of differentiation [287]. Insulin may play an important role in the growth regulation of U937 cells, since the number of insulin receptors is reduced after incubation with differentiation-inducing agents, and this may have a causal role in the observed growth inhibition. The association between differentiation induction and growth inhibition of U937 and HL-60 cells may also be linked to the ability of potent inducers of macrophage differentiation, such as phorbol ester, to induce cellular production of the

531 protein of molecular weight 17,000, termed tumor necrosis factor (TNF) [6, 309]. This activity is inhibitory to neoplastic cell proliferation, including that of leukemic cells and normal hematopoietic stem cells. The intriguing possibility of autoregulation of leukemic cells by production of an endogenous growth-inhibitory molecule (TNF) is suggested. More than 80% of cells from a human promyelocytic leukemic cell line (HL-60) possess the capacity for selfrenewal as evidenced by their ability to form large primary colonies in semisolid medium and the presence within these colonies of cells capable of subsequent colony formation. Colony development is independent of colony-stimulating factor. The observed autostimulation suggests the production of specific growth promoters by the cells. Differentiation, either to mature granulocytes or to macrophages, induced by various agents is associated with reduced cloning potential. Nevertheless, colonies containing differentiated cells can be developed either by cloning cells in the presence of suboptimal concentrations of inducer or by adding inducers to colonies developed in its absence. The loss of self-renewal was found to be one of the early properties that changed following the initiation of differentiation. The loss preceded not only the overt expression of maturation-specific functions but also cellular commitment to terminal differentiation; shorter contact with the inducer is required to cause loss of self-renewal than to induce an irreversible transition to differentiation. This results in cells that lose their self-renewal potential without being able to complete their program of differentiation [96]. Increased migration of tumor cells upon differentiation to form diffuse colonies may be the result of increased mobility and chemotactic responsiveness. In a modified chemotaxis assay, HL-60 cells increased their migration in response to fMLP after a 5-day of induction with db-cAMP or DMF [99, 388]. Phagocytosis of latex beads or opsonized yeast and the capacity to kill microorganisms are readily induced by a wide variety of differentiation-inducing agents. Phagocytosis is one of the earliest acquired effector functions of both granulocytes and monocytes. Phorbol ester or DMSO-induced HL-60 cells effectively kill staphylococcus [190]. It has been demonstrated that differentiation-induced HL-60 cells can mediate monocyte ADCC-like reaction against antibody-coated chicken erythrocytes [69]. The U937 cell line is capable of being induced to mediate antibody-dependent cellular cytotoxicity (ADCC) effector function. First reports of ADCC capacity against tumor markers used lymphokine, interferon, and phorbol ester preparations to activate or “induce” the

532 cells [147, 324, 365]. ADCC activity of U937 cells against erythrocytes can be induced by as little as ten units of γ-interferon and 300 units of α- and β-interferon. U937 cells have been studied for their ability to support the intracellular multiplication of Toxoplasma gondii. A small portion of U937 cells can spontaneously phagocytize these protozoans, and after 24 h those cells have either lysed or contain numerous trophozoites within their vacuoles. After a 3-day incubation with lymphokine preparations there is a significant decrease in the number of organisms found within the vacuoles, in spite of a generalized increase in phagocytosis [408]. In an effort to analyze the pathways involved in differentiation of cell lines such as HL-60, many attempts have been made to select sublines capable of sustaining exponential growth in the presence of a single inducer of differentiation but not in the presence of different, structurally unrelated inducers of differentiation. Neutrophilic granulocytic and monocyte/macrophage programs of HL-60 are mechanistically different and separable, and both agent-specific and common quantitative alterations contribute to the mechanism for resistance to granulocyte differentiation. Numerous studies have demonstrated that the oncogene c-myc is amplified 20- to 40-fold in HL-60 cells compared with normal human DNA and is associated with an elevated level of cellular myc-mRNA, and a decrease in this mRNA follows chemically induced differentiation [37, 132]. Within 5 days of addition of DMSO or phorbol ester to HL-60 cultures, transcription of the c-myc gene was markedly reduced when compared with control cultures. This decrease was not accompanied by alteration in either the bulk rate of transcription or the c-myc copy number, suggesting that decreased cellular myc RNA levels are due to decreased transcription of the myc protooncogene [132]. The expression of c-myc in HL-60 does not correlate with the proportion of proliferating cells, and the kinetics of decrease upon DMSO induction is paralleled closely by an increasing proportion of histochemically detected, differentiated myeloid cells and by a decrease in clonogenic potential, but not by changes in the proportion of proliferating cells [97]. Changes in c-myc expression subsequent to differentiation of HL-60 can therefore be directly related to the differentiation process rather than to a cell cycle-related phenomenon. Gallagher et al. [112] observed a little change or decrease in the amplification level of the known amplified c-myc gene in various drugresistant sublines in comparison with wild-type HL-60 cells and despite the existence of numerous double, minute chromosomes (indicators of amplified genes) in some drug-resistant sublines. Differential response to differentiation agents could still be related to amplification of

Growth and differentiation factors as cancer therapeutics genes other than c-myc; however, tests with several other oncogene probes, including N-ras, Ha-ras, Ki-ras, myb, and abl, showed no evidence of amplification or gross rearrangement of these genes in DNA in any HL-60 line or subline [65]. High levels of c-fos expression have been detected in macrophages, but not in uninduced HL-60 cells. However, when HL-60 cells were induced to macrophages, c-fos expression was readily detectable in the differentiated cells [272].

Vitamin A Analogues as Leukemia DifferentiationInducing Agents Retinoids (vitamin A and its analogues) and retinoic acid, in particular, are a family of fat soluble compounds that exert a potent effect on the cell growth and differentiation of various cell types including promyelocytic leukemia, neuroblastoma, teratocarcinoma, breast cancer, prostate cancer, melanoma, bladder cancer, squamous cell carcinomas of cervix, skin cancer, head and neck cancer and rhabdomyosarcoma [68, 342, 361, 434]. Their antiproliferative and differentiation-inducing properties have brought them under the close scrutiny of oncologists. A major development in the clinical application of retinoids was the discovery that ATRA could induce the mature phenotype in myeloid leukemia cells thus resulting in complete remission in patients with acute promyelocytic leukemia. [46, 72, 80, 90, 91, 170, 399] In light of high complete remission rates achieved (80–90%), ATRA is currently being used as a frontline therapy for treatment of APL patients. Retinoids exert their biological effects by interacting with two families of intracellular nuclear receptors: the retinoic acid receptors (RARs), which are activated by ATRA and 9-cis retinoic acid, and the retinoid X receptors (RXRs), which are activated by 9-cis retinoic acid only [32, 227]. Each of these receptor families consists of three subtypes (- α-β and -γ), and each subtype exists in multiple isoforms that arise due to alternative splicing and differential use of two promoters (Table 1).

Table 1. Retinoic acid (RAR) and retinoid X (RXR) receptors Receptor

Isoforms

Chromosomal location

RARα RARβ RARγ RXRα RXRβ RXRγ

α1, α2 β1, β2, β3, β4 γ1, γ2 α1, α2 β1, β2 γ1, γ2

17θ21.1 3p24 12q13 9q34.3 6q21.3 1q22–q23

Ligand ATRA & 9-cis RA 9-cis RA

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RARs and RXRs share – along with other members of the nuclear hormone receptor superfamily – a modular structure having a highly conserved DNA binding domain (DBD), a ligand-binding domain, and a less well conserved amino-terminal domain and a hinge region present between the DBD and the LBD. The DBD has two zinc fingers and is highly conserved between the members of the superfamily. The LBD is also involved in dimerization and has a carboxyl-terminal activation domain, called AF-2. The AF-2 domain has been shown to function as an autonomous transcriptional activation domain when fused to a heterologous DBD. The hinge region of the retinoid receptors has been shown to constitute the domain responsible for interaction with some co-repressors [51, 52]. The presence of intermediary factors that link nuclear receptors with basal transcriptional factors had been proposed based on transcriptional interference/squelching observed between transcriptional activation domains of different receptors. Two kinds of factors have been identified: (i) the corepressors, which bind to unliganded

a

receptors but dissociate upon ligand binding (Fig. 1a), and (ii) the co-activators that bind only to the liganded receptors (Fig. 1b). Two corepressors, SMRT and N-CoR, have been identified that associate with unliganded retinoid receptors and suppress the basal transcriptional activity. Another co-repressor, mSin3A, which is a homologue of the yeast global-transcriptional repressor Sin3p, has been shown to interact directly with SMRT and N-CoR1. Sin3A and SMRT, in turn, can interact with histone deacytylase 1 (HDAC1) to form a multisubunit repressor complex [277]. Similarly, a growing number of coactivator proteins have been identified, though their mechanism of action is not well understood except for it has been known that they bind to the liganded receptor at the AF-1 domain [208]. One factor that is known to potentiate the transcriptional activation of the retinoid receptors is the steroid receptor coactivator SRC-1 [412]. The formation of a RXR/RAR heterodimer is required for the high- affinity binding to specific DNA sequences known as retinoic acid response elements (RAREs) that is critical for subsequent

+0.1-1 µM ATRA RARα RXRα HL - 60 Cell/WT

+9-cis RA

RARα RXRα Differentiation

Apoptosis

b 1 µM ATRA

Differentiation blocked due to mutation in the RARα gene

HL-60R

c

0,1-1 µM ATRA

RARα-transfected HL-60 R cells regain differentiation potential in response to ATRA treatment

HL-60R/RARα

d 0.1 µM 9-cis-RA

RXR -transfected HL-60R cells undergo apoptosis in response to RXR ligation

HL-60R/RXRa

Figure 1. A model showing the basal repression and retinoid-induced transcriptional regulation of the target gene(s). (a) The RAR/RXR heterodimer binds to a specific direct-repeat regulatory element that is separated by 5 bp (DR5-RARE) in the regulatory site of the target gene. In the absence of ligand (ATRA), the RAR/RXR heterodimer recruits a transcriptional repression complex that comprises N-CoR, mSin3a, SMRT, and a histone deacetylase (HDAC-1). The HDAC-1 activity helps to suppress transcriptional activity. (b) Addition of ATRA results in a conformational change in the RAR/RXR heterodimer that leads to the release of the repressor complex and recruitment of a transcriptional activator complex that has histone acetyltransferase (HAT) activity. This activity destabilizes nucleosomes and creates a permissive state for promoter activation

534 retinoid-induced transcription of target genes. Most RAREs have been identified in the regulatory regions of genes whose transcription is induced by retinoids. These cognate response elements consist of direct repeats (DR) of two core motifs in the form of PUG(G/T)TCA(X) nPUG(G/T)TCA or similar but degenerate motifs. The most common spacing observed in RAREs is 5 bp(DR5); however, RAREs containing 1 (DR1) and 2 bp (DR2) are also common. HL-60 and NB4 cells have been extensively used to investigate retinoid-mediated signaling pathways during normal myelopoesis. Like normal granulocytes, they undergo terminal differentiation in response to retinoid treatment. Moreover, like normal granulocytes, ATRA-induced HL-60 and NB4 cells have a limited in vitro life span and undergo apoptosis in response to certain stimuli [233, 431]. Thus, Both of these cells can also serve as a model to investigate the molecular mechanisms of apoptosis in terminally differentiated hematopoietic cells. However, the interpretation of retinoids’ action on cell growth, differentiation, or apoptosis becomes complicated in view of the fact that retinoids can mediate these effects by binding and activating two different types of nuclear receptors: the RARs and the RXRs, which as described above, differ in their sequences and exhibit distinct ligand-binding properties. Since most of the blood cells, including HL-60 and NB4, express both types of receptors [144, 327], retinoid-induced effects in these cells may be a result of activation of either RARs, RXRs or both types of receptors. We addressed this problem by using a mutant subclone of the HL-60 cell line (HL-60R) in which retinoid receptor function has been abrogated as a result of a -trans dominant negative regulatory point mutation in the ligand-binding domain of the receptor [333]. HL-60R subclones expressing specific receptors were generated by retrovirusmediated transduction of RARα or RXRα specific coding sequences [334]. Our results suggested that the introduction of RARα into HL-60R cells completely restored their sensitivity to ATRA-induced granulocytic differentiation. In contrast, the introduction of RXRα cDNA rendered these cells remarkably sensitive to apoptosis in response to the RXR-specific legends [239]. These observations provided a direct evidence for RARα involvement in retinoid-induced differentiation and of RXRα in programmed cell death (Fig. 2). Using the receptor-selective retinoids, other investigators arrived at similar conclusion that specific retinoid receptors are involved in the regulation of differentiation and apoptotic events in HL-60 cells [278, 386]. More recently, it was demonstrated that ATRA induces post-maturation apoptosis of APL cells by regulating

Growth and differentiation factors as cancer therapeutics TRAIL (tumor necrosis factor related apoptosis inducing ligand) expression. Thus, induction of TRAILmediated death signaling may contribute to the therapeutic value of retinoids [8]. A two-step model has been proposed for induction of differentiation where early events anteceding precommitment regulate growth arrest and late events, and subsequent to precommitment regulate the choice of a specific differentiation lineage [414]. Thus the lineage specificity of cells treated sequentially with two discrete exposures to alternative inducers depends on the order of exposure. Retinoic acid exposure of HL-60 for 24–72 h followed by phorbol ester treatment produces monocyte-macrophage differentiation, whereas reversing the order of treatment favors granulocyte differentiation [425]. The precommitment phase has a characteristic duration following retinoic acid exposure, persisting for several cell cycles despite removal of retinoic acid [134]. This precommitment is paralleled by elevations in c-myc. Other events associated with retinoid induction of differentiation involve elevation of tyrosine kinase activity [74] and a protein kinase C cascade system. Sphinganine, a potent inhibitor of PKC, enhances differentiation of HL-60 induced by retinoids, and the granulocytes produced are more fully differentiated as indicated by enhanced superoxide production in response to fMLP [363]. A role for topoisomerase II in retinoidinduced granulocytic differentiation has been suggested [102]. In HL-60 differentiation, retinoic acid stimulates transient relocation of DNA supercoiling, and this is associated with the formation of small numbers of protein-linked DNA breaks (a characteristic of topoisomerase reactions). Both events are perturbed by VP16, which inhibits differentiation [102]. Combinations of differentiation-inducing agents have been explored in vitro with retinoic acid. Twenty-two of 24 patients with ANLL showed differentiation of bone marrow in cultures with a combination of retinoic acid (10−6 M), aclacinomycin A (80 nM), and dimethylformamide (100 mM) [145]. Enhancement of differentiation was seen with retinoid combined with interferons-α and -β [183, 193, 196], and -&gammabdot; [153, 383]. Tumor necrosis factor (TNF) at 2.5 U/ml inhibited growth and, synergistically with retinoic acid, induced differentiation in HL-60 and KG-1 cultures and in marrow cultures from four to nine patients with ANLL [378]. Retinoic acid reversed TNF inhibition of normal marrow myeloid colonies and leukemic growth marrow cultures from three to nine patients with ANLL. In this context, leukemia differentiation-inducing factor (GM-DF) produced by mitogen-stimulated human leukocytes acted synergistically with retinoic acid in inducing

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mSin3a

a

N-CoR

HDAC-1 C

Repression complex

SMRT

LBD

LBD

TRANSCRIPTIONAL REPRESSION

RARα

Histones DBD

DBD Closed chromatin (repressed) 5 bp AGGTCA

AGGTCA

DR-5 RARE ATRA

b

CPB/p300 LBD

SRCs

(HATs)

LBD

Ligated RARα DBD

TRANSCRIPTIONAL ACTIVATION

DBD Ac

Ac

Ac

AGGTCA

Ac Ac

Ac

Open chromatin (activated)

5 bp AGGTCA

DR-5 RARE

Figure 2. Retinoid-mediated differentiation and apoptosis of myeloid leukemia HL-60 cells (a) The RARα nuclear receptors in HL-60 cells when bind to and are activated by appropriate ligand (e.g. ATRA) results in granulocytic differentiation of the cells. The differentiation process is associated with induction of several new genes, including RXRα receptors. It is conceivable that ATRA is isomerized to 9-cis RA in situ, which can then bind and activate RXRα receptors, leading to the onset of apoptosis in differentiated HL-60 cells. (b) HL-60R cells harbor a functional mutation in the RARα gene that results in non-functional RARα protein and renders the cells resistant to ATRA. (c) Retrovirally-transduced expression of functional RARα in HL-60R cells restores the ability of these cells to differentiate in response to ATRA treatment. (d) However, transduction of RXRα cDNA, renders the HL-60R cells highly sensitive to RXR-selective ligand(s) (such as, 9-cis RA) and induce massive apoptosis in these cells without morphological differentiation.

maturation of the human leukemic lines U937 and HL-60 [292]. T-cell-derived GM-DF was subsequently shown to be due to the synergistic action of IFNγ, lymphotoxin, and GM-DSF [154]. Compounds elevating intracellular levels of cAMP, such as dibutryl cAMP, PGE, and choleratoxin, acted synergistically with retinoic acid to induce maturation of both cell lines. In contrast to the requirement for continuous presence of retinoic acid for up to 5 days in order to achieve terminal differentiation of HL-60 cells, differentiation proteins or cAMP-elevating compounds are active on leukemic cells primed with retinoic acid within 8–16 h [292]. Retinoid-mediated differentiation of myeloid leukemia cells is not a universal phenomenon. While the murine myelomonocytic leukemic cell line WEHI-3 can

be induced to mature neutrophil differentiation [74] and retinoic acid induces the human malignant monoblast line U937 to monocyte-like cells with the capacity to reduce nitroblue tetrazolium [292], the human myeloid cell lines KG-1 and K562 cannot be induced to differentiate [77]. The mouse myeloid leukemia M1 can be induced to increased levels of lysomal enzyme production without induction of phagocytosis, locomotive activity, or morphological maturation. Fresh leukemic cells from patients with various myeloid leukemias have also been exposed to retinoic acid in short-term primary suspension cultures, and morphological and functional maturation was observed only in cases of acute promyelocytic leukemia [113, 193]. The differential sensitivity of various leukemias to retinoic acid induction of terminal

536 differentiation is not dependent on the presence or absence of cellular retinoic acid binding protein [77, 371]. High-affinity retinoic acid receptors (RARs), predominantly alpha-type, were found in 12 leukemic cell lines and in marrow blasts from 32 patients with ANLL [203, 283, 396]. While some correlation was found between RAR expression and retinoid response with four leukemic cell lines [203], extensive analysis of marrow cultures from a large group of primary AML patients showed no correlation between receptor expression and ability of retinoic acid to inhibit colony formation or induce differentiation [396]. The potential for terminal differentiation may be irreversibly lost in many cases of acute myeloid leukemia, but this need not negate the therapeutic value of retinoic acid treatment in a wide range of leukemias, since considerable evidence has accumulated to suggest that retinoids can selectively inhibit leukemic cell self-renewal independently of activation of a differentiation program in the leukemic stem cell. Retinoic acid is a potent inhibitor of the clonal growth in vitro of myeloid leukemic cells, and a 50% growth inhibition of HL-60 was achieved by 2.4 nM retinoic acid. The human myeloid leukemic line KG-1, which is not inducible to differentiation, was nevertheless extremely sensitive to retinoic acid, with 50% of the colonies inhibited by 2.4 nM concentrations of the drug [77]. Thus, antiproliferative action of retinoids upon leukemic cells is more general than the incidence of induction of terminal differentiation, and is seen with retinoid concentrations readily attainable in vivo. The potential efficacy of retinoic acid in the treatment of human leukemia is further suggested by the observation that it enhances colony-stimulating factor (CSF) induced clonal growth of normal human myeloid and erythroid cells in vitro [78]. In long-term bone marrow culture it also enhanced progenitor cell production. Maximal stimulation occurred at a retinoic acid concentration of 3 × 10−7 M acid, which increased the mean number of colonies by 213 ± 8% over plates containing CSF alone [31]. Retinoic acid has no direct CSF activity, nor does it stimulate CSF production by the cultured bone marrow cells or marrow stroma. This stimulation may be mediated by increased responsiveness of the progenitor cells, possibly by increasing the number of growth factor receptors per cell. Retinoids are reported to enhance the binding of epidermal growth factor (EGF) to fibroblasts and epidermal cells by increasing the number of EGF receptors per cell [176, 188, 319]. Enhancement of normal myelopoiesis [31] and inhibition of myeloid leukemic cell proliferation by retinoic acid suggest that 13-cis-retinoic acid, which is significantly less toxic in vivo than retinoic

Growth and differentiation factors as cancer therapeutics acid, might be effective in the therapy of patients with myelodysplastic syndrome because of the possibility of prevention of progression to overt leukemia of these preleukemic patients, for whom no other effective treatment is currently available. Table 2 lists some of the clinical studies conducted in patients with myelodysplastic syndromes (MDS). These studies employed oral doses of retinoids ranging from 10–100 mg/m2/d for 4 weeks to 5 years of duration [171]. The percentages of response described in these studies have been encouraging, but toxicity often limited the duration of the therapy. Hepatotoxicity was dose-limiting but was completely reversible upon cessation of the therapy. In addition, cheilosis, hyperkeratosis, stomatitits, and increase in serum triglyceride levels were reported. For example, Besa et al. [29] in their study observed a 47% response including complete remissions in 17 patients with MDS receiving 100 mg/m2 13-cis-retinoic acid with an improved survival in responders of 33 months versus 10 months in the nonresponders. However, no beneficial effects were reported with either retinoic acid or isoretinoin in two studies of 14 MDS patients [146, 164]. N-4-Hydroxyphenylretinamide (Fenretinide) lacked clinical effect in 15 MDS patients and in some may have enhanced leukemic progression [115]. In a double-blind, placebo-controlled trial of 13-cis-retinoic acid in 68 MDS patients with 100 mg/m2 for 6 months, no significant differences were observed between treatment groups [195]. Approximately 30% of patients in both groups had progression of the disease and survival was virtually identical. Ninety percent of the treated patients developed mild to moderate skin toxicity. In view of these generally negative results, MDS therapy with retinoic acid has been extended to combinations with other agents with potential differentiationinducing capacity (Table 2). In a comparative study of such combination therapy in 62 MDS patients, 50% response was seen with a combination of retinoic acid, 1,25(OH)2 D3, and interferon, a response rate comparable to that obtained with low-dose cytosine arabinoside [151]. Combining all four agents proved too toxic. Combining retinoic acid with low-dose ara-C produced favorable results in one study [103], but in another study of 14 MDS patients the response was no better than either agent alone [160]. Combining a tocopherol (800 mg/m2) with retinoic acid (100 mg/m2) produced a 62% response in 13 MDS patients and reduced the toxicity involving liver damage, hyperkeratosis, and mucositis [29]. In poor-prognosis acute nonlymphoblastic leukemia, 13-cis-retinoic acid has some efficacy on its own in one

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Table 2. Clinical studies with retinoids in MDS patients Retinod

No. of Pts.

Dose (mg/m2/day)

Duration (weeks)

Response (%)

Ref.

15 18 15 10 8 33 30 35 66 34

20–125 50–100 100 100 20–100 20 50 100 100 10–60

7–30 >8 8 8 6 8 >4 6 6 >12 (5 years)

33 16 20 30 50 9 56 3 23 12

[125] [128] [368] [312] [183] [58] [205] [195] [28] [35]

2 14 29 23 10

45 30–90 10–250 45 45

8–10 12 8 >4 6

100 0 3 13 50

[392] [15] [15] [289] [393]

2 3 77 44 26 40 90 75 52–61 23

[160] [59] [103] [151] [152] [114] [235] [391] [93] [165]

13-cis RA

ATRA

Retinoid + others 13-cis RA + LD-Ara-C 13-cis RA + LD-Ara-C 13-cis RA + VCR 13-cis RA + Vit.D3 + IFNα 13-cis RA + Vit.D3 + LD-Ara-C ATRA + G-CSF ATRA + G-CSF + EPO + tocopherol IFNα2 + thymopentin + LD-Ara-C 13-cis RA + Vit.D3 + 6-TH ATRA + IFNα + G-CSF 6-TH, 6-thyoguanine; LD-Ara-C, Low dose Ara-C

small study [164], and clinical improvements were seen using combinations with low-dose ara-C [349] or vincristine or 6-thioguanine [103]. Evidence of differentiation-induction was obtained in one case of a patient who achieved a complete remission with cytogenetic evidence of persistence of an abnormal clone in the marrow [349]. Acute promyelocytic leukemias (APL) almost uniformly respond to retinoic acid treatment. APL constitutes approximately 5–10% of all cases of acute myeloid leukemia (AML) and is characterized by M3 morphology of FAB classification and chromosomal translocations fusing retinoic acid receptor alpha (RARα) gene on chromosome 17 and one of four different genes, including promyelocytic leukemia (PML), promyelocytic zinc finger (PLZF), nucleophosmin (NPM) or nuclear matrix associated (NuMA) gene [300]. The most common forms of translocations are t(15,17) (q22,q21) encoding PML-RARα and t(11,17)(q23,q21) encoding PLZF-RARα fusion receptor proteins, found

in 99% and >1% of APL patients, respectively. The translocations are usually reciprocal chromosomal translocations, leading to creation of reciprocal hybrid receptor proteins (X-RARα and RARα-X). APL expressing PML-RARα, NPM-RARα or NuMA-RARα are responsive to ATRA-induced differentiation effects with the exception of PLZF-RARα type APL that is resistant to ATRA. ATRA induces differentiation of APL blasts into terminally differentiated granulocytic cells that is associated with clinical remissions. ATRA-induced differentiation of APL blasts requires expression of PML-RARα receptor protein [360]. PML-RARα can heterodimerize with RXR or form homodimers and subsequently bind to retinoic acid response element (RARE), located in the promoters of the ATRA-responsive target genes. ATRA can bind to PML-RARα with an affinity comparable to RARα. In the absence of ligand, RAR-RXR in normal blasts and PML-RARα-RXR heterodimers in APL cells, recruit nuclear co-repressor proteins, N-CoR or SMRT,

538 and Sin3A or Sin3B which in turn form complex with histone deacetylase enzymes (HDAC1 or HDAC2), resulting in transcriptional repression or silencing [300]. The transcriptional suppression occurs because deacylation of histone protein creates conformational changes, limiting access and binding of transcription factors and RNA polymerase to related genes [198]. At physiologic concentrations of ATRA (10−9−10−8 M), the nuclear corepressors protein and HDAC complex are dissociated from RARα in normal blasts, which in turn results in recruitment of co-activators with histone acetyltransferase (HAT) activity, such as steroid receptor coactivator-1 (SRC-1), PCAF, p300/CBP, ACTR, TIF2 or P/CIP (Fig. 1b). Acetylation of lysine residues in the N-terminal of histones by HAT activity results in transactivation of responsive genes leading to differentiation. However, the physiologic concentration of ATRA does not cause dissociation of nuclear co-repressors protein and histone deacetylase complex from the PML-RARα fusion receptors in APL blasts, leading to differentiation block. The co-repressor complex is dissociated from PML-RARα at only pharmacological concentrations (10−7−10−6 M) of ATRA, resulting in removal of transcriptional repression and transcription of genes related to differentiation. In addition to release of transcriptional repression, the other possible mechanism involved in ATRA effectiveness in myeloid cell differentiation include expression of different class of genes [207, 212] including induction of expression of p21WAF1/Cip1 cyclin-dependent kinase inhibitor [45], upregulation of C/EBP- [270], interferon regulatory factor-1 (IRF-1) [308], PDCD4 [418] and regulation of the localization of PML oncogenic domains (PODs) [403]. More interestingly, the treatment of APL cells with ATRA inhibited the synthesis of vascular endothelial growth factor that in turn resulted in decrease in bone marrow microvessel density [185]. More recent results with acute promyelocytic leukemia have clarified the role of ATRA in this disorder. In multiple studies [54, 91, 364, 400], it is now clear that a dose of 45 mg/m2/day given by mouth in one or two divided doses until complete remission followed by anthrocyclinebased combination chemotherapy for consolidation, will induce a high response rate and improve survival in patients with APL. On the basis of these and other studies, ATRA has recently been approved by the FDA as standard treatment for APL. Although trans-retinoic acid is effective in the treatment of APL, resistance is common with the return of leukemia cells shortly after treatment in the absence of effective programs of consolidation. The role of chemotherapy during induction treatment of APL is still unclear. European approaches have utilized high doses

Growth and differentiation factors as cancer therapeutics of chemotherapy including an anthracycline plus cytosine arabinoside in patients with white counts exceeding 10,000/m2 at initiation of therapy. The clinical studies conducted over the past years have determined that the combination of ATRA and chemotherapy gives better survival than chemotherapy alone in newly diagnosed APL; the relapses were less and complete remissions are slightly higher. These studies also revealed that maintenance treatment with ATRA, and possibly with low dose of chemotherapy, can further reduce the incidence of relapse [92, 171]. Although ATRA therapy is usually well tolerated, two problems are frequently encountered. The first is leukocytosis, an increase in peripheral leukocytes to ≥20,000 cells/μL, which occurs in about half of the APL patients treated with tRA [106]. The second, more serious problem, retinoic acid syndrome (RAS), develops in about 20–30% of APL patients treated with tRA. RAS is characterized by high fever, respiratory distress, weight gain, pulmonary infiltrates, hypotension, pleural effusion, and sometimes renal failure [70, 126]; findings at autopsy show extensive infiltration of mature myeloid cells into lungs, skin, kidney, liver, and lymph nodes. Although leukocytosis is often present in RAS, one third of APL patients who have normal leukocyte counts also develop this syndrome. Steroids such as dexamethasone have ameliorated RAS [106]; possibly by affecting leukocyte activation, cytokine production, or endothelial reactions. Since RAS occurs only in patients with APL or AML who have been treated with ATRA, this syndrome is considered to reflect an aberrant interaction between maturing granulocytes and host tissues. Because the clinical signs of RAS resemble those of endotoxin shock and adult respiratory distress syndrome it has been suggested that ATRA treatment may affect the expression of cytokines by myeloid cells. Induction of IL-1 and G-CSF secretion by APL cells under the influence of ATRA may contribute to hyperleukocytosis in vivo. On the other hand, secretion of IL-1, IL-6, TNF-α, and IL-8 could contribute to the pathogenesis of RAS, since they are involved in leukocyte activation and adherence and have been implicated in the development of the adult respiratory distress syndrome. No evidence of synergism of 13-cis-retinoic acid with chemotherapeutic agents was found in the treatment of patients with chronic myeloid leukemia. Based upon in vitro observations that retinoic acid significantly reduced the recloning capacity of bone marrow myeloid progenitors in vitro in certain cases of this disease, Arlin et al. [12] added retinoic acid to an intensive chemotherapy protocol including daunorubicin, cytosine arabinoside, and thioguanine, and in 17 evaluable patients

Kapil Mehta et al. did not observe any increase in the incidence and duration of true Ph chromosome-negative remission in the chronic phase of the disease. The future of retinoid therapy resides in (a) development of analogues with enhanced differentiation inducing action with reduced toxicity, (b) developing combinations with either conventional chemotherapeutic drugs or other differentiation-inducing agents, applying retinoids more extensively as a maintenance therapy, and (c) developing approaches to reduce toxicity such as use of liposomeencapsulated retinoic acid. In this context, a recent study suggested that tyrosine kinase inhibitor ST1571 could augment the cyto-differentiating, antiproliferative, and apoptogenic activity of ATRA [120]. Moreover, in APL cell lines made resistant to ATRA, the STI1571 could relieve the resistance. Similarly, the use of liposomalATRA was shown to be superior to the oral ATRA in terms of maintaining higher plasma levels in animal models and in humans [86, 88, 238, 305]. These results demonstrated that encapsulation of ATRA in liposomes and I.V. administration generate better pharmacokinetic profile than oral ATRA by circumventing hepatic metabolism of ATRA. Evaluation of liposomal ATRA in phase I trial in patients with refractory hematological malignancies showed that in contrast to the decline in plasma AUC (area under the concentration time curve) of ATRA seen 3 to 4 days of initiation of oral ATRA, there were no difference between the AUC on day 1 and day 15 following liposomal ATRA treatment [34]. In the same study, liposomal ATRA was shown to be safe and toxicity profiles were similar to oral ATRA, although liposomal ATRA produced much higher AUC. I.V. administration of liposomal ATRA (90 mg/m2) monotherapy was shown to be effective in newly diagnosed APL patients, inducing PCR-negative molecular CRs in a high proportion of patients [76, 86] These studies supported the hypothesis that I.V. liposomal administration may improve activity of ATRA by altering its pharmacological profile and remain elevated following extended treatment, providing a basis for long-term remissions in APL patients. New synthetic retinoid compounds: These include Am-80, Am580 and 4-HPR, CD437, MX3350-1 and LGD1069 (Targretin, Bexarotene), a RXR selective retinoid. Am-80, a synthetic retinoic acid receptor (RAR)specific agonist, has been successful in patients with relapsed APL previously treated with ATRA, inducing CR in about 60% of patients in a limited study [435, 436]. Am80 is approximately ten times more potent than ATRA in terms of induction of differentiation in vitro, and more stable to light, heat, and oxidation than ATRA. Am80 has a low affinity for cellular retinoic acid binding protein,

539 and does not bind to retinoic acid receptor-gamma. Am80 has been shown to be effective in patient with APL that had relapsed from CR induced by ATRA. Of 24 evaluable patients, who received Am80, 6 mg/m2, orally alone daily until CR, 14 (58%) achieved CR [435]. The interval from the last ATRA therapy was not different between CR and failure cases. The outcome was well correlated with the in vitro response to Am80 in patients examined. Adverse events observed in patient treated with Am80 included retinoic acid syndrome, hyperleukocytosis, xerosis, cheilitis, hypertriglyceridemia, and hypercholesterolemia, but generally milder than those of ATRA, which all patients had received previously. Am80 is effective in APL relapsed from ATRA-induced CR and deserves further clinical trials, especially in combination with chemotherapy. Am580, a stable retinobenzoic derivative and a RAR-alpha agonist, is a powerful inducer of granulocytic maturation in NB4 and in freshly isolated APL blasts [437]. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF) at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. In addition, Fenretinide (N-(4-hydroxyphenyl) retinamide, 4-HPR), a synthetic derivative of ATRA [438], and nonretinoid compounds such as, 1,25-dihydroxyvitamin D3 [138, 438, 440] and vitamin K2 in combination with ATRA [441] have been shown to be effective in inducing differentiation in ATRA-resistant APL cell lines and in cases to whom ATRA cannot be used [442– 444]. 4-HPR inhibits cell growth through the induction of apoptosis in hematopoietic malignancies and variety of solid tumor cells including head and neck, cervical carcinomas, neuroblastoma, lung carcinoma and breast cancer cells [445–450]. Recently, 4-HPR received a significant amount of attention because of its promise as chemopreventive and therapeutic activity in breast, prostate and ovarian cancers in preclinical and clinical models with minimal toxicity [451, 453]. The ability of 4-HPR to induce apoptosis in ATRA-resistant tumor cell lines, such as acute myeloid leukemia (HL-60R) and promyelocytic leukemia (NB306) [446], neuroblastoma [449], head and neck carcinoma [455] and breast cancer cells [456] suggested that 4-HPR acts independently of retinoid receptors to mediate its biological actions. However, a potential role for RARs in 4-HPR induced growth inhibition or apoptosis has been supported by other studies [450, 457–459]. 4-HPR inhibits cell growth through the induction of apoptosis rather than differentiation in HL-60 and NB4 cells [103, 111].

540

Vitamin D Metabolites and Analogs as Leukemia Differentiation-Inducing Agents The term vitamin D is generally used to describe a number of chemically related compounds having common antirachitic properties, but differing in the rapidity of their action and the conditions under which biologic activity is observed. In humans, cholecalciferol (D3) produced in the skin and the fraction obtained from the diet undergo sequential hydroxylation reactions, first in the liver microsomes and then in the kidney mitochondria, resulting in the formation of 25-(OH) D3 and 1,25(OH)2 D3, respectively. The latter is thought to be the active form of D3 in enhancing bone resorption mediated by osteoclasts and in enhancing absorption of calcium and phosphorus by the intestine. In addition, it appears to act on the kidney in concert with parathyroid hormone to promote calcium resorption. It was formerly believed that 1,25-(OH)2 D3 was synthesized in placenta and by calvarial cell suspension containing osteoclasts, osteoblasts, fibroblasts, and endothelial cells [366]. The local production of active metabolites of vitamin D by target organs such as bone raises interesting questions as to the role of these tissues in mediating vitamin D action, and may provide indications of a new dimension to local actions of vitamin D metabolites on normal or leukemic marrow cell function, as well as more conventional aspects of mineral metabolism. Interest in vitamin D action in hematopoiesis was prompted by the observations that osteoclasts originate by fusion of circulating mononuclear precursor cells and almost certainly represent one of the end-stage cells of mononuclear phagocyte differentiation [43, 177, 178, 376]. Like osteoclasts, other mature, nonproliferating phagocytic mononuclear cells, such as monocytes and macrophages, possess the capacity to attach to and degrade bone matrix [178]. Monocytes and macrophages possess receptors for 1,25(OH)2 D3, and the culture of human monocytes with 10−8 M of this metabolite results in macrophage maturation [320]. In cultures of normal human bone marrow, 1,25(OH)2 D3 induces extensive macrophage differentiation [236]. Long-term cultures of human cord blood myeloid cells also terminally differentiate to monocytes and macrophages with vitamin D [344]. Direct evidence of 1,25-(OH)2 D3 induction of osteoclast development has been reported by Abe et al. [72], who demonstrated that a 1.2 nM concentration of the metabolite induced extensive fusion of mouse alveolar macrophages, and that in the presence of the lymphokine macrophage fusion factor, as little as 0.012 nM was active in producing multinucleated giant cells. Multinucleated giant cell formation

Growth and differentiation factors as cancer therapeutics has also been observed when the HL-60 cell line and the mouse macrophage-like J774.2 cell line were exposed to 10−7−10 M 1,25-(OH)2 D3 for 24–72 h [22]. In this context, in vivo injection of 1,25-(OH)2 D3 into patients with malignant osteopetrosis corrected bone binding and resorptive deficiencies in the patients’ monocytes [22]. Koeffler et al. [191] have shown that 10−7 to 10−9 M concentrations of either the active metabolite of vitamin D (1,25-(OH)2 D3) or certain fluorinated analogs of vitamin D can induce normal human myeloid progenitors (GM-CFC) in the presence of CSF to differentiate preferentially to macrophages in vitro. Marrow cultures exhibited an absolute, not just a proportional, increase in macrophage colonies, indicating that the action was not simply inhibition of granulopoiesis. The plasma concentration of 1,25-(OH)2 D3 in humans is approximately 7.7 × 10−11 M [148], and the concentration of 1,25-(OH)2 D3 inducing macrophage differentiation of progenitors in vitro is >10−9 M, raising the question of physiologic relevance of this observation. Certainly, patients receiving superphysiologic doses of 1,25-(OH)2 D3 have not been reported to have monocytosis; likewise, patients with vitamin D-resistant rickets have not been reported to have low monocyte or macrophage levels. Nevertheless, the possibility of local production of the active form of vitamin D by cellular components of the marrow microenvironment [385], possibly even by tissue macrophages, raises the possibility of much higher local concentrations of 1,25(OH)2 D3 in the environment of the progenitor cells than plasma levels may suggest. Furthermore, recurrent infections, impaired phagocytic function, and decreased mobility of leukocytes have been shown in vitamin D-deficient states. This defect in phagocytic function has been reversed by in vitro culture of macrophages with 1,25-(OH)2 D3 [22, 332]. The immediate biological precursor of 1,25(OH)2 D3, 25-(OH) D3, is without biologic effect unless used at 10- to 100-fold higher concentrations than its hydroxylated metabolite. Thus, evidence that macrophages themselves have 1-hydroxylase activity and can synthesize 1,25-(OH)2 D3 from its precursor suggests that this molecule may also be a monokine with important local actions in recruitment and activation of macrophages [332] and of osteoclasts, with which they share a common derivation from a marrow hematopoietic progenitor.

Action of Vitamin D Metabolites on Cancer Cells Besides playing a crucial role in maintaining the calcium homeostasis in the body, 1,25-(OH)2 D3 can also affect cell growth and differentiation in several cell types,

Kapil Mehta et al. including cancer cells. Based on these properties of 1,25-(OH)2 D3, it has been studied for its ability to inhibit and prevent the cancer growth. The results from various clinical studies were though encouraging but due to the calcemic adverse effects the therapeutic window of this compound was extremely narrow [36]. Abe et al. [46] were among the first ones to demonstrate that 1,25-(OH)2 D3 could induce macrophage differentiation of myeloid leukemic cells. Induction of phagocytes, lysozyme production, and locomotive activity were seen with concentrations as low as 10−10 M of the vitamin. The potency of this differentiation inducer was such that a 1,000-fold higher concentration of the next most potent inducer of M1 differentiation, dexamethasone, was required to achieve a comparable level of maturation. Simultaneous treatment of M1 cells with low, physiologic concentrations of 1,25-(OH)2 D3 (0.12 nM) and dexamethasone (10 nM) induced a degree of differentiation equivalent to the response obtained with higher concentrations of either agent alone [249]. The isolation of two variant clones of M1, one resistant to dexamethasone and the other to 1,25-(OH)2 D3, strongly suggests that these differentiation-inducing agents act in different ways and that combination therapy with both steroids may be useful in reducing leukemogenicity [249]. The mouse myelomonocytic leukemic cell line WEHI-3 is also inducible to macrophage differentiation when exposed to 1,25-(OH)2 D3 in a clonal assay system. The differentiation-susceptible D+ line was both growth-inhibited and macrophage-differentiated to the 50% level with 10−8−10−10 M 1,25-(OH)2 D3. Of particular interest was the observation that a subline of WEHI-3 refractory to other differentiation-inducing agents was exquisitely sensitive to 1,25-(OH)2 D3, with 50% growth inhibition and macrophage differentiation seen with 10−13 M vitamin. The first reports of the capacity of 1,25-(OH)2 D3 to induce differentiation of human leukemic cells indicated that HL-60 was growth-suppressed, and phagocytosis and C3-rosette formation were markedly induced in a dose-dependent manner over a range of 10−8 to 10−10 [248]. Unfortunately, these investigators concluded that HL-60 was induced to form granulocytes, as had previously been observed for retinoids and DMSO, rather than the uniform pattern of monocyte-macrophage differentiation reported in subsequent studies [189, 228, 234, 236, 275, 352, 356]. HL-60 cells following their treatment with 1,25-(OH)2 D3 acquire several phenotypic changes that were similar to the mature monoyctes/macrophages. These maturation features were induced in a dose-dependent (10−11 to 10−7 M) and time-dependent (1–6 days) manner, and resulted in a functional phenotype

541 and two-dimensional gel pattern of proteins close to, but not identical with, those of peripheral blood monocytes [251]. Cell division apparently is not required for expression of these differentiation features [94, 191]. The ability of low concentrations of 1,25-(OH)2 D3 to induce macrophage differentiation of HL-60 is similar to that reported for phorbol esters; however, this is not due to similar binding sites, since 1,25-(OH)2 D3 did not compete for phorbol diester binding sites as measured by (3H)phorbol dibutyrate binding on HL-60 [275]. Variants of HL-60 have been developed which are resistant to differentiation induction by DMSO, retinoic acid, TPA, and 1,25-(OH)2 D3. One variant resistant to phorbol esters was also resistant to 1,25-(OH)2 D3 [275], suggesting that mutants that cannot be induced to differentiate can involve common events following the receptor binding stage. The possibility of synergism between 1,25-(OH)2 D3 and phorbol esters or other differentiation inducers is definitely indicated in the case of HL-60, and has been reported with retinoic acid and with DMSO since low concentrations of 1,25-(OH)2 D3 in combination with DMSO produced cessation of proliferation of HL-60 cells within 2 days, as well as a greater expression of differentiation [352]. Simultaneous treatment of HL-60 with suboptimal concentrations of 1,25-(OH)2 D3 (0.12– 1.2 nM) showed additive effects in reducing nitroblue tetrazolium, a common marker for monocyte-macrophage and granulocyte differentiation [250]. The human monocytoid cell line U937 is also induced to macrophage differentiation with loss of plating efficiency in the presence of 10−10 M 1,25-(OH)2 D3 [245, 293]. Differentiation involves development of adherence, macrophage morphology, lysozyme production, capacity to reduce NBT, expression of β-glucuronidase and alkaline phosphatase, Fc receptor expression, phagocytosis, and reactivity with antimonocyte-specific monoclonal antibodies [293]. As in the case of HL-60, the differentiation of U937 is not blocked by inhibitors of DNA synthesis, but is by the calcium ionophore A23187 [175]. The existence of synergism between 1,25-(OH)2 D3 and other biological response modifiers has been established with retinoic acid. U937 cells can be primed by short incubation with 1,25-(OH)2 D3 to respond by maturation to agents such as cAMP, PGE, and cholera toxin, which alone do not induce differentiation [293]. 1,25-(OH)2 D3 or retinoic acid plus dibutyryl cAMP is effective in inducing a variety of differentiation markers in U937. Their actions on insulin receptors were the opposite, however. 1,25-(OH)2 D3 increased the binding, while retinoid decreased the binding. This effect was specific for insulin, since the transferrin receptors were reduced by both methods of differentiation [335].

542 Thus, changes in insulin receptors during maturation in vitro depend on the inducing agent and are not causally related to the differentiation process. Other differentiation-inducing agents have been compared with 1,25-(OH)2 D3, which was the most effective. The polar/planar compound HMBA was most effective in reducing cell recovery, but did not induce cell maturation. Retinoic acid-reduced cell and total blast cell recovery with an increase in neutrophil differentiation. Protein inducers of differentiation, α- and γ-interferon, showed slight activity in reducing cell and blast recovery, whereas a murine serum source of differentiation factor (GM-DF) was highly effective in inducing macrophage differentiation and reduction in recovery of immature cells. 1,25(OH)2 D3 or IFN-γ decreased blast cells and increased macrophage differentiation in suspension cultures of marrow from patients with myelodysplastic syndrome [380, 417]. As an alternative to the suspension culture technique for monitoring differentiation induction of fresh leukemic marrow, an agar cloning assay has been used, in which colony or cluster incidence was measured in 7-day cultures of 105 leukemic marrow cells. Unlike the clonal assay for differentiation-induction of HL-60 or WEHI-3 cells, primary human leukemia cultures formed small clusters of 10–20 cells (microclusters), 20–40 cells (macroclusters), small colonies with an excess of clusters (microcolonies), or colonies of normal size with a normal cluster-to-colony ratios [148, 191, 265]. In most cases, the clonal growth was diffuse and colonies or clusters failed to differentiate (with the exception of most patients with chronic myeloid leukemia and some patients with preleukemia) [250, 332, 336, 382]. In view of this growth pattern, clonal differentiation can only be measured by isolation and staining of individual clones, a laborious and inexact procedure at best. As an alternative, 1,25(OH)2 D3-induced reduction of leukemic cloning capacity was used as an index of differentiation. The legitimacy of this approach has been validated by studies showing that 50% growth inhibition of proliferation generally approximated to 50% growth inhibition when 1,25-(OH)2 D3 or its derivatives were used to induce differentiation. It should be stressed that this linkage between proliferation inhibition in clonal assay and differentiation induction generally does not hold true for other types of differentiation agents. For example, inhibition of the colony growth by most chemotherapeutic agents is not associated with differentiation, and protein sources of differentiation activity may not influence primary leukemic cloning capacity, but only recloning capacity. In a more extensive analysis of heterogeneity of responsiveness to the growth-inhibitory/differentiation

Growth and differentiation factors as cancer therapeutics capacity of 1,25-(OH)2 D3, it was observed that preleukemic marrows exhibiting colony formation analogous to normal marrow were least responsive to 1,25-(OH)2 D3, whereas preleukemic marrows with cluster-forming acute myeloid leukemia-type clonal growth were most responsive, being comparable to the clinical observations. These observations suggest the interesting, perhaps surprising, conclusion that the more acute the leukemia the more it is responsive to 1,25-(OH)2 D3. Koeffler et al. [191] have also shown that 1,25-(OH)2 D3 and two fluorinated analogs, 24, 24-F2-1,25-(OH)2 D3, inhibited colony formation of marrow from patients with ANLL (four cases) and chronic myeloid leukemia (four cases) at concentrations of 10−8 M, with 50–80% of leukemic colony-forming cells assuming a macrophage-like morphology. This result is comparable to the report of Moore et al. [265], with the exception that in the latter study more patients were investigated and the greater sensitivity seen in acute myeloid leukemia patients (50% inhibition at 4 × 10−12 M) may be explained by the use of total clonogenic units measured (i.e., colonies of >40 cells and clusters of 3–40 cells) rather than restricting inhibition analysis to colonies of >40 cells. The cells of the majority of patients with acute myeloid leukemia do not form colonies of 40 cells, and the leukemic cells in general form small clusters. A variety of metabolites and analogs of vitamin D3 have been developed and tested for biological activity and in vivo toxicity. Recent studies have extended biological screening to leukemia differentiation systems. HL-60 is induced to differentiate upon exposure to 10−7−10−10 M 1,24-(OH)2 D3 or 1,24R-(OH)2 D3 in a fashion comparable to that with 1,25-(OH)2 D3. A different analog of 1,25-(OH)2 D3 has been found highly active in stimulating intestinal calcium transport and bone calcium mobilization in vitamin D-deficient rats [374]. This 24, 24-F2-1,25-(OH)2 is highly active in induction of WEHI-3 and HL-60 differentiation and growth inhibition, with 50% activity at 10−14 M, thus making it the most active D3 analog tested in the leukemic assay system [258]. Unfortunately, this analog is considerably more toxic in vivo than is 1,25-(OH)2 D3. These compounds showed the same relative activities, in that normal marrow was always least sensitive to growth inhibition and acute myeloid leukemia marrow most sensitive, with preleukemia and chronic myeloid leukemia occupying intermediate positions. 24-Homo-, and 26-homo-1,25(OH)2 D3 and delta [42] analogues were ten-fold more potent than 1,25(OH)2 D3 in inducing differentiation of HL-60 [298]. The 24-homo-analogue was significantly less active in mobilizing calcium from bone.

Kapil Mehta et al. It is obvious that more extensive screening must be undertaken to determine if the calcium-mobilizing activity and consequent toxicity as a feature of D3 metabolites are invariably related to efficacy in induction of leukemic growth inhibition and differentiation. Obviously, as compared with a long in vivo half-life, low toxicity and retention of selective leukemia-cell differentiating activity would be highly desirable for clinical studies. Clinical trials with vitamin D have been limited because of intolerable hypercalcemia which often develops with this compound. Some responses have been seen in patients with myelodysplastic syndromes when treated at 2 mg/day [192]. With new analogues of vitamin D3 now available, a separation of the effects of calcium metabolism and those on cell differentiation is now possible and clinical investigations are underway [301].

Receptors for Vitamin D and its Metabolites The role of 1,25-(OH)2 D3 as an agent responsible for mineral homeostasis has been extensively studied. Its mechanisms of action within target cells is retinoid and steroid hormone-like in that the binding of 1,25-(OH)2 D3 to vitamin D receptor (VDR) induces conformational changes in the VDR which promote heterdimerization with retinoid X receptor (RXR) and recruitment of a number of nuclear receptor coactivator proteins including the SCR family members (Fig. 2) [224]. All known 1,25(OH)2 D3-responsive tissues, such as the intestine, bone, and kidney, contain VDRs. In addition, many other tissues and cultured cells have been shown to possess 1,25-(OH)2 D3 receptors, including some tumor cells. Breast cancer, melanoma, and osteogenic sarcoma cells possess a low density (8,000–15,000 per cell) of receptors with high affinity (Kd 10−10−10−11 M) for 1,25-(OH)2 D3 [84, 100, 105, 229, 186]. Receptors with similar affinity and density have been reported on human monocytes, malignant B and T leukemic cell lines, activated T-cells, Epstein-Barr virus-transformed B-lymphocytes, and cell lines such as K562, HL-60 and U937 [228, 229, 320, 352]. Identification of 1,25-(OH)2 D3 receptors by specific immunochemical reactivity and selective chemical dissociation has shown that nuclear binding of the vitamin receptor is a rapid event (minutes) in HL-60, whereas the cellular differentiation response is delayed (6–7 days) [228]. This may be reconciled if the receptor must be maintained within the nucleus over the long term. A differentiation-noninducible variant of HL-60 has been reported to have only 8% of receptor copy numbers of the parent line, suggesting that assay of

543 1,25-(OH)2 D3 receptors in leukemic patients may be predictive of the ultimate response of the patient to adjunct therapy with 1,25-(OH)2 D3 [228]. This possibility is supported by the observation that the equilibrium dissociation constant of the 1,25-(OH)2 D3 receptor on HL-60 is close to the vitamin concentration, causing 50% of HL-60 cells to reduce NBT [189]. Against this view, there is the fact that the human myeloblastic leukemic cell line KG-1 has approximately the same number of 1,25-(OH)2 D3 receptors as HL-60, yet cannot be induced to differentiate by the vitamin [189]. There is extensive evidence for a classic steroid receptor-DNA interaction for the 1,25-(OH)2 D3 receptor, with selective binding of the receptor to A + T-rich segments of double-stranded DNA. Franceschi [101] demonstrated that the receptor can also interact with RNA as well as DNA. The physiologic significance of this observation remains obscure. However, in other steroid hormone systems, steroids can influence certain nontranscriptional processes, such as the stability of hormone-dependent mRNA, as well as posttranscriptional processing of secretory proteins, the regulation of 5S RNA synthesis, and the processing of heterogeneous nuclear RNAs. Interaction of 1,25-(OH)2 D3 with receptors on normal or malignant target cells results in variable and complex changes in proliferation. A biphasic effect on the growth of breast tumor [116, 175] and osteosarcoma cells [229] has been reported, with physiologic concentrations of 1,25-(OH)2 D3 (10−10–10−11 M) stimulating growth, and higher concentration (10−7–10−8 M) inhibiting it. In contrast, a uniform pattern of growth inhibition is seen with the leukemic cell lines HL-60, U937 [294], M-1 and WEHI-3, and primary myeloid leukemia at concentrations as low as 10−12 M. No inhibition of normal marrow CFU-GM colony formation is seen with 1,25-(OH)2 D3 except at very high concentrations of 0.1–1 μg/ml; growth stimulation of normal mouse and human CFU-GM colony formation can be observed. Kuribayashi et al. [199] have reported on two variant clones of HL-60, resistant to differentiation and growth inhibition in the presence of 1,25-(OH)2 D3. One clone was also unresponsive to phorbol ester, actinomycin-D, and DMSO. The variant clones were found to possess reduced amounts of cytosol receptor protein, to which 1,25-(OH)2 D3 was specifically bound, but the hormonereceptor complex could be transferred to the chromatin acceptor site in both the wild-type and variant clones. This would indicate that 1,25-(OH)2 D3 resistance is due to a reduction in specific cytosol receptors. Freake et al. [105] reported that whole chronic myeloid leukemic cells specifically took up 1,25-(OH)2 D3 with

544

Growth and differentiation factors as cancer therapeutics

high affinity (Kd = 3.6 × 10−11 M) and low capacity. Subcellular fractionation of labeled cells showed that binding was restricted to cytosols and nuclei; however, chronic myeloid leukemic cells appeared to contain both the receptor for 1,25-(OH)2 D3 and an unknown substance that prevents its detection following the preparation of cytosol. Cells from patients in chronic phase specifically bound more vitamin (18 fmol/107 cells) than did those in the blastic phase (7 fmol/107 cells), or cells from patients with acute myeloid leukemia (2.6 fmol/107 cells). From observing that only cells from patients with chronic myeloid leukemia responded to 1,25-(OH)2 D3 by differentiation along the monocyte-macrophage pathway, it was concluded that differentiation induction was most likely dependent upon adequate levels of receptors, and that intact cells rather than cytosol preparations should be studied before cells of a particular tissue are designated as receptor negative. In view of the heterogeneity of the cellular composition of chronic myeloid leukemia and the heterogeneity of morphological type in blastic chronic myeloid leukemia (30% terminal transferase positive) and ANLL, general conclusions on receptor display with such small groups of patients should be treated with caution. The HL-60 genome contains several different oncogenes, but only one, c-myc, is significantly amplified and transcribed at high levels (×20). Westin et al. [404] reported that myc on mRNA is no longer present in HL-60 cells induced to granulocytic differentiation by DMSO or retinoic acid. Reitsma et al. [330] have shown that 1,25-(OH)2 D3 reduced myc nRNA levels in HL-60 within 4 h of exposure to the vitamin/hormone, and this change preceded the onset of other measurable phenotypic changes by at least 8 h. It remains to be determined whether the altered transcription is the consequence of the hormone interaction with the c-myc promoter or a spectrum of promoters, each associated with a gene or gene cluster involved in determining cell phenotype.

fibrous tissue is also mediated by monocytes and macrophages, and the number and activity of these cells are increased by 1,25-(OH)2 D3. Thus, the various actions of this vitamin contribute to a reduction in the collagen content; conversely, a deficiency of it may allow abnormal accumulation of collagen in the marrow. In this context, myelofibrosis in a rachitic infant regressed following vitamin D therapy [66], and a group of rachitic children with anemia and a blood picture typical of myelofibrosis also responded to vitamin D [415]. A novel immunoregulatory role has been proposed for 1,25-(OH)2 D3. Intracellular receptors that bind the vitamin and sediment at 3.35 were not detected in “resting” T or B lymphocytes, but T cells activated by EpsteinBarr virus produced the receptor, and the amount of the macro-molecule induced was the same as in normal monocytes. Since the D3-binding macro-molecule is seen in actively mitotic cells, it may exert an antiproliferative-differentiative influence in the immune system. 1,25-(OH)2 D3 at picomolar concentrations has also been shown to inhibit production of the T-lymphocyte growth-promoting lymphokine interleukin 2 (IL-2). Other metabolites of vitamin D3 were less effective, and their order of potency corresponded to their respective affinity for the 1,25-(OH)2 D3 receptor, suggesting that suppression of T-cell production of IL-2 was mediated by this specific receptor [384]. 1,25-(OH)2 D3 may selectively inhibit the action of IL-1 in stimulation of thymocyte proliferation [273]. It also modulates GM-CSF production by T cells by posttranscriptional reduction in the half-life of GM-CSF, nRNA in mitogen-activated T cells and T-cell lines [377]. In view of the well-established calcium-mobilizing effect of 1,25-(OH)2 D3 in its classic target tissues [226], it is possible that the suppressive effect of this hormone on IL-2 is mediated by an influence on calcium translocation and again indicates a physiologic role of this hormone in immunoregulation.

Action of 1,25-(OH)2D3 on other Aspects of Hematopoiesis

In vivo Effects of Vitamin D Metabolites

Myelofibrosis with myeloid metaplasia is considered a neoplastic disorder in which fibroblast proliferation and collagen synthesis in the marrow are increased by platelet-derived growth factor, or related substances, released by neoplastic megakaryocytes [48]. It has been postulated that 1,25-(OH)2 D3 may inhibit the formation of fibrous tissue (mainly collagen) in bone marrow and also may increase its degradation [237]. The hormone also inhibits the proliferation of megakaryocytes that normally promote collagen synthesis. Degradation of

Sato et al. [347] have evaluated the effect of 1-α-(OH) D3 on the growth of two solid tumors transplanted subcutaneously in mice. 1-α-(OH) D3 administered orally by stomach tube at daily doses of 0.1 and 0.2 μg/kg body weight for 114 days suppressed sarcoma 180 tumor growth by 37 and 64% respectively, without significantly affecting serum calcium levels. Similar oral treatment with 0.1 and 0.2 μg/kg of 1-α-(OH) D3 for 21 days resulted in a 75–80% decrease in pulmonary metastases in mice injected subcutaneously with Lewis lung carcinoma fragments. While the data illustrate a potential

Kapil Mehta et al. therapeutic anticarcinogenic effect of this metabolite, they should be considered as preliminary, since small numbers of mice (three to six) were tested in each treatment group. 1-α-(OH) D3 and 1,25-(OH)2 D3 have marked effects on the growth and differentiation of cultured murine myeloid leukemia cells (M1 cells), established from an SL mouse with myeloid leukemia. These results, and the fact that syngeneic SL mice inoculated with M1 cells all die of leukemia, prompted a recent evaluation of the in vivo effects of 1-α-(OH) D3 and 1,25-(OH)2 D3 by Honna et al. [166]. Thrice-weekly intraperitoneal injections of picomole amounts of 1-α-(OH) D3 and 1,25-(OH)2 D3 considerably prolonged the survival time of syngeneic SL mice inoculated with M1 cells. A similar, marked prolongation of survival time was observed in athymic nude mice with M1 leukemia and treated intraperitoneally with 1-α-(OH) D3. The results with the athymic mice suggested to the authors that T-lymphocyte-mediated immune responses were not directly involved in the effects of the vitamin D3 metabolite. Serum levels of calcium and phosphorus were not significantly affected in the nude mice given M1 cells and 1-α-(OH) D3 for 30 days, compared with mice given M1 cells alone. Attempts to duplicate these observations in BALB/c mice inoculated with syngeneic WEHI-3 myelomonocytic leukemia cells proved unsuccessful [143]. Neither dexamethasone nor a combination of it with 1,25-(OH)2 D3 prolonged the survival of WEHI-3 tumor-bearing mice [143, 166]. The disparity between the in vivo observations with M1 and WEHI-3 leukemias could have a number of explanations, which require further testing before dismissing the therapeutic potential of vitamin D derivatives in leukemia. The WEHI-3B grows more rapidly than M1, and 105 cells generally result in 100% mortality within 21 days. However, treatment with conventional cytotoxic agents, such as Cytoxan, produces an increase in life span, even producing cures in a dose-dependent fashion [143]. In retrospect, however, the explanation may reside in the relative resistance of the WEHI-3D+ line to 1,25-(OH)2 D3-induced growth inhibition (50% inhibition in vitro with 4 × 10−4 M), in contrast to the much greater sensitivity of the WEHI-3D−, the HL-60, M1, and fresh acute myeloid leukemic cells (10−9–10−12 M). Potter and Moore [317] have extended in vivo studies with successful growth and maintenance of the human myeloid leukemia lines U937, KG-1, and K562, as subcutaneously induced granulocytic sarcomas in nude mice. Studies involving routine and special histochemistry, enzyme histochemistry, immunocytochemistry, surface markers, cytogenetics, and electron microscopy have

545 demonstrated virtual identity of cells from the induced granulocytic sarcomas in nude mice with respective cells from the cultured lines. The validity and reliability of the living model have thus been established. Using this in vivo model, preliminary experiments were undertaken to evaluate the effect(s) of 1,25-(OH)2 D3 on the development of these subcutaneous leukemic tumors induced on nude mice from cultured cells. Based on the work of Hartmann and Moore [143], a dosage of 1 μg/ ml of 1,25-(OH)2 D3 was chosen as potentially the most effective, but least toxic. The method of administration involved subcutaneous implantation of an osmotic minipump (OMP) at a site remote from the concurrently inoculated tumorigenic cells. One group of mice received only washed cultured leukemic cells, another received cells and an OMP containing only solvent, and a third group of mice received leukemic cells and an OMP delivering 1 μg/ml of 1,25-(OH)2 D3. All mice were weighed on alternate days and observed for tumor development. Of the mice receiving cultured K562 or HL-60 cells only, almost all developed granulocytic sarcomas within an average of 4–5 weeks after inoculation. A similar result was seen in mice inoculated with cultured cells from either of these lines that also bore implants of an OMP containing only solvent. Granulocytic sarcomas either failed to develop, or developed infrequently and (on the average) later in the group similarly inoculated with cultured cells from these lines, and bearing OMPs containing 1,25-(OH)2 D3. Since each mouse underwent the same priming and received the same number of cultured human leukemic cells, it appears that the absence of, or delay in the development of, induced granulocytic sarcomas may be attributable to the influence of the administered 1,25(OH)2 D3 in the system. These results suggest that 1,25(OH)2 D3 might have an inhibitory effect on the proliferative capacity of human leukemic cells in vivo. Whether the mechanism involves induction of terminal differentiation on the human tumor cell inoculum requires investigation. The involved mechanism(s) of inhibition are also of interest. In those few treated mice demonstrating proliferation of xenogenic (human) leukemic cells, the possibility of emergence of 1,25-(OH)2 D3-resistant clones should not be overlooked, and the availability of receptors for 1,25-(OH)2 D3 should be determined on cells derived from these tumors. Recently developed analogues of 1,25-(OH)2 D3 notably 1,25 (OH)2-16-ene-23-yne-D3, were remarkably effective in “curing” mice bearing the WEHI-3B + leukemia [32]. Differentiation was induced at dosages that did not produce hypercalcemia.

546 Translating these observations into a clinical application, it is clear that in vitro screening for response to 1,25-(OH)2 D3 can be used to identify resistant and susceptible patients with leukemia and preleukemia. While it is unlikely that the vitamin could prove effective on its own, it could be combined with conventional chemotherapy or used as maintenance therapy in patients achieving remission by conventional protocols. The toxicity of 1,25-(OH)2 D3 in mice at dosages unable to produce leukemic regression in vivo would require precise dose-response analysis in humans. In this regard, 1,25-(OH)2 D3 has been used effectively in patients with post-menopausal osteoporosis [64]. Short-term treatment (6–8 months) with 0.5 mg/day restored calcium absorption to normal, and calcium balance improved and the bone resorption rate decreased. With long-term therapy (2 years) both bone resorption and formation rates increased. The lack of side effects in long-term treatment with 1,25-(OH)2 D3 provides a dosage guideline for studies in leukemia [111]. Oral administration of 1-α-(OH) D3 in two patients with AML and one with MDS was reported to reduce the number of leukemic cells in the marrow. In a study of 18 patients with MDS, 1,25-(OH)2 D3 produced a partial or minor response in blood in eight cases but with no significant improvement in blood or marrow blasts [192]. Seven of the patients developed leukemia before or by 12 weeks of treatment and half the patients developed hypercalcemia. It was not possible to sustain serum levels of 1,25-(OH)2 D3 at levels necessary to induce differentiation or growth inhibition without producing unacceptable toxicity. Future clinical trials await the use of recently developed analogues with reduced calcium mobilizing action and enhanced differentiating activity [32]. A further rationale for therapy with 1,25-(OH)2 D3 and its analogues is provided by the observation that the metabolic pathway of 1,25-(OH)2 D3 is defective in patients with MDS or AML [30]. Bone marrow plasma levels of 1,25-(OH)2 D3, but not its immediate precursor 25-(OH) D3, was decreased significantly in 50% of MDS and 30% of AML patients.

Polar-Planar Compounds as Differentiation Inducers Differentiation-inducing activity has been reported for various polar-planar compounds, most particularly DMSO, hexamethylene bisacetamide (HMBA), N-methylformamide and, cotylenin A [62, 63, 169, 427,

Growth and differentiation factors as cancer therapeutics 428]. The differentiation-inducing action has been most extensively analyzed in murine erythroleukemic (MEL) cells. Inducer-mediated differentiation is a multistep process characterized by a latent period when a number of changes occur [232, 348]. These include alterations in ion flux, increase in membrane bound PKC, appearance of CA2+ and phospholipid-independent PKC activity in the cytosol and modulation of the expression of genes such as c-myc, c-myb, c-fos and p53. Commitment to differentiation is seen within 12 h and increases stochastically over 48 h and is associated with suppression of c-myb expression and a 10- to 30-fold increase in globin gene expression [232, 331]. The levels of ornithine decarboxylase are also regulated by HMBA. HMBA induces a transient, genome-wide hypomethylation of DNA achieved by replacement of 5-methylcytosine with cytosine residues [327]. This may be a necessary but not sufficient step in triggering the whole program of differentiation. Superoxide dismutase activity is induced by HMBA in parallel with differentiation and enzyme levels are directly related to the degree of cytosolic hemoglobinization [24, 304]. Introduction of superoxide dismutase into MEL cells with liposomes induces differentiation as do other oxidative treatments (liposome amino acid oxidase, xanthine oxidase, potassium superoxide). In contrast, antioxidants inhibit HMBA-induced differentiation [24]. The induction of superoxide dismutase in MEL cells may also be a cellular response to oxidative stress from hemoglobin autooxidation. Potential improvements in efficacy of HMBA may be accomplished by changes in the chemical structure of the inducing agents and by increasing the sensitivity of tumor cells to inducers of differentiation. MEL cell lines that have acquired low levels of resistance to vincristine display a markedly increased sensitivity to HMBA [231]. A series of hybrid increased polar/apolar compounds have been produced that in certain instances are more active than HMBA in vitro and whose chemical structure makes it likely that they have different pharmacokinetics [231]. The first Phase I trials of HMBA involved continuous infusion of escalating doses of HMBA for 5 or 10 days in patients with refractory solid tumors [11, 83, 213, 340]. Dose limiting toxicity, specifically thrombocytopenia, was observed at 20–40 g/m2. The MTD of continuously infused HMBA was 28 g/m2/day and acidosis and CNS dysfunction were toxicities, as well as hemorrhage related to thrombocytopenia. The plasma half-life was 2.5 h and plasma levels of 1.42 nM or higher could be achieved [419]. In Phase I trials nasogastric or oral administration of HMBA at 30–36 g/m2/day

Kapil Mehta et al. was also associated with thrombocytopenia and neutrotoxicity. Attempts have been made to individualize patient dosage based on plasma levels and clearance rates of HMBA [64]. Plasma levels of 1.5–2.0 mM could be sustained but toxicity was seen. The cause of the thrombocytopenia is obscure but appears to be a production defect rather than a peripheral destruction or pooling of platelets. At the dosages of HMBA required to produce differentiation, significant inhibition of cloning of normal myeloid, erythroid, and megakaryocyte progenitors is seen in vitro in murine bone marrow culture. Fifty percent inhibition of cloning, and suppression myelopoiesis in long-term bone marrow cultures, are seen with doses of HMBA of 1.2 mM with an unusually steep dose response. This observation does not fully explain the observed clinical toxicity, since the thrombocytopenia was not usually associated with a neutropenia. The development of analogs of HMBA with differentiation-inducing capacity and reduced suppressive activity against myeloid progenitors is a like direction in development of an effective clinical differentiation protocol [231] that could be applied in leukemia and MDS as well as in a variety of solid tumor systems (colon, bladder, breast).

Chemotherapeutic Agents as Differentiation Inducers It is becoming apparent that of the variety of agents utilized in cancer chemotherapy, some may be effective because of their capacity to induce, selectively, tumor cell differentiation, and this property may be more important than cytotoxic potential. After extensive screening (for reviews, see [169, 189, 343]) a number of agents have been found to have differentiation capacity [63, 65, 169, 214, 246]: mitomycin, doxorubicin, bleomycin, daunomycin [169, 189, 214, 246], cytosine arabinoside [130, 167, 169, 189, 214, 274, 369], hypoxanthine [63], 3-deazauridine, 5-azacytidine, methotrexate [33, 351], 5-aza-2-deoxycytodine [51, 57, 254, 314], aphidicolin [125], and adenine arabinoside [274]. In all the preceding cases the differentiation action was observed at concentrations either below cytotoxic levels or well below maximum growth inhibition in suspension or clonal assays of leukemic cells. Most compounds have been tested against murine (M1) and human (HL-60) myeloid leukemic cell lines, in which differentiation into macrophages and granulocytes is reported. When tested in vitro against leukemic blast cells from

547 patients with acute myeloid leukemia, actinomycin D was reported effective in differentiation induction in all 14 cases studied [169]. In all three cases in another study cytosine arabinoside [246] and 5-aza-2-deoxycytidine [314] were also effective in inducing macrophage differentiation of leukemic blast cells. In the absence of an in vivo measure of leukemogenicity, the ability of these agents to eliminate the proliferative potential of the leukemic clone must be measured indirectly. In recloning studies of primary human leukemias cultured in methylcellulose, a decrease self-renewal potential of clonogenic cells (plating efficiency 2, PE2) can be observed with various chemotherapeutic agents, independent of their chemosuppressive action on primary leukemic cloning efficiency [41]. This action on PE2 is a likely differentiation index, since more primitive leukemic stem cells with extensive proliferative potential are “differentiated” into a non-self-renewing compartment. Furthermore, this PE2 parameter correlated with clinical response [42]. The combination of low doses of the above-mentioned chemotherapeutic agents with other differentiation-inducing agents may prove effective when neither type of agent can induce differentiation directly. In this regard, differentiation-resistant clones of M1 (D−) that failed to respond to protein differentiation factor or chemotherapeutic agents could be ‘sensitized’ by as little as 0.25 μg/ml of actinomycin D, daunomycin, mitomycin C, hydroxyurea, bleomycin, 5-fluorouracil, prednisone, or dexamethasone to terminal differentiation when combined with protein factor [149, 169]. Lotem et al. [219] report that in vitro screening for differentiation-inducing compounds and compounds that show toxicity to blast cells may be useful in selecting appropriate treatments. However, their results were ambiguous, in that with some patients studied both before and after in vivo chemotherapy there was a similar differentiation response, or an apparent loss, or a gain of response in vitro of the remaining leukemic cells tested. In further studies, using five compounds known to induce HL-60 differentiation (DMSO, hexamethylene bisacetamide (HMBA), hypoxanthine, actinomycin D, and 6-thioguanine), differentiation of fresh myelogenous leukemic cells was tested in vitro [192]. Of 12 patients studied, the blast cells in most cases showed little morphological, histological, or functional maturation after exposure to the various compounds, as compared with the blast cells cultured without the compound. Actinomycin D was the only agent capable of causing significant maturation. This study suggests that many compounds shown to differentiate HL-60 may not trigger differentiation of less mature myeloid leukemic cells.

548

Molecular Mechanism Implicated in Leukemia Cell Differentiation Cytosine arabinoside is one of the most effective single agents for the treatment of myeloid leukemia and is conventionally considered to act by incorporation into DNA and by inhibiting DNA replication through production poor primer termini. Cytosine arabinoside induces nonspecific esterase activity in HL-60 cells [107] and increases surface expression of the monocyte surface antigen MY-4. Aphidicolin, an analogue of deoxycytodine, also induces HL-60 differentiation and slows DNA synthesis, but, unlike cytosine arabinoside, it is not incorporated into DNA and acts as an inhibitor of DNA polymerase [130]. Using a purine rather than a pyrimidine antimetaboliteadenosinearabinoside, Monroe et al. [274] also observed differentiation of HL-60 that correlated with slowing of DNA synthesis by an agent known to act at the level of the DNA polymerase template complex. The relationship of differentiation to DNA synthesis is complicated by observations that terminal differentiation of HL-60 to macrophages induced by the tumor promoter TPA can occur in the absence of DNA synthesis [339] and that terminal differentiation to granulocytes without cell division is observed following treatment with actinomycin D, DMSO, and butyric acid [236]. Agents can, however, induce HL-60 differentiation without inhibiting cell proliferation [33], or inhibit proliferation without inducing differentiation [351]. Thus, it remains to be determined whether inhibition of DNA synthesis is causally or indirectly related to differentiation. Other mechanisms of action of chemotherapeutic agents directed toward differentiation induction may involve cell membrane effects. Anthracyclines may play a role in alteration of leukemic cell glycoproteins, and there is evidence that these drugs bind extensively to membrane-lipid domains, altering membrane fluidity and directly modifying the synthesis or expression of glycoproteins at the cell surface [350]. A more specific action has been suggested involving DNA methylation changes: for example, cytosine arabinoside may have a direct influence on methylation of the c-myc oncogene [130], the expression of which is considerably amplified in myeloid leukemic cells and rapidly reduced once the cells are exposed to differentiation-inducing agents [330, 404]. 5-Azacytidine and the less toxic 2-deoxy derivatives are also interesting candidates for in vivo use in leukemia differentiation therapy. These compounds have been shown to trigger gene expression in several systems, including globin gene expression, when given in vivo to thalassemic and sickle-cell anemia patients [206].

Growth and differentiation factors as cancer therapeutics The mechanism by which 5-aza-deoxycytidine induces leukemic cell differentiation most likely involves DNA hypomethylating ability [254, 314]. Synthesis of hypomethylated DNA takes place after incorporation of the drug into DNA and is due partially to the chemical structure of the compound and mainly to the trapping of DNA methyltransferases, thus blocking their action. The known differences regarding the molecular targets of the two drugs could account for the greater differentiation-inducing ability and lower toxicity of the 2-deoxy derivative. It is well known that 5-azacytidine is actively incorporated into mRNA and tRNA, thus producing its major toxic effects. A cautionary note should be introduced in considering the potential therapeutic role of 5-azacytodine. Motojo et al. [271] exposed blast cells from patients with acute myeloid leukemia to 5-azacytidine, 6-azacytidine, and the 2-deoxy derivative. Simple negative exponential colony survival curves were obtained for the three drugs, with the 5-aza-2-deoxy compound being most toxic and the 6-aza least toxic. Although confirming other reports of increased expression of certain antigenically defined phenotypic markers of leukemic blast cell differentiation, colonies surviving drug exposure to 5-aza and 5-aza-2deoxy compounds had increased secondary replating efficiency. This suggests that hypomethylation of DNA may promote leukemic cell self-renewal.

In vivo Induction of Differentiation with low-dose Cytosine Arabinoside or Azocytidine in Patients with acute Myeloid Leukemia and Myelodysplastic Syndrome (MDS) The preceding in vitro observations provide a strong case for the efficacy of low doses of ara-C in induction of terminal differentiation of myeloid leukemic cell lines and fresh leukemic blast cells, this action being either direct or by synergy with endogenous differentiationinducing factors. It was thus of interest to investigate the actions of low-dose ara-C in patients with acute myeloid leukemia and myelodysplastic refractory anemia with excess of blasts. Baccarani and Tura [17] reported the first remission in a patient with RAB and subsequently extended the study to 20 patients with myelodysplastic syndrome (MDS), observing one complete and two partial remissions. Complete or partial remissions in MDS have been reported by other groups [167, 253, 318, 402, 409], but no response to low-dose-ara-C was observed in a large cooperative study of refractory anemia with excess of blasts [280]. In acute myeloid leukemia,

Kapil Mehta et al. Housset et al. [168] reported remission in all three patients, and Weh et al. [402] had seven complete and two partial remissions in 12 cases. Encouraging results have been obtained by others [253], but no response was reported by Hagenbeek et al. [136] in four acute myeloid leukemia patients treated with the same protocol that produced responses in other studies [168, 402]. The reasons for variable results are unclear, but the patient population, particularly the MDS cases, was heterogeneous, and the dosage and timing of drug administration in different studies varied between 10 and 30 mg/m2, every 12 or 24 h, for 7–28 days. Indeed, the more encouraging results obtained by Housset et al. [168], compared with those of Baccarani et al. [16], may be due to the more frequent and longer duration of ara-C administration in the former study, thereby probably achieving a rather constant in vivo concentration of the drug. In one series of 21 patient (five with refractory anemias with an excess of blasts and 16 with acute leukemias) treated with small doses of ara-C (10 mg/m2/12 h for 15–21 days), improvement was noted in 15 cases (71%), and complete remission was observed in 12 (57%) [47]. Complete remission was obtained after one course of treatment in eight cases. The fact that these patients entered remission relatively slowly and did not suffer marrow aplasia suggests that low-dose ara-C was functioning by inducing differentiation. Generally, when clinical response has been obtained, the evidence points to a differentiating role for the drug rather than an antitumor effect, with progressive evolution of recovery, absence of aplasia, and the simultaneous presence of normal islets of promyelocytes and leukemic myeloblasts [168]. The availability of the in vitro assay for detection of leukemic cell responses to differentiation agents should mandate prescreening of patients for in vitro sensitivity before enrollment in a differentiation protocol such as low-dose ara-C. Toward this goal, leukemic marrow cultures were exposed to low doses of ara-C, and a reduction in blast cells and an increase in more mature granulocyte and macrophage elements were noted in two or three patients. Subsequent low-dose ara-C treatment in vivo resulted in complete remission in the two patients that showed a strong in vitro response [246]. In a large study of low-dose (10 mg/m2) ara-C in poor-risk ANLL, overall survival was comparable to that observed following conventional therapy with highdose (200 mg/m2) ara-C and anthrocyclin [71]. While the high-dose regimen produced more complete remissions (55%) than the low dose (33%), there were more early deaths in the intensive therapy group. Very-low-dose (3 mg/m2) ara-C produced hematologic improvement in the majority of patients in a study trial of 73 MDS

549 patients [71]. In other recent studies of low-dose ara-C in 73 patients with ANLL and MDS [10] and 40 patients with ANLL and MDS, complete remission rates of 24–31% were reported with 35–45% responders. In these studies evidence for leukemic cell differentiation was obtained. Cytogenetic and morphologic studies suggested that cytotoxicity rather than differentiation was responsible for remissions observed in two other studies with low-dose ara-C in poor-risk ANLL [19, 49]. Low-dose 5-azacytodine has also proved effective in MDS, and in one study of 44 patients, 48% responded with 11% complete and 25% partial remissions [357]. The median duration of remission was 53 weeks. 5-Aza2′-deoxycytidine in 134 patients with AML, CML in blastic crisis, and MDS produced two complete and four partial remissions. The drug appeared to modify the leukemic phenotype in vivo as well as producing a direct cytolytic effect [423]. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is an inhibitor of inosine5-monophosphate (IMP) dehydrogenase and is the enzyme in the rate-limiting synthesis of guanylate [401]. Tiazofurin has both cytotoxic and cytodifferentiation activities and has been used to induce responses in patients with blast crisis in CML [381]. Toxicity has been severe with nausea, rash, myalgia and serositis. Given the high toxicity, further clinical trials to this approach must be conducted to determine optimal dose and schedule. Combinations of tiazofurin with other differentiations may be of interest to investigate in the near future.

Cytokines and Hematopoietic Growth Factors active in Regulating Proliferation and Differentiation of Leukemic Hematopoietic Cells Recognition that physiologic inducers of differentiation of normal hematopoietic cells could also influence proliferation and differentiation of leukemic cell lines led to a series of studies over the last 2 decades involving characterization of hematopoietic growth factors, in many cases using leukemic cell lines as sources of growth factor and/or as target for growth factors in various bioassays. As discussed in other chapters, the genes for these factors have been cloned and recombinant factors have been tested in vitro and in vivo for biological activity. This large family of cytokines and polypeptides includes factors with pleotropic and overlapping activities, and additive or synergistic interactions are frequently observed [139, 209, 321, 426].

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CSF-Dependence of Myeloid Leukemic Progenitors The cloning of normal or leukemic human CFU-GM in either agar or methylcellulose has permitted analysis of both quantitative and qualitative changes in this cell compartment in leukemia and other myeloproliferative disorders. Changes observed include abnormalities in the maturation of leukemic cells in vitro, defective proliferation as measured by colon size or cluster-to-colony ratio, abnormalities in biophysical characteristics of leukemic GM-CFC, the existence of cytogenetic abnormalities in vitro, and regulatory defects in responsiveness to positive and negative feedback control mechanisms (for reviews, see refs. [255–258, 261, 263–265]). Detection of this spectrum of abnormalities has proved to be of clinical use in diagnosis of leukemia and preleukemic states, in classification of leukemias, and in predicting remission in acute myeloid leukemia. Variation has been reported among different groups investigating the characteristics of human acute myeloid leukemia cells in culture. These differences reflect, in part, the heterogeneity of the disease as well as variation in the culture criteria, the source and activity of CSF, and the timing of the culture. The preceding studies indicated that leukemic cells from patients with acute or chronic myeloid leukemia or preleukemic states were absolutely dependent upon a source of stimulatory factors for their clonal proliferation in culture at low plating densities. Analysis of the dose response of myeloid leukemia CFU-GM indicates that in the majority of cases they do not differ markedly from normal in their responsiveness to various sources of CSF, although occasionally, hyperresponsiveness of leukemic cells is seen [255, 257, 258, 261, 263, 265]. In an analysis of growth-factor responsiveness in suspension cultures of leukemic marrow from 25 patients, Lowenberg et al. [220] showed that 17/25 cases exhibited spontaneous “autocrine” proliferation, [362] and this was enhanced in 21 cases by IL-3, in 17 cases by GM-CSF of G-CSF, and in five cases by M-CSF. In four cases the cells responded to all factors and the remainder responded to three, two, or only one source of stimulus. As with normal progenitor assays, “spontaneous” leukemic cloning is observed as the marrow cell or peripheral blood leukocyte plating density increases, owing to endogenous production of CSFs by accessory populations, which may be residual normal cells of T-cell lineage or leukemic cell subpopulations. The autocrine concept of malignant transformation proposes that cells become malignant by the endogenous production of polypeptide growth factors that act upon their

Growth and differentiation factors as cancer therapeutics producer cells through functional external receptors. Support for the concept has been obtained by studies on oncogene action, since oncogenes may confer growthfactor autonomy on cancer cells by coding directly for autocrine polypeptide growth factors or their receptors, or by amplifying the mitogenic signals generated as a consequence of growth factor-receptor interaction. Strong evidence for an autocrine role for GM-CSF has been provided in a study of 22 cases of primary human acute myeloid leukemia in which Northern blot analysis revealed expression of the GM-CSF gene in 11 cases [420]. GM-CSF expression was not found in normal hematopoietic tissue, with the exception of activated T-cells, strongly indicating that the GM-CSF gene activation was intimately associated with the transformation event. Furthermore, in six cases, GM-CSF was secreted by leukemic cells of both early and late stages of differentiation, and activity was specifically neutralized by antiserum to GM-CSF [129]. The paradox of CSF dependence of primary human myeloid leukemias in clonal assay, and the autocrine production of GM-CSF, can be answered in part by the concentration at which the cells are plated: at high concentrations, ‘spontaneous’ leukemic colony or cluster formation is the norm. In the study of Young et al. [420], 9 of 22 cases showed autonomous growth of leukemic clusters when cells were plated at 5 × 104/ml, yet exogenous GM-CSF increased cloning number and clone size in 15 of 22 cases. It is possible that in some cases GM-CSF is not actually secreted by the leukemic cells, but is present in active form as a membrane-bound moiety. In this regard, Nara and McCulloch [281] showed that purified cell membranes from cells of some acute myeloid leukemia patients, but not normal bone marrow of ALL cells, could promote proliferation of other acute myeloid leukemic cells in short-term suspension culture, enhancing self-renewal. A direct link between constitutive GM-CSF expression and leukemic transformation was provided by Lang et al. [202], who transfected the murine GM-CSF gene into CSF-dependent, nonleukemic, myeloid cell lines and produced leukemic cell lines that constitutively secreted GM-CSF. To date, no example of human myeloid leukemia constitutively producing and responding to IL-3 has been reported, but in the mouse system, the well-characterized spontaneous murine myelomonocytic leukemia WEHI-3 constitutively secretes IL-3, apparently because of insertion of retroviral sequences adjacent to the IL-3 gene [416]. Retroviral insertion of the IL-3 gene with a viral long terminal repeat promoter produces leukemic transformation [140]. The fact that insertion of G-CSF, GM-CSF,

Kapil Mehta et al. or IL-3 genes into normal hematopoietic stem cells or in transgenic mice did not lead to leukemia [240] suggests that autocrine induction of factor-independence is necessary, but not sufficient, for converting normal cells to a transformed state. Genetic alterations may predispose them to undergo leukemic transformation by an autocrine mechanism. In this context the murine factordependent cell line FDC-P1 develops into fully leukemic cells when transplanted into whole-body-irradiated mice associated with development of autocrine GM-CSF or IL-3 production [80, 81]. Expression of mRNA for GM-CSF, G-CSF and M-CSF is ubiquitous in the blast cells of AML but not CML [55, 285, 397, 420, 421]. In a minority of these cases significant quantities of bioactive CSF were produced, sufficient to stimulate autocrine blast cell proliferation [329, 420]. The significance of these observations was questioned by studies showing that enhanced expression of the GM-CSF gene in ABL blast cells was a consequence of manipulations used to enrich blast cell populations [180, 367]. Constitutive expression of IL-1 on mRNA and bioactive protein production is a characteristic of the majority of AMLs and is not a consequence of manipulation of the cells. IN 10/17 cases of AML, IL-1β mRNA was identified [131], and this autocrine IL-1 can induce AML proliferation [247]. The proliferative effect of IL-1 on leukemic blast cells, like its action on normal cells, is mediated in synergy with growth factors such as IL-3, GM-CSF, and G-CSF [161, 162]. Since IL-1 induces G-CSF, GM-CSF, and M-CSF production by bone marrow stromal cells (endothelium, fibroblasts, macrophages; see Moore [260] for review), and these factors stimulate leukemic cell proliferation, a paracrine mode of stimulation of leukemic cell proliferation has been proposed for AML [131], and juvenile CML [18]. An IL-1-mediated autocrine growth stimulation was proposed in a study of 13 cases of AML [67]. In all cases, immunofluorescence showed that up to 80% of all fresh leukemic blast cells in all patients contained either the 33-kDa IL-1β propeptide or both the 33- and 17-dDa mature form. The bioactive IL-1α propeptide was also detected in all cases but was less frequently released. In six cases studied, anti-IL-1β and to a lesser extent anti-IL-1α inhibited spontaneous proliferation, and in 10/12 cases sufficient exogenous IL-1 was produced to stimulate significant proliferation. AML cells constitutively released as much IL-1 as did endotoxinstimulated normal monocytes, and 2/12 patients that did not respond to exogenous IL-2 were both high endogenous producers and presumably were maximally stimulated. In most instances, exogenous IL-1 supported the

551 establishment of continuous lines of AML cells that grew for >2 months [67]. A more complex autocrine loop is suggested by the studies of Bradbury et al. [36]. AML cells at low cell density were stimulated independently by exogenous GM-CSF or IL-1, and the response to both factors was inhibited by antibody to GM-CSF. Antibody to IL-1 inhibited spontaneous proliferation of these cells, and endogenous GM-CSF could be detected. Thus, IL-1 is produced by the leukemic cells, particularly from the more differentiated cells, and this in turn induces GM-CSF production, which directly mediates autocrine proliferation. Autocrine IL-1 induction of GM-CSF may account for the appearance of GM-CSF mRNA in cultured rather than fresh AML blasts [180]. Therapeutic strategy in the face of IL-1, GM-CSF-, or IL-3-dependent leukemogenesis, either autocrine or paracrine, would require intervention to block CSF or IL-1 production or action. High-affinity antibodies to the factor or its receptor, in the latter case coupled to some form of toxin, may be one possible strategy which is under active consideration as a therapy in the case of IL-2-dependent T-cell lymphoma (anti-IL-2 receptor antibody and IL-2 toxin conjugates). A second strategy envisages the use of GM-CSF to recruit resting leukemic stem cells into cell cycle and synchronize the population in S phase in conjunction with cycle-specific chemotherapy [87, 217, 322].

Colony-Stimulating Factors as Leukemia Differentiating Agents The G-CSF and GM-CSF induce granulocytic differentiation of HL-60 and WEHI-3B leukemic cell lines [24, 25, 108, 315], but leukemic blast-cell proliferation is the more general response in primary cultures of AML bone marrow. GM-CSF is a universal proliferative stimulus for AML cells, generally without differentiation, whereas G-CSF stimulates proliferation and in a proportion of cases granulocytic differentiation [73]. This area is reviewed elsewhere in this volume and the actions of G-CSF and GM-CSF in clinical trials in myelodysplastic syndrome and AML are discussed. M-CSF is also a proliferative stimulus for some AML cells, but in many instances it induces differentiation to adherent macrophages [397]. Most leukemic blast populations express the M-CSF receptor, fms, and M-CSF mRNA is found in about half the blast population. Marked heterogeneity of M-CSF response is seen with different patients, ranging from cases where blast populations were unresponsive to M-CSF, to examples where blast-cell self-renewal was stimulated, to instances where the major effect of M-CSF was the generation of

552 terminally differentiated cells with monocyte-macrophage characteristics. This heterogeneity in proliferative versus differentiation responses of leukemic blasts to various CSF species in different patients indicates the necessity of prescreening for factor response on an individual patient basis.

Interleukin 1 and Cell Proliferation and Differentiation Interleukin 1 has been implicated in an autocrine or paracrine mode of stimulation of myeloid leukemic cells in synergy with GM-CSF [36, 67, 131, 161, 162, 247]. It has also been reported to inhibit proliferation of a human myelomonocytic leukemic cell in the presence of low to intermediate, but not high, levels of GM-CSF [346]. The IL-1 inhibition of tritiated thymidine incorporation into murine M1 cells was first reported by Onozaki et al. [295]. On its own, IL-1 did not induce differentiation, but both growth inhibition and macrophage differentiation were induced synergistically by IL-1 and LPS coadministration. In further studies these investigators reported that while IL-1α, TNFα, and IFNβ1 all had antiproliferative, but not differentiation-inducing, action on M1 cells, as little as 1 unit of IL-1, in conjunction with TNF or IFNβ1, induced FcR, phagocytic activity, and morphological charge [297]. The differentiation induced by IL-1 plus TNF was inhibited by antibodies to IFNβ1, as was the antiproliferative effect of TNF (but not IL-1), indicating autocrine IFNβ1 is induced and mediates direct differentiation and antiproliferative effects. Thus IFNβ1 is one of two signals required for M1 differentiation, the other being IL-1, and the antiproliferative effect of IL-1 appeared to be a direct one. The differentiation-inducing factor for M1 cells in LPSstimulated peritoneal macrophage conditioned medium was identified as TNF, synergistically active with IL-1 [372]. Interleukin 1 induction of another cytokine, MG1-2 or IL-6, has also been implicated in IL-1 induction of M1 differentiation. Lotem and Sachs [215, 216], in contrast to the results of Onozaki et al. [297], showed that IL-1 on its own induced M1 differentiation as measured by FcR and C3R induction, lysozyme secretion, and morphology. In vitro, and in vivo in diffusion chambers implanted in mice, IL-1 induced granulocytic differentiation of M1 cells. IL-1 also acted synergistically with GM-CSF to induce differentiation of a GM-CSFresponsive, IL-1-unresponsive clone of leukemic cells. The in vivo action of IL-1 was associated with rapid induction of elevated serum levels of MGI-2/IL-6, but serum levels were not sufficiently elevated to account for the differentiation observed within the diffusion

Growth and differentiation factors as cancer therapeutics chamber. A more likely mechanism implicates IL-1 induction of autocrine production of IL-6 by leukemic cells, and this was observed within 6 h in IL-1-treated M1 cells, with production increasing by 2–3 days [215, 216]. The indirect differentiation induction mechanism was blocked using antibodies to IL-6. As with the autocrine IFNβ route for differentiation, the endogenous IL-6 may require a synergistic interaction with IL-1 to produce optimal differentiation. These results point to the importance of considering combinations of cytokines, both exogenous and endogenous, in designing optimal differentiation strategies. The ability of IL-1 to induce certain leukemic cells to differentiate in association with growth inhibition involves synergistic interactions with other cytokines, some of which are produced by leukemic cells in response to IL-1. In normal hematopoiesis, IL-1 also induces a spectrum of hematopoietic growth factors (G-CSF, GM-CSF, M-CSF) by an action on endothelial cells, fibroblasts, and macrophages and acts synergistically with these and other (e.g., IL-3) hematopoietic growth factors to stimulate proliferation of early hematopoietic stem cells [65, 79, 262, 266]. In vivo IL-1 alone or in combination with G-CSF was particularly effective in accelerating myeloid regeneration following high-dose chemotherapy or radiation [259, 262].

Interleukin-6 and Leukemic Cell Proliferation and Differentiation Interleukin 6 promotes the terminal maturation of B cells to antibody-producing cells, augmenting IgM, IgG, and IgA production in stimulated B cells [184]. It was originally identified as novel fibroblast-type interferon (IFNβ2) and was found to be identical to a B-cell differentiation factor, BSF2 [158], a hybridoma/plasmacytoma growth factor [390], and a macrophage-granulocyte inducer of leukemic cell differentiation (MGI-2) [354]. Its action on normal hematopoiesis includes stimulation of multilineage blast cell colonies in synergy with IL-6 [172]. It may act to induce the entry of Go stem cells into cell cycle [197]. Interleukin 6 potentiates thrombocytosis [155, 174]. Chronic stimulation of B cells was achieved in transgenic mice carrying the human IL-6 gene conjugated with the Ig enhancer [184]. These mice developed polyclonal nontransplantable plasmocytomas. Freshly isolated human myeloma cells also produce and respond to IL-6, indicating an autocrine role of this molecule in the proliferation, but not differentiation, of certain malignant B cells [13, 424]. Dependence of myelomas on IL-6 may also involve a paracrine mechanism involving marrow stromal cell production

Kapil Mehta et al. of IL-6 in response to myeloma cells [187]. This could involve malignant B-cell production of IL-1, which is a potent inducer of IL-6 gene transcription and translation [260]. In contrast to its action on normal B cells, IL-6 does not augment Ig secretion in myeloma cells [373]. The IL-6-induced proliferation and autocrine IL-6 action have also been reported for certain human B-cell lymphomas, and elevated serum levels of IL-6 were found in 50% of patients with active lymphoma [413]. Interleukin 6 was found to be identical to a macrophage-granulocyte inducer (MGI-2A) that caused differentiation of myeloid leukemic cells but lacked the ability to stimulate colony formation [218, 354]. It was also identical to a differentiation factor (DIF or D Factor) produced by Krebs ascites tumor cells, although it should be noted that this source contains a second D-factor, which is leukemia inhibitory factor (LIF, see following section). IL-6 produces complete growth arrest of the murine myeloid leukemia M1 within 48 h, and this is associated with morphologic maturation of the blast cells to macrophages with upregulation of cfms, FcR, C3R, lysozyme secretion, and development of phagocytic capacity [241, 251, 252]. In clonogenic assay of M1 cells diffuse differentiated colonies develop within 48 h of addition of IL-6 and the number of colonies is reduced [241]. The murine myelomonocytic leukemic WEHI-3 (D+ variant but not D−) was also induced by IL-6 to macrophage differentiation with up-regulation of FcR and cfms [50, 240]. Interleukin 6 did not reduce the W3 leukemic clonogenic capacity. Synergistic or additive interactions between IL-6 and other cytokines influence both proliferation and differentiation. The IL-6 and LIF or G-CSF have additive or supraadditive effects on M1 differentiation [240, 241, 251, 252]. The IL-1 and IL-6 were additive or synergistic in growth inhibition of U937, HL-60, and M1 [296, 316]. Interleukin 1 alone induced lysozyme production by M1 cells but had no direct effect on the expression of FcR or on morphology. Interleukin 6 independently induced FcR and lysozyme, but the combination triggered the entire sequence of differentiation markers [316]. The M-CSF also synergized with IL-6 to enhance differentiation of M1 cells but it counteracted the growth inhibition, resulting in increased size and number of macrophage colonies [240, 241]. The possibility existed that these interacting cytokines were in fact members of a cascade involving autocrine production of factors by leukemic cells. Interleukin 6, IL-1, LIF, and G-CSF are all differentiation-inducing factors, they are all endotoxin-inducible macrophage products, and they can be produced by myelomonocytic leukemic cells. The M1 cells produce IL-6, and levels are increased following

553 LIF treatment [240, 241]; however, antibody to IL-6 does not block LIF induction of M1 cells. In U937 cells, IL-6 did not induce IL-1, nor did IL-1 induce IL-6, and both factors appear to provide distinct signals for differentiation of this neoplastic macrophage cell line [296]. The action of IL-6 on human acute myeloid leukemic blast cells is observed only when it is combined with GM-CSF or IL-3. A heterogeneous response is seen involving synergistic blast cell proliferation [131, 161, 162]. Like IL-1, IL-6 is secreted by most leukemic cells where there is evidence of monocytic differentiation (12/15 patients with ANLL: of FAB type M4 or M5) [389]. The significance of this potential autocrine look is uncertain.

Leukemia Inhibitory Factor A spectrum of factors have been described that induce differentiation of the murine M1 myeloblastic leukemic line. This includes G-CSF, IL-1, IL-6, and TNF as well as a unique factor termed leukemia inhibitory factor (LIF) on the basis of its potent and selective inhibition of M1 cell proliferation in association with induction of macrophage differentiation [127, 118]. This factor had earlier been characterized in the conditioned media of L929 cells and termed D-factor, inducing morphological maturation in M1 cells with the development of locomotor activity, phagocytic capacity, and upregulation of FcR, C3R, and prostaglandin E [381]. The factor was purified from Krebs II ascites conditioned medium as a 58-kDa glycoprotein [118] and from mouse Ehrlich ascites cells as a 40-kDa activity [221]. The native molecule of 179 amino acids [118] or 180 amino acids [221] has a molecular weight of 20 kDa with seven N-linked glycosylation sites. The human gene is located on human chromosome 22q11–q 12.2 [366]. The murine and human molecules are active across species and are 78% homologous at the amino acid level. LIF was found to be identical to a variety of other cytokines that had been reported to possess diverse functions. Alloreactive human T cell clones obtained from rejected kidney, when stimulated with a specific antigen and IL-2, produced a factor that triggered the proliferation of a subline of the IL-3-sensitive murine factor-dependent cell line DA-1 [268, 269]. This factor, called HILDA (human interleukin DA), was a 38- to 41-kDa glycoprotein that was also reported to have eosinophil chemotactic and activating properties and erythroid burst-promoting activity [122]. In retrospect these latter activities were probably due to a minor contamination of the protein with GM-CSF and/or other lymphokines.

554 HILDA cDNA cloned from human lectin-stimulated T cells was shown to be identical to LIF [269]. A human macrophage differentiation-inducing factor (DIF) for M1 cells isolated from a human monocytic leukemia line, THP-1, was also shown by amino acid sequencing to be identical to LIF [3]. The growth of totipotential embryonic stem cells in vitro is sustained by a differentiation inhibitory activity (DIA) produced by a number of sources, including the 5,637 human bladder carcinoma cell line. Purified DIA and LIF were very similar in biochemical features, and purified recombinant LIF can substitute for DIA in the maintenance of totipotent embryonic stem cells that retain the potential to form chimeric mice [406]. A factor constitutively produced by human squamous carcinoma cells stimulated hepatocytes to produce the same spectrum of acute phase proteins as IL-6 [23]. This hepatocyte stimulating factor III was distinct from IL-6 but was identical to LIF. Rat heart tissue produces a cholinergic differentiation factor that controls the phenotypic choice in neurons without affecting their survival or growth [411]. This factor was also identical to LIF and acted on postmitotic rat sympathetic neurons to specifically induce expression of acetylcholine synthesis and cholinergic function and suppress adrenergic function. Receptor analysis with 125LIF indicated that M1 cells possess a single class of high-affinity (Kd 100–200 pM) receptors of relatively low frequency (300–500 per cell) [156]. In bone marrow, spleen, and peritoneum, monocytes, macrophages, and their precursors were the most obvious cells binding LIF. The in vivo action of LIF was revealed by studies in which mice were engrafted with cells of the murine hematopoietic cell line FDC-P1 multiply infected with a retroviral construct containing cDNA encoding LIF [242, 243]. The mice developed within 12–70 days a fatal syndrome characterized by cachexia, excess osteoblasts with new bone formation, calcification in heart and skeletal muscle, pancreatitis, thymus atrophy, and abnormalities in the adrenal cortex and ovarian corpora lutea. The development of this osteosclerotic syndrome, with marked increases in osteoblasts, indicates a role for LIF in osteoblast production and function and in regulation of bone formation and calcium metabolism. The presence of LIF receptors on osteoblasts further indicates the direct nature of the stimulation [242, 243]. The action of LIF on leukemic cells appears to be highly restricted, inducing M1 cell differentiation alone, or synergistically with G-CSF, IL-6, or M-CSF [244]. The growth inhibition and differentiation effect was rapid, being evident at 24 h and marked at 48 h. LIF was without effect on normal CFU-GM or WEHI-3

Growth and differentiation factors as cancer therapeutics B myelomonocytic leukemic cells. Abe et al. [3] reported a direct differentiation-inducing, and proliferationinhibiting, action on HL-60 and U937 cells. Maekawa and Metcalf [225] did not observe a direct action of LIF on HL-60 or U937 cells but reported clonal suppression of these leukemic cells by LIF in combination with GM-CSF or C-CSF. Sequential recloning of these cells was also suppressed by LIF-CSF combinations. Despite the relatively weak action of LIF on human leukemic cells, LIF clearly is able to exhibit suppressive actions. This, coupled with its lack of suppressive effects on normal hematopoietic cells, suggests a role in suppressing human leukemias, possibly in combination with other hematopoietic factors.

Tumor Necrosis Factor (TNFa) and Lymphotoxin (TNFb) Tumor necrosis factor was identified as a protein with antitumor activity in serum of mice infected with Bacillus Calmette-Guerin and treated with endotoxin [335]. TNFα has significant sequence homology to human lymphotoxin β and binds to the same receptor. The biological actions of TNF are multifaceted, involving both stimulatory and inhibitory actions depending upon the target cell population. In normal human hematopoiesis TNFα was reported to inhibit CFU-GM day 7 (ED50 = 10 U) and CFU-GM on day 14, BFU-E, CFU-E, and CFU-GEMM (ED50 = 50 U) [39, 223, 306]. Even greater inhibition of BFU-E, CFU-GEMM and CFU-E was reported with both TNF alpha or β in other studies [276]. TNF inhibition was enhanced in a synergistic manner by interferon γ [39, 276, 306, 307]. TNF inhibition was reported to be nonreversible within 60 min, suggesting a cytotoxic mechanism [39], but others have reported either full reversibility following a 24 h preincubation [276] or partial reversibility [279]. The variable results can be attributed to the use of different sources of CSF and the variable presence of accessory cells in the preparation. With G-CSF as a stimulus, the ED50 with TNFα or -β was 10 U whereas with a GM-CSF stimulus only a 20% inhibition was seen with 1,000 U TNF [20]. Thus in normal marrow one cell in 500 will form a colony with G-CSF or GM-CSF, but with TNF only one cell in 100,000 will respond to G-CSF, yet the frequency of cells responding to GM-CSF remains essentially unchanged [21, 194]. With highly purified marrow progenitors, the ED50 for TNF was 5 U with G-CSF-stimulated colonies, and 500 U with GM-CSF stimulation [267]. Using CD34-positive selection for CFU-GM, G-CSF-stimulated colonies were strongly inhibited by TNF, but an actual enhancement of

Kapil Mehta et al. GM-CSF or IL-3-stimulated colonies was recently reported [345]. In murine serum-free bone marrow cultures the ED50 for CFU-GM was 20–200 U or murine TNF and for BFU-E and CFU-GEMM it was 2,000 U [85]. Using human TNF, a lower degree of inhibition is generally found. In most studies, human TNF does not inhibit at doses below 10,000 U/ml, and indeed some slight potentiation of M-CSF stimulated colonies was reported [407]. In long-term murine or human bone marrow culture, TNF exhibits a dose-dependent (10–1,000 U/ml) inhibition of CFU-GM production and neutrophil generation [85, 267]. The inhibition induced by TNF appears to be selective for neutrophil differentiation and is not manifest in reduction of eosinophils or monocyte/macrophages. The interaction of TNF with leukemic cells is a complex one, variously resulting in differentiation, and growth inhibition or growth potentiation. A differentiating factor for HL-60 and ML-1 leukemic cells, produced by PHA-stimulated lymphocytes or the HUT 102 T cell line was shown to be TNF [370]. As little as 1, 10 U of TNF alone or in synergy with IFNγ promoted monocytic differentiation of HL-60, U937 and ML-1 [135, 194, 306, 346]. The action of TNF on fresh AML marrow cells has been investigated in clonogenic (PE1) and recloning (PE2) assays, and suspension culture. Early reports uniformly reported inhibition. In one study of ten patients 50% inhibition was seen with 15 pM TNF and there was synergy with IFNγ [306]. Inhibition to a degree comparable to normal was reported in seven patients [39], and in nine of ten patients 75% inhibition of leukemic cell cloning was seen with 100 U TNF compared to 44–48% inhibition in remission marrow. Enhanced differentiation to monocyte macrophages with enhanced NBT-reducing activity was seen with TNF or TNF plus IFNγ in suspension cultures of marrow from 1 AML and 5 CML in blastic crisis [119]. Differentiated macrophages were observed with Auer rods, indicating their leukemic origin. In other, more recent studies, a more complex picture emerges. Clonogenic assays stimulated with G-CSF or GM-CSF and exposed to high (1,000 U) or low (100 U) doses of TNFβ showed four types of response in AML and preleukemic myelodysplastic syndrome: inhibition greater than normal or remission inhibition equivalent to normal, no response, and significant enhancement. This latter pattern, seen in 4/13 cases, involved a synergism with either G-CSF or GM-CSF and clonogeneic capacity increased up to 60-fold. Assessment of plating efficiency (PE1, PE2) using 5637 CM as a source of stimulus revealed three patterns of response with TNF [279]; (a) PE1, PE2, and suspension cell were inhibited

555 90–100% with 100–1,000 U TNF; (b) PE1 and PE2 were not inhibited and possibly potentiated at low doses of TNF but suspension cells were markedly inhibited; and (c) marked inhibition of PE1 and PE2 but not of suspension cells. In no case was there evidence of TNF induction of differentiation. The nature of the stimulus may determine whether the TNF response is inhibitory or stimulatory. Proliferation of AML blast cells (ten patients), induced by G-CSF, was further stimulated by TNF, and clonogenic assays were potentiated 50–250% of maximum [163]. In contrast, IL-3- or G-CSFstimulated cultures were inhibited 100% and 5,637 CM-stimulated cultures by 30% with 600 pM of TNF. Autocrine stimulation of AML cell proliferation seen at high cell densities was also enhanced by TNF, suggesting synergism with leukemia cell-derived GM-CSF. The mechanism of TNF potentiation is probably indirect and was inhibited by antibodies to IL-1. Furthermore, IL-1 synergises with GM-CSF in stimulating leukemic blast cells [162], and TNF synergism was not seen in cultures in the presence of exogenous IL-1. IL-1 and TNF have been shown to be constitutive products of AML cells and production of bioactive IL-1 can be increased by TNF treatment [131, 163, 398]. The proliferative response of leukemic blast progenitors to TNF under conditions that favor autocrine stimulation may represent one property that allows leukemic cells to escape from negative regulation. In the chronic phase of CML, TNF substantially inhibits DNA synthesis in cultures of purified progenitors and inhibits day 7 and day 14 CFU-GM at doses of 10–500 U [82, 194]. Some patient-to-patient variation is seen with inhibition in the normal to less-than-normal range. Antibodies to TNFα blocked the TNF inhibition and enhanced GM-CSF-stimulated colony formation twofold in cultures of accessory cell depleted progenitors [82]. Autocrine production of TNF by immature CML cells was confirmed by Northern analysis and ELISA assays. The quantities of TNF produced by leukemic cells were sufficient to inhibit normal hematopoiesis and may provide a selective advantage for leukemic clones if CML progenitors are less sensitive to TNF inhibition than normal. In this context TNF production by hairy-cell leukemic cells has been identified as the possible cause of myelosuppression and neutropenia seen in this cancer [211].

Transforming Growth Factor b Transforming growth factor β (TGFβ) is a member of a group of polypeptide growth factors that regulate cell growth and differentiation. TGFβ exists as a 25-dK

556 disulfide-linked dimer, and subtypes include TGFβ1, TGFβ2, TGFβ1, 2, existing in homodimer and heterodimer forms, and TGFβ3. The factor is produced by most normal cells and its pleiotropic actions include growth stimulation of fibroblasts and growth inhibition of epithelial cells, endothelial cells, and various malignant cells. It also affects differentiation of cells as varied as adipocytes, myoblasts, chondrocytes, and epithelium and is involved in production of extracellular matrix, bone remodeling, and repair. TGFβ acts as a modifier of the immune response and may play an important role in hematopoiesis. It is a potent monocyte chemoattractant and induces these cells to produce IL-1 and TNF [394]. In vitro, TGFβ1 and -β2 (2–4 pM) inhibit megakaryocytopoiesis and CFU-MK [40, 173]. In both murine and human systems, pluripotential progenitors (high proliferative potential CFU, CFU-GEMM) and BFU-E are strongly inhibited [7, 137, 182, 299, 340, 358]. Erythropoietin-stimulated CFU-E are not inhibited [181, 182, 210, 341]. The action of TGFβ on in vitro myelopoiesis is determined by the stage of differentiation of the progenitor the type of CSF used, and the species. In the murine and human systems, G-CSF and M-CSF stimulated colonies are not inhibited [137, 181, 182, 358]. The GM-CSF-stimulated murine colonies are variously reported to be resistant to TGFβ [181, 182] or inhibited, but to a lesser extent than earlier progenitors, at doses of 0.2–0.5 ng/ml [75, 137, 157, 288, 341]. Human day 7 CFU-GM (predominantly G-CSF responsive granulocyte-committed progenitors) were enhanced 150–175% by TGFβ1 or -β2 even in the presence of plateau concentrations of G-CSF, GM-CSF, or IL-3 [56, 299]. Day 14 CFU-GM were variously reported to be unaffected by TGFβ [7, 157], or inhibited to a lesser degree than earlier progenitors [182, 358]. Synergistic or additive interactions occur between TGFβ and other cytokines. BFU-E were synergistically inhibited by TGFβ plus TNFα, and these cytokines additively inhibited CFU-GEMM and CFU-GM [359]. Interferon gamma and TGFβ synergistically inhibited CFU-GM and additively inhibited BFU-E and CFU-GEMM. In long-term bone marrow culture, TGFβ serves as a potent inhibitor of myelopoiesis, probably by inhibiting proliferation of early stem cells in the adherent stromal layer [44, 150]. Indeed, TGFβ production by marrow stromal cells may be an important negative regulator of steady-state hematopoiesis. Further evidence for a physiological role for TGFβ as negative regulator was provided in studies in which TGFβ1 (1–5 μg/mouse) was injected via the femoral artery into normal mice [123, 124]. The CFU-GEMM stimulated by IL-3 were completely inhibited in the femur, and CFU-GM were inhibited 50% by

Growth and differentiation factors as cancer therapeutics 24 h with reversal of inhibition at later times. This observation suggests that TGFβ, by reversibly inhibiting the cycling of early stem cells, may be effective in protecting such cells from damage inflicted by cell cycle-specific chemotherapy. The action of TGFβ on myeloid leukemic cells generally involves potent growth inhibition; however, an influence on differentiation is seen in some systems. TGFβ alone induced monocyte-macrophage differentiation of the U937 and THP-1 cell lines but was only a weak inducer of HL-60 [179]. Synergism between TGFβ and TNFα, IFNγ, dexamethasone and phorbol esters is seen in differentiation induction of U937, THP-1, and ML-1 in human monocytic or myeloblastic leukemias [133, 179]. Low doses (0.5 ng) of TGFβ induced hemoglobinization of K562 with growth inhibition [53]. Inhibition of proliferation of M1 murine myeloid leukemic cells is produced by low doses of TGFβ with enhanced adherence, but differentiation-associated properties such as phagocytic activity, lysozyme secretion, and morphologic change were not induced [290]. Indeed, TGFβ inhibited the dexamethasone-induced differentiation of this cell line. Potent inhibition of proliferation was seen when TGFβ was added to a variety of factor-dependent cell lines (NFS-60, 32D, FDCP-1, B6 S4t A, DA-3) and to the IL-3-producing WEHI-3 myelomonocytic leukemic cell line [181]. Inhibition was also seen with the human leukemic cell lines U937, THP-1, and KG-1 but not with various other lines (e.g., HL-60, various T and B leukemias and lymphomas) [182]. The response of the cell lines to TGFβ appeared to correlate with the extent of display of TGFβ receptors. In primary cultures of bone marrow from 15 patients with chronic myeloid leukemia, all showed TGFβ inhibition of D14 CFU = GM but only 4/15 showed the stimulation of D7 CFU-GM, which is the response seen with normal marrow, and in 11/15 TGFβ inhibited D7 CFU-GM [7]. Both TGFβ1 and -β2 inhibited 45% of CFU-GM stimulated by GM-CSF in marrow cultures from five CML patients but, in contrast to normal, G-CSF-stimulated colonies were also inhibited [358]. In studies of acute myeloid leukemia (18 patients), TGFβ suppressed both primary and secondary leukemic clonogenic capacity [282, 375]. These results showed that 1–10 ng/ml of TGFβ delayed progression of leukemic blast-cell progenitors from G1 to S phase in a cytostatic manner with no induction of differentiation. It is now apparent that a large number of factors responsible for growth and differentiation have been identified. In addition, their receptors on normal and neoplastic cells are also being identified and investigated. It is clear that some of these factors are produced

Kapil Mehta et al. by tumor cells and represent autocrine growth factors. Obviously, the blockage of these growth factors by reducing their production or by blocking their attachment to receptors could be an effective strategy for cancer treatment. Likewise, the production of stimulatory factors for angiogenesis by tumor cells has now been well characterized and the blockage of their paracrine effect on adjacent blood vessels in normal tissue is another potential method of therapy. Some tumors produce a considerable quantity of growth factor proteins and peptides and others produced smaller amounts or a wider diversity of these factors. In some, factors which promote the growth of tumor cells clearly play a role in cancer growth and metastasis and the autonomy that cancer seems to enjoy. An interruption of this process will clearly lead to further approaches in cancer treatment. A good example of the clinical significance of growth factors has been demonstrated in studies showing that approximately 50% of tumor tissue from breast cancer patients expresses epidermal growth factor receptor. These receptors appear to be inversely related to steroid receptors and therefore bring a poor prognosis to patients who are steroid-receptor negative and epidermal growth factor receptor positive. Since thousands of tumors have been studied with regard to this particular characteristic, it is abundantly clear that epidermal growth factor receptor should be explored as a target for cancer treatment. Monoclonal antibodies have been produced by various investigators which are being administered to patients with breast cancer in an attempt to block the epidermal growth factor receptor. Many of these studies are being done in association with chemotherapy to try to get an effective one–two punch in inhibiting tumor growth. Oncogenes such as erb and v-cis are known to encode for growth factor receptors. Soluble receptors with high affinity binding might be useful in therapy to bind with growth factors and reduce their activities on membranebound receptors present in the tumor cell or adjacent blood vessel. Factors which are structurally similar to growth factors but are inactive in inducing receptor function (blockers) are also being explored as treatment strategies. Growth factor interaction can also be blocked by certain drugs such as Suramin. Suramin and its analogues are currently in clinical trials for these effects. Anti-angiogenesis factors have also been identified and these are also in clinical trials. In summary, the application of techniques to block growth factors and/or the transduction of biochemical effects from growth factor receptor stimulation represent advent technologies for the millennium. As more is known about autocrine and paracrine effects of growth factors, more can be done with respect to altering their effects on tumor cells and

557 adjacent tissues. This approach, when employed in concert with the use of differentiation agents, may allow for the regulation and control of tumor growth in a manner very different than chemotherapy and radiation therapy. Perhaps one can regulate cancer in a manner similar to the use of insulin to regulate diabetes rather than attempting to destroy every last cancer cell with all the toxicity inherent in these historical approaches.

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17 Granulocyte colony-stimulating factor: biology and clinical potential MARYANN FOOTE and GEORGE MORSTYN

Introduction The study of hematopoiesis was greatly facilitated in the mid-1960s when techniques for studying hematopoietic cells in clonal culture were developed. Initially, serum or conditioned medium was added to cultures as a source of growth factors, i.e., the colony-stimulating factors (CSFs) [56, 57, 85]. One of the factors that was isolated, purified, cloned, and produced in commercial quantities was granulocyte colony-stimulating factor (G-CSF), a protein that acts on the neutrophil lineage to selectively stimulate the proliferation and differentiation of committed progenitor cells and activation of mature neutrophils (Fig. 1). A property that distinguished G-CSF from other CSF and facilitated its purification, molecular cloning, and large-scale production in prokaryotic cells was its ability to induce terminal differentiation of a murine leukemic cell line (WFHI-3B). After observing that serum from endotoxin-treated mice was capable of causing the differentiation of a WFHI-3B myelomonocytic leukemic cell line, Metcalf [55] named the activity GM-DF (granulocyte-macrophage differentiating factor). Further analysis showed that this serum contained G-CSF as well as granulocyte-macrophage colonystimulating factor (GM-CSF). Nicola et al. [66, 68] further purified G-CSF from medium conditioned by lung tissue of endotoxin-treated mice. This G-CSF could stimulate WFHI-3B cells as well as normal cells, supporting the formation of numerous small, neutrophilcontaining colonies at a concentration similar to that needed for WFHI-3B differentiation [67]. Subsequently, murine G-CSF was identified as a protein and was shown to have both differentiation-inducing activity for WFHI-3B and granulocyte colony-stimulating activity in bone marrow cells [68]. Other researchers, notably Asano et al. [2] and Welte et al. [102], found several human carcinoma cells that constitutively produce colonystimulating factors. One of these factors was purified to apparent homogeneity from the conditioned medium of bladder carcinoma 5637 cells [102] or a squamous carcinoma cell line [71]. The purified CSF selectively stimulated neutrophilic granulocyte colony formation from

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

bone marrow cells, so it was concluded that this factor was the human counterpart to mouse G-CSF. The protein initially identified as G-CSF also was called CSF-13 and pluripoietin (pCSF). The study of G-CSF progressed to the purification and molecular cloning of both murine and human forms and then to the first clinical trials of recombinant human (rHu) G-CSF in cancer patients [7–9, 26, 27, 60–62]. Because of its unique biologic activities, rHuG-CSF is used for reversing or ameliorating neutropenias of various causes, for allowing increased cancer chemotherapy dose intensity, and for mobilizing hematopoietic stem cells for transplantation. Filgrastim, the nonglycosylated rHuG-CSF, was the first hematopoietic growth factor approved for commercial use. A second form of rHuG-CSF, a glycosylated version, lenograstim, was developed and approved for commercial use in Europe. Nartograstim (also known as marograstim, KW-2228, or NTG), a mutein rHuG-CSF, was developed, but it had limited clinical development [89]. A polyethylene glycol-modified rHuG-CSF (pegfilgrastim) has been approved for commercial use [28]. Another pegylated rHuG-CSF is RO 258315; it is nartograstim with polyethylene glycol molecules at the amino terminus and the four lysine residues. The final product of pegylation of RO 258315 is a mixture of one, two, three, or four polyethylene glycol molecules attached to each molecule of KW-2228, with the dipegylated protein most common (60%) [94]. Table 1 summarizes what is known about endogenous G-CSF and Table 2 summarizes some characteristics of the rHuGCSFs. The molecule will be specified in this review when the term ‘rHuG-CSF’ is insufficient. G-CSF’ will be used to discuss the endogenous protein.

Biochemistry and Structure Native human G-CSF appears to exist as a mixture of two forms, one having 177 amino acids and the other having 174 amino acids [1, 76, 86]. The gene for human G-CSF is positioned at 17q11–22 and is approximately

569

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Granulocyte colony-stimulating factor: biology and clinical potential

Pluripotential Stem Cell

CFU-Blast

flk-2/flt-3 ligand

flk-2/flt-3 ligand

SCF

flk-2/flt-3 ligand

flk-2/flt-3 ligand

SCF

SCF

SCF

Lymphoid Stem Cell

CFU-GEMM

MGDF/TPO SCF IL-3

SCF IL-3

SCF IL-3 flk-2/flt-3 ligand

SCF IL-3

SCF IL-3

SCF IL-3

SCF IL-7

NK Precursor

SCF IL-7

flk-2/flt-3 ligand flk-2/flt-3 ligand

CFU-GM CFU-Meg

BFU-E SCF IL-3 GM-CSF EPO

SCF IL-3 GM-CSF IL-11 IL-6 MGDF/TPO

IL-3 GM-CSF

IL-3 G-CSF GM-CSF

CFU-M

CFU-E IL-3 GM-CSF EPO

IL-3 GM-CSF M-CSF

CFU-G

CFU-Eo

IL-3 GM-CSF

CFU-Ba

IL-3

CFU-Mast

Pre-B Cell

IL-3 SCF

IL-7

IL-3 GM-CSF G-CSF

Megakaryocyte

IL-6 Monocyte GM-CSF M-CSF

Reticulocyte

Pre-T Cell

B Lymphocyte

IL-2 IL-7

SCF IL-2

Proplatelet

Red Blood Cell

Platelet

Macrophage

Neutrophil

Eosinophil

Basophil

Tissue Mast Cell

Plasma Cell

T Lymphocyte

Figure 1. Hematopoietic tree (Figure courtesy of Amgen Inc., Thousand Oaks, CA)

Table 1. Summary of characteristics of human G-CSF Characteristic Location of gene Gene size Number of exons Protein produced Number of amino acids in mature protein Glycosylation site Disulfide bonds Basic structure Receptor structure

Chromosome 17q11-22 (human) Approximately 2500 nucleotides (approximately 2.5 kb) 5 Proform 174 (18.6 kDa) Thr 133 2 (between 36 and 42, and 64 and 74) 4 helices arranged in antiparallel fashion; and sheet pleating Single 150-kDa chain-forming homodimer

NK Cell

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Table 2. Characteristics of the forms of recombinant human G-CSF Generic name

No. of amino acids

Cell source

Other information

Marketing company

Filgrastim Lenograstim Nartograstim Pegfilgrastim RO 25–8315

175 174 174a 175 174

E. coli Chinese hamster ovary E. coli E. coli E. coli

Non-glycosylated Glycosylated Mutein form Non-glycosylated, pegylated Mutein form, pegylated

Amgen, Kirin, Roche Chugai, Rhône-Poulenc Kyowa-Hakko Amgen Roche

a

Changes to amino acids 1, 3, 4, 5, and 17

2.5 kbp [84]. It is encoded by the CSF3 gene. One chromosomal gene exists per haploid genome. The core protein of native human G-CSF has a molecular weight of 18.8 kDa and the glycosylated protein 20–23.5 kDa [76]. Filgrastim is a highly purified protein consisting of a single 175-amino acid polypeptide; lenograsfim, lacking an N-terminal methiorryl residue, has 174 amino acids. Lenograstim exists as two formulations, one using gelatin and one using human serum albumin as a stabilizer. The gelatin lenograstim formulation reportedly allows faster neutrophil recovery after chemotherapy than lenograstim [90]. Although nartograstim also has 174 amino acids, the molecule has been modified at the first, third, fourth, fifth, and 17th amino acids [70]. Nartograstim is conjugated with an average of two polyethylene glycol chains per protein molecule [23]. Pegfilgrastim has a 20-kDa polyethylene glycol molecule added to filgrastim, giving the protein an extended half-life in the serum [58]. Native human G-CSF and lenograstim are O-glycosylated [64, 86] whereas filgrastim and pegfilgrastim are not glycosylated. The sugar chains are responsible for the differences in molecular mass. The presence and type of glycosylation of the recombinant protein depends on the cellular source (i.e., yeast, mammalian cell, or bacterial cell) [62]. Lenograstim has heterogenous glycosylation: not all the sugar chains are identical. Not all structural components of native human G-CSF have been identified, but it is known that its sugar composition is not identical to that of lenograstim [72, 73]. Native human G-CSF does not bind to concanavalin, suggesting that it does not have mannose-containing carbohydrates [69]. When G-CSF was treated with neuraminidase, it showed reduced charge heterogeneity in isoelectric focusing, suggesting sialic acid-containing O-glycosylation [68]. Crystallography studies of filgrastim have shown that the glycosylation site (threonine 134) is attached to the C-D loop at a distance from the active biologic sites [43, 74]. Although the glycosylation does not seem to have a role in the biologic function of the molecule, it may partially protect the molecule from proteolytic degradation. The observation that a limited proteolytic degradation

of filgrastim results in the cleavage of the molecule near threonine 134 points to a role of glycosylation for proteolytic protection and indicates that the residues along this portion of the protein structure may serve as ‘handles’ for proteolytic degradation [76]. Although it has been suggested that glycosylation of lenograstim increases biologic activity in vitro compared with filgrastim [103], this advantage has not been verified in randomized doubleblind comparative studies [35]. Glycosylation possibly influences the antigenicity of recombinant proteins [48]. Several cell types in the human body produce G-CSF, including stromal cells, endothelial cells, fibroblasts, macrophages, and monocytes [19, 24, 61, 76, 79, 96, 105]. These cell types are widely distributed in the body, suggesting that G-CSF participates in the production and functional enhancement of neutrophils that occur in response to local infection or other causes. In a variety of pathologic conditions, including exposure to endotoxin, response to an infection or to neutropenia, the amount of circulating endogenous G-CSF in the blood increases [10, 40, 56, 98]. In humans, G-CSF is the primary factor for the upregulation of neutrophils in infection and in various pathologic conditions with decreased neutrophil counts [10]. It has been suggested that the Toll-like receptors on macrophages and dendritic cells detect gram-negative bacteria and induce IL-23 production that, in turn, induces IL-17 production by T cells, which then stimulates G-CSF production. Mechanistically, as G-CSF concentrations increase during bacterial infections and circulate in the blood, neutropoiesis is stimulated in the marrow. Hence, in cases of gram-negative and fungal infections, serum G-CSF concentrations are increased [40]. Indeed, the highest G-CSF concentrations have been found in patients with neutropenia and febrile neutropenia [10]. G-CSF selectively stimulates the proliferation and differentiation of neutrophil precursors by binding to a specific cell-surface receptor [18]. The G-CSF receptor is expressed on cells of the neutrophil lineage from myeloblast to the mature neutrophil and on a subset of cells of the monocyte lineage [56]. It has been reported

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Granulocyte colony-stimulating factor: biology and clinical potential

that G-CSF receptor expression on cell types is not usually associated with its activity including endothelial cells, activated T lymphocytes, and nonhemopoietic tumor cell lines. The role of these receptors is unclear. Both filgrastim and lenograstim, as well as native HuGCSF derived from a tumor cell line, have identical dosedependent affinity for G-CSF receptors (Fig. 2). The binding of G-CSF to its receptor decreases the number of available surface receptors as the surface receptor complex is internalized and degraded. Both endogenous and recombinant G-CSFs work on neutrophil precursor cells and mature neutrophils. rHuG-CSF stimulates the proliferation, differentiation, and activation of committed progenitor cells of the neutrophil lineage and reduces neutrophil maturation time from 5 days to 1 day, leading to a rapid release of mature neutrophils from the bone marrow into the circulation [53, 54]. Neutrophils previously treated with rHuG-CSF have normal intravascular half-life [8, 54]. In the presence of rHuG-CSF, neutrophils have enhanced superoxide production in response to chemoattractants [1, 100]. G-CSF also enhances chemotaxis by increasing the binding of fMLP (formyl-methionyl-leucyl-phenylalanine) [14] as well as enhancing anti-Candida activity. rHuG-CSF does not stimulate release of other cytokines by neutrophils and mononuclear cells [78].

Lenograstim also increases superoxide anion production in neutrophils in response to fMLP [93]. Neutrophils from healthy volunteers treated with lenograstim showed evidence of enhanced stimulation by agonists, including adhesion to nylon fibers and physiologic substrates [33, 36]. In vivo studies in animals and some studies in humans have shown that rHuG-CSF causes a dose-dependent increase in the number of neutrophils in the peripheral blood. This increase is thought to be a result of decreased maturation time, increased number of cell divisions, and accelerated release into the peripheral blood. The action of rHuG-CSF causes rapid dose-dependent increases in neutrophils and small or no effects on monocytes and eosinophils, respectively [50, 52, 63].

Physiology G-CSF is an indispensable cytokine for normal murine myelopoiesis, as has been shown by knock-out-mouse experiments [47]. Circulating neutrophil concentrations were reduced by 70–80% with less of a reduction in marrow stores of progenitors (50%) compared with those of normal mice. Despite appearing superficially healthy, these mice have a diminished ability to mount neutrophilia

Figure 2. Inhibition of labeled filgrastim, lenograstim, and tumor cell-derived human G-CSF binding to neutrophils. Those data demonstrate that the three forms have the same affinity for G-CSF receptors in vitro. (Figure courtesy of Amgen Inc., Thousand Oaks, CA)

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and monocytosis in response to infection and have a marked impairment in ability to control Listeria infections. The observations from this study indicate that G-CSF is required for maintaining the normal quantitative balance of neutrophil production during steady-state granulopoiesis in vivo and implicate rHuG-CSF in emergency granulopoiesis during infectious episodes.

Pharmacokinetics Pharmacokinetic/pharmacodynamic data from the different rHuG-CSF products are difficult to compare directly, as different study designs, doses, regimens, routes of administration, and populations were evaluated. Table 3 summarizes these data.

administration, and no dose sequence effects were observed in a crossover study [38]. Administration of single 10, 30, 60,100, or 150 μg/kg doses of RO 258315 to healthy men produced dosedependent increases in peak neutrophil count [94]. The time to reach peak concentration and the area under the serum concentration time curve increased 100-fold over the range of doses studied. A healthy volunteer study was reported with pegfilgrastim [58]. Pegfilgrastim was administered as a single injection of 30, 60, 100, or 300 μg/kg, and blood samples were taken at specific time points for 48 h. Neutrophil counts increased in a dose-dependent manner, and the peak neutrophil count attained and the length of the response were dose dependent.

Patients with Disease

Healthy Volunteer Studies Table 3 summarizes pharmacokinetic and pharmacodynamic data from healthy volunteer studies. Healthy men given single doses of 3.45 μg/kg filgrastim by 30-min intravenous (IV) infusion had a mean serum concentration of 20.8 ng/ml 5 min after the end of infusion [3]. The mean (SD) elimination half-life was 163 (±7.4) min, and G-CSF concentrations returned to normal values within 14–18 h. After single subcutaneous (SC) injections of lenograstim 10, 20, or 40 μg in healthy volunteers, Cmax values were 0.09, 0.18, and 0.48 μg/L, respectively, within 3.5–4.5 h, peak serum concentrations were maintained for almost 4 h, and lenograstim was almost eliminated from the serum by 48 h [81]. Lenograstim exhibits dose-dependent pharmacokinetic characteristics with peak serum concentrations after repeated doses (SC or IV) that are proportional to the administered dose [13]. Lenograstim does not appear to accumulate after repeated

Patients with cancer receiving filgrastim 11.5 μg/kg as a 30-min IV infusion had a peak serum concentration of 384 ng/ml [26, 27]. When patients with cancer received SC bolus or SC infusion doses, serum concentrations of filgrastim reflected rapid absorption [60]. The serum halflife of filgrastim has a t1/2 of 3.5 h [48]. With filgrastim, the maximum increase in neutrophil count can be achieved by all routes of administration tested [60, 80]. Patients with nonmyelogenous malignancies receiving lenograstim at 5, 10, or 20 μg/kg/day as a SC injection had peak plasma concentrations of 10.1, 35.0, and 49.7 mg/l, respectively [25]. Cmax was reached within 4–8 h. The serum half-life of lenograstim was 3.0, 4.8, and 6.0 h, respectively, for the 5-, 10-, and 20-μg/kg per day cohorts. Results of a randomized, dose-escalation study with pegfilgrastim in patients with nonsmall cell lung cancer receiving chemotherapy were published [39]. Thirteen patients were randomly assigned to receive daily injections

Table 3. Summary of pharmacokinetic/pharmacodynamic data in normal volunteers. Pharmacokinetic data are difficult to compare directly because of differences in study designs, doses, and populations Generic Name

Cmax

tmax (h)

AUC

Filgrastim (75 μg/kg dose) Lenograstimb (5 μg/kg dose) Nartograstimc (2 μg/kg dose) Pegfilgrastima (30 μg/kg dose) Ro 25–8315d

1.65±0.80 ng/ml 10.3–11.9 mg/L 1.52±0.58* 43.6±20.0 ng/ml 2.5±1.6 ng/ml

5.5±1.8 6.0 4.0±2.0 9.50±3.51 13±5.9

14.3±4.3 (0–24 h, ng/L×h) 89.8–102.3 (0–24 h, mg/L×h) 12.7±4.78 ng×hr/ml 887±336 (0–infinity, ng/L×h) 184±77 ng × h/ml

a

Cmax, peak serum concentration; tmax, time to Cmax; AUC, area under the serum drug concentration curve versus time curve * Units not provided a Amgen data on file; bDunn and Goa [24]; cSuzuki et al. [92]; dVan der Auwera et al. [97]

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of filgrastim 5 μg/kg per day or a single injection of pegfilgrastim at 30, 100, or 300 μg, 2 weeks before chemotherapy and 24 h after completion of chemotherapy. Peak serum concentrations of filgrastim and the duration of increased serum concentrations were dose dependent. The concentration of serum filgrastim remained high for a longer duration in the setting of chemotherapy-induced neutropenia until neutrophil recovery occurred. Patients treated with pegfilgrastim had a higher prechemotherapy neutrophil count than patients treated with filgrastim (who did not receive the study drug until after chemotherapy was completed). After chemotherapy, the neutrophil count nadirs were similar among patients receiving filgrastim 5 μg/kg per day or pegfilgrastim 30 μg; higher neutrophil count nadirs were seen in patients receiving the higher doses of pegfilgrastim.

Pharmacodynamics The exposure of filgrastim may be influenced by the increased neutrophils formed after administration of the cytokine [80]. Increased numbers of neutrophils were shown to be associated with increased clearance of filgrastim in patients with cancer, which suggests that a negative feedback mechanism is involved in maintaining neutrophil counts at optimal values [45]. The bioavailability of filgrastim was an average of 53% when administered at very low doses (1 μg/kg) to healthy volunteers [91]; when administered at therapeutic doses, its bioavailability has been reported as high as 80% [80]. Lenograstim has an absolute bioavailability of 30% after SC doses of 2–5 μg/kg, with an apparent volume of distribution of 1 L/kg [13]. The serum elimination halflife after SC administration is approximately 3 h, but it is shorter after repeated IV infusion (1–1.5 h) [38]. The absolute bioavailability of pegfilgrastim after SC administration is estimated to be approximately 15% [31]. The exposure of pegfilgrastim is affected by the neutrophil status; pegfilgrastim concentrations for patients who experienced severe neutropenia were sustained longer and higher than those who did not experience severe neutropenia. Based on pharmacokinetic/pharmacodynamic modeling, clearance of pegfilgrastim is almost completely (>99%) regulated by neutrophil-mediated clearance. While renal clearance plays a significant role in the elimination of filgrastim, it is absent for pegfilgrastim, based on results from a bilateral nephrectomy rat study [58].The predominant dependency of pegfilgrastim clearance on the neutrophil-mediated pathway allows pegfilgrastim to be under highly efficient ‘self-regulation’.

Clinical Implications Much clinical data have been published about the various forms of rHuG-CSF and their use in numerous therapeutic areas. To cite all the important references in this review paper is impossible, and we have concentrated on the pivotal phase 3 trials used in obtaining marketing authorization whenever possible. The interested reader is directed to the cited reviews [21, 49, 59, 88, 101].

Chemotherapy-induced Neutropenia Neutropenia and infection are common dose-limiting effects of cancer chemotherapy. It has long been known that the risk of infection is directly related to the depth and duration of neutropenia [6]. The severity of chemotherapy-induced neutropenia depends on the dose intensity of the therapy and other factors. Often, oncologists withhold or delay the next cycle of chemotherapy if a patient has low neutrophil counts. Such practice, although done to prevent infectious complications, can compromise otherwise effective cancer chemotherapy. Two randomized, placebo-controlled, double-blind studies involving more than 300 patients with small-cell lung cancer receiving cyclophosphamide, doxorubicin, and etoposide (CAE) chemotherapy showed that filgrastim decreased the incidence, severity, and duration of severe neutropenia [15, 92]. These two studies showed that, in the placebo control group, most of the febrile neutropenic events occurred in the first cycle. Two randomized placebo-controlled phase 3 trials with lenograstim showed significant reductions in the median duration of neutropenia in patients with inflammatory breast cancer [12] or non-Hodgkin’s lymphoma [29]. The median neutrophil nadir was higher in lenograstim-treated patients who had inflammatory breast cancer than in patients administered placebo. Treatment with lenograstim was associated with reductions in culture-confirmed infections during periods of neutropenia, shorter durations of hospitalization for infections, and reduced need for antibacterial drugs. In two double-blind randomized studies, use of pegfilgrastim reduced neutropenia and its complications in patients receiving chemotherapy [31, 37]. In one study, patients with high-risk stage II or stage III/IV breast cancer received a single 6-mg injection of pegfilgrastim or daily 5 μg/kg SC injections of filgrastim for up to four cycles of chemotherapy [31]. The single injection of pegfilgrastim was as effective as a mean of 11 daily injections of filgrastim with respect to the observed mean duration of grade 4 neutropenia. The incidence of febrile neutropenia was lower, but not statistically significantly,

M. Foote and G. Morstyn in the pegfilgrastim group compared with the filgrastim group. In the second study, patients received either 100 μg/kg pegfilgrastim once per cycle of chemotherapy or daily injections of 5 μg/kg filgrastim [37]. Again, single doses of pegfilgrastim were comparable to multiple doses of filgrastim in terms of duration of grade 4 neutropenia and depth of neutrophil nadir counts in all cycles. In both studies, pegfilgrastim and filgrastim were well tolerated. Since these pivotal randomized clinical trials were completed, additional studies were conducted. This has led to major organizations such as ASCO, EORTC, NCCN, and ESMO evaluating these data and producing guidelines. These data have been reviewed by an expert panel in Europe [32]. The ASCO evaluation was published in 2006. There is agreement in all studies that rhuG-CSF reduces the rate of febrile neutropenia by 50% what ever the background febrile neutropenia rate. Much discussion, however, ensued about the high background febrile neutropenia rate in the initial registration trials and whether the use of rHuG-CSF was appropriate in settings with a lower febrile neutropenia rate. One issue in the early studies was the frequent measures of neutrophil counts and temperature led to the identification of neutropenia that might have been missed if blood counts were performed less frequently. Nevertheless, recent studies were performed in settings in which the febrile neutropenia rate was approximately 20% and so current guidelines recommend the use of CSFs in settings in which the risk of febrile neutropenia is greater than 20%. The existence of risk factors such as age, treatment intent such as dose intensity, and prior toxicity from chemotherapy may increase risk.

Bone Marrow or Stem Cell Transplantation Bone marrow or stem cell transplantation allows the use of very high doses of chemotherapy, with or without radiotherapy, to eliminate cancer cells from patients with refractory tumors. After ablative therapy and before the patient’s bone marrow recovers full function, the patient often experiences profound pancytopenia that requires multiple transfusions of blood and blood products. In the pivotal phase 3 trial for filgrastim in the highdose chemotherapy and autologous bone marrow transplantation setting, patients were administered filgrastim 10 or 30 μg by continuous IV infusion or placebo for 5 days, starting 1 day after bone marrow infusion [87]. The dose of filgrastim was adjusted as needed during neutrophil recovery for a scheduled maximum of 28 days of administration. The median number of days of

575 neutropenia (neutrophil count <0.5 × 109/L) was 23 days for the control group, 11 days for the 10-μg/kg per day group, and 14 days for the 30-μg/kg per day group. For the control group, compared with both treatment groups combined, the difference in the number of days of neutropenia was statistically significant. Moreover, filgrastim reduced the duration of fever and febrile neutropenia. A phase 3 trial of lenograstim was performed in 298 patients receiving either autologous or allogeneic bone marrow transplantation [30]. The addition of lenograstim 5 μg/kg per day produced 30% reduction relative to placebo in the median time to reach neutrophil counts of 0.5 × 109/L. Other significant reductions compared with placebo were seen in lenograstim-treated patients for median time in hospital, duration of antibiotics, and duration of parenteral nutrition. The incidence of infections, febrile neutropenia, and septicemia were similar between the two groups, but the median duration of infection and febrile neutropenia were reduced with lenograstim. In another study with lenograstim [51], reduction of neutropenia was associated with a reduction in the number of days of fever and a shorter duration of parenteral antibiotic use after transplantation. Recombinant HuG-CSF alone or in combination with chemotherapy is an effective agent for recruiting peripheral blood progenitor cells (PBPC) with long-term reconstituting ability [11, 22, 41, 82, 83]. In a historically controlled study, filgrastim-generated PBPC, in conjunction with autologous bone marrow transplantation and daily filgrastim, accelerated recovery of neutrophil and platelet count [82]. In a study of 85 patients with relapsed Hodgkin’s disease [11], use of filgrastim for mobilization resulted in an accelerated time to recovery of granulocytes compared with nonmobilized PBPC recipients. The use of mobilized PBPC resulted in an accelerated time to platelet engraftment compared with nonmobilized PBPC recipients. Costs were lower in patients who received filgrastim-mobilized PBPC compared with patients who did not. Use of lenograstim for PBPC mobilization has primarily been reported in abstract form. Patients with lymphoma previously treated with lenograstim had a median time to neutrophil recovery of 11–12 days after chemotherapy and infusion of lenograstim-assisted autologous PBPC transplantation [99]. A comparison of lenograstim and filgrastim administered after chemotherapy to increase PBPC yield was reported as a 126-patient retrospective study [46]. No differences were observed between groups for median CD34 cells harvested or number of leukophereses needed to obtain the 3 × 106 CD34 cells/kg required for transplantation.

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Severe Chronic Neutropenia A phase 3 trial of filgrastim in patients with severe chronic neutropenia has shown long-term efficacy in boosting neutrophil counts, with hematologic and clinical benefits sustained during prolonged maintenance treatment [16, 17]. Of the 120 patients who received filgrastim, 108 had a median neutrophil count of >1.5 × 109/L during the study. Their bone marrow had increased proportions of maturing neutrophils. The incidence and duration of infection-related events was reduced by approximately 50% and the duration of antibiotic use was reduced by 70%. No phase 3 studies of lenograstim in patients with severe chronic neutropenia have been reported, but several small groups of patients reported have been treated with lenograstim [21]. One European phase 2 study reported that all 19 children treated with lenograstim had neutrophil recovery to >1.0 × 109/L [20]. Normal bone marrow cytology was attained by ten of the 19 children.

Bone Marrow Failure States Both filgrastim and lenograstim are licensed in Japan and China for the treatment of aplastic anemia [77]. Studies suggest that rHuG-CSF increases neutrophil counts in some patients with moderate aplastic anemia; however, patients with very severe hypoplasia generally do not respond to growth factors [5, 42]. The European Group for Bone Marrow Transplantation (EBMT) Working Party discourages the use of rHuG-CSF as a single agent in patients with newly diagnosed aplastic anemia [4]. Although published data do not fully support the use of growth factor as first-line treatment for aplastic anemia, rHuG-CSF in combination with other approaches for this disorder (e.g., with immunosuppressive therapy [76]) may have a role. The myelodysplastic syndromes are a group of neoplastic hematopoietic disorders. In a phase 3 randomized study involving 102 patients with RAEB or RAEB-t subtypes of myelodysplastic syndromes, filgrastim was efficacious in increasing neutrophil counts [65], although imbalances in patient characteristics made the overall benefit, if any, of the use of rHuG-CSF in this setting difficult to define. In a phase 2 study to demonstrate the efficacy of lenograstim, most patients responded to IV doses of 2 or 5 μg/kg per day [104]. Administration of lenograstim increased neutrophil counts in 18 Japanese patients and no patient progressed to acute myeloid leukemia.

AIDS/HIV Infection Filgrastim has been approved in Australia, Canada, the European Union, and Japan for use in patients with HIV

infection, for reversal of clinically significant neutropenia, and for maintenance of adequate neutrophil counts during treatment with antiretroviral therapy. A phase 3 study reported the effect of filgrastim on the incidence of severe neutropenia in patients with advanced HIV infection and its effect on the prevention of infectious morbidity [44]. In this 24-week study of 258 patients, filgrastim was administered daily at 1 μg/kg and adjusted to as much as 10 μg/kg, or was administered intermittently at 300 μg daily 1–3 days per week. Patients in a control group received filgrastim only if they developed severe neutropenia. Both daily and intermittent administration of filgrastim lowered the incidence of bacterial infection rates compared with patients in the control group. The filgrastim-treated patients had 31% fewer bacterial infections than did the control patients, suggesting that the use of filgrastim can reduce the risk of several bacterial infections and subsequent number of days of hospitalization. No phase 3 studies with lenograstim in the setting of AIDS/HIV infection appear to have been published as full reports. One study using lenograstim in the setting of AIDS showed that lenograstim could safely ameliorate the neutropenia associated with zidovudine treatment in patients with AIDS or AIDS-related complex [95].

Current Issues Although both filgrastim and lenograstim have been marketed for more than a decade and pegfilgrastim was approved in 2002, and are licensed for many uses in countries around the world, work continues to fully understand the mechanisms of action and long-term results of chronic administration and to discover other conditions and disease states where rHuG-CSF may be useful. One issue that may not be fully resolved in the perceived relationship between use of hematopoietic growth factors in several patient populations and the risk of developing leukemia [34]. It is not clear if the slightly increased risk of developing leukemia is due to the underlying disease concurrent with chemotherapy or if the use of a growth factor increases the risk. Surveillance is on going and the benefits appear to outweigh any potential risks.

Immunomodulation Immune reconstitution depends upon hematopoietic reconstitution as the first step, and hematopoietic reconstitution, of course, depends on the fine actions and interactions of hematopoietic growth factors. Hematopoietic growth factors, including G-CSF, may be important modulators of immune reconstitution because of their ability to increase the number of leukocytes, recruit

M. Foote and G. Morstyn immature leukocytes, cause maturation and augmentation of leukocytes, and suppress leukocytes [97].

Use in Patients with Sickle-cell Anemia Increases in leukocyte counts may be associated with worsened prognosis in patients with sickle-cell anemia. A study of 3,764 patients with sickle-cell disease, ranging in age from birth to 66 years of age, was done to determine the life expectancy, median age at death, and circumstances of death [75]. Patients with sickle-cell anemia who had hemoglobin values <7.1 g/dL (below the tenth percentile) had a slightly higher risk of death than those patients with increased white blood cell counts (>1.5 × 109/L).

Conclusions The discovery, isolation, and cloning of G-CSF and the subsequent production of rHuG-CSF have had profound effects on the practice of medicine, particularly oncology. The use of rHuG-CSF has allowed increased administration of cytodoxic drugs, thereby allowing increased efficacy of chemotherapy and eliminating the dose-limiting toxicity of myelosuppression; however, the treatment of many diseases, including nonmalignant disease and non-neutropenic infections, also has been altered by the use of rHuG-CSF. Although rHuG-CSFs have been commercially available for more than 2 decades, research continues to find new formulations that improve patient compliance or that have enhanced biologic activity.

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18 Granulocyte-macrophage colony-stimulating factor MARYANN FOOTE AND GEORGE MORSTYN

Introduction Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the colony-stimulating factors whose name is derived from its major target cell lineages. Since the original studies, which characterized its ability to stimulate the clonal proliferation of myeloid precursors, however, a broader range of biologic effects on mature, effector cells, and the immune system, have been identified. These expanded areas of activity have carried its more recent clinical application beyond that of ‘colony-stimulating factor’ into the realm of immunomodulation. GM-CSF was initially purified from media used to culture an HTLV-II-infected, T lymphoblastoid cell line (Mo) [5], but was subsequently found to be produced by a variety of cell types including macrophages, endothelial cells, and certain mesenchymal cells. Although basal serum concentrations of GM-CSF are often not measurable in normal adults, its elaboration is readily inducible in ‘producer’ cells by immune stimuli and many of the mediators of inflammation. In 1985, GM-CSF was the first myeloid hematopoietic growth factor, to have its gene sequenced and cloned [20, 92]. GM-CSF is used for the native form of the molecule or when it is not necessary to distinguish a specific form of the recombinant molecule, which is referred to as rHuGM-CSF.

Biochemistry and Structure The gene for GM-CSF has been mapped to the long arm of chromosome 5 (5q21–32) [51] in the cluster region of genes for other growth factor proteins and their cell-surface receptors, such as interleukin-3 (IL-3) [56], IL-4 [93], IL-5 [83], macrophage colony-stimulating factor and its receptor (the c-fms proto-oncogene product) [70], and the receptor for platelet-derived growth factor [93]. The gene for GM-CSF encodes a protein of 127 amino acids whose molecular weight varies from l4 to

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

35,000 daltons depending upon the degree of glycosylation. The gene has been expressed using recombinant DNA technology in bacteria (Escherischia coli), Chinese hamster ovary (CHO) cells, and yeast (Saccharomyces cerevisiae) resulting in the availability of large amounts of glycosylated and nonglycosylated material for clinical trials. Small differences in the amino acid sequence, as well as major differences in the degree of glycosylation, can be found among the preparations. The growth factor produced by expression of the cDNA in bacteria is not glycosylated [16], but that expressed in mammalian cells and yeast demonstrates variable degrees of glycosylation [20]. Both the glycosylated and nonglycosylated forms are active in vitro or in vivo, and have similar activity to the natural form of the protein, but the degree of glycosylation plays a role in inducing antibody formation, as well as altering some specific biologic activities and toxicities.

Physiology Both endogenous and recombinant GM-CSF generate predominantly granulocyte and monocyte colonies in semisolid culture systems. It acts as a potent stimulus for the growth of uncommitted and committed bone marrow progenitors including colony-forming unit–granulocyte, erythrocyte, monocyte, and megakaryocyte (CFU– GEMM) [61, 77]. It is a potent inducer of granulocytemonocyte differentiation, and potentiates the growth of human blast-forming units–erythroid (BFU-E) in the presence of erythropoietin in vitro. At the level of mature cells, GM-CSF enhances the function of neutrophilic and eosinophilic granulocytes, and of monocyte/macrophages. Exposure of neutrophils to GM-CSF produces increased expression of cellular adhesion molecules [5, 23, 41] such as CDllb, increased expression of class II MHC molecules [38, 63], increased number of FMLP receptors [88], and increased fMLPinduced superoxide production [87], chemotaxis, antibody dependent cellular cytotoxicity (ADCC), and phagocytosis. In vitro, it was reported to inhibit random

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582 neutrophil granulocyte migration (8,334) and in vivo, when administered by continuous infusion, it prevented neutrophils from migrating into areas of inflammation [69]. GM-CSF prolongs the survival of neutrophilic and eosinophilic granulocytes in vitro [60] by inhibiting programmed cell death [11], and enhances microbicidal activity and their leukotriene synthesis [79]. Exposure of macrophages to GM-CSF increases expression of cell-surface adhesion molecules such as CD11a, b, and c; and Fc RII (CDw32) receptors involved in phagocytosis [72, 91]. In vitro, it enhances ADCC, phagocytosis, microbicidal and tumoricidal activity [39, 44, 89], and the synthesis and secretion of other cytokines including those related to inflammation such as tumor necrosis factor (TNF) and IL-1. GM-CSF exerts its biologic activity by binding to specific transmembrane surface receptors, which are subsequently internalized, on the target cells [24, 33]. These receptors have been detected on mature neutrophils, monocytes/macrophages, some lymphocytes, normal bone marrow progenitors, dendritic cells (DC), fibroblasts, endometrial and endothelial cells, fresh leukemic cells, and leukemic cell lines [13, 14,18, 66]. GM-CSF itself can downregulate these receptors on cells such as neutrophils, monocytes, and normal bone marrow myeloid cells in vitro [18]. Crosslinking experiments have revealed molecular weights of 75–156 kD for the receptors. At least two types of GM-CSF receptors have been identified: one with high affinity (20–100 pmol/L) and one with low affinity (1 nmol/L), but as many as four types may exist on AML cells [13]. Two subunits (alpha and beta) comprise the heterodimer receptor and both have been cloned [46]. The alpha subunit is specific to the GM-CSF receptor. At least two distinct regions within the cytoplasmic domain of the common beta subunit have been shown to be responsible for different signals [74]. A membrane proximal region of approximately 60 amino acids has been shown to be essential for induction of c-myc and activation of DNA replication, which involves the tyrosine phosphorylation of a Janus kinase (JAK2) and activation of its in vitro kinase activity [71]. A second, distal region of about 140 amino acids is required for activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, and induction of c-fos and c-jun [74].

Pharmacology Both glycosylated and nonglycosylated forms of rHuGM-CSF have been studied clinically. Glycosylated rHuGM-CSF (sargramostim), produced in yeast, is available in the United States. Molgramostim, which is

Granulocyte-macrophage colony-stimulating factor not glycosylated, is available in Europe and other countries.

Pharmacodynamics and Pharmacokinetics Several early pharmacodynamic studies defined the pharmacology of rHuGM-CSF [58]. Radiolabeling of leukocytes suggested that a transient leukopenia is due to sequestration of the cells within the lung. In vitro and in vivo studies have suggested that this effect may be due to a change in cell surface adhesion molecules on the leukocytes such as those identified by CD11b [5, 80], which is upregulated on neutrophils, as well as LAM-1, which is downregulated, and adherence to endothelial cells of the blood vessels [41]. The major effect of rhGM-CSF on hematopoiesis is the leukocytosis that was reported to be dose dependent in most of the early studies [58] when either glycosylated or nonglycosylated rHuGM-CSF was administered daily subcutaneously (SC) or by prolonged intravenous (IV) or SC infusions. The pattern of the leukocytosis in many of these trials appeared as a biphasic response: an initial increase that frequently plateaued during the first few days of treatment, followed by a decline. With continued dosing, however, another increase in leukocytes, which often contained more immature myeloid cells such as myelocytes, promyelocytes, and myeloblasts, was observed. Appearance of monocytes and eosinophils generally occurred later in the course of treatment and at higher doses of rHuGM-CSF. Morphologic changes in neutrophils included toxic granulation with an increase in leukocyte alkaline phosphatase, prominent Döhle bodies, cytoplasmic vacuolization and, at higher doses, increasing numbers of hypersegmented neutrophils. At the higher doses of rHuGM-CSF in patients with normal marrows, the cellularity of the bone marrow was increased with a left-shift and an increase in the myeloid-to-erythroid ratio [50, 58]. An important observation made during these early trials, which later found significant clinical application, was the dose-dependent increase in peripheral blood erythroid and myeloid progenitors [59]. Bone marrow progenitors were found to be unchanged or decreased, probably somewhat dependent upon the degree of expansion of the non-colony forming myeloid mononuclear cells [62]. Pharmacokinetic studies were conducted using both glycosylated and nonglycosylated rHuGM-CSF by radioimmunoassay and by bioassay. Route of administration affected the peak serum concentration, area under the concentration-time curve, and the time during which GM-CSF was detectable in the serum.

Maryann Foote and George Morstyn

Clinical Implications Much clinical data have been published about the various forms of rHuGM-CSF and their uses in many therapeutic areas. It is impossible to cite all the important papers, and we have concentrated on reports of pivotal studies.

Chemotherapy-induced Neutropenia Many clinical trials have studied the ability of rHuGMCSF to abrogate the hematologic toxicity of chemotherapy (both standard and dose-intensified) and bone marrow/stem cell transplantation. Most of the early studies were not randomized studies, included relatively small numbers of patients, compared the results of cycles of chemotherapy given without growth factor to cycles given with growth factor, and cycles of chemotherapy given with growth factor compared with data of historical controls. In most studies, rHuGM-CSF did not completely eliminate profound leukopenia. In general, the neutrophil nadir occurred earlier in the courses of chemotherapy in which rHuGM-CSF was administered than in the courses without. Higher doses of the growth factor in some studies decreased the depth of the absolute neutrophil count (ANC) nadir, and frequently the duration of neutropenia and time to an ANC > 1.0 × 109/L was shortened at during the first cycle of chemotherapy administered with rHuGM-CSF. The results with respect to the ability to reduce the morbidity (hematologic, and in some cases the incidence of infection and mucositis) of dose intensification was variable. Furthermore, even in the larger randomized trials [6, 15, 35, 53], efficacy was variable. In most cases, duration of neutropenia was reduced at least during the first cycle; however, persistence of this effect on subsequent cycles, and impact on infections was much more variable.

Bone Marrow and Stem Cell Transplantation One of the areas of more intense research has been derived from the ability to mobilize peripheral blood stem cells (PBPC). Although it is difficult to collect PBPC from a patient who is in a hematologic steady-state, the number of circulating stem cells increases during blood cell count recovery after treatment with chemotherapy, especially after cyclophosphamide. The early trials showed that CSF alone could increase the number of circulating PBPC [80], and when administered after chemotherapy the number was enhanced even further [78]. The PBPC harvested by leukopheresis during rHuGMCSF priming without chemotherapy or while a patient is receiving growth factor to abrogate the myelosuppression

583 of chemotherapy have been used to supplement bone marrow after autologous bone marrow transplantation [36]; to support dose-intensified chemotherapy when growth factor alone is not adequate [76]; and when autologous bone marrow alone was used for hematopoietic support [27, 43]. Although the early trials were conducted primarily in patients undergoing autologous transplant, the technique has been applied to normal donors and for allogeneic stem cell transplant harvesting. PBPC were initially used in combination with autologous bone marrow, and were found to reduce the time to hematologic reconstitution [12, 36, 68]. Durable reconstitution was demonstrated using PBPC alone [37, 68, 76]. CSF-mobilized PBPC are preferred because of the ability to harvest great numbers of PBPC, the simplicity of harvesting, the ability to harvest from patients with a history of pelvic irradiation and/or other causes of poor bone marrow reserve, and the rapidity of engraftment [9]. For autologous, allogeneic, and syngeneic transplantation, rHuGM-CSF has been administered after the conditioning regimen and reinfusion of bone marrow and/or PBPC to shorten the duration and reduce the severity of neutropenia, and thus decrease the requirement for supportive care, and the duration of hospitalization [10, 55, 64, 65].

Bone Marrow Failure States GM-CSF has multilineage activity, in vitro and somewhat more variably in vivo. It can also enhance effector cell function, and induces differentiation of some leukemic cell lines. Based on these activities, it was thought to be a natural candidate to study as therapy for patients with myelodysplastic syndromes (MDS), who frequently have bi- and tri-lineage cytopenias, as well as dysfunctional granulocytes. The focus of most of these studies was improvement in blood counts, and determination if myeloblasts were stimulated [3, 26, 32, 40, 49, 84, 85]. A wide range of doses were administered by a variety of dosing schedules for all of the preparations of rHuGMCSF. The reported studies, for the most part, have included relatively small numbers of patients. Their disease status covered the spectrum from refractory anemia (RA) to the poorer prognosis refractory anemia with excess of blasts (RAEB), refractory anemia with excess of blasts in transformation (RAEB-t), and chronic myelomonocytic leukemia (CMMoL). In general, these studies reported comparable increases in neutrophil counts but no consistent improvement in the other hematopoietic cell lines [29]. An increase in bone marrow and peripheral blood myeloblasts and conversion to acute myelogenous leukemia (AML) occurred more often in patients with RAEB and RAEB-t, but in many cases, the number

584 of myeloblasts decreased and/or returned to baseline upon discontinuation of the growth factor. In a number of patients, the drug was discontinued due to progression of disease or to side effects. Several investigators tried to circumvent the issues of toxicity and myeloblast stimulation by manipulating the dosing of the GM-CSF [54].

Acute Leukemia Use of hematopoietic growth factors has taken a number of approaches in patients with acute leukemia: for facilitating recovery from the myelosuppressive effects of induction and/or consolidation chemotherapy to reduce morbidity; as a means of sensitizing myeloid leukemic cells to chemotherapy by recruitment of these cells into S-phase followed by treatment with a cycle-specific drug, for harvesting PBPC (after intensive chemotherapy) that may be used for stem cell support after bone marrow ablation; and as differentiation/maturation therapy in myeloid leukemia. There has been appropriate concern about possibly stimulating the leukemic population, especially in AML, with rHuGM-CSF since such activity has been reported in vitro [42, 62, 86]. Sargramostim showed reduction in early deaths due to fungal infections and reduction in neutropenia in a small randomized trial that led to its registration in this setting. The second approach taken in the use of myeloid growth factors in patients with AML has been an attempt to cycle activate leukemic cells, thus sensitizing them to cycle-specific chemotherapy, such as cytosine arabinoside, and potentially improving remission rates [4, 8, 17, 25, 30, 47]. None of the studies showed an absolute change >15% in the S-phase fraction of cells during treatment with rHuGM-CSF; in general the percent change was small. The use of rHuGM-CSF in acute lymphoblastic leukemia (ALL) has been less controversial, but even fewer studies have been reported. The complex nature of most ALL chemotherapy regimens, which include frequent dosing, and the difficulty in determining appropriate growth factor dosing, has undoubtedly contributed to the limited number of clinical trials.

Current Issues Use in Children Although G- and GM-CSF were placed into clinical trials in the late 1980s and approved by the FDA in 1991, most clinical trials have been conducted in adult patients. As is commonly the case with new agents, the evaluation

Granulocyte-macrophage colony-stimulating factor of these agents in the pediatric population was extremely limited by concerns about their long-term effects. One of the earliest areas of investigation for myeloid growth factors in pediatric patients was chronic severe neutropenia [31, 90]. Because of the limited numbers of studies using these agents, that permitted enrollment of children, the safety and efficacy was not established until considerably later than when the same was defined for adults. In pediatric oncology, these agents were used as adjuncts to chemotherapy and radiation to facilitate engraftment after transplantation, and to mobilize PBPC. Guidelines for their use in children were not available until a European expert panel convened to define them and finally published their recommendations in 1998 [75]. Until that time, guidelines published by ASCO were extended to children, despite the lack of clinical trials to support those recommendations [1, 2]. The ASCO guidelines for clinical use of CSFs: 2000 update made no significant changes to its previous recommendations for their use in the pediatric population [67]. Clinical trials using G- and GM-CSF as mobilizing agents for PBPC have demonstrated efficacy for collecting autologous stem cells that can reduce the period of neutropenia compared with bone marrow. Based on such studies, the European guidelines recommended the routine use of CSF in children for this purpose. On the other hand, despite the now common use of CSF to mobilize PBPC from normal donors in the adult population, there has been significant hesitation in extending that practice to the pediatric population. The limited information available regarding the long-term effects of CSF in normal pediatric subjects resulted in the statement by the European panel that such use was contraindicated.

Immunomodulation GM-CSF has a broad spectrum of activities that can contribute to the immune response in humans. Early in the studies characterizing this protein, it was recognized that GM-CSF could induce production of other cytokines and growth factors such as IL-1, IL-6, and tumor necrosis factor (TNF) that also contribute to the expansion of B and T cells, as well as induce or coinduce TNF-α gene expression with interferon (IFN)-γ in monocytes [19, 45] and in conjunction with IL-2, costimulates T-cell proliferation [73]. As the biology of DC has been better defined, it has become evident that in vitro and possibly in vivo, GM-CSF is a cofactor in the differentiation and functional activities of these cells. DC are antigen-presenting cells that have a critical role in T-cell immune responses

Maryann Foote and George Morstyn [7, 11, 15, 82]. DC can be differentiated from CD34+ hematopoietic progenitor cells in human bone marrow, peripheral blood, and cord blood [21, 22] when cultured with GM-CSF plus TNF. They can be cultured from monocytes using GM-CSF plus IL-4. Antigens are presented on their surface in association with class II MHC molecules, to be recognized by T4 helper cells. The CD4+ cells participate in the development of B cells, antigen-specific cytotoxic T8 cells; and macrophages, eosinophils, and natural killer (NK) cells, which are antigen nonspecific [7]. All these cells may be involved in an immune response. In addition to its activity on the function of DC, GM-CSF increases expression of class II MHC molecules on the DC, which are essential to recognition. It is an enhancer of costimulatory molecule expression – such as B7 and adhesion molecules like ICAM that are necessary for the interaction of antigen presenting cells and T cells.

Vaccine Therapy Since the mid 1990s, many nonclinical and clinical studies have used genetic engineering techniques to harness the immunomodulatory activity of GM-CSF to augment antitumor immunity. Using tumor cell lines or autologous tumor cells genetically modified to produce GM-CSF, investigators have shown that this immunostimulatory protein can enhance tumor immunogenicity in a variety of tumors. Replicating tumor cells are transduced with viral supernatants harvested from CRIP packaging cell lines transfected with MFG-S-human GM-CSF. They are irradiated and administered as vaccine to tumorcontaining animals or patients. Delayed-type hypersensitivity skin responses were demonstrated against irradiated autologous cancer cells after vaccination [52]. Biopsies of vaccine sites showed recruitment of DC, T cells, and eosinophils [48, 81], regression of metastatic lesions was observed in some studies [28, 52, 57] and prolongation of survival [25, 48].

Conclusions rHuGM-CSF has found limited adoption for the prevention of neutropenia and infections in the induction therapy of acute myeloid leukemia. It has been used for the mobilization of progenitor cells for PBPC transplantation. Its effects on other cells of the immune system acting as a vaccine adjuvant and in DC therapy are not approved by the FDA for clinical use. In the future, rHuGM-CSF in these settings may have great utility.

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587 64. Nemunaitis J, Singer JW, Buckner CD, et al. Use of recombinant human granulocyte-macrophage colony-stimulating factor in autologous marrow transplantation for lymphoid malignancies. Blood 1988;72:834–836. 65. O’Day SJ, Rabinowe SIN, Neuberg D, et al. A phase II study of continuous infusion recombinant human granulocyte-macrophage colony-stimulating factor as an adjunct to autologous bone marrow transplantation for patients with non-Hodgkin’s lymphoma in first remission. Blood 1994;83:2707–2714. 66. Onetto-Pothier N, Aumont N, Haman A, et al. Characterization of granulocyte-macrophage colony-stimulating factor receptor on the blast cells of acute myeloblastic leukemia. Blood 1990;75:59–66. 67. Ozer H, Armitage JO, Bennett CL, et al. Update of recommendations for the use of hematopoietic colony-stimulating factors: Evidence-based, clinical practice guidelines. J Clin Oncol 2000;18:3558–85. 68. Peters WP, Rosner G, Ross M, et al. Comparative effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) on priming peripheral blood progenitor cells for use with autologous bone marrow after high-dose chemotherapy. Blood 1993;81:1709–1719. 69. Peters WP, Stuart A, Affronn ML, et al. Neutrophil migration is defective during recombinant human granulocyte-macrophage colony-stimulating factor infusion after autologous bone marrow transplantation in humans. Blood 1988;72:1310–1315. 70. Pettenati MJ, Le Beau MM, Lemons RS, et al. Assignment of CSF-1 to 5q33.1: Evidence for the clustering of genes regulating hematopoiesis and for their involvement in the deletion of the long arm of chromosome 5 in myeloid disorders. Proc Natl Acad Sci USA 1987;84:2970–2974. 71. Quelle FW, Sato N, Witthuhn BA, et al. JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colonystimulating factor, and its activation requires the membrane-proximal region. Mol Cell Biol 1994;14:4335–4341. 72. Ross AA, Cooper BW, Lazarus HM, et al. Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques. Blood 1993;82:2605–2610. 73. Santoli D, Clark SC, Kreider BL, Maslin PA, Rovera G. Amplification of IL-2-driven T cell proliferative by recombinant human IL-3 and granulocyte-macrophage colony-stimulating factor. J Immunol 1988;141:519–526. 74. Sato N, Sakamaki K, Terada N, Arai K, Miyajimi A. Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling. EMBO J 1993;12:4181–4189. 75. Schaison G, Eden OB, Henze G, et al. Recommendations on the use of colony-stimulating factors in children: conclusions of a European panel. Eur J Pediatr 1998;157:955–966. 76. Shea TC, Mason JR, Storniolo AM, et al. Sequential cycles of high-dose carboplatin administered with recombinant human granulocyte-macrophage colony stimulating factor and repeated infusions of autologous peripheral-blood progenitor cells: A novel and effective method for delivering multiple courses of dose-intensive therapy. J Clin Oncol 1992;10:464–473. 77. Sieff C, Emerson SG, Donahue RE, et al. Human recombinant granulocyte-macrophage colony-stimulating factor: a multi-lineage hematopoietin. Science 1985;230:1171–1173. 78. Siena S, Bregni M, Brando B, et al. Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: Enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1989;74:1905–1914.

588 79. Silberstein DS, Owen WF, Gasson JC, et al. Enhancement of human eosinophil cytotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor. J Immunol 1986;137:3290–3294. 80. Socinski MA, Cannistra SA, Sullivan R, et al. Human granulocytemacrophage colony stimulating factor induces expression of the CD11 b surface adhesion molecule on granulocytes in vivo. Blood 1988;72:691–697. 81. Soiffer R, Lynch T, Mihm M, et al. Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocytemacrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma. Proc Natl Acad Sci USA 1998;95:13141–13146. 82. Steward WP, Scarffe JH, Dirix LY, et al. Granulocyte-macrophage colony stimulating factor (GM-CSF) after high-dose melphalan in patients with advanced colon cancer. Br J Cancer 1990;61: 749–754. 83. Sutherland GR, Baker E, Callen DF, et al. Interleukin-5 is at 5q31 and is deleted in the 5q- syndrome. Blood 1988;71:1150–1152. 84. Thompson JA, Lee DJ, Kidd P, et al. Subcutaneous granulocytemacrophage colony-stimulating factor in patients with myelodysplastic syndromes: Toxicity, pharmacokinetics, and hematologic effects. J Clin Oncol 1989;7:629–637. 85. Vadhan-Raj S, Keating M, LeMaistre A, et al. Effects of recombinant human granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndromes. N Engl J Med 1988;317: 1545–1551.

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19 Cancer gene therapy DONALD J. BUCHSBAUM, C. RYAN MILLER, LACEY R. MCNALLY, AND SERGEY A. KALIBEROV

Introduction Cancer is the product of a multi-step process involving an accumulation of genetic alterations in somatic cells [203]. Advances in the understanding of the molecular basis of neoplasia and host–tumor relationships have resulted in new cancer therapy strategies with translation into clinical trials. Gene therapy has emerged as a treatment strategy which relies on the introduction of genetic material to cure, slow the progression, or possibly even prevent a variety of diseases including genetic, infectious (HIV in particular), cardiovascular, and arthritic diseases [11, 66]. Gene therapy has shown potential for the treatment of cancer. A number of cancer gene therapy approaches have been developed based on direct correction of genetic lesions. These “mutation compensation” approaches, designed to correct the molecular lesions underlying neoplastic transformation and progression, have demonstrated efficacy in the context of in vitro and preclinical model systems. Although many therapeutic genes exist an efficient, non-toxic gene delivery system is not currently available. The efficiency and accuracy of gene delivery remain the most significant barriers to the success of cancer gene therapy [200]. However, preclinical studies have demonstrated approaches that may have promise in the clinical setting. The approaches that have been explored include genetic immunopotentiation with cytokine gene transfer and tumor cell vaccination, molecular chemotherapy with prodrug activation by suicide genes or an increase in tumor cell sensitivity to chemotherapy or radiation therapy, protection of bone marrow cells from the toxic effects of chemotherapeutic drugs, inhibition of activated oncogenes by antisense treatment, transfer of tumor suppressor genes, and pro-apoptotic gene therapy. Each of these approaches have been translated into clinical cancer therapy trials [32, 66]. Gene therapy is likely to play a prominent role in the treatment of cancer in the future. The purpose of this article is to provide an update on research in gene therapy for the treatment of cancer. Several excellent recent reviews of cancer gene therapy have been published [32, 48, 64, 66, 105, 168]. In this paper, current cancer

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

gene therapy strategies for the treatment of cancer are summarized in Table 1. However, insufficient information is available to evaluate the therapeutic efficacy of these strategies in most clinical trials. The first clinical gene transfer trial involved the transfer of gene-marked tumor-infiltrating lymphocytes into patients with advanced cancer. There are now approximately 190 active gene therapy protocols which involve thousands of cancer patients [39]. About 50% have been designed to augment an immune response against cancers by vaccine or direct cytokine gene transduction and about 20 protocols involve a drug-sensitization strategy. Approved protocols exist for overcoming multi-drug resistance in breast and ovarian cancer patients receiving myelosuppressive chemotherapy.

Gene Transfer Vectors Vectors are vehicles to carry the genetic material. The vectors for modification of tumor cells or normal tissues should allow definitive therapeutic or preventive interventions. To achieve this end, there is a need for gene delivery vectors capable of efficient and selective gene transfer to tumor cells in vivo. One of the major problems has been a low level of gene transfer in human gene therapy clinical trials. Recent advances in the development of vectors for gene transfer make it likely that gene therapy will have an increasing role in the clinical treatment of cancer. Vectors are being engineered that will target specific tumor cell types. Vector requirements depend on the specific approach and disease. Gene transfer vectors are either nonviral or viral [96]. Viruses are currently the most effective means of gene delivery and can be manipulated to express therapeutic genes or to replicate specifically in certain cells. Viral vectors used in in vivo pre-clinical studies have included retrovirus, adenovirus, adeno-associated virus, and lentivirus. Each has limitations, and studies leading to a better understanding of virus–cell interactions are needed to improve vector technology. Some viruses, such as adenovirus, only result in transient production of the therapeutic protein as they do not incorporate the

589

590

Cancer gene therapy

Table 1. Current strategies for cancer gene therapya Strategy Mutation Compensation Induction of tumor suppressor genes Pro-apoptotic and cell cycle gene therapy Inactivation of oncogenes Intracellular knockout of growth factor receptors Antisense Genetic immunopotentiation Genetic modification of tumor cells Genetic modification of immune effector cells Molecular chemotherapy Suicide gene therapy Chemoprotection of bone marrow Inhibition of angiogenesis Replicative vector oncolysis Chemosensitization and radiosensitization

Specific agent

Reference

P53, Rb, BRCA-1 bax, bak, Fas ligand, caspase-9, p21

[32, 33, 66, 141, 170] [88, 101, 148, 149, 211]

erbB-2 k-ras, c-myc, TGFß

[8, 43, 66] [66]

cytokines, B7, MHC Cytokines

[41, 50, 66, 201, 219] [66, 167]

HSV-tk, CD

[5, 9, 66, 72, 108, 146, 168, 169, 183, 216] [36, 126] [13, 52, 104, 174] [66, 102, 123, 187, 193] [14, 30, 31, 42, 47,51, 66, 70, 89, 110, 117, 143, 151, 154, 169, 180, 181, 216]

MDR flt-1, flk-1, endostatin, angiostatin Adp53, herpes virus bax, bcl-2, erbB-2, p53, cytokines, angiostatin, HSV-tk, CD

a

Reprinted with permission from Mary Ann Liebert, Inc.

Table 2. Characteristics of most commonly used vectorsa Type

Insert size

Genome

Cell division Duration of required expression Advantages

Disadvantages

Retrovirus

5–7 kb

RNA

Yes

Long-term

Insertional mutagenesis

Adenovirus

7–35 kb

DNA

No

Transient

Adenoassociatedvirus Herpes simplex virus

2–4 kb

DNA

No

Long-term

Up to 30 kb

DNA

Yes

Transient

Nonviral vectors No limitation RNA or DNA No

Transient

Potential to integrate into genome of target cell Relatively high transduction efficiency into normal and tumor cells; easy production and at high titers; tropism can be modified High transduction efficiency into muscle and brain High transduction efficiency Repetitive and safe administration feasible

Local tissue inflammation and immune response

Insertional mutagenesis; difficulties with production; do not work in all organs Difficult to obtain long-term gene expression; difficult to target Low efficiency gene transfer

a

Adapted from [66, 105]; Reprinted with permission from Mary Ann Liebert, Inc.

genes into the chromosomes of the target cells [125]. Other vectors, such as adeno-associated viruses, are limited in the size of therapeutic genes that can be incorporated or produce low levels of protein [208]. The mechanisms by which adeno-associated virus integrates into the human genome are unclear, and HIV lentiviral vectors may have biosafety issues. Major limitations of different vector systems include biosafety risk, random integration into the genome, need for cell proliferation, potential for immune responses to the vector, lack of a tissue-specific response, and unknown duration of effect.

The characteristics of the most commonly used vectors used in cancer gene therapy are summarized in Table 2.

Retroviral Vectors Retroviral vectors have been used for many gene transfer protocols because of their ability to produce stable, high level gene expression in many cell types as a consequence of their integration into the target cell genome. There are several limitations of retroviral vectors, including the size of the inserted gene, low titers, difficulty in

Donald J. Buchsbaum et al. large-scale production, and they can only insert their genes into the genome of actively dividing cells. Vectors derived from lentiviral retroviruses can transduce gene expression in both dividing and nondividing cells [96], and have been used for cancer gene therapy in animal models [45]. The issues and assays needed to ensure patient safety with this new vector system are being defined [22, 49, 156]. Efforts are ongoing to increase the infectivity of retroviruses through the development of retroviral vectors which display ligands on their surface for which receptors are present on target cells. Serious adverse events in some clinical trials have highlighted safety concerns when retroviral viral vectors are used for gene transfer [142].

Adenoviral Vectors Adenoviral vectors for gene transfer are adenoviruses (Ad) that have been genetically modified through deletions of the viral genome to create space for insertion of a foreign transgene [96]. Both replication-incompetent and replicative Ad vectors have been used in clinical cancer gene therapy trials. There is no risk for insertion mutagenesis since they do not incorporate into the genome. An entire issue of Human Gene Therapy was devoted to Ad vector safety and toxicity [134]. Ad vectors produce higher levels of gene expression and can be produced in greater quantities than retroviral vectors [96]. Another advantage of Ad vectors is their ability to infect both dividing and nondividing cancer cells. A disadvantage is that Ad vectors tend to be recognized as foreign and therefore elicit host cellular and neutralizing humoral immune responses directed against the viral capsid. These inflammatory reactions limit the efficacy of repetitive administrations of Ad vectors and compromise the persistence of transduced cells in vivo [48]. A solution to overcoming the immune response has been to produce Ad vectors deleted of all Ad gene products. Although gene expression is transient following Ad infection, since the transferred genes do not integrate into the genome, this produces a biosafety advantage. To sustain transgene expression levels, repeat administrations of the Ad vectors are required. Analysis of a variety of primary human tumors has demonstrated deficiencies in the primary receptor for Ad, coxsackie and Ad receptor (CAR), which explains the lack of gene transfer in many human clinical trials. Modification of Ad vectors may reduce expression of endogenous Ad proteins and diminish immune responses to Ad vectors. Second generation Ad vectors have been developed to reduce the host immune response against viral proteins and to increase the duration of therapeutic

591 gene expression [66, 105]. The recognition of CAR deficiency in most human carcinomas has resulted in the generation of Ad vectors capable of CAR independent gene transfer [36, 66]. Strategies that have been developed to achieve this utilize retargeting ligands as well as genetic capsid modifications. This has allowed infection and gene transfer to otherwise refractory tumor cells, and resulted in therapeutic gain in preclinical models. These new vectors are currently being evaluated in human clinical trials [107]. Replication-competent adenovirus vectors, which cause cytolysis as part of their natural life cycle, represent an emerging technology that shows considerable promise as a novel treatment option, particularly for armed vectors which carry therapeutic genes [135]. The use of replication competent viruses is an attractive strategy for tumor therapy because the virus can replicate and spread in situ, exhibiting oncolytic activity through a direct cytopathic effect, and produce higher levels of therapeutic gene expression achieved as a result of adenovirus replication.

Adeno-associated Viruses Adeno-associated viruses (AAV) have received considerable attention for gene transfer studies and cancer gene therapy [95, 197]. AAV are capable of infecting both dividing and nondividing cells, producing stable and efficient integration of viral DNA into the host genome, and achieving long-term gene expression. Only minimal viral gene products are retained in current vectors so that they are not very immunogenic and do not markedly elicit host cytotoxic T-cell immune responses against vector-transduced cells. AAV vectors may be useful for antiangiogenic gene therapy and cytokine gene transfer and in vivo immunization approaches [140, 157]. However, major obstacles in the development of effective recombinant AAV mediated gene therapy are infection specificity and gene targeting.

Herpes Simplex Virus The herpes simplex virus (HSV) has been used to deliver therapeutic genes to some forms of brain cancer and prostate adenocarcinoma [96, 202]. HSV displays a broad host cell range and its cellular receptors, heparan sulfate (HS), herpes virus entry mediator (HVEM), and nectin-1 and 2, are widely expressed on the cell surface of numerous cell types. Herpes viruses have been engineered to replicate selectively in tumors but poorly or not at all in normal tissues. The problems with HSVmediated gene transfer include the cytopathic nature of

592 HSV, the difficulty in maintaining long-term expression of inserted genes, and low infection efficiency [105]. This suboptimal result may reflect viral gene deletions, which can reduce the replicative potential of viruses in tumor cells. For example, deletion of the γ34.5 gene significantly reduced viral growth even in rapidly dividing cells [194]. Biosafety concerns of HSV relate to the fact that wild-type virus is highly pathogenic and cerebral injection causes fatal encephalitis. Toxic and/or pathogenic properties of the virus must, therefore, be disabled prior to its use as a gene delivery vector.

Other Viral Vectors Vaccinia viruses, poxviruses, baculoviruses, and RNA replicons derived from poliovirus [12] have also been investigated for use in the delivery of genes for therapeutic purposes. A variety of replicative viruses have been employed for gene transfer including parvovirus, herpes virus, reovirus, and Ad [66]. The antitumoral effect is achieved by virtue of the replicative cycle of the virus to produce oncolysis selectively in tumor cells. An attractive aspect of replicative viruses is their ability to achieve an amplified effect as a result of their capacity to spread and infect tumor cells within solid tumors [7, 37, 65]. Replicative viral vectors have been tested in human clinical gene therapy trials. Vector modifications to overcome viral receptor deficiency and to achieve tumor replicative specificity are being investigated.

Nonviral Vectors Nonviral vectors have been another promising area of vector development. Several lipid-, peptide-, and polymer-based systems are being investigated for gene delivery [34, 73, 144]. Liposomes have been used for gene transfer of foreign MHC genes in a clinical trial. The liposomes can be modified to provide for selectivity in cell targeting. However, the levels of gene expression are limited by the liposomal degradation of the internalized particles. Systemic administration of cationic lipid/ plasmid complexes yields predominantly transfection of the lungs, with the major transfected cell type being endothelial cells [13]. Improved cationic liposomes delivered the p53 tumor suppressor gene to primary and metastatic murine and human lung tumors in mice and this was associated with suppressed tumor growth and prolonged animal survival with minimal toxicity [160]. Direct injection of naked DNA into tumors using mechanical methods has been shown to result in gene transfer and expression [105]. A problem is the inability to transduce a large number of cells and the fact that the

Cancer gene therapy DNA is only transiently maintained. This approach has also been used for the generation of cancer vaccines. Nonviral vectors are attractive with respect to ease of large-scale production and lack of specific immune response [111].

Targeting of Viral Vectors Transductional Targeting Several studies have shown that viral vectors can be targeted to specific cell types after attachment of ligands (e.g. transferrin, folate, epidermal growth factor, fibroblast growth factor, peptides, and single chain antibodies (sFv) that bind to tumor cell surface receptors) to the viral capsid, either by chemical conjugation employing the Fab fragment of an anti-knob monoclonal antibody, or by genetic engineering for transductional regulation of vector infection (Fig. 1) [16, 36, 68, 86]. Tropismmodified Ad vectors can infect cells that are refractory to transduction by the native Ad, resulting in enhanced gene transfer and therapeutic benefit in vivo [162]. Efforts have been made to genetically engineer the viral capsid proteins to contain cell-targeting ligands. Our group and others have modified the carboxy terminus of the Ad fiber protein to incorporate peptides or growth factors with specificity for tumor cellular receptors [36, 66]. Ad vectors which have been engineered to incorporate a RGD motif at the carboxy terminus or in the HI loop for binding to tumor cell surface integrins have shown markedly enhanced transduction of human cancer cell lines and primary tumor cells, which express low levels of the CAR receptor [36, 65, 94]. Combined targeting of Ad to glioma cell surface integrins and epidermal growth factor receptors increased gene transfer into primary glioma cells [66]. It has been shown that Ad vectors can be targeted to vascular receptors by using peptide-based molecular adaptors [196]. Alternatively, the therapeutic gene can be placed under the control of a tissue- or tumor-specific promoter which is activated in tumor cells but not normal cells, and therefore restrict expression to the tumor cell.

Transcriptional Targeting Targeted expression of therapeutic genes has been obtained using transcriptional regulatory sequences from tumor-specific genes that are ectopically expressed in cancers, viral genes expressed in virus-associated cancers, and tissue-specific genes expressed in cancers and their tissues of origin [125]. Examples include the

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Gene Expression Figure 1. Strategies to target Ad vector gene transfer include (a) transductional targeting to modify Ad binding tropism to the CAR receptor in which a ligand that binds to tumor cell surface receptors is conjugated to a Fab of an anti-knob monoclonal antibody, (b) transductional targeting by genetic engineering of a peptide with specificity for tumor cellular receptors into the knob of the Ad fiber protein, and (c) transcriptional targeting using a tumor-specific promoter (TSP) to produce targeted expression of a therapeutic gene in a tumor cell with specific transcriptional regulatory sequences in combination with genetically modified Ad. Reprinted with permission from Mary Ann Liebert, Inc.

alpha-fetoprotein promoter for hepatocellular carcinoma, the hTERT promoter in telemorase positive tumors, the PSA, PSMA, and probasin promoters for prostate cancer, and the MUC1 and erbB-2 promoters for breast cancer [61, 125]. The incorporation of these promoters into gene therapy vectors allows selective expression of the transduced therapeutic gene. Other examples of inducible promoters that have been investigated are hypoxiaresponsive elements that are induced by hypoxia which is present in many solid tumors [82], and radiation responsive promoters that provide a three- to four-fold induction in gene expression in the radiation field [19]. Regulatory sequences from genes expressed in tumor endothelial cells and from cell cycle-regulated genes are also candidates for transcriptional targeting [125]. For example, we have developed Ad vectors encoding apoptosis-inducing (bax or TRAIL) genes under control of the VEGF or flt-1 promoter, respectively, and angiogenesis-suppressing soluble VEGF receptor 2 gene under control of the VEGF promoter element. Using VEGF and VEGFR1 promoter for targeting of gene expression in VEGF/VEGFR1 positive tumor cells as well as tumorassociated blood vessel endothelial cells significantly increased therapeutic efficacy of gene therapy in human prostate tumor xenograft models [90–92].

Mutation Compensation Induction of Tumor Suppressor Genes The p53 protein is a multifunctional regulator of cell growth and plays a critical role in regulating the cell cycle. When the p53 gene is mutant or absent, the lack of control of p21 results in lack of inhibition of cyclin dependent kinases and unregulated cell growth. The p53 protein also plays a central role in the apoptosis pathway. A large proportion of human cancers carry impaired p53 gene function. In certain tumor types, dysfunctional p53 has been associated with more aggressive tumor growth, enhanced tendency for tumor metastases, and poor survival rates. The approach of p53 gene transfer has been used by a number of investigators [141]. When p53 was transfected into tumor cells and overexpressed, apoptosis and growth arrest has been observed. It was shown that transfecting normal p53 to cells with deleted or mutated p53 decreased anchorage independent growth in vitro and tumorigenicity in animals [32]. The approach taken by Clayman et al. [32] to p53 gene replacement therapy has been to inject head and neck squamous cell cancer with an Ad vector encoding wild-type p53. Patients with head and neck cancer have

594 accessible lesions allowing for direct delivery of vectors. Intratumoral injection of Ad containing wild-type p53 produced growth inhibition of established head and neck squamous cell tumors in animal models [32]. The Ad vector was not toxic to normal squamous oral epithelial cells or normal fibroblasts in culture, and minimal toxicity was produced in animal models. Studies performed in mouse models of non-small cell lung cancer also have shown a significant therapeutic effect of Adp53 gene therapy [32]. Studies have shown that vascular endothelial growth factor (VEGF) expression is reduced in cancer cells following Adp53 infection, resulting in inhibition of angiogenesis in vivo [32]. Phase I clinical trials of Adp53 gene transfer have been completed [32, 33]. The studies demonstrated that Adp53 gene transfer is safe and well tolerated. Transient p53 expression occurred in solid tumors despite high levels of antibodies to Ad. The most frequently observed toxicities were fever, chills, and injection site pain [32]. A Phase II study of 170 patients with recurrent head and neck squamous cell cancer further established the safety and lack of toxicity of direct intratumoral Adp53 injections [32]. A small percentage of patients had stable disease. Randomized trials of Adp53 gene therapy in head and neck cancer patients are currently ongoing. Other clinical trials have confirmed the safety and efficacy of retroviral or Ad p53 gene replacement therapy in non-small cell lung cancer, prostate cancer, recurrent ovarian carcinoma, and esophageal cancer [32, 170, 177]. The results of a recent Phase II study indicated that intratumoral Ad p53 provided no additional benefits in patients receiving first-line chemotherapy for advanced non-small cell lung cancer [176]. In addition, other replacement strategies that are being investigated clinically include induction of Rb and BRCA-1 gene expression [65]. Preclinical studies showed expression of the wild-type Rb and BRCA-1 proteins after gene transfer and reversion of the malignant phenotype, often associated with the induction of apoptosis in tumor cells [141]. Another tumor suppressor gene that has been investigated is PTEN [129]. Multiple gene replacements have been examined as a potential treatment of cancer and have generally resulted in additive or synergistic effects. Combination delivery of both p16 and p53 into cancer cells by adenovirus resulted in an additive or synergistic apoptotic effect for treatment of cancer [100, 173].

Pro-apoptotic and Cell Cycle Gene Therapy Another approach that is receiving attention is pro-apoptotic cancer gene therapy in which the ratio of pro-apop-

Cancer gene therapy totic and anti-apoptotic proteins is modified in order to increase apoptosis. Human tumor cells have been transduced with bax, bak, and Fas ligand genes in Ad vectors and shown to undergo apoptosis [88, 101, 148, 149]. In addition, the gene for caspase-9 has been used in an Ad vector to induce apoptosis in human prostate cancer cells and suppress tumor growth in nude mice [211]. A tumorspecific apoptotic effect of TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) has been shown, in which the TRAIL gene was delivered using Ad or AAV and shown to have significant anti-tumour efficacy in animal models of aggressive primary and metastatic cancer [119]. A limitation of these approaches is that every cell of the primary tumor and metastases be affected by the treatment, unless these approaches are used in combination with other therapeutic modalities. Induction of apoptosis following p21 gene transfer which was originally identified as a molecule that regulates transition from the G1 phase to the S phase of the cell cycle has been reported [87].

Inactivation of Oncogenes Although most oncogenes are silenced after fetal development to prevent abnormal tissue growth, cancer cells may propagate by activating or amplifying oncogenes. One form of cancer gene therapy is the targeted disruption of tumor oncogenes by: (1) inhibition of oncogene transcription; (2) reduction of mRNA translation into protein; and (3) interference with protein function. Inhibition of erbB-2, and blockade of k-ras, c-myc, c-fos, TGFß and insulin-like growth factor 1 are approaches that are being investigated clinically [66]. Transcription of dominant oncogenes has been inhibited using triplex-forming oligonucleotides. Ad gene E1A that inhibits transcription of the human c-erbB-2 promoter suppressed tumorigenicity and metastatic potential induced by the erbB-2 oncogene. Translation of oncogene messenger RNA has been blocked using specific antisense sequences [66]. These include antisense treatment against k-ras in lung cancer, c-myc in breast and prostate cancer, and TGFß in glioma. The erbB-2 oncoprotein has been inhibited with the use of intracellular antibodies. In this regard, Deshane et al. [43] from our group showed that intracellular expression of an anti erbB-2 sFv following Ad gene transfer resulted in downregulation of cell surface erbB-2 expression, and cytotoxicity in human ovarian cancer cells both in vitro and in vivo in an animal model. This approach was translated into a clinical Phase I trial in patients with ovarian cancer [8]. Of the 13 patients evaluable for response, 5 (38%) had stable disease and 8 (62%) had progressive disease.

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Genetic Immunopotentiation Genetic immunopotentiation strategies are designed to achieve active immunization against tumor associated antigens by gene modification of tumor cells to enhance their immunogenicity, or enhance the anti-tumor activity of immune system cells [53]. Genetic immunopotentation is most utilized for the treatment of melanoma. Tumor vaccines are used to initiate an immune response against an unrecognized or poorly antigenic tumor. Treatment with unmethylated cytosine–phosphate–guanine (CpG) activates plasmacytoid dendritic cells, thus releasing IFN alpha and boosting T-cell and natural killer cell responses as well as activating conventional myeloid dendritic cells to treat early stage melanoma [131]. Alternatively, metastatic melanoma treatment with retroviral vectors armed with IFN-gamma is currently in phase I clinical trials as a tumor cell vaccine [60].

Genetic Modification of Tumor Cells It has been hypothesized that vaccination against the tumor by exposing tumor antigens to the immune system in a more favorable context will result in tumor rejection [66]. A major focus of research is the identification of new tumor antigens and the development of cancer vaccines and new anticancer drugs. The first clinical trials with cDNA vaccination with carcinoembryonic antigen demonstrated limited benefit. Cytokine or co-stimulatory molecule B7 gene modified tumor cells and defined tumor antigens have also been used. Numerous studies have shown that tumor cells can be transfected with various cytokine genes and become targets for specific immune rejection [201]. Transduction of a cytokine gene into tumor cells elicits an inflammatory host reaction that impairs tumor growth [124, 207]. This approach has been used in clinical trials [63, 201]. Several investigators have shown that exposure to tumor cells induced to express B7 can be highly effective at enabling normal animals to reject subsequent challenge by tumor cells, but this approach has been much less effective at enabling tumor-bearing animals to kill their pre-existing tumor [50]. In a melanoma trial with autologous irradiated tumor cells transduced with GM-CSF, 20% of patients who received three vaccinations were alive after a follow-up of 3–5 years [41]. Live attenuated viruses have been used in vaccines. Cationic liposomes that encapsulate DNA expression plasmids which encode allogeneic MHC molecules have been used to generate tumor vaccines in situ [219]. In these approaches the genetic modification of tumor cells and effector immune cells can be performed ex vivo, thus enhancing

595 the level of gene transfer and avoiding toxicity compared to in vivo gene transfer. However, the level of gene transfer into tumor and immune effector cells in clinical trials has so far been limited [66].

Genetic Modification of Immune Effector Cells Cells of the immune system including lymphocytes, NK cells, and dendritic cells have been genetically modified to augment their capacity to kill tumors [66]. Tumor infiltrating lymphocytes were the first immune cells to be genetically transduced and used in a human gene therapy trial against melanoma [167]. Although attempts have been made to modify their binding tropism [66], the modest localization and toxicity of these lymphocytes remains a limitation for this form of therapy. Using dendritic cell vaccines requires the cells to be loaded with antigen for presentation to elicit an immune response to tumor [175]. The first reported dendritic cell vaccination study was for indolent lymphoma with other trials for lymphoma [78]. In the case of glioma, tumors have been targeted by bone marrow-derived dendritic cells loaded with glioma cell mRNA resulting in a specific T cell response to protect against intracerebrally implanted glioma tumor cells [114]. Numerous studies continue to evaluate the effectiveness of dendritic cell vaccines in preventing tumor relapses and extending patients’ survival [218].

Molecular Chemotherapy Evolution of Molecular Chemotherapy Paradigm The narrow therapeutic index of drug toxicity to tumor versus normal tissues, has significantly limited conventional systemic chemotherapy and necessitated further drug development research aimed at designing more selective chemotherapeutic agents. Because systemic toxicity is the main limitation to cytotoxic chemotherapy, one approach towards increasing its therapeutic index is through research on alternative forms of drug delivery. One promising alternative employs gene transfer to engineer either (1) increased tumor cell sensitivity or (2) decreased normal tissue sensitivity to systemically administered cytotoxic agents. This approach, which has been termed molecular chemotherapy, gene-directed enzyme-prodrug therapy (GDEPT), or suicide gene therapy involves delivery of a specific enzyme that can produce cell death through the conversion

596 of an inactive non-toxic prodrug into a cytotoxic drug metabolite. Specifically targeted expression of the prodrug-activating enzyme avoids systemic toxicity, and results in high drug concentration in the tumor mass and an improved therapeutic index compared to systemic drug administration. The key element of a GDEPT therapy system is a gene that encodes an enzyme, which converts a prodrug to an active cytotoxic drug. Importantly, prodrug-activating enzymes are normally absent or poorly expressed in mammalian cells. This means tumortargeting of gene therapy, using specific delivery vehicles, restricts enzyme expression to the transduced tumor cells and adjacent surrounding tumor cells through diffusion of the drug metabolite to generate a bystander effect.

From Concept to Clinical Trials A number of enzyme-prodrug (EP) systems have been investigated for cancer gene therapy over the last 20 years. Of the nine classes of traditional chemotherapeutic agents, alkylating agents, anthracyclines, antibiotics, antimetabolites, camptothecins, platinum agents, podophyllotoxins, taxanes, and vinca alkaloids, EP systems have been described for five. A comprehensive overview of these systems and their development in cell culture and animal model systems was outlined in the previous edition of this text [21]. Here, we will focus on the clinical development of these approaches over the past 5 years. One of the most widely studied suicide gene-prodrug systems for cancer gene therapy utilizes the herpes simplex virus-thymidine kinase (HSV-tk) in combination with anti-herpetic drugs such as a 9-[2-hydroxy-1(hydroxymethyl)-ethoxy]-methylguanine (ganciclovir; GCV) or its analogues (acyclovir and valacyclovir). The HSV-tk/GCV system is characterized by the effective phosphorylation of GCV into a toxic compound that is incorporated into DNA during its replication. This incorporation into guanine sites in newly synthesized DNA chains causes termination of synthesis and the selective killing of dividing cells by activation of apoptosis pathways. The first report of GDEPT for cancer appeared in the literature in 1986 and involved HSV-tk and the prodrug GCV [132]. HSV-tk GDEPT was tested in the first clinical gene therapy trial for cancer in humans, which began patient accrual in 1993 [145] and paved the way for a series of similar clinical trials involving retrovirusmediated delivery of HSV-tk. Initial studies in animal models suggested that the main limitation to this strategy was the limited HSV-tk gene transfer efficiency to tumor cells mediated by murine fibroblast-based retroviral vector producer cells. These findings were later confirmed in a large, multinational phase III prospective, open-label,

Cancer gene therapy randomized controlled trial, the most comprehensive cancer gene therapy clinical trial to date, published by the GLI-328 International Study Group in November 2000 [159]. This study assessed the efficacy of standard therapy, consisting of surgery plus fractionated external beam radiotherapy, versus standard therapy plus adjuvant, intratumoral HSV-tk retroviral gene therapy at the time of surgery in 248 patients with previously untreated (grade IV) glioblastoma multiforme (GBM). No significant differences were found between the two treatment arms in either overall median survival (354 versus 365 days, respectively, p > 0.05) or progression-free survival (183 versus 180 days, respectively, p > 0.05). At autopsy or post-therapy biopsy, 7 of 17 (41%) tumors and 1 of 13 (8%) normal brains tested positive for retroviral vector DNA by PCR. Thus, anecdotes and case reports of response notwithstanding [198], 20 years of preclinical and clinical testing of retroviral vector-mediated, HSVtk-based cancer gene therapy failed to achieve significant clinical benefit in patients with GBM. As of April 2008, 121 clinical trials of enzyme-prodrug cancer gene therapy had been initiated in 14 different countries for over 20 different tumor types [1]. Despite the major setbacks to the field in the early 2000s, the number of new enzyme-prodrug clinical trials worldwide continues to grow at approximately the same rate (six new trials per year). The majority (77 of 90, 86%) have utilized either retroviruses or adenoviruses to effect gene delivery. Although retroviruses remain the single most common gene transfer vector employed worldwide (Fig. 2a), its use has plateaued in the United States since 1996 (Fig. 2b) and no new enzyme-prodrug trials for cancer have been presented at the NIH Recombinant DNA Advisory Committee (RAC) since 2000 [2]. Problems with retrovirus vectors are wellestablished [190] and alternative vectors, including replication-competent adenovirus and herpes simplex viruses (HSV) are becoming increasingly employed. HSV-tk/GCV remains the most actively investigated enzyme-prodrug combination, but alternative systems, including cytosine deaminase (CD) and the combination of HSV-tk and CD either as separate or fused transgenes, are becoming more frequent, both in the US and worldwide (Fig. 3). CD/5-fluorocytosine (CD/5-FC) was proposed as an alternative enzyme-prodrug system in 1992, shortly after the initiation of phase I clinical trials with HSV-tk/GCV [133]. In contrast to GCV, 5-FC, a non-toxic antifungal agent utilized clinically for CNS mycoses, produces a well-characterized, highly effective chemotherapeutic agent, 5-FU, upon intratumoral conversion by CD. Moreover, 5-FU is freely diffusible and, unlike GCV, does not require gap junction-mediated

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Figure 2. Enzyme-prodrug cancer gene therapy clinical trials worldwide (a) and in the United States (b). The cumulative number of clinical trials were determined from the gene medicine [1] and NIH Recombinant DNA Advisory Committee (RAC) databases [2] and stratified by gene transfer vector employed. Abbreviations: Ad, adenovirus; HSV, herpes simplex virus

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Figure 3. Enzyme-prodrug cancer gene therapy clinical trials worldwide (a) and in the United States (b). The cumulative number of clinical trials were determined from the gene medicine [1] and NIH Recombinant DNA Advisory Committee (RAC) databases [2] and stratified by transgene delivered. Abbreviations: CD, cytosine deaminase; CYP1B1, cytochrome P450 1B1; HSV, herpes simplex virus; HSV-tk*, genetically modified HSV-thymidine kinase transgene or co-delivery of HSV-tk and other transgenes; TK, thymidine kinase

Donald J. Buchsbaum et al. intracellular communication to elicit a potent bystander effect [67, 179]. 5-FU is also a well-established, clinically useful radiosensitizing agent commonly employed for chemoradiation of gastrointestinal (GI) malignancies [189]. We have previously demonstrated that adenovirus (Ad)mediated expression of E. coli CD (AdCMVCD), together with 5-FC, a combination termed virus-directed EP therapy (VDEPT), and external beam radiation (XRT) significantly enhanced in vitro cytotoxicity and in vivo tumor growth control in both GI [151, 152] and non-GI [128] tumors relative to those receiving AdCMVCD/5-FC alone. Moreover, incorporation of AdCMVCD/5-FC into clinically appropriate fractionated XRT schemes significantly enhanced local tumor growth control relative to single high-dose XRT [181]. One promising approach towards generating more potent enzymes for EP gene therapy involves utilizing either homologous enzymes from different microbial species or mutated endogenous enzymes that possess improved kinetics, facilitating enhanced prodrug conversion. For example, CD/5-FC EP systems have been described using CD from at least three different microorganisms: the bacterium E. coli [133], the yeast Saccharomyces cerevisiae [98] and Candida albicans [10]. Lawrence and colleagues demonstrated the catalytic superiority of recombinant CD derived from the yeast Saccharomyces cerevisiae (yCD) relative to E. coli CD (bCD) [98] and that the favorable enzyme kinetics of yCD translate into (1) enhanced tumor regression after systemic 5-FC [98], (2) 19F-NMR-based detection of CD activity [182], and (3) radiosensitization [99]. We have recently described benefits of the second approach, specifically mutation of E. coli CD [93]. Substitution of alanine (A) for aspartic acid (D) at position 314 of CD increased the relative specificity of the mutant CD-D314A enzyme to 5-FC in comparison with wild-type CD [121] and increased prodrug conversion and cytotoxicity to human glioma cells in vitro [93]. We constructed the AdbCD-D314A vector, encoding mutant bCD-D314A gene and investigated mutant bCD gene transfer in an Ad-directed molecular chemotherapy approach for treatment of human glioma xenografts. This study demonstrated that AdbCD-D314A infection resulted in increased 5-FC-mediated cell killing, compared with AdbCDwt. Therapy with a replication-incompetent adenovirus encoding CD-D314A with 5-FC and XRT showed significant reduction in clonogenic glioma cell outgrowth in vitro and subcutaneous xenograft growth in vivo compared to wild-type CD/5-FC/XRT. Finally, increased 5-FC conversion efficiency of this CD-D314A system led to improved survival of mice

599 bearing orthotopic, intracranial xenografts of human glioblastomas after 5-FC therapy and efficacy was further potentiated by concomitant fractionated external-beam XRT [93]. Co-delivery of cDNAs encoding multiple prodrug metabolizing enzymes, either as separate or fused coding sequences, is another promising approach first described for the two most common EP systems, HSV-tk/GCV and CD/5-FC. The CD-TK fusion gene proved to be synergistic when co-expressed in tumor cells from two separate coding sequences [6]. Freytag and colleagues extended this approach by combining enzyme-prodrug and oncolytic viral therapy through use of a CD-TK fusion construct delivered by a replication-competent adenovirus (Ad5-CD/TKrep) [166]. Freytag, Kim, and colleagues at Henry Ford Hospital have initiated three phase I clinical trials with Ad5-CD/TKrep in patients with prostate carcinoma, either alone [54], with concomitant conformal XRT [59], or with intensity-modulated radiation therapy (IMRT) [54]. CD-TK gene therapy proved to be safe in all three trials. Although the first trial was a phase I doseescalation trial and was not powered to determine efficacy, preliminary analyses of 5-year follow-up data from 16 patients treated with direct in situ injection of 1010– 1012 Ad5-CD/TKrep particles and a 1 week course of GCV/5-FC showed an increase in the prognostically meaningful surrogate endpoint of mean prostate-specific antigen (PSA) doubling time (PSADT) [59]. The authors demonstrated a significant increase in PSADT from 16 to 31 months. Androgen suppression therapy (AST) is a palliative approach for patients with treatment-refractory prostate carcinoma and is associated with significant patient morbidity. The authors demonstrated that the projected onset of AST in the treated cohort of patients was delayed by 2 years [59]. The exact mechanisms of the apparent therapeutic effect will require additional clinical trials and follow-up correlative molecular studies. However, at a time of decreased pharmaceutical company interest in enzyme-prodrug gene therapy and tight funding of biomedical research, these results have been hailed in the gene therapy and urological communities [191] as evidence that this line of research merits continued financial support and multi-institutional testing of this approach in phase II clinical trials.

Inhibition of Angiogenesis Extensive preclinical and clinical data support the concept that tumor growth is dependent on angiogenesis and that VEGF plays a central role in this process. Inhibition of angiogenesis is one of the major targets for

600 anti-cancer gene therapy strategies [52]. An advantage of the anti-angiogenic approach is the highly amplified death of a large number of tumor cells when deprived of their vasculature, which can partially overcome current limitations in the number of tumor cells modified by gene transfer in vivo, as well, it offers an alternative means of tackling multidrug-resistant tumors that have proved intractable to conventional chemotherapies because unlike cancer cells, endothelial cells are stable and do not mutate [20]. An additional advantage is the direct access of vectors delivered systemically to vascular endothelial cells. Both suppression of cellular angiogenic signals and augmentation of inhibitors of angiogenesis have proven to be feasible strategies in preclinical tumor models [104]. It has been shown that Ad-mediated anti-VEGF therapy using a gene encoding a soluble VEGFR1/flt-1, a naturally encoded, alternatively spliced form of flt-1 VEGF receptor, can be used to control tumor growth [52, 122]. Treatment with AdVEGF-sKDR, encoding a soluble VEGF receptor 2 under control of the VEGF promoter, significantly inhibited the proliferation and migration of human vascular endothelial cells and prostate cancer cells. AdVEGF-sKDR also sensitized cancer cells to ionizing radiation. In vivo tumor therapy studies demonstrated significant inhibition of tumor growth in mice that received combined AdVEGF-sKDR infection and radiation versus AdVEGF-sKDR alone or radiation therapy alone [92]. Similarly, suppression of other angiogenic factors such as basic fibroblast growth factor (bFGF) by Ad mediated expression of antisense basic fibroblast growth factor (bFGF-AS) resulted in significant inhibition of transitional cell carcinoma growth [83]. RNasin, the placental ribonuclease inhibitor, is known to have anti-angiogenic activity through the inhibition of angiogenin and bFGF. A plasmid-mediated gene therapy approach employing Rnasin in B16 murine melanoma cell lines significantly inhibited angiogenesis and tumor metastatic progression [18]. Angiopoietins regulate blood vessel assembly and mediate their activity through the receptor tyrosine kinases Tie-1 and Tie-2. Administration of an Ad vector expressing a soluble Tie-2 receptor (AdExTek) an endothelium-specific receptor tyrosine kinase, which is capable of blocking Tie-2 activation, significantly inhibited the growth rate of tumors in mice with two different well established primary tumors, a murine mammary carcinoma (4T1) or a murine melanoma (B16F10.9), 64% and 47%, respectively. It has also been shown that administration of AdExTek therapy inhibited tumor metastasis when delivered at the time of surgical excision of primary tumors [116].

Cancer gene therapy Several immuno-cytokines and or chemokines have been employed in anti-angiogenic gene therapy strategies. Interferon alpha (IFN-alpha) gene delivery by electroporation produced regression of squamous cell carcinoma (SCVII) tumors in 50% of the mice and increased survival time more than two fold [112]. Similarly, inhibition of tumorigenicity and metastasis of human bladder carcinoma in athymic mice by Ad-mediated IFN-beta gene therapy is also attributed to the inhibition of angiogenesis [85]. IFN-gamma gene therapy employing retrovirus completely eradicated intracranial C6 glioma tumors in an immunocompetent mouse model [171]. The IFNgamma mediated tumoricidal activity is due to an apparent interplay of B and T cell components of the immune system, as well as the inhibition of tumor angiogenesis. Further, Ad-mediated gene therapy of proliferin-related protein (PRP), a strong inhibitor of endothelial cell migration or interferon inducible protein (IP10), a suppressor of capillary tube formation, significantly suppressed murine melanoma tumor growth [163]. A recent study also reported that gene transfer of human IP10 using replication-competent retroviral (RCR) vectors markedly reduced growth in xenograft and syngeneic mouse models associated with a marked reduction in microvessel density [185]. Interleukin-8 (IL-8) is a mediator of angiogenesis. Based on this mechanism, an Ad-mediated antisense IL-8 gene therapy significantly inhibited human bladder carcinoma in athymic nude mice [84]. Similarly, treatment of subcutaneous and intracranially established rat C6 cell glioma by retroviral delivery of interleukin-4 (IL-4) in situ, resulted in tumor growth arrest and was associated with eosinophil infiltration and inhibition of angiogenesis [172]. Matrix metalloproteinases (MMPs) play a critical role in degradation of endothelial basement membrane which are required to initiate angiogenesis, thus promoting angiogenesis and cancer progression. TIMP-2 is a natural MMP inhibitor. Ad-mediated TIMP-2 gene therapy significantly reduced tumor growth rates by 60–80%, angiogenesis by 25–75%, and increased survival by 90% in murine lung, colon, and human breast cancer models in mice [108]. TIMP-1 gene transfer delivered by an AAV vector inhibited angiogenesis via reduced endothelial cell migration and invasion in vitro and inhibited tumor growth in a murine xenotransplant model [222]. Treatment with this low dose of AdhTIMP-2 produced a synergistic effect in combination with TNF/IFN-gamma using the murine B16BL6 melanoma model [199]. Angiogenesis is regulated by several angiogenic agents and at multiple levels [103]. The anti-angiogenic molecules function by different mechanisms, including

Donald J. Buchsbaum et al. endothelial cell proliferation, migration, protease activity, and tubule formation. Therefore, it can be inhibited by different methods either by a combination of angiogenic inhibitors or combination of tumor suppressor agents and angiogenic inhibitors, and/or combination of immunotherapy and angiogenesis inhibitors. For example, retroviral mediated angiostatin and endostatin combination gene therapy showed synergistic anti-tumor activity and survival in murine leukemia and melanoma models with complete loss of tumorigenicity in 40% of animals in the leukemia model [174]. Thrombospondin I has been shown to inhibit angiogenesis. However, a synergistic inhibition of MDA-MB-435 breast cancer tumor growth has been shown with a combination gene therapy of liposomes complexed with p53 and thrombospondin I encoding plasmids BAP-TSPf and BAP-p53 when compared to either BAP-p53 or BAP-TSPf alone [214]. Antigen specific cancer immunotherapy and antiangiogenesis have emerged as two attractive strategies for cancer treatment. An innovative approach that combines both mechanisms which has potent anti-tumor activity has been reported using Calreticulin (CRT) which has the ability to enhance MHC class I presentation and exhibit an antiangiogenic effect [28]. Another study reported that treatment of murine breast carcinoma with combined Ad-mediated murine angiostatin and Ad-mediated murine IL-12 resulted in 96% of the animals developing initial regression with 54% undergoing total regression of the tumor. Further, mice were resistant to rechallenge and developed strong CTL response [71]. One novel form of gene therapy has been reported wherein a cell based anti-angiogenic gene therapy approach has been employed. Fibroblasts were retrovirally transduced to overexpress thrombospondin-2 to inhibit human squamous cell carcinoma, malignant melanoma, and Lewis lung carcinoma growth [184]. However, a similar study using hematopoietic stem cells transduced with retrovirus encoding a secretable form of endostatin did not show inhibition of neoangiogenesis or anti-tumor activity [150]. Another study employed a gene therapy approach with murine bone marrowderived cells encoding soluble flk-1 to inhibit tumor growth. Significant reduction in tumor size was reported when challenged with tumor within 3 months after transplantation with bone marrow-derived cells encoding truncated soluble flk-1 [40]. The induction of several inhibitors of angiogenesis has been carried out by transfection of cells with the Thrombospondin-1 gene, or by using viral vectors that encode the genes for soluble platelet factor-4 and angiostatin [52]. Cationic lipid based delivery of endostatin gene sequences and its expression in muscle suppressed

601 primary tumor growth and development of metastases in the lungs of mice [13]. Further, retroviral-mediated delivery of angiostatin and endostatin in both murine leukemia and melanoma models produced enhanced antitumor efficacy [174]. Also, Ad-mediated murine endostatin gene therapy prolonged survival and in 25% of the mice completely prevented tumor growth in a prophylactic human colon/liver metastasis xenograft murine model [25]. However, high dose intravenous delivery of Ad-mediated human endostatin was associated with severe acute toxicity in mice that included loss of weight, bleeding, death of animals [204]. Although there is convincing proof of concept in animal models that an anti-angiogenesis gene therapy approach can be used to treat cancer, realization of its full potential for the treatment of a broad array of diseases will require several challenging technical hurdles such as duration of expression, induction of immune response, cytotoxicity of the vectors and tissue specificity to be overcome and safety concerns to be alleviated [24, 94, 113, 168, 206].

Replicative Vector Oncolysis One approach to overcome suboptimal tumor transduction of non-replicative viral vectors is the use of conditionally replicative viral vectors, in which replication competent virus selectively replicates within tumor cells but not in normal tissues. Release of progeny virions from the initially infected tumor cells would infect neighboring tumor cells and thereby spread throughout the tumor volume. In addition, the use of viruses that have a lytic life cycle would result in virus-mediated oncolysis [66]. The currently available replicating vectors for cancer oncolytic virotherapy include Ad or HSV-1 and can be divided into two large groups: genetically engineered viruses with enhanced selectivity and/or decreased toxicity (including a wide variety of vectors with specific deletions in viral genes or that encode essential genes under control of tumor specific promoters) and vectors engineered to function as therapeutic gene delivery vehicles by incorporating an expression cassette containing a transgene to improve the antitumor efficacy of oncolytic virotherapy (including GM-CSF or CD/TK genes). Importantly, some of these vectors have already been explored in extensive preclinical studies with clinical evaluation in patients showing significant antitumor effects. As of April 2008, 28 clinical trials had been initiated employing Ad, HSV-1, Vaccinia Virus and Poliovirus for different tumor types [1].

602 Since the E1B 55kD gene product in Ad vectors is responsible for p53 binding and inactivation, it was hypothesized that an E1B 55kD deletion mutant Ad would be unable to inactivate p53 in normal cells and thus would be unable to replicate efficiently. Because p53 is absent in many tumors, the replication of a mutant Ad would be selective in tumors. A selectively replication-competent E1B 55kD gene-deleted Ad, dl1520 (ONYX-015) has been injected into solid tumors of patients whose tumors carry mutant p53. Clinical trials using ONYX-015 alone produced minimal tumor responses in patients with head and neck cancer while combination treatment with chemotherapy produced a 63% response rate [102]. In recent years, two major strategies have been developed for specific retargeting of conditionally replicating Ad vectors. In the first one, transcription of essential viral genes has been controlled by replacing the native viral promoters with tumor or tissue-specific promoters. A number of oncolytic Ads have been generated in which the gene essential for viral replication is under the control of exogenous promoters that are preferentially active in tumor cells [77, 158, 197]. One group has produced a conditionally replicative Ad in which the expression of E1A is controlled by the midkine promoter that induced tumor cell killing of neuroblastoma and Ewing’s sarcoma cells [3]. CN706 is an oncolytic conditionally replicating Ad vector, encoding the E1A gene under the control of an exogenous minimal enhancer/promoter construct derived from the 5′ flank of the human PSA gene promoter [165]. CN706 was tested in a phase I clinical trial, in which virus was intratumorally injected into patients with locally recurrent prostate cancer without dose-limiting toxicity [44]. Another example of a specifically targeted replication-competent Ad vector, Ad-hOC-E1, which contains a single bidirectional human osteocalcin (hOC) promoter to drive both the early viral E1A and E1B genes was constructed. This vector selectively replicated in OC-expressing prostate cancer cells and viral replication was enhanced at least ten-fold with vitamin D3 exposure. Unlike Ad-sPSA-E1, an Ad vector with viral replication controlled by a strong super prostatespecific antigen (sPSA) promoter which only replicates in PSA-expressing cells with androgen receptor (AR), Ad-hOC-E1 retarded the growth of both androgendependent and androgen-independent prostate cancer cells irrespective of their basal level of AR and PSA expression [60]. CG7870, a prostate selective replication-competent Ad with improved efficacy, contains the prostate-specific rat probasin promoter, driving the E1A gene, and the human prostate-specific enhancer/promoter,

Cancer gene therapy driving the E1B gene. In nu/nu mice carrying LNCaP xenografts, a single tail vein injection of CG7870 eliminated tumors within 4 weeks [221]. Also, this agent was evaluated in a phase I trial of patients with hormonerefractory metastatic prostate cancer. Systemic injection of CG7870 produced a transient minor decrease of prostate-specific antigen levels [178]. Studies using an Ad vector encoding the E1A gene under transcriptional control of the human telomerase reverse transcriptase promoter have shown that viral genome replication and productive infection is primarily restricted to telomerase-positive tumor cells. Administration of the virus into nude mice bearing human prostate xenografts produced significant tumor reduction [81]. The second strategy is to genetically modify the fiber protein to present a tumor-specific binding ligand. Progress in this direction has included the successful insertion of small peptides such as an RGD motif in the Ad fiber, resulting in Ad retargeting [15]. It was shown that the efficacy of a replicating Ad can be improved by incorporating a RGD peptide motif into the fiber protein [186]. Also, an Ad that secretes a fusion molecule consisting of the extracellular domain of CAR (sCAR) and epidermal growth factor (EGF) was constructed. Infection of tumor cells with a sCAR-EGF-retargeted replication-competent virus system resulted in increased oncolysis in vitro and a therapeutic benefit against tumor xenografts [76]. In an attempt to improve both the efficacy and safety of oncolytic virotherapy, a replication-competent Ad vector encoding a CD and HSV-tk fusion gene was constructed. A single injection of a replicative HSV-tk vector in established s.c. human glioma xenografts resulted in a significant reduction of tumor growth. The addition of ganciclovir produced an additional slowing of tumor growth and prolonged survival [138]. Phase I studies demonstrated the safety of intraprostatic administration of this Ad in combination with conventional-dose threedimensional conformal radiation therapy in patients with intermediate to high risk prostate cancer [54, 57, 59]. Herpes simplex virus 1 (HSV-1) vectors have also been developed that replicate conditionally in dividing tumor cells based on mutations engineered in the viral genome [193]. So far, four oncolytic HSV vectors have been tested in several clinical trials with encouraging results. G207 is an attenuated/replication-competent HSV-1 mutant that lacks both copies of ICP34.5 (RL1) gene and contains an insertion of lacZ in the ICP6 (UL39) gene [130]. These gene modifications offer a new modality in cancer therapy through the ability of G207 to selectively replicate within and kill malignant cells with minimal toxicity to normal tissues [106].

Donald J. Buchsbaum et al. G207 induced a significant inhibition of the tumor growth alone and in combination with radiation in human prostate tumor xenograft models [202]. Based on positive results in preclinical brain tumor models, G207 has been tested in clinical trials in patients with glioblastoma, with some evidence of disease stabilization [123]. A gamma-34.5 gene deleted HSV1716 vector was also evaluated in patients with recurrent malignant glioma following surgical resection in phase I/II trials [74, 147, 161] and in patients with metastatic melanoma [120]. NV1020 HSV-1 mutant vector has been tested followed by chemotherapy in 12 patients with colorectal cancer hepatic metastases with some minor responses [114]. All the patients in these clinical trials tolerated the treatment well. The safety of ICP34.5-deleted HSV has been shown in multiple clinical studies. Recently, a phase I clinical trial with a second-generation oncolytic HSV expressing granulocyte macrophage colony-stimulating factor (Onco VEXGM-CSF) has been conducted. This virus was administered intratumorally in patients with cutaneous or s.c. deposits of breast, head and neck and gastrointestinal cancers, and malignant melanoma [79]. Improved oncolysis was achieved through the use of a more potent clinical isolate of HSV for construction of the virus, and, in addition to the deletion of ICP34.5, the expression of the US11 gene as an immediate early rather than late gene was used since this has previously been shown to boost tumor-selective virus replication [23]. Also, coexpression of GM-CSF has shown promising preclinical and clinical results [192, 205]. Incorporation of a gene encoding a cell membrane fusion glycoprotein of the gibbon ape leukemia virus into the HSV genome significantly increased the antitumor potency in mouse models of primary and metastatic human prostate cancer [137].

Chemosensitization and Radiosensitization The limitations of surgery and radiation therapy concern treatment of cancer metastases. The problem with chemotherapy is its low therapeutic ratio for many tumors and the development of multi-drug resistance. Gene therapy offers the potential to enhance radiobiological effects through various mechanisms, and combination treatment with radiation therapy might amplify tumor cell killing with selected gene transfer events. Chemosensitization can be achieved by genetic induction of apoptosis, by inhibition of molecules involved

603 in tumor cell resistance, or by enhancing intratumoral production of cytotoxic drugs [154, 223]. Ionizing radiation and many chemotherapies depend on wild-type p53 function for their cytotoxic effect. Thus, restoration of wild-type p53 function in tumor cells can be used to potentiate the effects of radiation therapy and chemotherapy [47, 213]. Animal studies have shown that the effects of cisplatin can be synergistic in combination with Adp53 gene transfer in human lung cancer cells in vivo. A trial was carried out in non-small cell lung cancer patients receiving Adp53 alone, or preceded by cisplatin. Clinical responses were observed, and progression-free survival was prolonged with the combined treatment [169]. Colon and nasopharyngeal cancer cells transfected with Adp53 showed increased sensitivity to radiation [110, 169]. Studies of Adp53 gene therapy combined with surgery, chemotherapy, or radiation therapy have been initiated in patients with head and neck cancer and non-small cell lung cancer [17, 51, 139, 188]. Results show that Adp53 is well tolerated and produces efficacious treatment in combination with radiation and/or chemotherapy agents [62]. Restoring or enhancing the capacity of tumor cells to undergo apoptosis through genetic modification of Bax or Bcl-2 expression has resulted in tumors being more sensitive to chemotherapeutic drugs and radiation therapy [14, 64, 210]. The cellular transcription factor E2F1 promotes apoptosis. Intratumoral injection of E2F1expressing Ad vector in combination with gemcitabine produced a significant reduction in pancreatic tumor xenograft size [164]. Transduction of the caspase-3 gene in rat hepatoma cells in the liver with an Ad vector induced extensive apoptosis and reduced tumor volume when combined with etoposide administration [215]. A HSV type 1 mutant in combination with mitomycin C exhibited a synergistic cytotoxic effect against nonsmall cell lung cancer cells in vitro and an additive effect against tumor xenografts [195]. Transcriptional targeting via radiation inducible promoters in a vector can selectively produce gene transfer in a tumor in combination with radiation therapy [31]. Promoters triggered by radiation confine the cytotoxic effect to the high dose target volume. Examples of radiation inducible promoters include various early response genes (c-jun, c-fos, Egr-1, and NFκB), which can be coupled to genes which produce proteins that enhance radiosensitivity. Similarly, the Egr-1 promoter in an Ad vector carrying the TNF-α gene has been activated by several chemotherapy drugs [118]. Tumor injection of an Ad expressing IL-12 and B7.1 following fractionated radiation therapy resulted in a greater therapeutic effect in murine tumor models than

604 with either treatment alone [117]. Expression of IL-3 in transfected murine tumors increased their response to radiation, and systemic administration of IL-3 gene transduced tumor cells in combination with local radiation therapy resulted in enhanced efficacy [30]. Rat glioma cells transduced to express HSV-tk and treated with prodrug acyclovir or bromovinyldeoxyuridine had enhanced sensitivity to radiation in vitro and in vivo [31]. Weichselbaum et al. showed that irradiation mediates enhancement of HSV replication and produces enhanced antitumor activity [4, 127]. It was shown that HSV-tk/ganciclovir suicide gene therapy, vector-based TNF-α expression, and radiosurgery was more effective in controlling human glioblastoma xenografts than single or dual-component protocols [143]. Triple therapy with Ad.Egr-TNF, radiation, and temozolomide produced increased survival in mice bearing glioma xenografts compared with dual treatment [217]. A phase I trial of a replication-deficient Ad vector expressing TNF-α under control of the Egr-1 promoter in combination with radiation therapy in patients with soft tissue sarcomas of the extremity was well tolerated and produced a high number of objective responses [136]. The combination of radiation therapy and angiostatin gene therapy produced enhanced antitumor effects in a rat glioma model [70]. The combination treatment with anti angiogenic agents and chemotherapy or radiation therapy has been shown to produce an enhanced anti-tumor effect in preclinical models [89, 212]. Based on this observation, genetic modification of tumor vascular endothelial cells would be expected to produce an enhanced therapeutic effect in combined modality therapy. Several chemotherapy drugs are proven radiosensitizers. One such drug, 5-FU, when produced by the CD/5-FC suicide gene therapy approach following intratumoral injection of an Ad encoding the CD gene, has been shown by our group to enhance the effects of radiation therapy in preclinical models of cholangiocarcinoma, glioma, pancreatic cancer, and colon cancer [93, 151, 181]. Thus, strategies to increase chemosensitivity and radiosensitivity by gene transfer have potentially wide applicability for the treatment of cancer clinically. Freytag et al. evaluated the efficacy and toxicity of replicative Ad-mediated suicide gene therapy in combination with radiation therapy in a preclinical model of prostate cancer, and in prostate cancer clinical trials [54–56, 58, 59]. Replication-selective oncolytic Ad have been constructed to target tumor tissue for selective replication and amplification at the tumor site with limited replication in normal cells, minimizing toxic side effects. Although low efficacy was documented with oncolytic

Cancer gene therapy Ad as a single agent therapy, favorable interactions with the cytotoxic drugs cisplatin and 5-FU were reported in phase II and III trials with recurrent squamous cell carcinoma of the head and neck [29, 58, 59, 97, 209]. Synergistic effects between oncolytic Ad vectors and chemotherapeutic drugs (e.g. paclitaxel, docetaxel, doxorubicin, cisplatin, and 5-FU) have been reported in animal models and in clinical trials [75, 102, 220]. Combination with radiation therapy allowed a 50-fold decrease in the dose of the oncolytic Ad in a prostate xenograft model [26, 44]. Combination of oncolytic Ad with radiotherapy significantly increased antitumor efficacy against prostate cancer xenografts compared to either agent alone [46].

Current Limitations and Future Directions of Cancer Gene Therapy The development of sophisticated molecular biological techniques over the past 25 years and their application to the study of the molecular mechanisms of cancer have made gene therapy both a logical and practical extension of classic treatment paradigms that is poised to make major advances in new treatments for cancer in the near future. The major limitation of gene therapy for the treatment of cancer arises from the relative inefficiency of current vectors in transducing target cells, the inability to transfer therapeutic genes into sufficient numbers of target cells in situ to elicit the desired biological effect, the development of both vector- and transgene-induced humoral and cellular immunity, and the inability of vectors to selectively localize and transduce tumor cells following systemic administration. Both viral and non-viral vectors are rapidly cleared from the circulation, primarily by hepatic sequestration, following intravenous injection. These four limitations pose significant problems for the development of cancer gene therapy employing systemic vector administration. Future research is likely to be focused on generating modified vectors with reduced toxicity and immunogenicity, increased transduction efficiency, increased duration and regulation of gene expression, and enhanced vector specificity and targeting [66, 105]. Refinements directed towards these goals will be made with replicative viral vectors capable of both therapeutic gene delivery and direct oncolysis [102]. A universal gene delivery system has yet to be identified, so that further optimization of existing vector delivery systems is likely to occur.

Donald J. Buchsbaum et al. Small interfering RNA (siRNA) has been used to knock down or silence gene expression. This has been accomplished using chemically synthesized oligonucoleotides delivered in nanoparticles and liposomes, or vector-based siRNA [80, 155]. Targets have included oncogenes, and genes involved in angiogenesis, metastasis, anti-apoptosis, and resistance to chemotherapy. Further research into this promising gene therapy technique is required. Gene-modified stem cells have been used for cancer therapy [27, 153]. Their ability to traffic to tumor sites makes them an attractive gene delivery vehicle. They may be useful in conjunction with immunotherapy and gene therapy for treating a variety of cancers. The targeting of tumor blood vessels should be a useful approach for the treatment of a variety of cancers. Issues that need to be addressed are highly efficient gene delivery and long-term expression of the antiangiogenic genes in metastatic cancer. The development of targeted vectors should aid in the development of this approach. Gene-directed enzyme prodrug therapy uses transcriptional differences between normal and cancer cells to drive the selective expression of a metabolic “suicide gene” that is able to convert a nontoxic prodrug into its toxic metabolite. A variety of enzyme prodrug systems have been evaluated in phase I/II clinical trials using either retrovirus or adenovirus to deliver the enzyme to tumor cells [38]. The evaluation of gene therapy in combination with surgery, radiation therapy, and chemotherapy is likely to undergo further development. Gene therapy will continue to be applied to many fields of medicine, and particularly to the treatment of cancer. Whereas human gene therapy trials for cancer have not yet yielded clear benefit, these studies highlight the deficiencies of current approaches. Gene therapy will likely become an integral component of a multimodality strategy for the treatment of cancer. Improvements in vectors that increase the specificity and duration of gene expression, the reduction of immunogenicity and toxicity, and targeted vector delivery are required for advancement of the clinical practice of gene therapy with an increase in response rate. As knowledge of the molecular basis of cancer accumulates, and as genetic screening programs are introduced to identify high-risk patients, it might be possible to use gene therapy for early treatment, including vaccination against cancer. Acknowledgements We acknowledge research support from the National Institutes of Health (P20 CA101955). We are grateful to Sally Lagan for preparation of the manuscript.

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612 209. Xia ZJ, Chang JH, Zhang L, Jiang WQ, Guan ZZ, Liu JW, Zhang Y, Hu XH, Wu GH, Wang HQ, Chen ZC, Chen JC, Zhou QH, Lu JW, Fan QX, Huang JJ, and Zheng X. [Phase III randomized clinical trial of intratumoral injection of E1B gene-deleted adenovirus (H101) combined with cisplatin-based chemotherapy in treating squamous cell cancer of head and neck or esophagus]. Ai Zheng 2004; 23: 1666–1670. 210. Xiang J, Gomez-Navarro J, Arafat W, Liu B, Barker SD, Alvarez RD, Siegal GP, and Curiel DT. Pro-apoptotic treatment with an adenovirus encoding Bax enhances the effect of chemotherapy in ovarian cancer. J Gene Med 2000; 2: 97–106. 211. Xie X, Zhao X, Liu Y, Zhang J, Matusik RJ, Slawin KM, and Spencer DM. Adenovirus-mediated tissue-targeted expression of a caspase-9-based artificial death switch for the treatment of prostate cancer. Cancer Res 2001; 61: 6795–6804. 212. Xu F, Ma Q, and Sha H. Optimizing drug delivery for enhancing therapeutic efficacy of recombinant human endostatin in cancer treatment. Crit Rev Ther Drug Carrier Syst 2007; 24: 445–492. 213. Xu L, Pirollo KF, Tang W-H, Rait A, and Chang EH. Transferrinliposome-mediated systemic p53 gene therapy in combination with radiation results in regression of human head and neck xenografts. Hum Gene Ther 1999; 10: 2941–2952. 214. Xu M, Kumar D, Stass SA, and Mixson AJ. Gene therapy with p53 and a fragment of thrombospondin I inhibits human breast cancer in vivo. Mol Genet Metab 1998; 63: 103–109. 215. Yamabe K, Shimizu S, Ito T, Yoshioka Y, Nomura M, Narita M, Saito I, Kanegae Y, and Matsuda H. Cancer gene therapy using a pro-apoptotic gene, caspase-3. Gene Ther 1999; 6: 1952–1959. 216. Yamamoto M, Alemany R, Adachi Y, Grizzle WE, and Curiel DT. Characterization of the cyclooxyygenase-2 promoter in an adeno-

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20 Regulatory process for approval of biologicals for cancer therapy ANTONIO J. GRILLO-LÓPEZ

Abstract The tenet ‘Above all do no harm’ is necessary and important but not sufficient [1]. Today’s physicians have a responsibility to actively participate in making new therapies available to the cancer patient. We know that cancer can be cured. However, biologicals and all new anticancer agents must be efficiently and rapidly evaluated to find those that are most promising. They must then be incorporated into combination regimens that provide the greatest opportunity for cures. Our regulatory agencies and processes were originally established with the goal of protecting the patient from unscrupulous investigators. Now it appears as if they exist to protect the cancer and not the patient. Where did we go wrong? What is the track record of the regulatory agencies? How can regulatory obstacles be overcome? How can regulatory challenges be turned into opportunities? What is the optimal process for the development of new anti-cancer therapies? Keywords Biological therapy • targeted therapy • antibodies • rituximab • FDA • EMEA • regulatory • IND • NDA • BLA • ODAC • accelerated approval • regular approval • clinical trials • endpoints

The Doctor–Patient Relationship: Duty and Responsibility As to diseases, make a habit of two things – to help, or at least to do no harm. Hippocrates

Primum non nocere (above all do no harm) is an important consideration for the physician caring for the cancer patient [1]. The extreme, inaction for fear of doing harm, is simply not acceptable particularly when challenged by a potentially curable illness. In cancer therapy all new agents present benefits and risks that must be weighed with the patient’s best interest in mind. In the face of a serious illness like cancer, patients are generally willing to take greater risks and accept more toxicity in

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

the expectation of a therapeutic benefit that will make their efforts worthwhile. Likewise, Oncologists have traditionally been more willing to accept a higher level of adverse events in treating their patients than physicians in other specialties. In cancer therapy primum non nocere really implies a very delicate weighing of risk versus benefit where the balance weighs somewhat more heavily on the benefits side while accepting more risk than one would accept for drugs in other therapeutic areas. A patient may be denied the possibility of a cure because of therapeutic nihilism or therapeutic conservatism. One extreme is the nihilistic approach that says – we have nothing that can cure you and therefore we will ‘watch and wait’. This approach, originally devised by Portlock and Rosenberg [2, 3] for patients with early stage Follicular Non-Hodgkin’s Lymphoma (NHL) is today obsolete as we develop potentially curative combination regimens for this disease [4]. At the other extreme is the urge to persist in administering one chemotherapy regimen after the next to a patient who is refractory and at the latter stages of the disease. Hippocrates said, first – to help, and then (when you can no longer help) – to do no harm. Physicians are bound by a sacred oath to care for their patients, to offer them the best that modern science provides for their disease, complications, and general wellbeing [5]. This duty, this responsibility, is critical to the patient with an incurable illness such as cancer. It is even more important when the potential for cure exists for a cancer that was previously held to be incurable. When that potential exists for a new agent still under investigation, time is of the essence. Developing that agent then becomes a race where the losers (patients) will perish because that curative therapy took too long to be evaluated and approved. The physician directly caring for the patient must seek the most effective therapies even when investigational and provide his patient with access to those via participation in clinical trials or referral to centers where those therapies are available. This duty, this responsibility extends beyond the physician directly caring for the patient. Physicians in

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the pharmaceutical industry must do everything within their reach to ensure that new agents are developed and submitted for approval in the most expeditious fashion. Physicians in our regulatory agencies [Food and Drug Administration (FDA), European Medicines Evaluation Agency (EMEA) and others] must likewise understand that they have similar duties and responsibilities to the patient. Being employed by a bureaucracy does not mean that your sacred oath is no longer valid. In fact, physicians in regulatory agencies could be an important catalyst in making new promising therapies available to patients in a timely fashion. However, the opposite has occurred and as a result, regulatory agencies and their bureaucratic processes are today the most important impediment to the timely development and availability of new (some of them potentially curative) anticancer therapies. The patient has great expectations of his personal physician but may not understand the role that physicians in the regulatory agencies are playing in defining their destiny. Regulatory agencies need to recognize that new anti-cancer agents are not truly available to all that can benefit from them until approved and marketed. Many types of expanded access and ‘compassionate use’ programs have been tried but these will never substitute for approval and marketing. The fact is that, prior to marketing, anti-cancer therapeutics are available only to a select minority of patients that qualify for these programs while the majority are deprived of this opportunity. Thus, regulatory agencies have a very serious responsibility to expedite the approval of new anti-cancer agents and make them broadly available. It must be made clear that the majority of physicians in the regulatory agencies are good professionals and well meaning individuals who work very hard for less than adequate salaries. They deserve respect and support. It is a minority that, strategically placed, has created and enforced the obstacles that impede progress. However, single agent approval although necessary and important is not sufficient as the majority of effective therapies (and specifically, curative therapies) are not based on single agents but on combination regimens [6–9]. The regulatory agency’s responsibility, as established by law, is the review and approval of new anticancer agents (single agents). Pharmaceutical industry is responsible for the discovery and development of new single agents and this is what the agency is required to regulate. Once the single agent is approved, it is the oncology community’s responsibility to find its optimal use in combination with other agents. The oncology community is then responsible for defining the role of the new agent within a combination regimen and its

place in the therapeutic armamentarium for the target disease. Regulatory agencies have no important role in this. Pharmaceutical industry can be a partner but should play a secondary role. Thus, investigators in academia and in the community (including academic institutions, cancer centers, cooperative groups, consortia, and others) have very important duties and responsibilities to the patient in the development and optimization of combination regimens.

Development of Cancer Therapeutics The mistakes are all there, waiting to be made. Chessmaster S.G Tartakower

Progress towards more effective and less toxic therapies and progress towards curative therapies depends on the very complex process of drug development and approval. It is only after approval that a new agent will be widely available to the patients who will benefit from its use. However, even after a new agent is approved it is still necessary to continue the clinical research process that will determine its optimal use within a combination regimen. On the average, it takes about 12 years for a new agent to proceed from discovery to approval (Fig. 1). Preclinical development accounts for the first 4 or 5 years; and clinical development, regulatory review and approval take over 7 years. It is thought that regulatory time accounts for 4 to 5 of these 12 years (FDA imposed delays, clinical holds, excessive and unreasonable requirements, waiting for meetings to be scheduled, review time, waiting for FDA responses, etc.). The clinical development process begins prior to the first patient being treated. The Phase I protocols must be written, a clinical plan defined, and all preclinical data submitted to the regulatory agencies before one can proceed. All of these documents are reviewed by the regulatory agency and a determination is made as to whether or not the Phase I (PI) clinical trials can be initiated. Once a recommended dose for PII has been established in PI, the PII trials can commence. Ideally this occurs with some overlap with the PI studies. Both the PI and the PII trials should include, as early as possible, combination regimens. These pilot combination studies are not necessary for the evaluation of the safety profile or the activity of the single agent; however, they are important for two reasons: (a) getting an early start towards treatment optimization within a combination regimen, and (b) evaluating possible combinations suitable for

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Figure 1. Timeline: New anticancer agents

PIII studies. It is unusual to find the optimal combination regimen this early in development. Following PII, the PIII trials are then carried out with a view to regulatory approvals. The clinical development plan incorporates all activities from PI to approval and beyond. It is usually written by a physician (project clinician) with support from other professionals. The extent and overall scope of this critical road map depends heavily on projections of the resources that will be available over the 6 to 10 years it will take to complete clinical development. Most new agents are developed by pharmaceutical companies. A large and well established company is more likely to dedicate substantial resources than a small start-up biotechnology company that depends, at least initially, on venture capital. Regardless of available resources, the clinical development plan must clearly define the path to a regulatory approval. Given a new agent with reasonable efficacy and tolerable toxicity, this is the main objective. It is the objective not only for the pharmaceutical company and its investors but also for the patient and all who have a role in the care of the patient. The road to therapeutic success in cancer is paved with many compounds that failed to show the desired efficacy or were too toxic. One must, however, wonder how many active compounds were trampled under and became part of that pavement due to simple mistakes, inappropriate development plans, technical problems in the conduct of clinical trials, companies going bankrupt, inadequate resources, misinterpretation of clinical data, and/or any combination of the myriad problems and challenges that plague the clinical phases of development. One important problem is

the shortage of well qualified and experienced physicians to assume the role of project clinician (or of Chief Medical Officer, CMO) in a pharmaceutical company. It used to be that any physician considering such a role would unequivocally be given the message, by his peers in academia, that this would be a black mark on his curriculum vitae and that his career might not survive that experience. This has changed over time and today there are many more qualified and experienced physicians, many with an academic background, in the pharmaceutical industry. However, over time, the need has also increased. As an example, in San Diego, California there are over 400 bio-medical companies. They all need a CMO and most also need one or more project clinicians depending on how many new agents they have in clinical trials. There are simply not enough physicians with clinical trials and regulatory expertise to satisfy the need. Simple mistakes can have major impact in the lengthy and complex process of cancer drug development. As an example, one small company contracted with a contract research organization (CRO) to have them create a database, perform data entry, and carry out the statistical analysis for one of their clinical trials. Despite the company’s extraordinary efforts to make sure that the CRO’s database was identical to the company’s database for their other studies, one simple mistake was made. Due to this, the summary data tables for safety and efficacy cutting across all studies were very obviously incorrect. After extensive investigation (double checking, and painstaking review of source documents, data entry, programming, queries, dictionaries, coding conventions, quality control, and of all the data), it was determined that the CRO had reversed the

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coding conventions followed by the company for gender. This one simple mistake cost the company a 2 months delay (or $3.0 million for this particular company) in filing the regulatory dossier. At the other extreme (more serious mistakes) some new agents have been turned down by the FDA for incomplete or inadequate data collection. When this happens at the end of PIII after many years of development, the losses are measured in the hundreds of millions and more importantly in thousands of lives when an effective product (with some curative potential) is lost to the cancer patient. Some may marvel at how well we have performed in terms of cancer drug development over the past few decades given all the challenges, mistakes and problems. Such complaisance is unacceptable. I would prefer to tackle the challenges and problems, avoid/preempt the mistakes and find solutions that will enable us to perform at peak efficiency as we head towards better and hopefully curative therapies. We owe that and more to the patients that we serve.

Biologicals and the Treatment of Cancer Rationale for Developing Biological Therapies According to the theory of aerodynamics, as may be readily demonstrated through wind tunnel experiments, the bumblebee is unable to fly. But the bumblebee, being ignorant of these scientific truths, goes ahead and flies anyway – and makes a little honey every day! Ross E. Hutchins

Back in the late 1980s and early 1990s, antibodies (MAbs) were the bumblebees of cancer therapeutics. Practically no one, after years of frustrating clinical experiences, believed that MAbs would ever fly. The 1994 review by Dillman listed the many MAbs taken to clinical trials with disappointing results [10]. Frustration and disappointment were understandable. After all, it had been nearly 100 years since Paul Ehrlich first spoke about MAbs and ‘magic bullets’ [11]. And yet some of us knew, even then, that harnessing the power of MAbs for therapeutic applications was a question of time [7]. Anticancer therapies, such as radiotherapy and chemotherapy, had been the standard for too many years, as we sought additional and better alternatives. They had served us well; however, their toxicity was tolerated, only because we lacked other options. MAbs had arrived; it was just a question of time!

The rationale is relatively simple. The intent is to make the patient’s immune system an ally. Not to impair it but to find ways of working with and enhancing the immune system. This is readily understood in the case of MAbs. They work either by utilizing the patient’s immune system (complement, effector cells, etc.) to kill the tumor cell or by binding to the target antigen and transmitting an apoptotic signal. MAbs are also used as per Ehrlich, as ‘magic bullets’. They are used to deliver to the tumor cell a payload consisting of a toxin, chemotherapeutic agent, or a radioisotope. Other biological therapies, in the broad definition of the term (treatments that are or resemble natural body substances or that are made from natural body substances), have very specific molecular targets (targeted therapies). The end of the last millennium ushered in a new era in cancer therapeutics. MAbs, led by rituximab, had arrived. Some other MAbs, as well as a variety of other targeted therapies, are now approved (Herceptin, Mylotarg, Campath, Zevalin, Bexxar, Erbitux, Avastin) and many are under investigation. We witnessed the development and approval of the first MAbs for the treatment of cancer, rituximab [12, 13]. Early in this new millennium, we witnessed the approval of the first radio immunotherapy [14, 15]. Other firsts will soon follow: the first vaccine for cancer, the first antisense product, the first genomicsderived therapeutic, and others. This will be the era of MAbs, of biologicals, of targeted therapies. Gone are the days when chemotherapy was the only and, unfortunately, usually the last resource. These new therapeutics open the door for new combination regimens and new multimodality therapies with curative potential. We now have much more selective and specific therapeutics. We now have agents with very different mechanisms of action that are ideal for combination therapies. Importantly, never before, in the history of cancer therapy, have we been able to harness, control, and direct the immune system as we can today. The immune system can now become an important and effective partner in our efforts to cure cancer. It is only the beginning. There is a bright future ahead for the many cancer patients who will benefit from all the promising advances that we envision today and that will become reality in this new era.

Rituximab as an Example The human mind treats a new idea the way the body treats a strange protein, it rejects it. P. B. Medawar

The development of rituximab began at IDEC Pharmaceutical Corporation’s (IDEC) laboratories in San Diego, California in 1991. Mice were injected with human

Antonio J. Grillo-López CD20 antigen and the anti-CD20 MAbs they produced were isolated and characterized. One of those, the murine IDEC-2B8, was chosen for chimerization. The chimeric MAb (IDEC-C2B8) was favored in order to reduce the immunogenicity of the murine parent MAb, to utilize a human framework which allowed for complement and effector cell binding, and to selectively effect B-cell depletion. The technology necessary for humanization was not yet fully developed at that time and thus was not pursued. A vector was engineered that enabled high MAb yields when inserted in CHO cells and used in a high volume fermentation process. The appropriate preclinical pharmacology and toxicology studies were carried out including B-cell depletion studies in monkeys. Mechanistic studies confirmed the desired complement binding, complement dependent cytolysis (CDC), effector cell binding and antibody dependent cellular cytotoxicity (ADCC). The MAb was clearly shown to induce apoptosis. All of this pre-clinical work was carried out within a year at IDEC’s laboratories [16, 17] (Table 1). Synergism studies were completed in collaboration with scientists at UCLA [18]. All the preclinical development work was performed at IDEC’s laboratories in San Diego, California with no government or NIH support. This MAb was ‘made in the USA’, at a small biotechnology company, a start up funded by venture capital, and with no taxpayer’s monies. Rituximab is an example of American entrepreneurship at its best. IDEC-C2B8 was ready for clinical trials by late 1992. The Investigational New Drug application (IND) was submitted to the FDA who placed the project on a clinical hold due to their concerns about the effect of B-cell depletion on immunoglobulin levels and immunocompetence. Finally, after months of scientific discussions, the FDA allowed the PI clinical trials to proceed (February 1993). The desired B-cell depletion was observed. The MAb Table 1. IDEC’s pre-clinical scientists responsible for the development of rituximab (IDEC-C2B8)a Name

Responsibilities

Nabil Hanna, Ph.D.

VP, Pre-Clinical R&D – in charge of all preclinical development Isolation and characterization of the murine parent antibody, IDEC-2B8 Gene identification and cloning experiments; antibody engineering Engineering of the chimeric antibody IDEC-C2B8 and vector engineering VP, Manufacturing – in charge of antibody production

Darrel Anderson, Ph.D. Roland Newman, Ph.D. Mitchell Reff, Ph.D. Chris Burman, Ph.D.

a

Key scientists. Many others at IDEC contributed and are not listed for space reasons.

617 was doing what it was intended to do. Significant reactions (primarily fever and chills) were seen even in the first patient treated. These were biologic manifestations of MAb induced B-cell depletion. The total absence of such reactions might indicate the MAb was not effective. Two patients had objective responses, to a single infusion of MAb, which lasted for months [19]. The PI/ II study was designed to define the pharmacokinetic profile [20], confirm the dose to be used in a four infusion schedule [21] and to establish a response rate in patients with relapsed or refractory Non-Hodgkin’s Lymphoma (NHL) [22, 23]. The PIII (pivotal trial) completed enrollment in 1 year, the data was collected and analyzed, and the complete filing (including all preclinical and clinical studies) was submitted simultaneously to the US FDA (electronic and paper copy) and in Europe to the EMEA in February 1997 [24–27]. The clinical work including the design of the overall clinical plan; writing and implementing all of the protocol for the initial eight studies; establishing collaborations with numerous academic institutions in North America and Europe; collecting, analyzing and interpreting the data; writing all reports, abstracts and publications; generating the electronic dossier; filing, presenting, and defending the data with regulatory agencies in the US and Europe; and many other activities critical to the ultimate success of this project were carried out by the Medical and Regulatory Affairs Division at IDEC [7] (Table 2). The clinical phase of development was completed in 3 years (first patient enrolled in Phase I to last patient enrolled in PIII), a new record in NHL as even the NCI’s cooperative groups will take at least 3 years to complete one PIII study. It took 1 year (total time from last patient enrolled in PIII to BLA filing) to complete the necessary observation time on the Phase III patients, and prepare and file the regulatory dossier (same day filing of a US BLA and an European dossier). Regulatory review and approval, in the US, took 6 months plus a 3 months delay imposed by the FDA while resolving manufacturing issues. Clinical/regulatory development time was a record setting 4 years and 9 months as compared to the average of over 7 years for cancer drugs in general (Fig. 2). The five things that made this clinical development program successful were: an active agent, the right people, good leadership, ironclad data and peer level relationship with the FDA. Clearly rituximab was bound to be successful. It worked exactly as it had been designed. IDEC had the right people empowered by good leaders. The company had a core group of professionals who had years of experience in clinical development. They were not inventing the wheel. IDEC was not a ‘virtual company’ outsourcing a lot of work. It was a small company and yet

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Table 2. IDEC’s clinical scientists responsible for the development of rituximab (IDEC-C2B8)a Name

Responsibilities

Antonio J. Grillo-López, M.D.

Chief Medical Officer and Senior VP Medical and Regulatory Affairs – in charge of all clinical development and regulatory affairs Senior Director, Clinical Operations – conduct of clinical trials including strategy, timelines, budgets and resources Senior Director, Project Planning and Regulatory Affairs – planning and timelines for all clinical trials; communications with the regulatory agencies; filings and dossiers Director, Medical Writing – documentation (protocols, clinical trial reports) for each study, regulatory filings (INDs, periodic reports, regulatory dossiers); publications Head, Clinical Immunology Laboratory – analysis of all patient samples for bcl-2 (pcr), pharmacokinetics; and other specialized tests Senior Director, Biometrics – data entry, programming, queries, tracking, statistical planning and strategy, data analysis, interaction with regulatory agency’s statisticians Director, Clinical Trials Monitoring – protocol design, study implementation, clinical trial monitoring and GCP, data collection and clean-up, interaction with clinical sites Senior Director, Regulatory Affairs - planning and strategy for regulatory evens, communications with regulatory agency’s staff; filings and dossiers Senior Director, Hematology and Oncology – safety officer; interactions with clinicians and their staff at study sites; publications and presentations

Brian K. Dallaire, Pharm. D. John Leonard, Ph.D. Anne McClure, M.S. Jay Rosenberg, Ph.D. David Shen, Ph.D. Chester Varns Alice Wei Christine White, M.D.

a Many others at IDEC Pharmaceuticals, as well as investigators and staff at investigational sites, made important contributions and are not listed due to space constraints.

Figure 2. Rituxan: Clinical development timeline

able to handle the work load for this program mostly internally. Virtual companies generate virtual data. That was not the case at IDEC. The clinical program was subject to careful prospective planning, meticulous study implementation and conduct, rigorous adherence to GCP and GLP requirements, strict auditing procedures, precise statistical methodology, and painstaking attention to correct response and duration adjudication. The clinical data, the cornerstone critical to the approval of any new agent, was ironclad. Not a single patient’s response or duration was

ever contested by reviewers, auditors, peers, or editors. The FDA contested only one patient, a patient in the Phase III study that had been classified as inevaluable. The result of the joint review by he FDA and the clinical group at IDEC was that the patient’s classification was revised to evaluable, response was a CR, and duration was long. The FDA had actually added a CR and increased the overall response rate (not their original intent). Lastly, a peer level relationship with the FDA is of the utmost importance. Establishing a relationship that is amicable,

Antonio J. Grillo-López

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collegial, professional and based on mutual respect is the key to success. It requires openness, timeliness, and the most professional and scientific approach. In such a situation the FDA staff can be your allies and partners. Ideally you end up with what amounts to a joint development team as happened with the rituximab CALA (Table 3). On a personal note, there was one new idea in the development of rituximab that was rejected by several prominent lymphoma experts. I knew that rituximab’s ultimate contribution to the care of the lymphoma patient would be determined by its contribution within a curative combination regimen such as CHOP in the treatment of Diffuse Large Cell B-Cell Lymphoma (DLBCL). Thus, one of the first clinical trials I designed was the Phase II pilot study of rituximab plus CHOP (R + CHOP) [28]. The definitive goal was to treat patients in a disease where CHOP was known to be curative (about 40% cure rate). Synergism between rituximab and doxorubicin had already been shown [29]. However, the toxicity of the combination and its impact on the cure rate of CHOP were unknown. Therefore, we chose to perform the first study of R + CHOP in patients with Low Grade NHL. Several prominent lymphoma experts rejected this new idea of the simultaneous administration of rituximab and CHOP as they might reject a foreign protein. Their objection was that ‘antibodies have limited efficacy and should be used only after chemotherapy, to treat minimal residual disease’. They argued for sequential rather than simultaneous administration. My new idea, that the synergism shown in cell lines would lead to enhanced efficacy in clinical trials and that we would miss that opportunity if we gave the antibody in sequence rather than simultaneously with chemotherapy, was not considered valid. One investigator did accept my new idea as valid (did not reject it as a foreign protein) and enthusiastically participated in this study. The study revealed a 100% response rate (7+ years median TTP). Side effects were similar to those of CHOP and rituximab with no additive toxicity. We conducted a similar study in DLBCL and found a 94% ORR [82% Table 3. Rituximab’s CALA Team (1996–97) Responsibility

FDA

Medical

Bernard Parker, M.D.

IDEC

Antonio Grillo-López, M.D. Susan Jerian, M.D. – Patricia Keegan, M.D. – Regulatory – Alice Wei – Augusta Cerny Product Reviewer Mark Brunswick, Ph.D. – Statistical Jawahar Tiwari, Ph.D. David Shen, Ph.D. Technical Support Robin Jones Ken Fite CALA Coordinator Michael Fauntleroy –

progression free survival (PFS) at 5 years] [30, 31]. These studies led to the GELA study, led by Bertrand Coiffier, which showed that the R + CHOP combination almost quadrupled the event free survival of CHOP [32]. This study established R + CHOP as the gold standard for the treatment of DLBCL. Rituximab had reached its curative potential in combination with CHOP. No other agent, alone or in combination, has ever achieved a significant improvement over CHOP alone.

The USA’s Food and Drug Administration Want of foresight, unwillingness to act, lack of clear thinking, confusion of counsel – these are the features which constitute the endless repetition of history. Sir Winston Churchill

The FDA was created in 1938 by an act of congress (FDAC Act) and charged originally with protecting the consumer by evaluating the safety of new products [33]. The role of the FDA has evolved over time. The act was revised in 1962 and thereafter the FDA required that, in addition to safety, efficacy be established in two adequate and well-controlled clinical trials. In 1997, the FDA’s Modernization Act (FDAMA) was approved and it allowed the FDA to accept one clinical trial and other supportive studies as evidence of efficacy [34]. However, in practice the FDA generally continues to operate as if efficacy should be shown in ‘two adequate and well controlled clinical trials’ and as if the only acceptable endpoint is overall survival. One unwritten rule is that short of the ‘two adequate and well controlled clinical trials’ the one Phase III study would have to reach high statistical significance for its primary endpoint at the p = 0.01 level (rather than 0.05 in the case of two studies). The presence of good supportive trials is of no consequence in this situation. Any application needs to contain ‘two adequate and well controlled clinical trials’ that show significance at the p = 0.05 level or one that that shows significance at the p = 0.01 level. The acts of Congress that created and, over the years, have modified the FDA and its mandates have been interpreted by the agency in their own manner. These interpretations are often issued in the form of guidelines which are not always followed by the FDA itself. All of this has evolved into a bureaucratic process that is complex, costly and time consuming. Each subsequent law, each subsequent guideline is one more band aid on a gaping wound that in reality can only be addressed by major reconstructive surgery. A few within the agency

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contribute to this bureaucracy. The majority try to do their best to work within this flawed system. Leadership is a major problem. The agency has for long periods of time operated under interim leaders. The commissioner of the FDA is appointed by the President and must be sanctioned by Congress. This is a highly political, drawn out process for a job that few want. In the absence of true leadership, the agency falls back on the more primitive instincts of self protection. Any publicity is bound to be negative and any misstep can affect their Congress approved budget. Any action taken by the FDA will be subject to intense scrutiny and criticism. Adverse events occurring post approval will be assumed to have happened because of the FDA’s negligence in scrutinizing safety data. In this environment it is easier to say no or not to act at all. Nevertheless, FDA reform is a buzzword that one hears mostly from politicians prior to elections. After that, it seems like no one dares tackle this subject.

The Oncologic Drugs Advisory Committee of the FDA Life is short, Art is long, Opportunity is fleeting, Experience is deceitful, Judgment is difficult. Hippocrates

The US Congress has enacted legislation authorizing the establishment of committees of experts to advise government agencies in discharging responsibilities. The US Food and Drug Administration (FDA), in turn, created the Oncologic Drugs Advisory Committee (ODAC) whose structure and function is defined in the Code of Federal Regulations (CFR), in the charter published by the FDA, and other documents [35–37]. According to the charter, the committee is expected to “review and evaluate data concerning the safety and effectiveness of marketed and investigational human drug products for use in the treatment of cancer and make appropriate recommendations to the Commissioner of Food and Drugs”. The FDA requests advice from the committee members (members) on a variety of matters, including various aspects of clinical investigations and applications for marketing approval of drug products. They receive summary information about the applications and copies of the FDA’s review of the application documents. Based on this information, the committee may recommend approval or disapproval of a drug’s marketing application. The FDA generally follows the committee’s recommendation, but is not bound to do so. The FDA must notify affected persons of their decision within 90 calen-

dar days of the committee’s recommendation. However, the FDA requests the committee’s advice via a series of questions that are delivered to committee members usually the day before the meeting [38, 39]. The list does not usually include the key question – should this product be approved; or, more appropriately, will this new agent benefit the patients who may receive it. The questions usually bring up technical issues regarding study conduct, data collection, choice of endpoints, or statistical analysis. The FDA’s desire for the committee to provide a certain answer or recommendation is many times transparent, equivalent to a jury being asked – do you find this guilty person guilty. Sometimes a question is asked in a negative way that could serve to lead the committee to respond negatively. Many times questions are asked that require an in-depth understanding of regulatory law at which committee members are not experts. One could argue that the committee is set up to fail. The members are academicians with a wealth of academic knowledge. There is not a single community Oncologist on the committee. In the US, the majority of cancer patients are treated in the community, not at academic centers. Members have experience mostly with Phase II carried out in their own institution and Phase III trials conducted by the cooperative groups. Not one committee member has ever been responsible for taking a new agent all the way through clinical development (Phase I to III) and to an approval. Not one committee member has ever had to produce, negotiate, file and defend a regulatory dossier submitted to the FDA and had that product approved. The one member of the committee with this kind of experience is the Industry Representative who has a voice but not a vote. Committee members are not trained on their role at ODAC or on regulatory law. There is information on an FDA website but no active training is provided. Members are not allowed to meet in the absence of an FDA representative. Why not? This would allow for an exchange of experiences and ideas that could only have positive results. ODAC’s recommendations are just that, the agency can accept them or not. They should probably be mandatory. After all, the agency brought the agent in question to the committee because they felt that en expert recommendation was required. Why should they then decide not to follow that recommendation? Members do not see all of the applications that are filed with the FDA. The agency selectively presents those where there are issues that require the committee’s expertise. How do we know that there were no similar issues with the other applications? Why can’t the committee learn from the discussion of those other applications? What precedents are set in the approval or denial of applications never seen by the committee?

Antonio J. Grillo-López Wouldn’t it be better for the committee to review all applications? For these and many other reasons judgment is difficult. And yet, the FDA makes it look like their questions are reasonably addressed by the committee as if judgment were simple. Each committee member should ask – am I being manipulated by the FDA? Actually, judgment could be simple as will be shown below.

Regulatory Review and Approval Process There are risks and costs to a program of action. But they are far less than the long-range risks and costs of comfortable inaction. John F. Kennedy

The current process for preclinical development entails meeting all regulatory requirements (for pharmacology, toxicology, and manufacturing), filing reports on all the data on an ongoing basis and having periodic meetings with the agency. At the end of this process following another meeting with the FDA, the IND is filed. The first clinical trial can begin after a mandatory 30 days wait provided that the agency does not impose a clinical hold. During clinical development periodic meetings are held with the FDA, data on all studies is filed (clinical study reports), study conduct is subject to GCP and end of Phase II or pre-licensing application (BLA or NDA) meeting is scheduled. At this meeting all plans for the content of the application (BLA) are presented and discussed with particular emphasis on the protocol, endpoints, and statistical analysis. The company may also submit a Special Protocol Assessment (SPA) where the FDA reviews the Phase III protocol and an agreement is reached as to its validity for approval purposes (given the appropriate results). Phase III is then implemented and conducted meeting all GCP requirements and with the appropriate audits during and at the end of the study. Nowadays, most Phase III studies will include provisions for a Data Safety Monitoring Board (DSMB) or a Data Safety and Efficacy Monitoring Board (DSEMB), a mechanism for histopathological review of biopsies and a mechanism for third party clinical review and classification of responders or of radiological exams. Procedures for blinding and for reporting are carefully defined. Interim analyses require additional rigorously documented processes. The cost of all of this has risen exponentially over the past few years. It is said that the cost of bringing one new drug to market exceeds a billion dollars and in a Phase III solid tumor study the per-patient, fully allocated, costs can exceed $100,000.00.

621 The BLA is filed with the FDA upon the completion of Phase III after the necessary patient observation period. Completing all of the documentation necessary for the BLA will take an average of 6 to 8 months from last patient observation date (last patient last visit) or, depending on the primary endpoint, from the median patient’s last visit. The FDA then has a certain period of time to review and approve the application. In the case of an application for accelerated approval that might be 6 months. Nevertheless, the FDA can ‘stop the clock’ at any time or occasionally even ‘restart the clock’. The agency can also request that the new agent be presented to ODAC. Again, a long and drawn out process that favors inaction over action. There are numerous possibilities for improvement. An action oriented process is sorely needed. A different way of looking at the regulatory review and approval process is by considering its overall place in the larger scheme leading to treatment optimization. It is through combination therapies that patients get the most benefit. The optimal role of any new agent within a combination regimen takes many years to define. Nitrogen mustard was available and used effectively as a single agent (despite its toxicity) for over 20 years before its role in the MOPP regimen was defined and found to constitute a curative therapy for Hodgkin’s Disease (Fig. 3). Many patients benefited from Nitrogen Mustard during those 20 years. Thus, one could envision a process that begins with discovery, followed by preclinical development, clinical development, regulatory review and approval, and reaching completion with treatment optimization. The FDA’s role is modest when viewed from this perspective. Their responsibility should only be that of ensuring the safety of new anticancer agents and thus limited to the review and approval of data generated during clinical development. Everything else they regulate today is really the responsibility of the company developing the new agent (discovery to approval) or of the oncology community (approval to completion of treatment optimization) (Fig. 4).

Accelerated Versus Regular Approvals Take calculated risks. That is quite different from being rash. George S. Patton

The two mechanisms for the approval of new anticancer agents of approval are (1) Regular Approval and (2) Accelerated Approval. Requirements for Regular Approval include evidence of clinical benefit as measured by endpoints reflecting improved quality or quantity of life or the use of established surrogate endpoints for clinical benefit such as survival, disease free survival,

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Figure 3. Nitrogen mustard

Figure 4. New anticancer agents: Optimization

and improvement in tumor-related symptoms. In contrast, Accelerated Approval requirements allow the use of surrogate endpoints reasonably likely to predict clinical benefit (for example, durable response) provided there is “substantial evidence” of efficacy from wellcontrolled clinical trials [40, 41]. This mechanism is specifically for situations involving “serious and lifethreatening” illness where patients’ diseases are unresponsive or patients are intolerant of available therapies (i.e. medical need). The new therapy must provide an advantage over existing therapies. Importantly, an additional requirement is the implementation of postapproval, confirmatory studies. The FDA’s approval is contingent on subsequent confirmation of efficacy pro-

vided by these studies, and approval can be withdrawn depending on their results. Accelerated approval was instituted because Regular Approvals took too long and there was a desire to bring new agents earlier to the patients who needed them. Clearly there is an element of risk as Accelerated approval occurs earlier when data is not as mature as for a Regular approval. Regular Approval, however, signifies that large numbers of patients may have to go untreated because an effective new agent is not available. Accelerated Approval implies a calculated risk. This risk is minimized when safety is acceptable and when mature data will be available in a reasonable period of time.

Antonio J. Grillo-López The FDA, as will be seen in 8.0 below, has a less than desirable track record with Accelerated Approvals. They are bound by their own regulations to accept this mechanism and yet they oppose it and have impeded its use. A recent example is the case of satraplatin. This is an oral platinum compound that was presented at the ODAC meeting of July 2007. Satraplatin has been evaluated in a large Phase III clinical trial (randomized, double blind) for the indication of androgen independent Prostate Cancer in patients who have failed at least one prior chemotherapy regimen [42]. The company applied for and obtained an SPA (reviewed and approved by the FDA). They then applied for an Accelerated Approval based on the protocol and SPA defined endpoint, progression free survival (PFS), for an interim analysis. The median PFS was 9.7 weeks for the control arm (prednisone) and 11.1 weeks for the satraplatin + prednisone arm. This 15% increase in PFS is a modest but statistically significant improvement in PFS and valuable to the patient that has already failed at least one chemotherapy regimen. The FDA asked ODAC several questions, some were endpoint related technical issues and the last was whether or not they should wait until completion of the study and availability of OS data for consideration of the application. At the end of the day, the committee voted only on this last question agreeing unanimously that the FDA should postpone any decision on the application pending completion of the Phase III study and availability of OS data [43]. I believe that the consequences of this decision are not well understood by the committee, the media or the public. The situation is as follows: 1. The question was totally inappropriate. The committee is not there to rule on whether an Accelerated or a Regular Approval should be considered. This is a regulatory issue to be decided by the FDA. The application was for an Accelerated Approval and that is what the FDA should have asked the committee – is there a medical benefit to patients and should this drug be granted Accelerated Approval. This question needed to be addressed independently of the availability, at some later point, of OS data. 2. Why was the question asked? The FDA had several options regarding the application: (a) They could have denied it and yet leave the door open for re-submission when OS data was available. This option presented relatively little risk for the FDA. (b) They could have approved; but then, they would have to face the uncertainty as to whether the OS data would support approval. This option is feared by the FDA because of the negative publicity it generates if the data does not support approval. And yet, this has

623 happened only exceptionally. There is some risk but this is the risk that the Accelerated Approval mandate recognized and intended be taken. Most Accelerated Approvals will make useful new agents available earlier. A minority will later fail to have a confirmed advantage and will need to be withdrawn. The FDA is fully authorized to withdraw approvals in such cases. (c) The FDA could ask the question of ODAC and thus shift the responsibility and the possible negative fallout from the agency to the committee, and this is what they chose to do. 3. What response was sought? The FDA must have thought that there was some merit to this new agent; otherwise they would have denied the application outright. However, it was much less risky for them to wait until there was confirmatory OS data. They wanted the committee’s support and wanted the committee to be responsible for their decision to wait. The ODAC was manipulated by the FDA to achieve their desired end result. Unfortunately, all of this means that satraplatin will not be available for approximately another 2 years even if all goes well with the re-submitted application (based on OS data). This does not hurt the FDA, the agency doesn’t seem to care and doesn’t want to take the risk. This is not understood by committee members, they have been manipulated before. Most of the media misinterprets the situation. The public is biased against pharmaceutical industry because of high drug prices and any setback for a company is not viewed negatively. It is only the patients who could benefit from satraplatin who will have to wait and suffer. It is difficult to understand how the availability of an oral platinum, the first of its kind, could be anything other than a positive outcome. The oncology community should have the opportunity to evaluate satraplatin in different combinations and find the optimal treatment regimen.

Clinical Trial Endpoints We will never have all the facts to make a perfect judgment, but with the aid of basic experience we must leap bravely into the future. Russell R. McIntyre

The agency has developed guidelines for the conduct of clinical trials and the endpoints to be measured in determining efficacy. They have reviewed their performance regarding endpoints for approval of new anti-cancer agents in 2003 [44] and more recently they have issued new guidelines [45]. The bottom-line is that the FDA continues to favor the most conservative approach. They favor

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OS as the ‘most reliable…, precise and easy to measure …, preferred endpoint’ and state that it should be ‘evaluated in randomized controlled studies’ [45]. It is obvious that they do not favor Accelerated Approval because it allows for single arm trials with endpoints other than OS. Nevertheless, one may still be able to negotiate and come to an agreement with the FDA to utilize TTP or PFS as primary endpoints. In fact, some new agents have even been approved with RR (response rate) as the primary endpoint. What the FDA does not realize is that OS is plagued by multiple factors that introduce bias when this endpoint is utilized. They claim that (in the case of OS) ‘bias is not a factor in endpoint measurement’. Obviously, prolongation of survival when cure is not possible is a most desirable objective for the individual patient as well as for those around him. Even so, this is not a strong argument for OS as an endpoint because survival as a goal for the patient is an entirely different issue than survival as an endpoint for clinical trials. Using OS as an endpoint has major drawbacks. These studies require more patients, more time, more resources, cost much more, and delay the availability of useful anti-cancer agents. Using OS as an endpoint prolongs clinical trials, delays cancer drug approval and is not in the better interests of all patients, current and future. Shouldn’t we be trying “to help or at least to do no harm?” That is not achieved by using OS as an endpoint. OS feels comfortable to the FDA as it appears to decrease the risks that they take whenever they approve a new agent. It only requires two time-points: date on study and date of death. Death is easily confirmed via a death certificate. OS is tantalizingly deceptive as it gives the impression of being so firm, simple, well defined and definitive that little judgment might be required in its interpretation whereas other endpoints might appear to be more problematic [34]. Actually, TTP and PFS are better endpoints even though they do require the performance of CTs, bone scans, or MRIs. These endpoints are not subject to the multiple factors affecting OS, particularly subsequent therapies, which are unrelated to the experimental therapy under evaluation and make OS a poor endpoint even for randomized and controlled clinical trials. Studies utilizing OS as the primary endpoint require more patients, more time, more resources, cost much more, and delay the availability of useful anti-cancer agents. Using OS as an endpoint prolongs clinical trials, delays cancer drug approval and is not in the better interests of the cancer patient. Instead of OS we should use endpoints with clear cause and effect. In the case of TTP, the cause is the biologic activity of the anti-cancer agent being studied, and the effect is lack of progression, an effect that ends when progression occurs. Both the onset and the end of the effect are easily measured (on-study date to date when progression is

identified) and occur during the study before any other therapy is administered. In the case of OS, the effect is survival duration. The cause is the biologic activity of the anti-cancer agent being studied as influenced by subsequent therapies, supportive care, etc. The effect, as modified by all of these factors, ends when death occurs. Death occurs after the study has ended and patients may have gone on to other studies with experimental drugs and/or received additional conventional chemotherapy. Endpoints such as TTP and PFS are much better endpoints for clinical trials than OS. In fact, the FDA has approved more new anti-cancer agents in the past 15 years based on these than based on OS. Choosing the perfect endpoint is not easy but ‘with the aid of basic experience we must leap bravely into the future’ and the future lies with the shorter timeframes of TTP and PFS.

The FDA’s Track Record Statistics is a tool, it is meant to serve, not to enslave [9].

In the past 15 years, since Accelerated Approval regulations have existed, the FDA has granted approval for around 30 anticancer indications [46–48]. If one counts only the first indication for each new agent, there have been a total of 23 new agents that have been granted Accelerated Approval. However, only 12 of these 23 agents were approved based on response as the sole endpoint and based on a single arm trial. This is the subset that truly represents the intent of “Accelerated Approval”. In the case of the other 11 agents there were multiple indications, and randomized trials were available for some, or the endpoints included some measure of response duration. Twelve agents in 15 years, less than one agent per year, is hardly a record of which to be proud and clearly indicative of the FDA’s bias against Accelerated Approval. Accelerated approval has been made more and more difficult since it became available in 1992. It appears as if the intent is to dissuade applicants from utilizing this mechanism. Pharmaceutical companies have become increasingly reluctant to use this mechanism. The FDA’s track record to date represents a failure to capitalize on an opportunity to bring many more promising new agents early-on to the patient. They do use their statistics to make it look otherwise. But then, the FDA does use statistics in different ways such as a tool in their decision making. However, they do go to the extreme of expecting that statistics will substitute for common sense and good judgment [9]. Some believe that Statistics is precise, an exact, science. They would have you believe that an analysis is correct if the p-value is less than 0.05 or better yet, less than 0.01 and that truth lies in a favorable hazard ratio, a significant

Antonio J. Grillo-López log-rank test, a pair of Kaplan Meier (KM) curves that do not meet, or a median PFS projection from such curves. The truth is that statistics are fallible and often inaccurate. A p-value of 0.0298 (or any p-value with four decimal places) is a joke in spite of the apparent accuracy conferred by the four decimal places. A p-value is worth only as much as the data from which it is derived. There is truth in the saying that Statistics is the science of producing reliable fact from unreliable figures. Clinical data is never 100% accurate, relevant, or specific. Patients and patient populations are remarkably heterogeneous biological entities that change and evolve constantly. It is impossible to reliably quantify an endpoint that depends on multiple and variable factors. Patient populations even in the most carefully stratified, randomized and balanced clinical trials will be heterogeneous. You may think that prognostic factors are balanced, but, as an example, two arms balanced on the number of prior therapies can be quite different depending on the nature of the therapies and the patient’s response. How can you stratify or balance for immune system responsiveness or for bone marrow reserves when we can’t even measure those very accurately? Some are concerned when two curves on a Kaplan Meier graph for PFS come together at the tail end. The only way that two KM curves will not come together at some point [in a TTP, PFS or OS graph] is when one of the two therapies confers immortality. A median PFS, from a Kaplan Meier graph, will be just a “projected or estimated PFS” until all events prior to that median have occurred or there are no remaining temporarily censored patients before that median. Only then will that median be fixed, before that there is every probability that it will change. The shape of the curve also changes over time. And, likewise, it will be fixed only when all events affecting the curve have occurred. Few clinicians truly understand Kaplan Meier graphs, their intricacies or shortcomings. The FDA errs in the opposite direction. They expect too much of statistics.

Is there a Simpler Approach to the Development of New Therapies? A Proposal I don’t know the key to success, but the key to failure is trying to please everybody. Bill Cosby

A simpler approach would be to approve new anti-cancer agents based on reasonable safety and efficacy in Phase II clinical trials [6]. This would reduce average clinical development time from 7 to 3 years. Clinical cancer

625 drug development and clinical cancer treatment development are two related but distinct objectives. It is critically important to understand this distinction. The FDA is intended to regulate drug development and not treatment development.

Drug Development Versus Treatment Development Clinical cancer drug development may be defined as the process required for the development of a new anticancer agent (single agent). It is usually carried out by a pharmaceutical company, is tightly regulated and influenced by the FDA, and has drug approval and marketing as its goal. Clinical cancer drug development consists of a lengthy series of activities that require expert professional staff and complex and costly equipment, systems and procedures. Clinical cancer treatment development may be defined as the process required for the development of a new anticancer treatment (combination or multimodality). It is usually carried out by academic institutions, consortia, or cooperative groups, is loosely regulated and not significantly influenced by the FDA, and has the development of new combinations or multimodality treatments as its goal (Fig. 5). The FDA’s responsibility, as established by law, is the review and approval of new anti-cancer agents. Pharmaceutical industry is responsible for cancer drug development and this is what the agency is required to regulate. Clinical cancer drug development should consist of the appropriate Phase I and Phase II clinical trials (sponsored by a pharmaceutical company) to determine the safety and clinical activity of a new anti-cancer agent as monotherapy. Development should be collapsed to the shortest possible timeframe and allow for early termination of ineffective agents. This would also allow for the expeditious development of the truly worthwhile drugs and decrease overall development time and cost (Fig. 6). The responsibility for cancer treatment development should be squarely on the shoulders of the oncology community. It is the oncology community (including academic institutions, cancer centers, cooperative groups, consortia, and others) that, in collaboration with pharmaceutical industry, should take a new anti-cancer agent through the appropriate Phase II and Phase III clinical trials to find its optimal use in combination with other agents. It is the oncology community that should be responsible for defining the role of a new agent within a treatment regimen and its place in the therapeutic armamentarium. This task is complex and requires multiple clinical trials of different combinations. The Phase III

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Regulatory process for approval of biologicals for cancer therapy

Figure 5. Cancer treatment development

Figure 6. Cancer drug approval: FDA

studies require large numbers of patients and take a long time to complete. It can take decades before the ultimate use of a new anti-cancer agent, within combination regimens, can be elucidated (Fig. 4). Nevertheless, during this time, patients can benefit from treatment with the new agent even though its optimal use in combination is still in the process of being defined. This process, cancer treatment development, should not be regulated by the FDA.

Kinds of Useful Anticancer Agents It is practical to classify clinically useful anti-cancer agents in three categories from the development as well

as from the regulatory viewpoint. These are: (a) agents with significant activity as single agents, (b) agents with some activity and/or synergy with other agents, and (c) agents with no significant clinical activity as single agents but with significant synergism with other agents. Agents with significant activity as single agents have a response rate that compares favorably with that of other available agents (when used as monotherapy). They should be approved on the basis of Phase II trials with data from historical controls. The entire application need not include more than 300 to 350 patients. Some measurement of response duration, such as TTP or PFS, would be useful but should not be an absolute requirement. After all, these new anti-cancer agents

Antonio J. Grillo-López are most frequently combined with other agents rather than used as monotherapy. It is the response duration of the combination that is of interest. However, the optimal combination may take years to define. In the meantime, these agents could be approved and made available to patients who may benefit from them. What is the downside of such an approach? At the extreme, the FDA may approve a drug that later shows toxic effects not seen in the original experience included in the application, and/or the response rate may turn out to be lower than originally determined, and/or no additive effect is seen when used in combination regimens. It is unlikely that all of these will be observed. If this extreme situation does occur, then approval can be withdrawn or, alternatively, the drug will die on its own as it will not be prescribed (a sort of market induced apoptosis). There are risks but they are small relative to the potential benefit of having an active agent approved earlier rather than much later. Agents with some activity and/or synergy with other agents have a response rate lower than that of other available agents (when used as monotherapy). They may or may not have synergism with other drugs or combinations. Their approval requires a risk benefit determination. The FDA will not approve such agents unless they show superiority or non-inferiority to a standard regimen in a Phase III trial. Thus, in most instances, such an agent will live or die based on its activity within a combination that will probably not be the optimal combination. These agents should be approved based on Phase II trials and the oncology community allowed to conduct the multiple studies that will eventually show how they may best be utilized in combination regimens. As above, there are risks, but not much to lose in approving such an agent. Agents with no significant clinical activity as single agents but with significant synergism with other agents are inactive as monotherapy but exhibit significant synergy with other drugs or combinations. In this case, there is no choice but to carry out controlled, randomized Phase III trials to show the synergistic effect. Two of these three kinds of useful anti-cancer agents can be approved based on reasonable safety and efficacy in Phase II clinical trials. They would be available to patients earlier, development costs and drug prices should decrease and the oncology community could proceed with combination studies earlier so that new curative regimens might be identified. A criticism might be that Pharmaceutical Industry will benefit from these earlier approvals. This should be tempered by the additional risk and cost that early approvals entail including the occasional approval withdrawal or failure in the marketplace. All in all, it seems like a win–win situation for the patient.

627 It is hard to please everybody but this does seem like a reasonable and simple approach.

Conclusions We cannot avoid meeting great issues. All that we can determine for ourselves is whether we shall meet them well or ill. Theodore Roosevelt

There is a regulatory process in place for the approval of biologicals for cancer therapy and, whether we like it or not, it must be followed if new anticancer agents are to be developed and approved. The process is unduly complex and the requirements many times unnecessary or overly rigorous. The FDA’s interpretation of their mandate has gone above and beyond what was originally envisioned; but then, they themselves interpret the laws by which they must abide. Additionally the current process significantly delays the availability of new agents to the patients who can benefit from them. This must change. The proposal discussed in section 9.0 above (approval based on reasonable safety and efficacy in Phase II clinical trials) is a possibility that should be seriously considered. It would serve to reduce average clinical development time from 7 to 3 years (Fig. 6) making the new agent available to patients earlier. Also, it would make the new agent available to the oncology community earlier for the broad combination studies that would eventually lead to the identification of the agent’s role within the optimal treatment regimen (Fig. 5). Every person interested in the wellbeing of the cancer patient must help effect this change. These are great issues and no one can avoid meeting them.

Curing Cancer Destiny is not a matter of chance, it is a matter of choice; it is not a thing to be waited for, it is a thing to be achieved. William Jennings Bryan

We know that we can cure cancer. Rituximab is an example of a biological that has significantly increased the cure rate in an important cancer (DLBCL). Every year there are in excess of 15,000 NHL patients in the US, another 15,000 in Europe, and a total of over 50,000 worldwide who are potentially curable if treated with R + CHOP. This also means that every year delay in getting rituximab to the market cost over 50,000 lives. We need more efficient and faster processes for the approval of cancer therapies. In 2004 there were over 150 new anticancer agents in clinical trials, including 59 biologicals. Of those, 13 were in Phase III. Of the 59 biologicals only

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Regulatory process for approval of biologicals for cancer therapy

5 have been approved. We need to do better than this. The destiny of the cancer patient is a matter of choice and the choice must be to act faster, decrease bureaucracy and delays, approve earlier and make promising new agents available to those who need them. Just like rituximab there may be new agents in development that can extend lives or be curative. They must be evaluated earlier with a view to their optimal role in combination regimens. Regulatory review and approval processes should help and not impede this ultimate objective.

The Patient’s Plight Were we directed from Washington when to sow, and when to reap, we should soon want bread. Thomas Jefferson

The patient is at the receiving end of drug development. The system is such that it takes a long time for effective drugs to go from discovery to being broadly available to patients. It is the government that imposes the delays, hurdles and restrictions. The patient has to wait and yet, the patient does not have the luxury of time. FDA reform must stop being a buzz word heard only prior to elections. It must become reality.

What does the Future Hold? I believe that the essence of government lies with unceasing concern for the welfare and dignity and decency and innate integrity of life for every individual. Lyndon B. Johnson

Today’s physicians have a responsibility to actively participate in making new therapies available to the cancer patient. We know that cancer can be cured. However, biologicals and all new anticancer agents must be efficiently and rapidly evaluated to find those that are most promising. They must then be incorporated into combination regimens that provide the greatest opportunity for cures. Physicians in the pharmaceutical industry must do everything within their reach to ensure that new agents are developed and submitted for approval in the most expeditious fashion. Physicians in our regulatory agencies (FDA, EMEA, and others) must likewise understand that they have similar duties and responsibilities to the patient. Being employed by a bureaucracy does not mean that your sacred oath is no longer valid. In fact, physicians in regulatory agencies could be an important catalyst in making new promising therapies available to patients in a timely fashion. Government must come to understand that they can correct the issues that we face given the current regulatory process for the approval of biologicals and other cancer therapies. They

need to act and show they are truly ‘concerned for the welfare and dignity and decency and innate integrity of life for every individual’.

References 1. Smith CM (2005). Origin and uses of primum non nocere – above all, do no harm! J Clin Pharmacol 45(4):371–377. 2. Portlock CS and Rosenberg S (1979). No initial therapy for stage III and IV non-Hodgkin’s lymphomas of favorable histologic types. Ann Intern Med, 90:10–13. 3. Horning S and Rosenberg S (1984). The natural history of initially untreated low-grade non-Hodgkin’s lymphomas. N Engl J Med 311(r):1471–1475. 4. Grillo-López AJ (2001). The important role of monoclonal antibodies in the treatment of non-Hodgkin’s lymphomas. Onc Spec 2:700–705. 5. World Medical Association International Code of Medical Ethics (2006). Modern version of the Hippocratic oath. http://www.wma. net/e/policy/c8.htm 6. Grillo-López AJ (2005). The ODAC chronicles: Part III. The FDA’s philosophy and process for cancer drug evaluation and approval (Editorial). Expert Rev Anticancer Ther 5(1):1–5. 7. Grillo-López AJ (2007). Curing cancer: A historical perspective on the development of rituximab. Book chapter in: Rituximab-Mediated Molecular Signaling and Interaction with Chemotherapeutic Drugs (B Bonavida, Ed.), Transworld Research Network, Kerala, India, pp. 1–7. 8. Grillo-López AJ (2001). Curative therapies for NHL: A question of time! (Editorial) in Monoclonal antibodies in the treatment of hematologic malignancies – A special issue of Curr Pharm Bio 2(4):1–2. 9. Grillo-López AJ (2004). The ODAC chronicles: Part II. Statistics and clinical medicine in the USA – the triumph of science over art? (Editorial). Expert Rev Anticancer Ther 4(6):941–944. 10. Dillman RO (1994). Antibodies as cytotoxic therapy. J Clin Oncol 12(7):1497–1515. 11. Ehrlich P (1957). The Collected Papers of Paul Ehrlich, Vol II (IF Himmelweite, M Marquardt, and H Dale, Eds.), Pergamon Press, London, England, pp. 550–557. 12. Grillo-López AJ, Dallaire BK, Varns CL, et al. (2000). Rituximab: The first monoclonal antibody approved for the treatment of lymphoma. Curr Pharm Biotechnol 1(1):1–9. 13. Grillo-López, AJ (2003). Rituximab: The first decade (1993–2003). Expert Rev Anticancer Ther 3(6):767–779. 14. Grillo-López AJ (2002). Zevalin: The first radioimmunotherapy approved for the treatment of lymphoma. Expert Rev Anticancer Ther 2(5):485–493. 15. Grillo-López AJ (2005). 90Y-ibritumomab tiuxetan: Rationale for patient selection in the treatment of indolent non-Hodgkin’s lymphoma. Semin Oncol 32(1, Pt. 2):44–49. 16. Reff ME, Carner K, Chambers KS, et al. (1994). Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 83(2):435–445. 17. Anderson DR, Grillo-López A, Varns C, et al. (1997). Targeting cytotoxic immunotherapy: Targeted anti-cancer therapy using rituximab, a chimaeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin’s B-cell lymphoma. Biochem Soc Trans 25:705–708. 18. Demidem A, Lam T, Alas S, et al. (1997). Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B-cell lymphoma cell line to cell killing by cytotoxic drugs. Cancer Biother Radiopharm 12(3):177–185.

Antonio J. Grillo-López 19. Maloney DG, Liles TM, Czerwinski DK, et al. (1994). Phase I clinical trial using escalating single-dose infusion of chimeric antiCD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma. Blood 84(8):2457–2466. 20. Berinstein NL, Grillo-López AJ, Bence-Bruckler I, et al. (1998). Association of serum rituximab (IDEC-C2B8) concentration and anti-tumor response in the treatment of recurrent low-grade or follicular non-Hodgkin’s lymphoma. Ann Oncol 9:995–1001. 21. Grillo-López AJ (2000). Rituximab: An insider’s historical perspective. Semin Oncol 27(6, 12 Suppl):9–16. 22. Maloney DG, Grillo-López AJ, Bodkin DJ, et al. (1997). IDECC2B8: Results of a Phase I multiple-dose trial in patients with relapsed non-Hodgkin’s lymphoma. J Clin Oncol 15(10):3266–3274. 23. Maloney DG, Grillo-López AJ, Bodkin D, et al. (1997). IDECC2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood 90(6):2188–2195. 24. McLaughlin P, Grillo-López AJ, Link BK, et al. (1998). Chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: Half of patients respond to a 4-dose treatment program. J Clin Oncol 16:2825–2833. 25. McLaughlin P, Hagemeister FB, and Grillo-López AJ (1999). Rituximab in indolent lymphoma: The single-agent pivotal trial. Semin Oncol 26(5, 14 Suppl):79–87. 26. Grillo-López AJ, Varns C, Shen D, et al. (1999). Overview of the clinical development of rituximab: First monoclonal antibody approved for the treatment of lymphoma. Semin Oncol 26(5, 14 Suppl):66–73. 27. Wei A, Shen D, Kim D, et al. (2003). The Electronic Solution to the FDA Submission – Notes From the Field. Appl Clin Trials 12(10):60–64. 28. Czuczman M, Grillo-López AJ, Saleh M, et al. (1995). Phase II clinical trial of IDEC-C2B8/CHOP combination therapy in low grade lymphoma: Preliminary results. Proc Am Soc Clin Oncol 14:401 (#1261). 29. Demidem A, Lam T, Alas S, et al. (1997). Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs. Cancer Biother Radiopharm 12(3):177–185. 30. Vose JM, Link BK, Grossboard ML, et al. (2001). Phase II Study of Rituximab in combination with CHOP chemotherapy in patients with previously untreated, aggressive non-Hodgkin’s lymphoma. J Clin Oncol 19:389–397. 31. Vose JM, Link BK, Grossbard ML, et al. (2005). Long-term update of a phase II study of rituximab in combination with CHOP chemotherapy in patients with previously untreated, aggressive nonHodgkin’s lymphoma. Leuk Lymphoma 46(11):1569–1573. 32. Coiffier B, Lepage E, Briere J, et al. (2002). CHOP chemotherapy plus rituximab compared with CHOP alone in elderly

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patients with diffuse large B-cell lymphoma. N Engl J Med 346(4):235–242. FDA History. http://www.fda.gov/oc/history/default.htm Grillo-López AJ (2004). The ODAC chronicles – Part I: My first ODAC experience (Editorial). Expert Rev Anticancer Ther 4(5):579–582. Establishment of standing technical advisory committees for Human Prescription Drugs. Code of Federal Regulations. Title 21, Volume 1 [Revised as of April 1, 2003], Part 14, Subpart I, Section 14.160. Committee Charter: Oncologic Drugs Advisory Committee. http:// www.FDA.gov. Guidance for Industry Advisory Committees: Implementing Section 120 of the Food and Drug Administration Modernization Act of 1997. http://www.fda.gov/cder/guidance/index.htm Grillo-López AJ (2005). The ODAC chronicles: Part VIa. ODAC’s structure and function (Editorial). Expert Rev Anticancer Ther 5(4):573–577. Grillo-López AJ (92005). The ODAC chronicles: Part VIb. ODAC’s structure and function (Editorial). Expert Rev Anticancer Ther 5(5):753–756. New Drug, Antibiotic, and Biological Drug Product Regulations; Accelerated Approval, 57 Fed. Reg. 58,942 (1992) [codified as amended at 21 CFR 314.500-.560, 601.40-.46 (1999)]. Subpart E – Accelerated approval of biological products for serious or life-threatening illnesses. 57 Fed. Reg. 58959 (1992) (Cite 21CFR601.40). Briefing documents for the ODAC meeting of July 24, 2007. http:// www.fda.gov/ohrms/dockets/ac/07/briefing/2007-4309b1-00-index.htm US panel: FDA needs more data on GPC cancer drug (2007). Reuters News Agency 24 July 2007. http://www.reuters.com/article/ governmentFilingsNews/idUSN2437083920070724 Johnson JR, Williams G, Pazdur R (2003). Endpoints and United States Food and Drug Administration approval of oncology drugs. J Clin Oncol 21:1404–1411. Guidance for industry: Clinical trial endpoints for the approval of cancer drugs and biologics (2007). http://www.fda.gov/cder/ guidance/7478fnl.pdf FDA Approval Statistics (2007). FDA/Center for Drug Evaluation and Research Last Updated: 08/20/2007 07:47:37. Originator: OTCOM/DLIS. HTML by SH. http://www.accessdata.fda.gov/ scripts/cder/onctools/statistics.cfm Dagher R, Johnson J, Williams G, et al. (2004). Accelerated Approval of oncology products: A decade of experience. J Natl Cancer Inst 96:1500–1509. Grillo-López AJ (2005). The ODAC chronicles: Part IV. Hurdles pre- and post-accelerated approval (Editorial). Expert Rev Anticancer Ther 5(2):197–200.

21.1 Cancer biotherapy: 2009 disease-related activity ROBERT K. OLDHAM AND ROBERT O. DILLMAN

In the last decade, we have witnessed a burgeoning number of biologicals under clinical investigation, either singly or in combination with other biologicals or chemotherapeutic agents. Biotherapies have demonstrated efficacy against certain malignancies and are approved for general use in medicine. It is expected that their assimilation into our standard anticancer armamentarium will continue to broaden in the next decade, leading to the dominance of biotherapy in cancer treatment soon after the year 2010. This chapter will summarize disease-related activity for selected biotherapies as we move into the second decade of the new millennium. Historically, cancers have been classified by histopathological features, based on the pathologist’s ability to discern similarities and differences among cancers by scrutinizing tissue sections under the microscope. Over 100 types of cancers have been so categorized. This pathology-based classification scheme, upon which the previous standard modalities of cancer treatment – surgery, radiation therapy, and chemotherapy – were structured, has contributed to major advances in the treatment of selected malignancies. As cancer biology and cellular genetics, including cancer susceptibility genes become better understood, we are learning that the unregulated growth of tissues, the activation of oncogenes, mutations, the persistence of cells that normally become senescent, and maturational aberrations may segregate in patterns not amenable to simplistic histopathological classification. Early evidence of such conflicts in categorization is provided by the presence of similar antigens on cancer cells arising from very different tissues. For example, antibodies have been described that react very clearly with breast cancer, colon cancer, and certain other adenocarcinomas. As such, these antibodies cross histopathological and anatomic borders. Similarly, there may be common reactivities among tumors of lymphoid origin, of squamous differentiation, and of mesenchymal derivation. A better understanding of oncogene activation, genomics and of normal and aberrant regulation of growth and differentiation may provide for a dynamic classification scheme rather than a static descriptive one

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

we now use. As new approaches in biotherapy are tested, scientists and clinicians must be constantly aware that our current histological anatomic classification system is largely artificial and that clues to the clinical activity of biotherapy may allow us to reclassify neoplasms according to biological features and behavior patterns. Indeed, recent advances in the molecular analysis of tumors by gene expression profiling and proteomics have begun to lead to a new paradigm for the classification of malignancies based on the molecular signature of an individual’s tumor derived from the global assessment of thousands of genes. By examining the clustering of gene expression, new categories of malignancy have begun to emerge that go beyond traditional distinctions based on histology alone [6]. Gene profiling and microarray analysis have recently been shown, by retrospective studies, to correlate with prognosis. Current efforts are focused on using this kind of genetic information to design clinical trials in breast, colon and lung cancer. The hypothesis in breast cancer is that a certain profile might predict for a better prognosis using hormone therapy alone vs. adding chemotherapy for those individuals having a gene profile consistent with more aggressive disease. Once such a hypothesis is validated through current prospective clinical trials, the adjuvant therapy of breast cancer will be much improved, given that we can focus chemotherapy on the most likely subgroup to benefit [2, 3, 4, 7, 13]. Such considerations transcend all aspects of classical developmental therapeutics, in which surgery, radiation therapy, and chemotherapy evolved along disease-specific and anatomic lines. Thus, although phase I toxicity trials are conducted across histological boundaries, phase II and III studies progress within a fairly rigid disease-specific format. As a consequence, antitumor activities in lung cancer, breast cancer, colon cancer, etc. are discussed and debated. While such a testing paradigm may have served oncologists well for protocol construction and for regulatory oversight, this mindset of classical pathology oriented phase II–III studies may actually inhibit the development of biotherapy [8]. Furthermore, the development of biological therapies and molecularly targeted

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632 anticancer agents poses special challenges to the design of clinical trials to include models that assess the effect of the agent on its putative target and the validation of new surrogate endpoints [12]. The traditional objectives of phase I and II studies (maximally tolerated dose and response rate, respectively) developed for cytotoxic agents are less relevant to biological and targeted therapies where a “biologically effective dose” may prevent tumor growth and induce stabilization of disease that is of important clinical benefit but is not reflected by a radiographically measurable objective response. With these new modalities, we must be open to the possibility that the biological activity of these agents and approaches may sort unpredictably and may require a more individualistic orientation. It is abundantly clear that monoclonal antibodies, proteomics, and gene expression profiling technology can allow for the precise description of an individual patient’s cancer using panels of antibodies, mass spectroscopy signatures, and microarrays that represent all of the expressed genes in the human genome [1, 5, 10, 11]. The use of the laboratory to develop patient-specific therapies may allow scientists and clinicians to approach the cancer problem from a completely different and more individualized perspective [9]. Many biotherapies are supportive or ancillary to the anti-tumor activity of standard approaches such as chemotherapy. Use of colony-stimulating factors, erythropoietin, blood transfusions, marrow transplantation, etc. has broad application in oncology. These techniques were described in earlier chapters. While recognizing the limitations of organ-specific categorization and the likely reclassification of tumors based on either gene expression clusters or the expression of common molecular therapeutic targets, it remains a standard and useful exercise to describe the clinical activities in biotherapy within such a historical framework. Therefore, in this chapter, a brief summary of the activity of various forms of biotherapy will be provided by cancer type. This chapter is designed to describe

Cancer biotherapy: 2009 disease-related activity these activities briefly with references into 2009 and is for quick reference only. The reader is encouraged to refer to specific, subject-oriented chapters and references for detailed information on specific methods of cancer biotherapy.

References 1. Avner BP, Liao SK, Avner B, DeCell K, Oldham RK. Therapeutic murine monoclonal antibodies developed for individual cancer patients. J Biol Response Mod 1989; 8:25–36. 2. Brenton JD, Carey, LA, Ahmed AA, et al. Molecular classification and molecular forecasting of breast cancer: Ready for clinical application? J Clin Oncol 2005; 23(29):7350–7360. 3. Cheang MCU, Voduc D, Bajdik C, et al. Basal-like breast cancer defined by five biomarkers has superior prognostic value than triplenegative phenotype. Clin Cancer Res 2008; 14(5):1368–1376. 4. Cronin M, Sangli C, Liu M-L, et al. Analytical validation of the oncotype DX genomic diagnostic test for recurrence prognosis and therapeutic response prediction in node-negative, estrogen receptorpositive breast cancer. Clin Chem 2007; 53(6):1084–1091. 5. Lakhani SR, Ashworth A. Microarray and histopathological analysis of tumours: the future and the past? Nat Rev Cancer 2001; 1:151–157. 6. Liotta L, Petricoin E. Molecular profiling of human cancer. Nat Rev Genet 2000; 1:48–56. 7. Loi S, Sotiriou C, Buyse M, et al. Molecular forecasting of breast cancer: Time to move forward with clinical testing. J Clin Oncol 2006; 24(4):721–722. 8. Oldham RK. Biologicals and biological response modifiers: design of clinical trials. J Biol Response Mod 1985; 4:117–128. 9. Oldham RK. Custom-tailored drug immunoconjugates in cancer therapy. Mol Biother 1991; 3:148–162. 10. Oldham RK, Lewis M, Orr DW et al. Adriamycin custom-tailored immunoconjugates in the treatment of human malignancies. Mol Biother 1988; 1:103–113. 11. Orr D, Oldham R, Lewis M et al. Phase I trial of mitomycin C immunoconjugates cocktails in human malignancies. Mol Biother 1989; 1:229–240. 12. Tan AR, Swain SM. Novel agents: clinical trial design. Semin Oncol 2001; 28:148–153. 13. van ‘t Veer LJ, Mongyue D, van de Vijver MJ, et al. Expression profiling predicts outcome in breast cancer. Breast Cancer Res 2003; 5:57–58.

21.2 Biological therapy of melanoma ROBERT K. OLDHAM

Melanoma is among the human solid tumors that best exemplify the application of biological therapy to the treatment of cancer. In the first decade of the twenty-first century, more than 4 million people will be diagnosed with malignant melanoma, and the incidence continues to increase in developed countries worldwide [9]. Although primary cutaneous melanoma is highly curable, with 85% of patients enjoying long-term survival after surgical excision, disseminated melanoma was amenable only to palliation with radiation therapy and chemotherapy [41]. Although short term responses to chemotherapy are not uncommon, particularly in skin, lung and subcutaneous lesions, significant long-term benefit in visceral disease is infrequent, with 5-year survival remaining less than 5% for disseminated disease. Because melanoma frequently affects young individuals, half of the patients who die of melanoma had a life expectancy of greater than 25 years, and the productive years of life lost exceed those of any other solid tumor [121].

Melanoma and the Immune System Melanoma has long been known to be a tumor with unusual behavior in certain patients. Spontaneous, complete regression occurs in one patient in 1,000–10,000 with lesser degrees of temporary spontaneous partial regression in as high as 0.5% of patients. Such observations indicate that the body has some inherent capacity to induce regression of this disease in selected instances. Despite considerable investigation, the mechanisms of such spontaneous regressions remain unclear, and formal proof of the involvement of an innate or adaptive antimelanoma immune response is still lacking. Several features of malignant melanoma suggest that the in vivo biological responses that play a role in the progression and regression of this form of cancer involve the immune system. For this reason, melanoma is an attractive and well-examined model for the therapeutic use of biological agents, particularly those designed to enhance or manipulate the host immune response. First, the infrequent spontaneous regression of cutaneous and disseminated

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

melanoma has been carefully documented [92]. Indeed, approximately 5% of patients with disseminated melanoma do not have an identifiable primary lesion, suggesting that the cutaneous disease may have spontaneously regressed before the growth of metastases is detectable [94]. Paradoxically, partial regression of primary melanoma lesions has been reported by some observers to be associated with a worse prognosis; however, this is likely due to the difficulty that this feature adds to the assessment of risk in the pathological specimen. This phenomenon has recently been shown to involve cytolytic T-lymphocytes [37, 71]. Second, paraneoplastic depigmentation events, such as the development of vitiligo and “halo” formation around primary skin melanomas and nevi are thought to indicate an immune mediated reaction against the pigmented cells in these lesions, and the development of vitiligo in malignant melanoma during biotherapy carries an improved prognosis [24]. The corollary of this clinical observation may have therapeutic implications: if the immune recognition of melanoma can lead to the destruction of normal melanocytes, immunization with melanocyte antigens may lead to the rejection of melanomas [7]. Third, the presence of tumor infiltrating lymphocytes in primary melanoma lesions indicates a host immune response, and the degree of lymphocyte invasion is of prognostic significance [15, 25]. Fourth, there are patients whose melanoma may remain dormant for many years, suggesting innate immune surveillance and control of micrometastatic disease, who subsequently relapse and die from disseminated melanoma 10 or more years after the diagnosis of the primary lesion [108, 110]. Finally, the incidence of melanoma is higher and the prognosis worse among immunosuppressed kidney transplant recipients, providing additional support for the role of immune surveillance in the evolution of malignant melanoma [23, 34].

Biotherapy of Malignant Melanoma Early observations indicated that the immune system could be harnessed therapeutically to treat malignant melanoma. Intratumoral injection of bacterial extracts such as

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634 Bacille Calmette-Guerin (BCG) or Corynebacterium parvum frequently induced the regression of the injected melanoma and occasionally lead to tumor regression at sites of lymphatic drainage and rarely distant metastases [90]. The natural history of malignant melanoma and these early therapeutic observations provide a compelling rationale for the systematic evaluation of biological therapy in malignant melanoma. In this light, many agents have now been evaluated to enhance or mimic the host immune response against melanoma. These agents have included effector cells such as lymphocytes derived from resected tumors or the blood of melanoma patients; antibodies directed at various epitopes on the melanoma cell; vaccines composed of natural or synthetic components of the melanoma cell or derived from mimic “anti-idiotype” antibodies; non-specific stimulators of the immune system such as BCG and the cytokines. Until recently, only the cytokines have consistently shown clinical, anti-tumor activity in melanoma, and two of these cytokines, interferon alpha and interleukin-2, have emerged as useful in clinical practice for the treatment of patients with malignant melanoma [10, 53, 62]. Although durable responses are rare, cytotoxic therapy for melanoma leads to tumor regression in a substantial number of patients, and the activity of single agents and chemotherapy combinations is well established. As a result, treatment regimens that combine chemotherapy and biological therapy agents, usually cytokines, are referred to as biochemotherapy [46, 96, 109, 119]. Monoclonal antibodies that influence the immune response are currently in clinical trials. Chief among these are anti-CTLA-4 antibodies, which have been promising in early-phase clinical trials with the induction of both partial and complete responses in patients with advanced melanoma. Toxicities have been substantial with bowel inflammation and severe colitis as well as iritis being seen in many of these patients. Vitiligo also is a side effect of this form of biological therapy and may predict a good response to the monoclonal antibody therapy [6, 27, 33, 42, 91, 107]. The gene therapies for malignant melanoma are new topics of interest; in these strategies, a therapeutic gene is introduced into an effector cell or a gene encoding a strong antigen is introduced directly into the melanoma cell. Clinical trials with gene therapies are now underway.

Immunostimulation and Vaccines Attempts to augment native immunologic defenses using various non-specific immunostimulators, particularly Bacillus Calmette-Guerin (BCG), have been intensively

Biological therapy of melanoma studied in melanoma. Up to 90% of melanoma nodules injected intralesionally with BCG will demonstrate regression in immunocompetent patients. In 15–20% of such patients, associated distant shrinkage of nodules may be seen [77]. Tumor regression by the local application of BCG has also been successfully applied to the treatment of superficial bladder cancer, and the mechanism of tumor regression has been investigated in this model. The local application of BCG sensitizes host T-cells to peptides in the extracts, and the presentation of these peptides by host antigen presenting cells within the injected tumor bed triggers local T-cell cytokine production that can activate host cells that can directly recognize and kill tumor cells [98, 99, 108]. Preparations of cellular constituents, such as cell-wall skeleton, may also serve as effective adjuvants [123]. Systemic effects have been demonstrated by the emergence of antimelanoma antibodies and by lymphocytic infiltration of regressing noninjected lesions [77]. Vaccines derived from allogeneic melanoma cells have long been of interest. In one early report, an enriched tumor cell vaccine was infused intra-lymphatically, providing proof of principle for the concept of inducing host antitumor immunity with antigens from allogeneic melanoma cells by demonstrating that the effects of such intra-lymphatic immunotherapy were not limited to regional lymph nodes and that systemic response were seen [1]. These initially encouraging results have lead to intense study of this strategy of immunomodulation for at least the last quarter century, yet these efforts have not significantly improved the survival of melanoma patients [78]. However, immunizing melanoma patients with a pure Gm2 ganglioside, known to be on melanoma cell membranes, has induced circulating IgG and IgM antibodies. The induction of such antibodies has been reported to be accompanied by prolonged survival [69, 75, 80]. While definitive proof of the success of vaccines derived from allogeneic melanoma cells is lacking, at least one allogeneic cell lysate vaccine (Melacine) has undergone rigorous analysis and was approved in 1999 for the treatment of disseminated melanoma in Canada based on phase III results demonstrating superior quality of life during active therapy for disseminated melanoma compared to a four-drug chemotherapy combination [39]. The two therapies achieved similar efficacy results. Survival data was also compared from the Phase III study with a comprehensive meta-analysis of therapies for Stage IV melanoma showing that the median survival of 11 months among evaluable patients receiving this allogeneic vaccine was better than that achieved by other available therapies [59]. Further, a significantly longer median survival of 18.2 months was observed in

Robert K. Oldham

635 has yielded a response rate of approximately 20% [51, 81]. On occasion, these responses have been complete, but activity in visceral disease has been infrequent. Perhaps as many as 30% of patients achieving a complete response enjoy an enduring remission. Studies using alpha-interferon in the adjuvant surgical setting demonstrated a reduced recurrence rate with substantial doses of interferon. This study lead to FDA approval of interferon for the treatment of melanoma. Beta and gamma interferon were also evaluated in melanoma with both now approved for non-cancer indications. A suggested potentiation by cimetidine of human leukocyte interferon activity in malignant melanoma [11, 47] has not been confirmed by later studies using natural or recombinant alpha-interferon [51, 68]. There has been great interest in exploring the benefits of adjuvant IFN for high risk, surgically excised melanoma. Based on the findings in metastatic disease, it was hoped that IFN would be most active when the tumor burden was low and would increase the overall survival of patients at high risk of relapse after surgery for melanoma. Multi-institutional, randomized controlled trials (RCTs) have been conducted in the United States and Europe in an attempt to demonstrate a benefit to adjuvant IFN. The inter-group United States trials, lead by the Eastern Cooperative Oncology Group (ECOG), E1684, E1690, and E1694, have received the

patients who were clinical responders to Melacine therapy. Melacine has not been approved by the FDA for use in the U.S. as of 2008. More recent studies using genetically engineered vaccines have been performed. Details can be found in the chapter on vaccines and the chapter on gene therapy. This work was prompted by previous studies using partially purified tumor vaccines (Table 1). More recent studies have used highly purified or genetically engineered vaccines with a variety of antigens and adjuvants which are immunogenic in humans (Tables 2 and 3). Clinical trials with genetically engineered vaccines are ongoing and while there is evidence of stimulation of cytotoxic T cells, enhancement of antibody titers and augmentation of DTH responses, the anti-tumor activities of these vaccines remain to be confirmed.

Interferon In phase I trials with alpha-interferon, an occasional melanoma patient experienced objective tumor regression. Subsequent phase II studies using partially purified human leukocyte or lymphoblastoid alpha-interferon showed response rates as high as 12% [43, 95]. The composite experience using recombinant alpha-interferon

Table 1. Tumor antigen vaccine trials Stage of disease

No. of patients Vaccine preparation

Adjuvant

Resected II

25

BCG

Disseminated

56

Disseminated 13 Resected stage II 94

Autologous soluble membrane extract Specific TAA

Controls

Results

Ref.

Historical Improved survival over historical growth Freund’s complete None Regression seen in approximately 25% of patients Polyvalent TAA None None CR 1 of 13 PR 1 of 13 Polyvalent melanomatumor 40 pts – Alum Historical Improved survival over antigen vaccine 17 pts – Cytoxan historical controls

Table 2. Adjuvants: effects on immunogenicity Mechanisms of action Approach

Depot effect

Macrophage

CD4+ T cell and B cell

CTL

Conjugate vaccines Recombinant vector vaccines BCG Vaccinia Immunological adjuvants Alum or oil (squalene) BCG or BCG CWS Endotoxin (lipid A) Liposomes MDP derivatives

+

−

++

−

+ +

+ −

+ +

++ ++

+ + − + −

− + ++ − +

− + + − +

− − − + +

162 137 36 38

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Biological therapy of melanoma

Table 3. Clinical trials with immunogenic antigens Antigen

Vaccine

No. of patients responding Ref.

GM2 GM2 GD2 Mage 1 Mage 3

BCG KLH KLH – –

50/58 30/30 4/6 Ongoing trial Ongoing trial

37 131, 217 131, 217 361 106

Tyrosinase

–

Ongoing trial

29

most attention and scrutiny in North America. E1684 enrolled 287 patients and randomly assigned patients to observation versus 1 year of high dose IFN for 1 year (“high dose” defined here as at least 10 million units per square meter) versus observation. Disease free and overall survival was both improved by IFN treatment [55], and long-term follow-up data from this study was confirmatory [55, 56]. In follow-up, E1690 accrued 642 patients to observation or treatment with high dose IFN or low dose IFN. Although median survival for high dose IFN was essentially identical to the median survival for high dose IFN on E1684, overall survival was not improved by treatment with high dose IFN (or low dose IFN) [54]. The lack of benefit for IFN in E1690 appears to derive from an unexplained improvement in the median survival of the observation group in E1690 compared to E1684. A significant number of patients on observation on E1690 received IFN at the time of recurrence, which may confound interpretation of the results of IFN treatment. Finally, E1694, enrolled 774 patients to treatment with high dose IFN or a vaccine, and demonstrated that the high dose IFN significantly prolonged both disease free and overall survival [54]. The vaccine treatment arm of E1694 had a similar survival compared to the control arm of E1690, indicating that the improvement in disease free and overall survival resulted from the treatment with IFN rather than any detrimental effect of the vaccine treatment. Other large studies have explored the role of adjuvant IFN in melanoma. For example, among the more recently reported, the AIM High Study randomized 654 patients to treatment with low dose IFN for 2 years or observation. No significant effect was observed on disease free or overall survival [12]. Several other important studies, also using low dose IFN, have also been performed and failed to convincingly demonstrate an overall survival benefit for adjuvant therapy for melanoma [18]. Definition of the high risk population has differed among randomized studies, primarily depending on the inclusion of patients with intermediate or deep primary lesions or involved regional lymph nodes. In the E1684 study that initially demonstrated a benefit to adjuvant IFN, the benefits of adjuvant IFN were limited

to patients with stage III disease that had involved regional lymph nodes [55]. In addition, the dose and duration of therapy differ and among studies and this issue remains contentious. Data from the United States intergroup studies suggest that higher doses of IFN in the adjuvant setting result in greater effectiveness. A metaanalysis of ten randomized controlled trials was recently conducted and concluded that there was a clear benefit to adjuvant IFN for prolonging disease free survival but the benefits were less clear in terms of overall survival [125]. In this meta-analysis, high dose versus low dose IFN was compared, and there was no statistically significant difference between high dose and low dose IFN treatment. However, a systematic review of eight randomized controlled trials of systemic adjuvant interferon as monotherapy versus no treatment (and thus excluded E1694 from analysis) for high risk melanoma concluded that there was no clear benefit of IFN therapy on overall survival and that heterogeneity between the trials made meta-analysis inappropriate [64]. Thus, despite fairly rigorous and intensive study, controversy remains regarding the benefits of IFN in the adjuvant setting [56]. Studies of interferon alpha in metastatic melanoma patients have shown response rates ranging from 0–30% with an overall average of 16% [15, 19, 20, 32, 35, 52, 63, 112, 117]. As such, about one in six patients with metastatic melanoma will benefit from interferon alpha therapy. However, it is notable that a small percentage of the total group responding to this treatment will achieve a complete response, and occasionally, long remissions have been observed. A review of 11 phase II studies of single agent IFN conducted in the 1980s and comprising 315 patients described an overall response rate of 15% and a median survival of 8 months [61]. The dose, route and schedule of IFN administration differed among the studies, but intramuscular injections given three times weekly were usually used, and doses of 10 million units per square meter or higher (up to 50 million units per square meter) were used in all cases. Complete remissions were observed in 0–4% of patients. It is difficult to extrapolate the optimal dose and frequency of administration of interferon alpha from the available data; a very wide range of doses and frequencies have been evaluated in the reported clinical trials (Tables 4 and 5). It is also not clear that a strong dose–response relationship exists for interferon alpha in malignant melanoma. It is important to compare these responses to those observed for single agent chemotherapy (also in the 15–20% range). Higher response rates were seen with drug combinations and the best three drug combinations report responses in the 30–40% range and generally include cisplatin and dacarbazine. However, even the

Robert K. Oldham

637

Table 4. Clinical trials with recombinant interferon Study

Dose (MU) route/ schedule

Evaluable patients

CR/PR (%)

Creagen [56]

12/M2 i.m. 3 times per. week 50/M2 i.m. 3 times per week 20/M2 i.v. daily × 5 50/M2 i.m. 3 times per week 3–36 i.m. daily or 3 times per week

30

20

31

23

15 18

0 11

62

8

Creagen [58] Coates [49] Hersey [134] Legha [178]

Interleukin 2/lymphokineactivated Killer (LAK) Cells

Table 5. Biochemotherapy treatment programs Study

Evaluable patients

Interferon + DTIC Kirkwood [159] 23 Gundersen [124] 15 Mulder [220] 31 Thompson [354] 86 Sertoli [322] 72 Interferon + platinum/combinations Hamblin [130] 12 Richards [286] 74 Legha [179] 30 Pyrhonen [271] 45 Richner [288] 20

activity for the two drugs [3, 36] while a third showed no improvement over dacarbazine alone [120]. A very encouraging randomized phase II trial has demonstrated that bevacizumab (Avastin) has shown activity in metastatic melanoma when compared in a randomized phase II trial with interferon. A large randomized phase III study is in progress to compare bevacizumab plus interferon with interferon alone.

CR/PR (%) 4 20 35 21 26 83 57 56 53 35

best chemotherapy combinations have failed to demonstrate a survival advantage, and the duration of response is usually less than 6 months [60]. A recent meta-analysis explored the net benefit of IFN therapy in metastatic malignant melanoma by compiling all randomized trials that compared a treatment regimen with IFN to a nonIFN containing treatment regimen. Eleven studies were identified (five unpublished) that collectively comprised 1,164 patients. The meta-analysis showed that the regimens including IFN-alpha improved response rates and overall survival compared with regimens without IFNalpha with odds ratios of 0.65 (for response) and 0.69 (for survival). In melanoma, the response rate for the IFN containing regimens was 24% compared with 17% (range, 5–30%) for those without IFN [45]. Taken together, these data indicate that IFN is an active agent and an important component of treatment regimens for metastatic melanoma. The combination of interferon alpha with chemotherapy met with early encouragement. Several phase II studies showed the combination of dacarbazine and alpha interferon to induce responses in 25–30% of patients with metastatic melanoma [15, 36, 93, 113]. In randomized studies comparing the combination to single agent dacarbazine, two showed superior

Interleukin-2/lymphokine activated killer (LAK) cells have been reported to provide a response rate of 50% for patients with advanced metastatic melanoma [100, 124] (Table 6). Most of these responses have been partial and of only several months duration, although occasional patients do experience complete responses. Subsequent studies with interleukin 2/LAK cells and studies using interleukin 2 in combination with interferon continue to demonstrate that melanoma is a neoplasm sensitive to these kinds of biological approaches [11, 28, 31, 58, 106, 114]. Overall, response rates have been in the 20–30% range. Responses continue to be most frequent in patients with skin, lymph node, and lung metastasis and less frequent with abdominal and bony metastasis. The propensity of melanoma to metastasize to the brain has been a limiting factor, since many of these patients have responded peripherally only to relapse and die with central nervous metastases. The major challenge from these studies using interleukin-2-based biotherapies is to determine why one patient will respond with a complete remission and another patient will not respond at all. Is this related to the state of immunological activation inherent to the patient or the state achieved by these biotherapies or does it somehow relate to the sensitivity of the melanoma cells to biotherapy? If it is possible to summarize all the available data into a single response rate by combining a very heterogenous group of clinical trials, IL-2 would appear to benefit about one melanoma patient out of every five treated with an overall response rate of 18% (Table 7). Complete responses with long-term remissions, rare in chemotherapy treated patients, are occasionally seen with IL-2. Many studies have shown a higher percentage of complete responders with high dose IV bolus IL-2, but no difference in survival compared to lower dose regimens. Similar to the situation with alpha interferon, the optimal dose and schedule of administration have not been determined, and a clear dose–response relationship has

638

Biological therapy of melanoma

not been demonstrated. IL-2 has been used alone or in combination with lymphokine-activated killer (“LAK”) cells. The addition of LAK cells, while of historical importance in the original description of IL-2 therapy for advanced malignancies, provides, at best, marginal improvement over IL-2 alone [104, 105]. Good preclinical models support the combination of IL-2 and interferon alpha, but studies, both phase II and randomized trials, showed no benefit for this combination in metastatic melanoma [115]. Of historical interest is the combination of chemotherapy and IL-2. Although the clinical trials combining single agent dacarbazine with IL-2 did not demonstrate a benefit for combining these modalities (Table 8) [29, 38, 40, 118], cisplatin based regimens were more promising [2, 8, 26, 50, 63, 97]. Four trials with cisplatin and IL-2 have shown overall response rates in excess of 50%, and all of these also included alpha interferon. Randomized studies have not confirmed these data [48].

Interleukin 2/T-cells As further evidence for the interleukin-2 mediated effect on melanoma, Rosenberg et al. reported a 50% response rate using T-cells isolated from melanoma nodules and grown to large numbers in vitro, another example of adoptive immunotherapy of cancer [101, 105]. These tumor-infiltrating lymphocytes (TIL) appear to have a higher level of specific activity and, in large part, are MHC-restricted T cells specifically reactive with the individual’s melanoma from whom the T cells were derived. Concomitant studies by Dillman and Oldham [30, 65] have confirmed the feasibility of this approach using tumor-derived activated cells (TDAC) although with much lower response rates. This approach of growing T cells from the tumor and/or draining lymph nodes is

technologically demanding and very expensive [65, 66, 83]. To date, the results are not clearly superior to the use of interleukin 2 alone or interleukin with peripheral blood cells (LAK) activated in vitro. On the other hand, these cells are exquisitely more specific for the melanoma cells. Clearly, this approach validates the belief of many investigators that specific T-cell reactivity occurs in advanced cancer and demonstrates the feasibility of applying such approaches as a clinical biotherapy [29, 66, 82, 107]. Rosenberg has pioneered the use of TIL cells plus interleukin-2 in the treatment of cancer utilizing IL-2/ TIL with and without cyclophosphamide and in various schedules and doses in several clinical trials. In summary, approximately 86 patients were treated; 28 of whom had had prior IL-2 and 58 had no prior IL-2. A 34% response rate was seen for the total group of patients with the same response rates in the both the IL-2 pretreated patients and the IL-2 naive patients. Both the response rate and duration of response were equivalent in the two groups and some of the remissions were of very long duration. 111 In labeled TIL cells were given to selected patients and with nuclear imaging the presence of these labeled cells in tumor tissue was verified. Thus, both based on the in vitro studies showing enhanced killing by T cells compared to LAK cells and in studies using TIL cells clinically, where a higher response rate was seen, the evidence is accumulating that these more specific T cells are more powerful when used with interleukin-2 in the treatment of advanced melanoma. It’s of particular interest to note that patients resistant to interleukin-2 respond to interleukin-2 plus TIL cells, indicating a role for the cells alone as a therapeutic approach in treating advanced melanoma. Curiously, no one has performed the kind of T cell dose response studies normally done with drugs in patients with advanced cancer. The major question

Table 8. The activity of combined IL-2 + chemotherapy in malignant melanoma First author

Year

Chemotherapy

Papadopoulos [244] Flaherty [138] Dillman [102] Stoter [435] Fiedler [131] Blair Demchak [93] Hamblin Richards [372] Legha [244] Khayat [212] Atkins [13]

1990 1990 1990 1991 1992 1991 1991 1991 1992 1992 1993 1994

DTIC DTIC DTIC DTIC DTIC CDDP, DTIC CDDP CDDP, DTIC CDDP, BCNU CDDP, DTIC, VLB CDDP CDDP, DTIC

Other therapy

No. of patients

CR + PR

IFN IFN, TAM IFN IFN TAM

30 32 27 25 16 28 27 12 74 30 39 38

33% (10) 22% (7) 26% (7) 24% (6) 13% (2) 43% (12) 37% (10) 83% (10) 57% (40) 56% (17) 54% (21) 42% (16)

Robert K. Oldham that remains is what would be the effect of giving repeated massive doses of activated T cells to patients with advanced melanoma? What would be the influence of dose, schedule, type of T cell, etc.? These studies need to be done before conclusions on the efficacy of T cell therapy are drawn. Studies conducted by Oldham and co-workers reported a series of patients with various types of cancer treated with a combination of interleukin-2 and tumor-derived activated cells (TDAC) [65–67, 72, 73, 82, 84, 85, 87, 88]. These tumor-derived T cells are similar to the TIL cells described by Rosenberg and in these studies some 366 tumor biopsies were tested with the T cells grown successfully in 75% of the cases. Sixty-three patients were treated with a minimum of at least 1010 TDAC by intravenous infusion and partial responses were seen in eight patients, including the dramatic complete regression of scalp nodules in a patient with renal cancer. The cells were well tolerated, responses were seen in patients with renal cancer and melanoma, most of whom were previously resistant to interleukin-2 and all of whom had bulky, advanced cancer [66]. Taken together, the results of T-cell therapy indicate that these cells are active in advanced cancer. The observation that they are active in patients previously resistant to Interleukin-2 demonstrates that the cells alone have consistent activity in a minority of patients with advanced cancer. Over the last few years, Rosenberg and a few other investigators in Europe have begun to look at ways to manipulate the immune system prior to infusing the activated T-cells. Very aggressive therapy such as combinations of high-dose chemotherapy in the setting of an autologous transplant plus whole body radiation therapy followed by the infusion of autologous T-cells and Interleukin-2, has resulted in a greater than 75% response rate in the first few patients treated with this approach at the NCI. Many of these patients had bulky, Interleukin-2 resistant disease, and the induction of complete responses in such patients is very encouraging [70, 103].

Gene therapy A new category of biological therapy has become recognized, utilizing gene engineered cells. By definition, gene therapy is the introduction of genetic material into cells for therapeutic purposes. The gene therapy of cancer has focused on advanced melanoma and renal cancer models for the development of this new therapeutic approach. The first patient treated on an approved gene therapy protocol was a patient with malignant melanoma

639 who on May 22, 1989, received an infusion of tumor infiltrating lymphocytes (TILs) marked with the neomycin phosphotransferase gene (a gene which carries neomycin resistance in bacteria). While this gene transfer was not designed for the therapeutic enhancement of TIL activity, the ten melanoma patients who consented to this gene “therapy” demonstrated that foreign genes could be effectively and safely transferred into human cells and administered to patients without apparent adverse effects on the patient nor risk to the caregivers or public [102]. This study also confirmed that the engineered T cells trafficked to the melanoma nodules. These results allowed Rosenberg and colleagues to proceed to alter the TILs with therapeutic intent. On January 29, 1991, the first patient treated on a true gene therapy protocol was an advanced melanoma patient who received an infusion of TILs that contained the gene for tumor necrosis factor (TNF). Several metastatic melanoma and other advanced cancer patients were treated in this early study, and responses have been observed [101]. The rationale for this strategy is to provide high local concentrations of TNF at tumor sites. TNF is quite toxic when systemically administered, but presumably the TILs provide sufficient local concentrations of TNF for an antitumor effect while sparing systemic toxicity. Another gene therapy approach initially described by Nabel and colleagues is to introduce the HLA B-7 gene directly into cutaneous melanomas of non-HLA B-7 patients with metastatic disease [79]. The concept was to induce a cytotoxic immune response against a foreign major histocompatibility complex (MHC) class I antigen, leading to the recognition of the injected cutaneous melanoma cell as foreign and ultimately generating a systemic anti-melanoma immune response. In the initial study, the five patients treated locally injected with the HLA B7 gene all showed evidence of safe and successful gene transfer, cytotoxic T-lymphocytes were shown to invade the lesions, and one patient showed tumor regression not only of the lesion that had the HLA B7 gene delivery but also of distant cutaneous and pulmonary metastases [79]. The initial success of this strategy has lead to a number studies with the HLA B7 gene (commercialized as “Allovectin 7”), now most commonly administered intramuscularly as naked DNA. Further details of this line of investigation may be found in the chapters on gene therapy and vaccines elsewhere in this book. Other genes under evaluation include granulocyte macrophage colony stimulating factor (GM-CSF) and B2-microglobin, whose gene products will induce an antitumor immune response when inactivated autologous tumor cells are reinfused into the patient. The cytokine gene therapy concept has been expanded in other clinical

640 trials that will evaluate the feasibility of genes for IL-2, gamma interferon, TNF, and IL-4 transduced into autologous tumor cells or fibroblasts to produce active cytokines effective in the treatment of melanoma and other cancers [22]. To date, the clinical benefit and activity of gene therapy for melanoma remains to be fully delineated [5, 17, 49, 74, 76].

Antibodies Relatively specific visualization of tumor nodules with radiolabeled antibody has been reported [13] and antibody given intravenously can subsequently be demonstrated on the melanoma cell surface by histopathologic techniques [111]. However, only minor clinical activity has been observed with either unconjugated antibody or with preparations of radiolabeled, drug-conjugated, or toxin-conjugated anti-melanoma monoclonal antibodies. The 9.2.27 antimelanoma monoclonal antibody recognizes a melanoma-associated antigen (a 250-kD chondroitin sulfate proteoglycan core glycoprotein) that is found on 90% of melanoma cells and relatively few non-melanoma cells [86]. Oldham et al. reported the selective targeting of this antibody to biopsied melanoma nodules in eight patients [86]. The 9.2.27 antibody does not activate complement, poorly activates ADCC, but may have application because its target antigen modulates very little. Their studies suggest that quantities on the order of 0.5 to several grams of antibody are necessary for saturation of target antigens in vivo. Schroff et al. have observed that an interrupted infusional schedule appears to compromise the in vivo localization of antibody to tumor, presumably by the development of human anti-murine antibodies (HAMA), which impede antibody localization and accelerate clearance [111]. Goodman et al. later administered MAb96.5 and MAb48.7 to four patients and MAb96.5 alone to a fifth patient. MAb96.5 is an IgG2a immunoglobulin that recognizes p97, a transferrin-like cell surface glycoprotein of 97 kD [44, 45]. MAb48.7 is an IgG1 immunoglobulin recognizing a melanoma-associated cell surface proteoglycan. Although the infusion of antibody was well tolerated, there were no objective remissions nor histopathologic changes found in biopsied tissue, although impressive antibody binding to melanoma cells was observed. Perhaps the lack of activity reflected the lack of in vitro activation of human complement and very modest ADCC. Studies by Vadhan-Raj et al. [122] are of interest in that the IgG3 anti-GD3 mouse monoclonal antibody R24, directed against the sialoganglioside membrane

Biological therapy of melanoma antigen GD3, appears to have induced clinical responses (four partial and two minor responses among 21 patients) using unconjugated antibody [122]. These studies are in contrast to previous studies using other anti-melanoma antibodies. MAb R24 provoked a clear inflammatory reaction with increased number of mast cells with evidence of their degranulation, an influx of polymorphonuclear cells, complement deposition, particularly C3, C5, and C9, and infiltration with T3+/T8+/Ia+ lymphocytes. MAb R24 has been studied in conjunction with other biological modalities such as IL-2 [4], interferonalfa-2a, with total lymphoid irradiation (which did not interdict the human anti-murine antibody response as had been hoped) and by novel routes of administration (e.g., isolated limb perfusion) [16]. Cheung et al. have studied another IgG3 anti-ganglioside MAb, 3F8, in a phase I trial [14]. Two of nine patients with metastatic malignant melanoma achieved partial responses, one lasting 22 weeks and the other continuing at 56 weeks. The most prominent toxicities consisted of severe pain, hypertension, and focal urticaria; a decrease in serum complement activity was observed when dosages equaled or exceeded 20 mg/m2. Houghton reported that the combination of MAb R24 and MAb 3F8 yielded one complete remission among 13 patients with melanoma. Goodman et al. observed no anti-tumor activity among eight patients given MAb MG-21, another antibody recognizing a GD3 surface antigen commonly displayed by human melanoma cells [44, 45]. In an intriguing report, Livingston and associates reported one complete response among 13 patients with metastatic malignant melanoma who received murine IgG2a MAb ME-36.1, which recognizes the melanoma-associated gangliosides GD2 and GD3. This patient’s posttreatment B lymphocytes, when stimulated with polyclonal goat anti-idiotypic ME-36.1, were found to synthesize human antibodies preferentially and specifically recognizing GD2, suggesting to the researchers that the induction of human anti-melanoma ganglioside antibodies provoked by ME-36.1 may have mediated the observed clinical response. These observations that murine monoclonal antibodies can induce complement activation, active antibody-dependent cellular cytotoxicity, and generate human anti-melanoma antibodies may support a further role for the investigation of unconjugated antibody in malignant melanoma. A murine monoclonal anti-melanoma antibody-ricin A chain immunotoxin (XOMAZYME-MEL) was studied in several clinical trials, since the initial report of its use in 22 patients in early 1987 [113]. The monoclonal antibody moiety of this immunotoxin is an IgG2A antibody recognizing melanoma-associated proteoglycan

Robert K. Oldham membrane antigens of 220 kD and greater than 500 kD. One patient demonstrated complete disappearance of detectable melanoma (a retrocardiac pulmonary metastasis) over an 8-month period following a single infusion of immunotoxin, with maintenance of a complete remission for greater than 2 years until relapse occurred in the brain. However, no major responses were seen in the remaining 21 patients. The treatment was accompanied by a transient reduction of serum albumin with associated fluid retention and weight gain, alopecia, fever, fatigue, and malaise, with mild allergic reactions occurring in three patients. In a phase II trial with this immunotoxin, three partial responses were observed among 43 patients, these responses lasting for 10+, 13+, and 15+ months [87, 116]. Cyclophosphamide at a dose of 1 g/m2 administered following the infusion of immunotoxin has not inhibited the generation of host immune responses directed against the immunotoxin preparation. Given the studies by various investigators using unlabeled antibodies, which have been marginally useful in the treatment of melanoma, several investigators have pursued studies using antibody in combination with cytokines [4, 21] with no evidence of activity over and above using the cytokines alone. Similarly, early trials with radiolabeled antibodies for therapy of melanoma have not proven useful [57]. Since antibodies can induce inflammatory infiltrates and activities around melanoma nodules, further work is ongoing with regard to using these antibodies to produce inflammatory effects and combining these effects with cytokines and chemotherapy. Engineered subfragments of antibody as well as humanized antibodies are currently in clinical trials and it is felt some of these improved molecules may improve the delivery of immunotoxins and/or radionuclides to melanoma deposits [89].

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47. 48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60. 61. 62.

63.

64.

cell carcinoma: an overview of randomized trials. J Immunother 1999; 22:145–154. Hill NO, Pardue A, Khan A et al. Interferon and cimetidine for malignant melanoma. N Engl J Med 1983; 308:286. Ives NJ, Stowe RL, Lorigan P et al. Chemotherapy compared with biochemotherapy for the treatment of metastatic melanoma: a meta-analysis of 18 trials involving 2,621 patients. J Clin Oncol December 1, 2007; 24(34):5426–5434. Johnson LA, Heemskerk B, Powell DJ et al. Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. J Immunol 2006; 177:6548–6559. Khayat D, Borel C, Tourani JM et al. Sequential chemoimmunotherapy with cisplatin, interleukin-2, and interferon alfa-2a for metastatic melanoma. J Clin Oncol 1993; 11:2173–2180. Kirkwood JM, Ernstoff M. Potential applications of the interferons in oncology: lessons drawn from studies of human melanoma. Semin Oncol 1986; 13:48–56. Kirkwood JM, Harris JE, Vera R et al. A randomized study of low and high doses of leukocyte alpha-interferon in metastatic renal cell carcinoma: the American Cancer Society collaborative trial. Cancer Res 1985; 45:863–871. Kirkwood JM, Ibrahim JG, Sondak VK et al. High- and low-dose interferon alfa-2b in high-risk melanoma: first analysis of intergroup trial E1690/S9111/C9190. J Clin Oncol 2000; 18:2444–2458. Kirkwood JM, Ibrahim JG, Sosman JA et al. High-dose interferon alfa-2b significantly prolongs relapse-free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with resected stage IIB-III melanoma: results of intergroup trial E1694/ S9512/C509801. J Clin Oncol 2001; 19:2370–2380. Kirkwood JM, Strawderman MH, Ernstoff MS, Smith TJ, Borden EC, Blum RH. Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the Eastern Cooperative Oncology Group Trial EST 1684. J Clin Oncol 1996; 14:7–17. Kirkwood JM. Building upon the standard of care in adjuvant therapy of high-risk melanoma. J Clin Oncol December 1, 2005; 23(34):8559–8563. Larson SM, Carrasquillo JA, Krohn KA et al. Localization of 131I-labeled p97-specific Fab fragments in human melanoma as a basis for radiotherapy. J Clin Invest 1983; 72:2101–2114. Lee KH, Talpaz M, Rothberg JM et al. Concomitant administration of recombinant human interleukin-2 and recombinant interferon alpha-2A in cancer patients: a phase I study. J Clin Oncol 1989; 7:1726–1732. Lee ML, Tomsu K, Von Eschen KB. Duration of survival for disseminated malignant melanoma: results of a meta-analysis. Melanoma Res 2000; 10:81–92. Legha SS. Current therapy for malignant melanoma. Semin Oncol 1989; 16:34–44. Legha SS. The role of interferon alfa in the treatment of metastatic melanoma. Semin Oncol 1997; 24:S24–S31. Legha SS, Papadopoulos NE, Plager C et al. Clinical evaluation of recombinant interferon alfa-2a (Roferon-A) in metastatic melanoma using two different schedules. J Clin Oncol 1987; 5:1240–1246. Legha SS, Ring S, Eton O, Bedikian A, Plager C, Papadopoulos N. Development and results of biochemotherapy in metastatic melanoma: the University of Texas M.D. Anderson Cancer Center experience. Cancer J Sci Am 1997; 3 Suppl 1:S9–S15. Lens MB, Dawes M, Goodacre T, Newton-Bishop JA. Elective lymph node dissection in patients with melanoma: systematic review and meta-analysis of randomized controlled trials. Arch Surg 2002; 137:458–461.

Robert K. Oldham 65. Lewko WM, Good RW, Bowman D, Smith TL, Oldham RK. Growth of tumor derived activated T-cells for the treatment of cancer. Cancer Biother 1994; 9:211–224. 66. Lewko WM, Hall PB, Oldham RK. Growth of tumor-derived activated T cells for the treatment of advanced cancer. Cancer Biother Radiopharm 2000; 15:357–366. 67. Lewko WM, Smith TL, Bowman DJ, Good RW, Oldham RK. Interleukin-15 and the growth of tumor derived activated T-cells. Cancer Biother 1995; 10:13–20. 68. Lipton A, Harvey HA, Simmonds MA. Lack of enhanced activity of systemic interferon by cimetidine in malignant melanoma. Proc Am Soc Clin Oncol 1984; 3:56 (abstract). 69. Livingston PO, Ritter G, Srivastava P et al. Characterization of IgG and IgM antibodies induced in melanoma patients by immunization with purified GM2 ganglioside. Cancer Res 1989; 49:7045–7050. 70. Lizee G, Radvanyi LG, Overwijk WW et al. Improving antitumor immune responses by circumventing immunoregularity cells and mechanisms. Clin Canc Res August 13, 2006; 12:4794–4803. 71. Mackenson A, Cercelain G, Viels S. Direct evidence to support the immunosurveillance concept in a regressive melanoma. J Clin Invest 1994; 93:1402. 72. Maleckar JR, Friddell CS, Lewko WM, Yannelli JR, West WH, Oldham RK. Tumor-derived activated cells: culture conditions and characterization. Immunol Ser 1989; 48:159–173. 73. Maleckar JR, Friddell CS, Sferruzza A et al. Activation and expansion of tumor-derived activated cells for therapeutic use. J Natl Cancer Inst 1989; 81:1655–1660. 74. Michalek J, Buchler T, Hajek R. T lymphocyte therapy of cancer. Physiol Res 2004; 33:463–469. 75. Mitchell MS, Abrams J, Thompson JA et al. Randomized trial of an allogeneic melanoma lysate vaccine with low-dose interferon alfa-2b compared with high-dose interferon alfa-2b for resected stage III cutaneous melanoma. J Clin Oncol May 20, 2007; 25(15):2078–2085. 76. Morgan RA, Dudley ME, Yu YYL et al. High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. J Immunol 2003; 171:3287–3295. 77. Morton DL. Active immunotherapy against cancer: present status. Semin Oncol 1986; 13:180–185. 78. Morton DL, Mozzillo N, Thompson JF et al. An internation, randomized, phase III trial of bacillus Calmete-Guerin (BCG) plus allogeneic melanoma vaccine (MCV) or placebo after complete resection of melanoma metastatic to regional or distant sites. J Clin Oncol 2007 ASCO Annual Meeting Proceedings June 20, 2007 supplement; 25(185):8508. 79. Nabel GJ, Nabel EG, Yang ZY et al. Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans. Proc Natl Acad Sci U S A 1993; 90:11307–11311. 80. Neidhart JA, Murphy SG, Hennick LA, Wise HA. Active specific immunotherapy of stage IV renal carcinoma with aggregated tumor antigen adjuvant. Cancer 1980; 46:1128–1134. 81. Oldham RK. Biologicals for cancer treatment: interferons. Hosp Pract 1985; 20:72–91. 82. Oldham RK, Maleckar, JR, Wart WH & Yanelli JR IK2 and Cellular Therapy In: Cytokines in Hemapoirsis Oncology and AIDS Eds Freevel CR et al Springer-Verlag, Berlin pp 661–671 1990. 83. Oldham RK. Cancer cures: by the people, for the people, at what cost? Mol Biother 1990; 2:2–3.

643 84. Oldham RK. Therapy with interleukin-2 and tumor-derived activated lymphocytes. Immunol Ser 1994; 61:251–271. 85. Oldham RK, Dillman RO, Yannelli JR et al. Continuous infusion interleukin-2 and tumor-derived activated cells as treatment of advanced solid tumors: a National Biotherapy Study Group Trial. Mol Biother 1991; 3:68–73. 86. Oldham RK, Foon KA, Morgan AC et al. Monoclonal antibody therapy of malignant melanoma: in vivo localization in cutaneous metastasis after intravenous administration. J Clin Oncol 1984; 2:1235–1244. 87. Oldham RK, Lewko WM, Good RW, Sharp E. Cancer biotherapy with interferon, interleukin-2 and tumor-derived activated cells (TDAC). In Vivo 1994; 8:653–663. 88. Oldham RK, Maleckar JR, Friddell CS, Lewko WM, West WH, Yannelli JR. Tumor-derived activated cells: preliminary laboratory and clinical results. Clin Chem 1989; 35:1576–1580. 89. Oldham RK, Dillman RO. Monelonal antibodies in Cancer Therapy: 25 years of Progress JCO 2008; 26(11):1774–1777. 90. Paterson AH, Willans DJ, Jerry LM, Hanson J, McPherson TA. Adjuvant BCG immunotherapy for malignant melanoma. Can Med Assoc J 1984; 131:744–748. 91. Peggs KS, Quezada SA, Korman AJ et al. Principles and use of anti-CTLA4 antibody in human cancer immunotherapy. Curr Opin Immun April 2006; 18(2):206–213. 92. Prehn RT. The paradoxical association of regression with a poor prognosis in melanoma contrasted with a good prognosis in keratoacanthoma. Cancer Res 1996; 56:937–940. 93. Pyrhonen S, Hahka-Kemppinen M, Muhonen T. A promising interferon plus four-drug chemotherapy regimen for metastatic melanoma. J Clin Oncol 1992; 10:1919–1926. 94. Reintgen DS, McCarty KS, Jr., Cox E, Seigler HF. Malignant melanoma in the American black. Curr Surg 1983; 40:215–217. 95. Retsas S, Priestman TJ, Newton KA, Westbury G. Evaluation of human lymphoblastoid interferon in advanced malignant melanoma. Cancer 1983; 51:273–276. 96. Ribas A, Hanson DC, Noe DA et al. Tremelimumab (CP675,206), a cytotoxic T lymphocyte associated antigen 4 blocking monoclonal antibody in clinical development for patients with cancer. Oncologist July 1, 2007; 12(7):873–883. 97. Richards JM, Mehta N, Ramming K, Skosey P. Sequential chemoimmunotherapy in the treatment of metastatic melanoma. J Clin Oncol 1992; 10:1338–1343. 98. Robinson E, Bartal A, Cohen Y, Haasz R, Mekori T. Treatment of lung cancer by radiotherapy, chemotherapy, and methanol extraction residue of BCG (MER): clinical and immunological studies. Cancer 1977; 40:1052–1059. 99. Roeslin N, Lang JM, Morand G, Wihlm JM, Witz JP. Regional immunotherapy in resectable squamous cell lung carcinoma. Analysis of a randomized study. Cancer Immunol Immunother 1982; 13:174–175. 100. Rosenberg SA. The adoptive immunotherapy of cancer using the transfer of activated lymphoid cells and interleukin-2. Semin Oncol 1986; 13:200–206. 101. Rosenberg SA. Karnofsky Memorial Lecture. The immunotherapy and gene therapy of cancer. J Clin Oncol 1992; 10:180–199. 102. Rosenberg SA, Aebersold P, Cornetta K et al. Gene transfer into humans – immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med 1990; 323:570–578. 103. Rosenberg SA, Dudley, ME. Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes. PNAS1 10.1073; September 20, 2004.

644 104. Rosenberg SA, Lotze MT, Yang JC. Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer. J Natl Cancer Inst 1993; 85:622. 105. Rosenberg SA, Lotze MT, Yang JC et al. Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 1989; 210:474–484. 106. Rosenberg SA, Lotze MT, Yang JC et al. Combination therapy with interleukin-2 and alpha-interferon for the treatment of patients with advanced cancer. J Clin Oncol 1989; 7:1863–1874. 107. Rosenberg SA, Packard BS, Aibersold PM et al. Use of tumorinfiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Engl J Med 1988; 319:1676–1680. 108. Ruotsalainen TM, Halme M, Tamminen K et al. Concomitant chemotherapy and IFN-alpha for small cell lung cancer: a randomized multicenter phase III study. J Interferon Cytokine Res 1999; 19:253–259. 109. Sanderson K, Scotland R, Lee P et al. Autoimmunity in a phase I trial of a fully human anti-cytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and montanide ISA 51 for patients with resected stages III and IV melanoma. J Clin Oncol February 1, 2005; 23(4):741–750. 110. Schmid-Wendtner MH, Baumert J, Schmidt M et al. Late metastases of cutaneous melanoma: an analysis of 31 patients. J Am Acad Dermatol 2000; 43:605–609. 111. Schroff RW, Woodhouse CS, Foon KA et al. Intratumor localization of monoclonal antibody in patients with melanoma treated with antibody to a 250,000-dalton melanoma-associated antigen. J Natl Cancer Inst 1985; 74:299–306. 112. Sertoli MR, Bernengo MG, Ardizzoni A et al. Phase II trial of recombinant alpha-2b interferon in the treatment of metastatic skin melanoma. Oncology 1989; 46:96–98. 113. Sertoli MR, Quierolo P, and Bajetta E. Dacarbazine (DTIC) with or without recombinant interferon alpha 2a at different dosages in the treatment of stage IV melanoma patient: preliminary results of a randomized trial. Proc Am Soc Clin Oncol 1992; 11:345 (abstract). 114. Sosman JA, Hank JA, Sondel PM. In vivo activation of lymphokine-activated killer activity with interleukin-2: prospects for combination therapies. Semin Oncol 1990; 17:22–30.

Biological therapy of melanoma 115. Sparano JA, Fisher RI, Sunderland M et al. Randomized phase III trial of treatment with high-dose interleukin-2 either alone or in combination with interferon alfa-2a in patients with advanced melanoma. J Clin Oncol 1993; 11:1969–1977. 116. Spitler LE, del Rio M, Khentigan A et al. Therapy of patients with malignant melanoma using a monoclonal antimelanoma antibodyricin A chain immunotoxin. Cancer Res 1987; 47:1717–1723. 117. Steiner A, Wolf C, Pehamberger H. Comparison of the effects of three different treatment regimens of recombinant interferons (r-IFN alpha, r-IFN gamma, and r-IFN alpha + cimetidine) in disseminated malignant melanoma. J Cancer Res Clin Oncol 1987; 113:459–465. 118. Stoter G, Aamdal S, Rodenhuis S et al. Sequential administration of recombinant human interleukin-2 and dacarbazine in metastatic melanoma: a multicenter phase II study. J Clin Oncol 1991; 9:1687–1691. 119. Tahini AA, Kirkwood JM, Gooding WE et al. Durable complete responses with high-dose bolus interleukin-2 in patients with metastatic melanoma who have experienced progression after biochemotherapy. J Clin Oncol September 1, 2007; 25(25):3802–3807. 120. Thomson DB, Adena M, McLeod GR et al. Interferon-alpha 2a does not improve response or survival when combined with dacarbazine in metastatic malignant melanoma: results of a multi-institutional Australian randomized trial. Melanoma Res 1993; 3:133–138. 121. Tsao H, Rogers GS, Sober AJ. An estimate of the annual direct cost of treating cutaneous melanoma. J Am Acad Dermatol 1998; 38:669–680. 122. Vadhan-Raj S, Cordon-Cardo C, Carswell E et al. Phase I trial of a mouse monoclonal antibody against GD3 ganglioside in patients with melanoma: induction of inflammatory responses at tumor sites. J Clin Oncol 1988; 6:1636–1648. 123. Vosika GJ. Clinical immunotherapy trials of bacterial components derived from mycobacteria and nocardia. J Biol Resp Modif 1983; 2:321–342. 124. West WH, Tauer KW, Yannelli JR et al. Constant-infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. N Engl J Med 1987; 316:898–905. 125. Wheatley K, Hancock B, Gore M, Suciu S. Eggermont AMM. Interferon-α as adjuvant therapy for melanoma: a meta-analysis of the randomised trials. Proc Am Soc Clin Oncol 2001; 20:1394 (abstract).

21.3 Biological therapy of genitourinary cancer ROBERT K. OLDHAM

Kidney Cancer Overview Kidney cancer remains one of the best examples of a disease that responds to the immune system and is amenable to intervention with biological therapy. Cancer of the kidney and renal pelvis was estimated to account for 51,200 new cancer cases and cause 12,900 cancer deaths in the United States in 2007. In the United States, kidney cancer is the 12th leading cause of cancer death, comprising 3% of all malignancies in men and 2% in women. The 5-year relative cancer free survival has improved over the last 2 decades, from 52% in the years 1974– 1976 to the current rate of 61% in the years 1989–2007, causing 2% of all cancer deaths in this country [32]. Internationally, kidney cancer has the highest incidence in North America and northern Europe, the lowest incidence in India, China, Japan, and South America, and accounts for 2% of all cancers worldwide [43, 56, 79]. The incidence of kidney cancer has increased by approximately 2% per year over the past 2 decades. The greatest rise in incidence is in patients with localized disease (3.7% per year] and may be ascribed in large part to the increase in abdominal imaging in the population at large. However, the incidence of regionally advanced and distantly metastatic kidney cancer has also increased over this time period, indicating that factors in addition to the increase in abdominal imaging may be contributing to the rising incidence of this disease [38]. Currently, approximately 25% of patients with kidney cancer present with disseminated disease while an additional one third to one half of patients without dissemination at initial diagnosis develop metastases [22, 38]. Thus, more than half of all patients with kidney cancer will present with or develop disease for which systemic anticancer therapy is needed. Five year survivals of over 90% have recently been reported for patients with small, incidentally detected kidney cancers [100]. However, relapse rates of 50% or higher in patients with larger or more extensive tumors provide a strong rationale for the development of a systemic adjuvant post-surgical therapy for patients with resectable kidney cancer. Since traditional forms of cytotoxic chemotherapy remain ineffective for

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

kidney cancer, these statistics illustrate the large potential patient population that can benefit from an active biological and targeted therapy of cancer. Recent progress in targeted therapy for renal cancer has lead to the identification of several agents, which target either vascular endothelial growth factor or a tyrosine kinase enzyme. These molecular findings have lead to the development of what is now being called “targeted therapy” of kidney cancer and many other types of cancer [15].

Cytotoxic Chemotherapy is Ineffective Kidney cancer remains one of few modern examples of a malignant disease for which currently available cytotoxic chemotherapy agents are essentially inactive. An exhaustive review of 143 published or nationally presented single agent chemotherapy trials concluded that no agent achieved significant activity in the treatment of renal cell carcinoma. This review included studies reported from 1975–1995 involving almost 4,000 patients treated with 80 different single agents, showing a 4% overall response rate [2]. Newer cytotoxic agents have had similarly disappointing results in renal cell carcinoma. A review of chemotherapy trials reported from 1990–1998 included 33 chemotherapy agents given to 1,347 patients on 51 phase II clinical trials lead to the conclusion that no chemotherapy has produced response rates that justify use as a single agent, although synergy may exist for certain combinations that warrant further study [63]. Responses to chemotherapy in renal cell carcinoma must be evaluated in light of the low but finite level of background noise created by spontaneous regression of metastases. The lack of an active cytotoxic agent for this disease provides further rationale for developing and optimizing biological and targeted therapy for kidney cancer.

Kidney Cancer is Responsive to the Immune System Several clinical observations similar to those described for malignant melanoma indicate a role for the immune system to influence the natural history and treatment of

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646 kidney cancer. First, patients with established metastatic kidney cancer may have long disease or treatment free intervals, irrespective of the treatment received, suggesting a degree of innate immune control. In a large, retrospective, single institution analysis, 5-year survival was observed in 4.5% of 670 patients with advanced kidney cancer treated on one of several cytokine, chemotherapy, or hormonal therapy trials from 1975–1996, including 9 of 294 patients (3%) treated with chemotherapy or hormonal therapy agents subsequently demonstrated to lack activity in this disease [61]. Similarly, patients may relapse many years, even decades after a nephrectomy with curative intent for renal cell carcinoma, indicating a potential role for immune surveillance to control micrometastatic kidney cancer effectively for many years [99]. Second, immunosuppression increases the risk of the development of kidney cancer, as seen in the increased risk of the development of renal cell carcinoma in kidney allograft recipients [46]. Third, immune stimulation with cytokine therapy can induce tumor regression and prolong survival in patients with metastatic kidney cancer and will be described in detail in this chapter. Fourth, spontaneous regression of metastatic kidney cancer has been carefully documented [102]. Indeed, the response rates for observation, placebo, and “placebo-equivalent” therapy in phase II and III trials of metastatic renal cell carcinoma have been as high as 7%. The most notable example of a clinical study showing a high rate of apparently spontaneous regression of metastatic kidney cancer was a phase II trial was performed on 73 patients with metastatic renal cell carcinoma who were identified prospectively and treated with observation until evidence of progression. Five patients (7%) achieved a partial or complete remission on observation and 12% were progression free for at least 1 year [68]. Recently reported phase III trials in metastatic renal cell cancer also demonstrated a spontaneous remission rate of greater than 5%. A randomized trial comparing interferon gamma to placebo in 197 patients with advanced renal cell carcinoma showed a 7% response rate in the placebo group that was higher than the response rate achieved in the treatment group [35]. Another recent phase III trial has demonstrated modest rates of regression with what is likely to be “placebo-equivalent” treatment in comparison to interferon alpha. Medroxyprogesterone acetate has been evaluated for single agent activity in renal cell carcinoma in a number of trials, is generally considered to be inactive, and may be thought of as a “placebo-equivalent” in this setting [29]. A trial comparing interferon-alpha to medroxyprogesterone demonstrated the superiority of interferon alpha, but a 3% overall objective response rate was observed in the medroxyprogesterone arm, and the

Biological therapy of genitourinary cancer authors reported a 7% response rate to medroxyprogesterone in the subset of patients who remained on treatment for at least 6 months [57]. Taken together, the results of these studies demonstrate the potential role of the innate immune system in mediating the spontaneous regression of established metastases from renal cancer. Based on these clinical observations, renal cell carcinoma, like melanoma, appeared to constitute an ideal human tumor system for biotherapy. Early attempts to harness the immune system in the treatment of kidney cancer are of historical interest and employed a variety of active specific immunization techniques. Tykka, in 1974, reported a 17% complete response rate of pulmonary metastatic lesions in patients receiving a monthly vaccine composed of homogenized tumor plus either tuberculin or Candida every 4 weeks [101]. Subsequently, three separate trials using either tumor homogenate plus tuberculin, enzymatically digested tumor tissue plus Corynebacterium parvum, or whole tumor cells plus attached dimethyldioctadecyl ammonium bromide (DDA) as vaccine have all reported responses in 15–25% of patients [54, 66, 70]. Furthermore, immune RNA has been extracted from lymphocytes of guinea pigs specifically immunized with renal tumor cells and coincubated with autologous lymphocytes; these reinfused lymphocytes induced a response in three of six patients [96]. Thymosin fraction V, a partially purified extract of calf thymus glands, has induced objective responses in approximately 15% of advanced renal cancer patients. These early studies provided the impetus for developing biological therapies for renal cell carcinoma. Important laboratory observations provide evidence of an immunologic response to RCC and have been reviewed elsewhere in this book. Several lines of evidence indicate that cytotoxic T-lymphocytes (CTLs) play a central role in mediating the regression of RCC. First, lymphocytic infiltrates within renal tumors are comprised primarily of CD3+ T-lymphocytes, and molecular analysis of these lymphocytic infiltrates has demonstrated clonally expanded populations of T-cell receptor alpha and beta restricted T-cells that are generally not found in peripheral blood, normal kidney or lymph nodes [47, 72]. Second, tumor-specific cytotoxic T-cell lines from tumor infiltrating lymphocytes have been developed from patients with RCC that are capable of recognizing and lysing autologous tumor cells [4]. Third, specific T-cell defined tumor associated antigens have been identified from patients with RCC, including HER-2/neu, PRAME, and RAGE [65]. Taken together, these correlative laboratory observations demonstrate the central role of the CTL in mediating the immune response to RCC.

Robert K. Oldham

Cytokine Therapy The treatment of MRCC with the cytokines interferonalpha and interleukin-2 results in objective responses in about 15% of patients. A recent retrospective analysis of a large, single institution patient database provides an example of the benefits that derive from treatment of MRCC [60]. This review of 670 patients with advanced RCC analyzed the outcomes of patients treated with cytokine therapy versus chemotherapy or hormonal therapy in clinical trials from 1976–1996. About 60% of patients were treated with cytokine therapy with interferon-alpha, interleukin-2, or interferon-alpha plus interleukin-2 versus and about 40% were treated with chemotherapy or hormonal therapy. A highly significant and clinically meaningful survival advantage was demonstrated for cytokine therapy versus chemotherapy or hormonal therapy, with median survival times of 13 versus 6 months (p < 0.001, 95% confidence intervals of 12–15 months versus 5–8 months). The survival advantage for cytokine therapy extended to all prognostic groups, with median survivals of 27 versus 15 months for the favorable subset of patients, although patients with the poorest prognosis survived less than 6 months even with treatment. This important analysis demonstrated that in each risk group, there was an approximate doubling of the median overall survival when patients were treated with cytokine therapy compared with chemotherapy or hormonal therapy. Trials of cytokine therapy in metastatic renal cell carcinoma, have demonstrated wide variations in response rate, including some trials that failed to demonstrate responses to high dose interleukin-2 [1] while others have demonstrated responses as high as 33% [86]. From these observations, several conclusions can be drawn. First, advanced kidney cancer has a variable natural history determined at least in part by the innate immune response to renal cell carcinoma. Second, pre-treatment patient and tumor characteristics have a large impact on the outcome of a given treatment or observation strategy. Third, a measure of caution must be exercised when evaluating the results of clinical trials in renal cell carcinoma, particularly when the results indicate marginal activity of a particular treatment. Several recent reports have identified some of the important prognostic variables that help to predict clinical outcomes in kidney cancer, including TNM stage, tumor grade, performance status, and certain indicators of disease burden (lactate dehydrogenase level, presence of anemia, and presence of hypercalcemia). It is possible to identify a favorable subset of patients with advanced kidney cancer whose median survival exceeds 2 years with cytokine therapy and an unfavorable subset of patients with a median survival of approximately 6

647 months or less for whom cytokine therapy may be less beneficial [60, 109]. Another important variable in the outcome of patients with metastatic kidney cancer treated with cytokine therapy is the presence of the primary kidney tumor [26, 27, 58]. It is also becoming increasingly evident that not all histological types of kidney cancer are equally capable of response to immune modulation. It is important to recognize that conventional (clear cell) renal cell carcinoma and its histological variants (conventional renal cell carcinomas may show a mixture of cytoplasmic features, including variants that have formerly been classified as “granular cell” and “sarcomatoid” renal cell carcinomas) represent a specific clinico-pathological disease entity characterized by typical findings on light microscopy and loss of the von Hippel-Lindau tumor suppressor gene. This disease entity, now classified as conventional renal cell carcinoma, comprises approximately 60% of all epithelial tumors arising from the kidney [78]. It is clear that the other histological types of tumors of renal epithelial origin (e.g. papillary, chromophobe, oncocytoma, and collecting duct tumors) represent distinct clinico-pathological diseases with different clinical behavior, but it is not clear whether other histological types of kidney cancer are capable of responding to immune manipulation [69]. Recognition of known prognostic indicators as determinants of eligibility and stratification is a critical component in the design of clinical trials and the evaluation of their results. Furthermore, because of the major contribution of prognostic factors on the outcome of patients with metastatic kidney cancer (MRCC) irrespective of treatment, randomized trials are required to demonstrate the relative merits of a particular therapy. The overall response rates observed with cytokine therapy for kidney cancer have been relatively low, and the natural history of patients with MRCC varies widely independent of treatment. As a result, the impact of treatment with these cytokines on survival has been controversial and can only be directly assessed in phase III clinical trials. Until recently, phase III trial data have been lacking, but several important phase III studies have recently been completed and reported that shed light on the impact of cytokine therapy on the survival of patients with MRCC, the role of “cytoreductive” nephrectomy prior to cytokine therapy in with metastases at initial presentation, and the incremental benefits that may be achieved with intensive therapy with high dose interleukin-2 [32, 45, 92].

Interferon-alpha Interferon-alpha has produced objective responses in 10–20% of patients with MRCC. An earlier review of

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Biological therapy of genitourinary cancer

the data from multiple phase II studies showed a response rate of 12% in 1,042 patients treated with a variety of doses and schedules of interferon, and concluded that longer survival could not be demonstrated in patients with MRCC treated with interferon-alpha [9, 105]. The mean time to response may be as long as 3–4 months. It can be argued that an optimal dose and schedule for alpha-interferon has never been defined for renal cell carcinoma. The anti-tumor activity of interferon can be attributed to its ability to modulate cellular immunity, to its direct (non-immunologically mediated) anti-proliferative or pro-apoptotic activity, or to an effect on tumor angiogenesis [52] It is not clear that all of these properties of interferon can be evoked optimally and simultaneously from a single dose and schedule of interferon in vivo. Some data appear to demonstrate a dose–response relationship for interferon, suggesting that the clinical responses induced by interferon are likely to be due to its known anti-proliferative activity rather than to immunomodulation. However, evidence for a dose–response relationship for interferon has not been consistently observed among investigators. While some toxicities from interferon do appear to be dose-related, not all antitumor responses appear to be dose-related. From his extensive review of the literature relating to the treatment of renal cell carcinoma with interferon, Muss [64] concluded that toxicity from interferon was dose-related and that the highest therapeutic index for interferon occurred at doses ranging from 5–10 million units/m2. However, it is interesting to note that some studies have demonstrated responses using very low doses of interferon. For example, a single-arm phase II trial conducted by the Southwest Oncology Group [63] accrued 40 fully evaluable, previously untreated patients with advanced renal cell carcinoma treated with alpha-interferon 1 million units subcutaneously daily continually until there was

evidence of disease progression, resulting in 6 responses (response rate, 15%; 95% confidence interval, 5.7–30%). The ability of interferon-alpha to prolong survival has now been evaluated in several randomized trials in comparison to a non-cytokine containing control arm [32, 48, 57, 73, 97]. The results are presented in Table 1. The two smaller studies showed no survival benefit for treatment with interferon-alpha, but included few patients and allowed crossover in one case. The two larger studies both demonstrated a statistically significant improvement in survival for the interferon-containing arm at doses of 10–18 MU three times weekly. Because the addition of vinblastine has been shown not to improve survival over interferon alone, the improvement in survival of interferon plus vinblastine over vinblastine alone can be attributed to the interferon. Taken together, the results of the four randomized, prospective trials comparing cytokine therapy versus non-cytokine therapy in several hundred patients, demonstrated an objective response rate of 16% versus 2% and average median survival of approximately 12 versus 8 months. These results also closely resemble the results of the large, retrospective single-institution analysis of cytokine versus noncytokine therapy described above in which median survivals were 13 versus 6 months [32]. A randomized trial involving interferon alpha was conducted through the Southwestern Oncology Group and was designed to answer the important question of whether nephrectomy in the setting of known metastatic disease is of clinical benefit [27]. Almost 350 patients with metastatic kidney cancer were randomly assigned to receive interferon alpha immediately upon enrollment into the trial or following a nephrectomy. Interestingly, the objective response rate to interferon alpha was unexpectedly low and not different between the two arms of the study (less than 4%), yet the median

Table 1. Randomized trials comparing interferon alpha with non-cytokine treatment Treatment

N

Response (%)

Median survival (months)

P value

Publication

IFN MPA IFN + VBL MPA IFN + VBL VBL IFN MPA Total IFN +/− VBL or MPA

30 30 44 45 79 81 167 168

6 3 20 0 16 2 16 2

7 7 16 10 17 10 8.5 6

NA

Steineck 1990(97)

0.19

Kriegmair 1995(48)

0.0049

Pyrhonen 1999(73)

0.011

MRC 1999(57)

320 324

16 2

11.5 7.7

IFN interferon-alpha, MPA medroxyprogesterone acetate, VBL vinblastine

Robert K. Oldham survival was significantly improved (by 3 months) in the patients who underwent nephrectomy before immunotherapy, independent of other prognostic factors. More rigorous assessment of objective response was proposed to explain the low response rate observed in this trial; dose and schedule were comparable to studies reporting much higher responses. A similar improvement in survival for nephrectomy in the setting of metastatic disease was also observed in a somewhat smaller study of similar design, and responses to interferon were in the usual range (12–19%) [58]. A similar European trial demonstrated an improvement in the time to disease progression as well as the median survival for patients who had a nephrectomy compared to those who did not prior to initiating interferon-alpha therapy. In addition, there are some studies that indicate resection of solitary or a small number of metastasis may lead to long-term survival in approximately 30% of patients with metastatic renal cancer. Thus, surgery plays an important role in MRCC, especially in conjunction with effective systemic therapy [22].

PEG-interferon The treatment of kidney cancer with inferon alpha therapy requires daily or thrice weekly subcutaneous administration. The preparation of the interferon with polyethylene glycol (PEG) sustains the absorption of interferon from a subcutaneous injection, slows its clearance, and improves its pharmacokinetic parameters. This improvement in pharmacokinetics allows for less frequent administration, for example once weekly, and may also improve the effectiveness and side effect profile by avoiding the frequent peaks and troughs associated with the shorter half-life of conventional interferon. PEGinterferon alpha has become commercially available for the treatment of hepatitis C, and studies were done to evaluate the effectiveness and tolerability of this agent in renal cell carcinoma. Preliminary studies suggest that PEG-interferon retains anti-tumor activity in renal cell carcinoma and has a spectrum of side effects similar to conventional interferon alpha, but improves the convenience of administration to a once weekly dose [62].

Other Interferons The role of beta-interferon in the treatment of renal-cell carcinoma remains to be established. A trial of combination interleukin-2 and beta-interferon evoked objective responses in 6 of 22 evaluable patients, with one complete remission and five partial remissions. Two of these responses lasted for almost 2 years [32]. Although this

649 27% overall response rate is similar to that achieved with interleukin-2 and lymphokine-activated killer (LAK) cell therapy and to higher doses of single agent interleukin-2, the contribution of beta-interferon is uncertain. Early experience with gamma-interferon suggested little if any activity [75, 80]. Gamma-interferon is a product of activated T cells and is unique in its ability to stimulate macrophages. Although approved for chronic granulomatous disease, it has demonstrated some activity in metastatic renal cancer and in one study using a low-dose weekly subcutaneous injection of 100 mg, a 30% response rate was observed in patients with metastatic renal cancer [5]. A second study reported a 15% response rate [19]. Combinations of alpha and gammainterferon have shown additive toxicities without added therapeutic benefit [74]. An effort to improve response by combining alpha-interferon and gamma-interferon failed to achieve this intent, perhaps attributable to additive toxicities and inability to suitably escalate dosages [28]. Some of the same principles regarding dose and schedule of alpha-interferon, discussed above, pertain to gamma-interferon. A dose–response relationship for gamma-interferon may not be straightforward [6]. Foon et al. conducted a phase I trial of gamma-interferon in which anti-tumor responses were observed. However, the responses occurred at the lowest doses tested [28]. Follow-up trials with both beta and gamma interferon have yielded equivocal results and only interferon-alpha is approved for use in MRCC.

Interleukin-2 In the United States, high-dose interleukin-2 was approved by the Food and Drug Administration for the treatment of advanced kidney cancer, whereas in Europe, lower doses and infusional IL-2 were approved and are more commonly used in MRCC. Kidney cancer cells in culture can grow in spite of the presence of high concentrations of interleukin-2, demonstrating that interleukin-2 has no directly cytotoxic activity. The action of interleukin-2 appears to be the activation of lymphocytes and natural killer cells that mediate the antitumor activity of this agent. About 10–20% of patients treated with interleukin-2 will achieve an objective response, and a small, but important fraction of these patients will achieve a complete remission, which may be quite durable. A review of over 1,900 patients treated with single agent interleukin-2 demonstrated an overall response rate of 15% that included complete remissions in 4% of the total patient population (Table 2) [7, 8]. This review included patients treated on phase I and II studies using various dose levels, schedules, and routes of administration.

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Table 2. Results of therapy with interleukin-2 Route of Administration

N

% responding

% complete responders

Intravenous bolus Continuous intravenous infusion Subcutaneous Total

733 922

16 13.3

5 2.5

290 1945

15.0 15.0

3 3.6

Table 2 adapted from(7,8).

It is likely that the dose intensity of interleukin-2 administration influences the proportion of patients that respond to treatment as well as the quality and durability of the responses, but a linear dose-response relationship has not been demonstrated for interleukin-2, and questions of dose and schedule have been the subject of several important clinical studies in the late 1990s. Interleukin-2 was approved by the United States Food and Drug Administration in 1992 based on a data set of over 450 patients treated on a series of phase II studies with high dose, intravenous bolus interleukin-2 with and without lymphokine-activated killer (LAK) cells, demonstrating an overall response rate of 21% with 6% complete and 15% partial responses [18, 25, 53]). Subsequently, studies have demonstrated that the benefits of high dose interleukin-2 plus LAK cells are derived from the interleukin-2 (as discussed below and reviewed in [48]), and of the over 450 patients analyzed, 255 were treated with high dose interleukin-2 without LAK cells. The results of treatment in these 255 patients have been rigorously scrutinized and followed long-term, updated reports have been published periodically [24, 25, 30, 31]. These reports are instructive in an analysis of the outcome of treatment of metastatic kidney cancer with interleukin-2. Objective responses following high dose interleukin-2 have been confirmed in 15% (7% complete and 8% partial), with a median response duration for the entire group of 15 months. Considering this group as a whole, the results of treatment with high dose interleukin-2 appear to be fairly similar to the results presented above for treatment with interferon alpha in which the overall response rate of 16% and median survivals of 7–16 months have been observed in rigorously conducted trials. However, the importance of aggressive immunotherapy with high dose interleukin-2 treatment may be the duration and quality of the responses observed. In this 255 patient data set treated with high dose interleukin-2, the median survival for responding patients is over 84 months, and the median response duration for completely responding patients has not been reached, which is tantamount to cure [24].

The administration of interleukin-2 at high doses requires hospital admission to an intensive care unit or a dedicated, specialized nursing unit capable of close monitoring and the administration of vasopressors to approximately half of patients, a skilled treatment team, and in spite of the careful attention of this skilled team is nonetheless associated with significant toxicity and occasional mortality. A 4% treatment related death rate was noted in the original 255 patient dataset, although more recent series have found the treatment related death rate to be 1% or less [34, 44, 107]. Because of the toxicity associated with the capillary leak syndrome and the potential cardiac toxicity associated with high dose interleukin-2, patient selection is very important for its safe administration [44]. The toxicity of high dose interleukin-2 has limited its application to patients that are relatively young, have limited co-morbidities, an excellent performance status, and access to one of the relatively few treatment centers that offer this intensive therapy. Because lower intensity and outpatient regimens of interleukin-2 lead to responses in a fairly similar proportion of patients in phase II studies, it has been tempting to replace high dose interleukin-2 with low dose, intravenous or subcutaneous, outpatient regime ns of interleukin-2. While considerably easier to administer and applicable to a much broader patient population, it is not clear that the response rate and quality of responses are preserved with this approach. In order to directly compare the results of therapy with high dose interleukin-2 compared to lower intensity and less toxic regimens, two large, prospective, randomized trials have completed patient accrual at the National Cancer Institute (NCI) and at multiple institutions through the Cytokine Working Group (CWG) comparing high dose interleukin-2 with lower dose regimens [55, 107]. In the former study from the NCI, patients were randomized to high dose intravenous bolus interleukin-2, low dose intravenous bolus interleukin-2, or subcutaneous interleukin-2. In the latter study through the CWG, patients were randomized to receive high dose intravenous bolus interleukin-2 or subcutaneous interleukin-2 plus interferon alpha. The response rates with high-dose interleukin-2 are approximately twice that observed with lower doses of interleukin-2 with or without interferon alpha in groups of patients that have been comparably screened and selected on the basis of their ability to undergo the rigors of treatment with high dose interleukin-2 [55, 107]. The survival and the response duration from these trials await further maturation of the data; however, the trend from both studies appears to indicate that the durability of the responses in patients treated with high dose interleukin-2 is superior to that achieved

Robert K. Oldham

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with low dose interleukin-2 [55, 107]. Overall survival was not significantly different in the two groups. [55, 107]. In summary, the consistent results of the numerous studies of the treatment of metastatic kidney cancer with cytokine therapy are that a relatively small and consistent fraction of patients, 10–20%, will achieve evidence of tumor regression with occasional complete remissions and long survival noted for a few patients. For the subset of patients who are able to receive aggressive immunotherapy with high dose interleukin-2, this intensive therapy may result in a higher percentage of complete and partial remissions and more durable responses(Table 3). Even enthusiasts of high dose interleukin-2 hoped to replace this labor intensive and toxic treatment with a more widely applicable but therapeutically equivalent regimen (in designing the randomized trial described above, the Cytokine Working Group had expected equivalent therapeutic results from the low dose regimen and performed this trial “before accepting the low dose regimens as the standard”). However, at the time of this writing, high dose interleukin-2 cannot be dismissed and remains one of the standard treatment approaches for appropriately selected patients treated at centers with expertise in the administration of this therapy.

Cytokine Combinations Pre-clinical studies of interleukin-2 and interferon alpha suggested additive or synergistic activity and led to a number of phase I and II studies to investigate the clinical activity of this combination. A review of over 1,400 patients has been published demonstrating response rates of approximately 20% and complete remissions in 5% of

patients irrespective of the route of interleukin-2 administration [8]. Because of this apparent slight but potentially significant improvement in the response rate, several randomized trials have been performed comparing combination versus single agent cytokine therapy. The largest of these, reported by Negrier et al., comprised over 400 patients and demonstrated an improved response rate for interleukin-2 plus interferon alpha (18.6%) compared to interleukin-2 (6.5%) or interferon alpha (7.5%) alone [65]. Unfortunately, the improvement in the response rate did not lead to a significant improvement in median survival, although the crossover design for patients failing to respond to interleukin-2 or interferon alpha monotherapy makes comparison of the survival times difficult. It is important to note that the overall response rate to cytokine monotherapy in this study was lower than what has generally been observed, again underscoring the importance of patient selection and the importance of randomized trials for comparing the outcomes of different treatments. An important observation resulted from the crossover design of this study: responses to interleukin-2 after interferon failure or interferon alpha after interleukin-2 failure are rare [20]. Thus, at present, monotherapy with either interleukin-2 or interferon alpha at modest doses remains the standard of cytokine care for most patients with kidney cancer and can be expected to result in tumor regression in 10–20% and overall median survivals of slightly greater than a year [32]. A cytokine therapy must be reinterpreted in light of the newer molecularly targeted agents described in a subsequent section. Perspective randomized trials of cytokine therapy versus targeted agents, and/or combinations thereof, are currently underway [32].

Table 3. Randomized trials comparing high dose versus low dose interleukin-2 Study/treatment

N

Complete responses (%)

Partial responses (%)

Objective response rate (%)

115 112 53

9 (8) 5 (4) 3 (6)

13 (11) 6 (5) 3 (6)

22 (19) 11 (10) 6 (11)

99 94

8 (8) 2 (2)

17 (17) 10 (11)

25 (25) 12 (13)

214 259

17 (8) 10 (4)

30 (14) 19 (7)

47 (22) 29 (11)

a

NCI Study(107) HD-IL-2 LD-IL-2 (IV) LD-IL-2 (SC) CWG Study(55) HD-IL-2 LD-IL-2 (SC) + IFN (SC) Totals HD-IL-2 LD-IL-2 (IV or SC) +/− IFN

HD-IL-2 high dose interleukin-2 by intravenous bolus injection; LD-IL-2 low dose interleukin-2 by intravenous bolus injection (IV) or subcutaneous injection (SC); IFN interferon alpha by subcutaneous injection a The NCI study was initially designed to evaluate high dose versus low dose intravenous bolus injections of interleukin-2. After the first 117 patients were randomized (56–57 per arm), the third arm of the study was added to compare subcutaneous interleukin-2 administration. For a statistically valid comparison, these data have been reported comparing only concurrently randomized patients [104]. For informational comparison in this table, the data have been presented as if all patients had been randomized concurrently.

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Adoptive Cellular Therapy Lymphocyte Activated Killer Cells and Tumor Infiltrating Lymphocytes Historically and for the purposes of review, cancer immunotherapy has been classified as “active,” including agents such as cytokines that activate the host anti-tumor immune response, and “passive,” including agents such as monoclonal antibodies or immune effector cells that directly (or sometimes indirectly) mediate the anti-tumor response themselves. The transfer of immune effector cells with direct anti-tumor reactivity has also been termed “adoptive cellular therapy.” In kidney cancer, the use of lymphokine-activated killer (LAK) cells is of great historical significance, since the development of modern cytokine therapy using interleukin-2 for renal cell carcinoma was originally based on the observation, in the early 1980s, that LAK cells are lymphoid cells that circulate in the peripheral blood, are activated in vitro by exposure to high concentrations of interleukin-2, and have the ability to selectively lyse neoplastic cells [44]. Early clinical trials of interleukin-2 were therefore done in combination with LAK cells, or more accurately stated, early trials of LAK cells required the systemic administration of interleukin-2 to demonstrate efficacy of the LAK cells [36, 85, 98, 104]. An initial response rate in excess of 75% was reported [85], but a series of phase II studies including over 500 patients with renal cell carcinoma demonstrated that this treatment approach lead to anti-tumor responses in 22% of patients (range 9–35%) [40]. Moreover, most of these responses have been partial and transient, typically lasting no more than several months. Subsequently, randomized trials at the National Cancer Institute [86] and elsewhere [40]) comparing interleukin-2 plus or minus LAK cells failed to demonstrate a contribution to response rate or survival by the LAK cells. In the hope of reducing the number of cells needed for passive transfer and decreasing the requirement for the co-administration of high dose interleukin-2, tumor infiltrating lymphocytes (TIL) were discovered in the search for a more potent lymphoid effector cell [108]. Whereas LAK cells are nonspecific natural killer cells, TIL are activated cytotoxic T cells, which show a greater propensity for target-specific killing. TIL are isolated from a primary or metastatic tumor, and, similar to LAK cells, are expanded ex vivo in the presence of interleukin-2, then reinfused in combination with systemic cytokine therapy. In murine models, TIL were found to be 50–100 times more potent on a per cell basis in the treatment of established lung micrometastases than LAK cells [87]. Sufficient studies utilizing acceptable doses of interleukin-2 along with escalating doses of activated cells have not

Biological therapy of genitourinary cancer been done. Perhaps the most extensive dose-related studies have been accomplished by Oldham and co-workers [51] in utilizing as many as ten doses of activated T cells in treatment of patients with advanced malignancy. With regard to patients with renal cancer, only four doses have been used, giving total cell doses up to 2 × 1011 cells. A series of phase I–II trials were performed, including 114 patients with renal cell carcinoma, treated with TIL plus IL-2, and demonstrating an objective response rate of 23% (range 0–35%) [40]. Again, in a randomized trial comparing interleukin-2 therapy alone to the combination of interleukin-2 plus TIL in 178 patients with renal cell carcinoma, there was no advantage in terms of response rate, duration of response, or survival to the addition of the TIL compared to interleukin-2 alone [23]. The addition of an expanded population of autologous tumor killing cells in the form of TIL or LAK cells to interleukin-2 has been carefully studied and in its present form offers no advantage to the treatment of kidney cancer over cytokine therapy alone. Nonetheless, early observations demonstrated that melanoma patients treated with TIL plus interleukin-2 were capable of responding even when a prior response to interleukin-2 alone was not observed [88]. The administration of an expanded population of highly selected T cells derived from autologous TILs in combination with high dose IL-2 and following a non-myeloablative chemotherapy conditioning regimen mediated the regression of metastatic melanoma and MRC patients refractory to standard therapy, including high dose IL-2. This study demonstrated that prior lymphodepletion improves the persistence and function of adoptively transferred cells [17]. Ongoing studies at the National Cancer Institute and in Europe indicate that TIL cells can be genetically engineered to enhance their activity against both melanoma and kidney cancer. Such genetically retargeted cells sometimes, accompanied by lympho-depletion strategies, have shown high response rates in a small series of patients over the last 5 years [17, 50].

Allogeneic Stem Cell Transplantion A novel approach to the biological treatment of metastatic renal cell carcinoma has employed allogeneic hematopoietic stem cell transplantation in an attempt to mediate anti-tumor activity from donor T-cells from an HLA matched sibling donor. This treatment strategy represents a form of adoptive cellular immunotherapy. Based on data that the transfer of allogeneic stem cells in the treatment of hematalogic malignancies can induce a graft-versus-leukemia reaction that may improve the cure of these malignancies, a similar graft-versus-tumor

Robert K. Oldham effect has been sought in the treatment of metastatic kidney cancer. Childs et al. established proof-of-principle for the existence of the GVT effect in metastatic kidney cancer [10, 11] and have recently reviewed their experience at the National Institutes of Health using this treatment modality [12]. Over 50 patients have been treated, and 47% have achieved evidence of tumor regression, including 18 (38%) partial responses and 4 (9%) complete responses [12]. Three of the four complete responders remain disease free, including the first patient treated whose remission exceeds 5 years. Interestingly, the majority of patients that eventually respond to this treatment experience initial tumor progression in the first few months following the procedure, and tumor regression occurs late in the course of the transplant at a median of 5 months. Disease regression typically occurred after the withdrawal of immunosuppression and after the conversion of the host to full donor T-cell chimerism, implying that these responses resulted from the GVT activity of donor T-cells. These results demonstrate the proof-ofprinciple that a clinically meaningful graft-versus-tumor response can be achieved in the setting of metastatic renal cell carcinoma. The wide applicability of this approach may be limited by the long interval of time required from the initiation of the evaluation of the patient for treatment with stem cell therapy until the withdrawal of immunosuppression when a graft-versustumor effect may be expected. This time interval has so far required many months, during which time patients with even a moderate rate of tumor progression may worsen clinically and become ineligible for aggressive intervention or succumb to their disease, particularly in light of the fact that most patients will have been treated with a traditional treatment approach before consideration of this novel form of immunotherapy. Indeed, as expected, of the first 19 patients reported by Childs et al., eight patients died of disease progression within approximately 1 year while awaiting the GVT effect during follow-up after transplant [10]. As a result, treatment of patients with stem cell transplantation requires careful selection of a subset of kidney cancer patients who are fit enough to undergo an aggressive treatment and who have a slowly progressive form of the disease. The role of stem cell therapy for the treatment of metastatic renal cell carcinoma will require further study with large numbers of patients and confirmation of these intriguing results by other investigators at other sites [84].

Vaccines and Gene Therapy Vaccines and gene therapy approaches to the treatment of kidney cancer have generally attempted to improve

653 tumor antigen presentation and patient T-lymphocyte activation by enhancing tumor antigenic peptide presentation in the context of major histocompatibility complex (MHC) molecules, by restoring co-stimulatory signals that are frequently deficient in tumor cells, and by amplifying recruitment of the patient’s immune effector cells. Gene therapy strategies for kidney cancer are based on the premise that genetic modification of immune cells can enhance function or that the introduction of a particular gene into tumor cells can increase their immunogenicity and induce an effective immune response not only against the gene-modified tumor, but also against the unmodified tumor cells that are present elsewhere in the individual [39, 67]. Tumor cells may be modified in vitro or in vivo to express cytokines, co-stimulatory molecules, or foreign MHC molecules.

Vaccine therapy Dendritic cells (DC) are the most potent, professional antigen presenting cells (APC) of the immune system and may be uniquely able to stimulate T-cells because they possess all of the required co-stimulatory molecules. Dendritic cells can be isolated from peripheral blood mononuclear cells and expanded and modified ex vivo, making vaccination with DC feasible. Strategies to induce the expression of tumor antigens on dendritic cells include gene transfer into DC using viral or non-viral vectors, tumor cell lysates, apoptotic bodies, tumor-derived RNA, and DC-tumor cell hybrids. Alternately, dendritic cells may be expanded in vivo by the systemic administration of combinations of cytokines such as GM-CSF and IL-4 [90]. These approaches indicate there is a need to reach a balance between immunity and tolerance [16]. A study in patients with metastatic renal cell carcinoma demonstrated the potential effectiveness of DC vaccines using electrofusion to generate autologous tumor cell-allogeneic DC hybrids [49]. Seventeen patients were vaccinated subcutaneously, and after vaccination, four patients achieved complete responses and two patients had partial responses. Fever and injection site pain were the only important toxicities reported. Controversy exists regarding the effectiveness and reproducibility of the electrofusion technique used in this study, but other human clinical trials employing dendritic cell vaccines in patients with metastatic renal cell cancer have also shown anti-tumor responses [33, 41]. The field of cancer vaccination has been advanced by evidence that both cytotoxic and helper T cells recognize intracellularly degraded peptides that are processed by specialized antigen presenting cells via the proteasome apparatus, inserted into the endoplasmic reticulum, and transported to the cell surface for association

654 with major histocompatibility (MHC) molecules [103]. Taking advantage of this finding, a specific approach to improving effective antigen presentation for the treatment of renal cell carcinoma involves the use of peptide vaccines derived from heat shock protein–peptide complexes from kidney tumors. Heat shock proteins (HSP) are molecular chaperones implicated in “loading” immunogenic peptides onto MHC molecules for presentation by antigen presenting cells to T-cells. In this vaccination strategy, autologous tumor derived peptides associated with HSP administered to patients are taken up by antigen presenting cells such as DC and processed by the APC so that tumor antigen is “re-presented” to naïve T-cells, leading to an antigen specific T-cell response [94]. Amato et al. have treated patients with metastatic renal cell carcinoma with autologous gp96 heat shock protein–peptide complex vaccination in a phase I–II trial [3]. The vaccine, at one of three doses, was administered once with a week for 4 weeks with follow-up doses at weeks 12 and 20 depending on response. Responses were observed in 4 of 16 patients (25%) at the 25 ug dose level, including one patient with a complete response; three additional patients experienced prolonged stabilization of disease in excess of 1 year. Follow on phase III randomized studies have not confirmed these earlier trials, and there has not been a significant difference seen between the HSP arm and the placebo arm in these adjuvant studies [95].

Gene Therapy Human gene therapy generally involves the insertion of functional DNA into a human cell either in vivo or ex vivo to replace a defective gene or to provide a new function to the cell. Traditionally, the therapeutic DNA has been packaged in a viral or lipid vector for delivery of a functional gene into human cells. Alternately, naked DNA, generally in the form of a plasmid, may be directly injected into skin or muscle or adsorbed onto microparticles and forcibly bombarded into the skin in vivo or other cells ex vivo. Naked DNA injections have been alternately termed “polynucleotide vaccines.” Human gene therapy approaches to the treatment of kidney cancer that have advanced into clinical trials have included altering immune effector cells with a gene designed to improve their function or altering tumor cells with a gene designed to improve their immunogenicity [33]. The “polynucleotide vaccine” strategy of transferring a gene encoding a tumor antigen into endogenous antigen presenting cells has yet to be exploited in the clinical model of renal cell carcinoma, although this approach has been attempted clinically in other solid tumors [89]. In an example of the former approach, investigators

Biological therapy of genitourinary cancer used natural killer cells transfected with the interleukin-2 gene for the adoptive immunotherapy of renal cell carcinoma and other malignancies [91]. This trial demonstrated the feasibility of this approach, the relative lack of toxicity, a significant increases in other immunostimulatory cytokines (interferon gamma, transforming growth factor beta, and granulocyte-macrophage colony stimulating factor), and clinical response in one patient with lymphoma. There are several examples of the latter gene therapy approach of genetically modifying tumor cells to increase their immunogenicity in the treatment of kidney cancer. Autologous renal cell carcinoma cells have been retrovirally transduced with the gene for granulocyte-macrophage colony stimulating factor and re-administered as a vaccine to 16 patients with metastatic renal cell carcinoma [93]. No significant toxicities were observed, and one patient experienced a partial remission. Another strategy to make tumor cells more immunogenic has involved the transfer of the gene encoding human leukocyte antigen (HLA) molecule B7 into autologous renal cell carcinoma cells. In one study, lipidDNA complexes (commercially called “Allovectin-7”) were transferred into accessible metastatic lesions in 15 HLA B7 negative renal cell carcinoma patients [83]. Conceptually, the intent of these intratumoral injection studies is to develop an in vivo autologous tumor vaccine that will lead to a systemic disease response. In this example, neither toxicity nor responses were observed. A similar example of this approach includes the direct transfer of the lipid complexed interleukin-2 gene into accessible metastatic lesions in patients with renal cell carcinoma. Further details of gene therapy are presented in Chapter 19.

Targeted therapy It has been known for many years that renal cancer overexpresses vascular endothelial growth factor (VEGF). Because of this finding, recent efforts have targeted VEGF in an attempt to block vascular supply and blood supply to MRCC. Bevacizumab (Avastin) is a recombinant human monoclonal antibody against VEGF [32, 71]. In a phase II trial or more than 100 patients with MRCC, randomized placebo versus a low-dose (3 mg/kg) or high-dose (10 mg/kg) of bevacizumab given intravenously in treatment-resistant patients every 2 weeks (over 90% had received prior IL-2) demonstrated an improved time to progression for the bevacizumab group [106]. As a result of this interesting phase II trial, there are ongoing studies with bevacizumab plus other agents active in MRCC [81]. Bevacizumab plus interferon is

Robert K. Oldham more active than interferon alone [106], and bevacizumab combined with erlotinib (Tarceva), an EGRF inhibitor, has shown objective responses in patients with resistant, advanced renal cancer [37]. Many clinical trials are underway using various combinations of cytokines and targeted therapies, but no specific combination has proven superior to an alternative combination to-date. In addition to a large molecule inhibitor of VEGF, such as the monoclonal antibody bevacizumab, there are small molecule inhibitors, which work through the super family of tyrosine kinases. Three of the agents are currently approved for the treatment of MRCC and are being used alone and in various combinations with the other active agents. Sorafenib (Nexavar) is an oral agent that inhibits Raf kinase. Sorafenib also is a direct inhibitor of the VEGF receptor family. In a phase II randomized study with over 200 patients with MRCC, patients received 400 mg twice a day of sorafenib versus placebo. The median progression free for survival was longer in the sorafenib group.. This study was followed by a phase III randomized trial or sorafenib versus placebo in cytokine-refractory MRCC [21]. Although only a small percentage of patients had a complete or partial response, and many of the patients exhibit stable disease while receiving the drug. The median progression free survival for the sorafenib group was 24 weeks, double that of placebo. Sorafenib was well tolerated, but does have significant side effects which are generally manageable, including dermatological, gastrointestinal and neurologic symptoms, along with fatigue and elevates blood pressures [76]. Another oral tyrosine kinase inhibitor of VEGF receptor is sunitinib (Sutent). This drug was effective in phase II trials and was subsequently approved based on randomized phase III trial in patients with MRCC [59]. Again, the median progression free for survival was approximately double in the treated group versus the placebo group. A third small molecule, temsirolimus (Torisel), has recently been approved for poor risk MRCC patients. This drug is given intravenously and is an inhibitor or rapamycin (mTOR), a molecule important in tumor-promoting intracellular signaling pathways. Studies comparing this agent to interferon-alpha demonstrated an improvement in survival amounting to 3–6 months [42] Other agents, such as lenalidomide [13], tykerb lapatinib [77] and axitinib [82], are underway. While it is clear that these small molecule and large molecule agents targeting various receptors and molecules that support neovascularization and/or promote tumor growth are important individually, it is unknown how best to combine them with other agents active in

655 MRCC. The daunting task of working through this will require many randomized trials for patients with MRCC [14]. While the best combination is not known, the future looks much brighter for these patients than it did a mere 5 years ago.

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657 75. Quesada JR, Kurzrock R, Sherwin SA, Gutterman JU. Phase II studies of recombinant human interferon gamma in metastatic renal cell carcinoma. J Biol Response Mod 1987; 6:20–27. 76. Ratain MG, Eisen T, Stadler WM, et al. Phase II placebo-controlled randomized discontinuation of trial of sorafenib in patients with metastatic renal cell carcinoma. J Clin Oncol 2006; 24:2505–2512. 77. Ravaud A, Gardner J, Hawkins R, et al. Efficacy of lapatinib in patients with high tumor EGFR expression: results of a phase III trial in advanced renal cell carcinoma (RCC) [abstract]. J Clin ONcol 2006; 24(Suppl):4502. Abstract 4502. 78. Reuter VE, Presti JC. Contemporary approach to the classification of renal epithelial tumors. Semin Oncol 2000; 27:124–137. 79. Ries LAG, Harkins D, Krapcho M, et al. eds. SEER Cancer Statistics Review, 1975–2003, Bethesda, MD: National Cancer Institute; 2006. 80. Rinehart JJ, Malspeis L, Young D, Neidhart JA. Phase I/II trial of human recombinant interferon gamma and renal cell carcinoma. J Biol Resp Modif 1986; 5:300–308. 81. Rini BI, Halabi S, Taylor J, et al. Cancer and Leukemia Group B 90206: a randomized phase III trial of interferon-alpha or interferon-alpha plus anti-vascular endothelial growth factor antibody (bevacizumab) in metastatic renal cell carcinoma. Clin Cancer Res 2004; 10:2584–2586. 82. Rini BI, Rixe O, Bukowski RM, et al. AG-013736, a multi-target tyrosine kinase receptor inhibitor, demonstrates anti-tumor activity in a phase 2 study of cytokine-refractory metastatic renal cell cancer (RCC) [abstract]. J Clin Oncol 2005; 23(Suppl):4509. Abstract 4509. 83. Rini BI, Selk LM, Vogelzang NJ. Phase I study of direct intralesional gene transfer of HLA-B7 into metastatic renal carcinoma lesions. Clin Cancer Res 1999; 5:2766–2772. 84. Rini BI, Zimmerman TM, Gajewski TF, Stadler WM, Vogelzang NJ. Allogeneic peripheral blood stem cell transplantation for metastatic renal cell carcinoma. J Urol 2001; 165:1208–1209. 85. Rosenberg SA, Lotze MT, Muul LM et al. Observations on the systemic administration of autologous lymphokine- activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 1985; 313:1485–1492. 86. Rosenberg SA, Lotze MT, Yang JC. Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer. J Natl Cancer Inst 1993; 85:622. 87. Rosenberg SA, Spiess P, Lafreniere R. A new approach to the adoptive immunotherapy of cancer with tumor- infiltrating lymphocytes. Science 1986; 233:1318–1321. 88. Rosenberg SA, Yannelli JR, Yang JC et al. Treatment of patients with metastatic melanoma with autologous tumor- infiltrating lymphocytes and interleukin 2. J Natl Cancer Inst 1994; 86:1159–1166. 89. Rousseau RF, Hirschmann-Jax C, Takahashi S, Brenner MK. Cancer vaccines. Hematol Oncol Clin North Am 2001; 15:741–773. 90. Roth MD, Gitlitz BJ, Kiertscher SM et al. Granulocyte macrophage colony-stimulating factor and interleukin 4 enhance the number and antigen-presenting activity of circulating CD14+ and CD83+ cells in cancer patients. Cancer Res 2000; 60:1934–1941. 91. Schmidt-Wolf IG, Finke S, Trojaneck B et al. Phase I clinical study applying autologous immunological effector cells transfected with the interleukin-2 gene in patients with metastatic renal cancer, colorectal cancer and lymphoma. Br J Cancer 1999; 81:1009–1016. 92. Schrodter S, Hakenberg OW, Manseck A, et al. Outcome of surgical treatment of isolated local recurrence after radical nephrectomy for renal cell carcinoma. J Urol 2002; 167:1630–1633. 93. Simons JW, Jaffee EM, Weber CE et al. Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo

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21.4 Biological therapy of colon cancer ROBERT O. DILLMAN

Colon Cancer Non-specific Immune Stimulants Levamisole Levamisole is a synthetic phenylimidazothiazole oral antihelminth that exhibited non-specific immunostimulatory properties in animal models [82]. Interest in this agent as a cancer therapy dates back to 1971. Small exploratory clinical studies were inconclusive, but considered encouraging enough to lead to randomized phase III trials in colon cancer. Two randomized trials failed to demonstrate a benefit for levamisole alone compared to placebo in the adjuvant setting. In one small double-blind U.S. trial 78 patients were randomized to receive 18 months of levamisole 2.5 mg/kg/day given on days 1 and 2 of each week, or placebo following resection of Dukes B or C colorectal cancer [8]. No benefit for levamisole was observed. In a double-blind randomized European trial comparing levamisole at 100 to 250 mg twice a week to placebo as adjuvant therapy for 297 patients with node-positive colon cancer, there again was no demonstrable benefit for levamisole [1]. Other trials combined levamisole with 5-fluorouracil (5FU). In a large adjuvant trial 1,296 patients, who had resected colon cancer that was either locally invasive (n = 318) or metastatic to regional lymph nodes (n = 929), were randomized to observation, or to 5FU plus levamisole for 1 year, or to levamisole alone [58]. Patients were assigned to observation, or to levamisole alone (50 mg p.o. three times a day for 3 days, repeated every 2 weeks for 1 year), or to this regimen of levamisole plus 5-FU (450 mg/m2 i.v. daily for 5 days and then, beginning at 28 days, weekly for 48 weeks). Once again levamisole alone provided no benefit compared to observation, but after a median follow up of 3 years, the combination of 5-FU plus levamisole was associated with a significantly reduced risk of recurrence and death for patients who had lymph node metastases (p = 0.006). After a median follow up of 6.5 years the advantages of levamisole + 5FU persisted in stage III disease, with a 40% reduced recurrence rate (p < 0.0001) and 33% reduced death rate (p = 0.0007) [59]. No benefit was

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

demonstrated in the smaller subset of patients with stage II disease [60]. On the basis of this trial, 5FU plus levamisole became standard therapy for the adjuvant treatment of stage III colon cancer during 1990–1997. A major limitation of the early levamisole trial was that it did not have a control arm of 5FU plus folinic acid (leucovorin), which was shown to be more active than 5FU alone while the trial was being conducted. Two large U.S. trials subsequently showed that levamisole did not add benefit to such a regimen. During 1989–1990 2,151 patients who had stage II or III colon cancer were randomized to receive weekly 5FU plus leucovorin, 5FU plus levamisole, or 5FU plus leucovorin plus levamisole [98]. After more than 7 years of follow up, the 5-year survival rates were 74% for 5FU plus leucovorin, 73% for all three drugs, and 70% for 5FU plus levamisole. During 1988–1992 3,794 patients were randomized in a four-arm trial to receive 5FU plus levamisole for 1 year or 6 months of low-dose leucovorin plus 5FU (“Mayo Clinic regimen”), or high-dose leucovorin plus 5FU (“Roswell Park regimen”) or the Mayo clinic regimen plus levamisole, plus FU (LDLV plus LEV) regimen [33]. After 10 years of follow up there were no differences in survival among the arms, and the 5FU plus leucovorin regimens replaced the levamisole regimens as standard therapy when the results were first reported in 1997. Other randomized trials of adjuvant therapy in colon cancer confirmed beyond a doubt that the addition of levamisole provided no benefit in the adjuvant therapy of colon cancer. In a trial in which 379 patients received 5FU plus folinic acid, and 374 received 5FU plus levamisole, after a median follow up of 6.8 years, there was a 72% survival rate in both arms [63]. In a four-arm trial in which 598 patients were randomized to receive 1 year of weekly 5FU, or 5-FU plus levamisole, or 5-FU plus IFN-α or all three agents, recurrence rates and survival were worse in the combined arms that contained levamisole (p = 0.003) [77]. In another randomized trial there was no difference in survival for 92 patients who received 5FU plus levamisole compared to 92 who received 5FU alone, but leucopenia and hepatic toxicity were more frequent in the levamisole arm [5]. In a two-arm

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Biological therapy of colon cancer but no responses in previously treated patients [89–91]. Unfortunately, other investigators typically reported much lower response rates for this combination in colorectal cancer, with a range of 20% to 50% [12, 49, 68, 92, 93]. Randomized trials were unable to confirm response rates any higher than 20% to 25%, which is no different that 5FU alone. In a randomized trial for patients with previously untreated advanced colon cancer, the response rate was 21% for 104 patients who received 5FU alone (750 mg/m2 as a 4-h i.v. infusion on days 1–5 and then i.v. bolus weekly) compared to 25% for 101 patients who received 5FU plus IFN (3 to 9 MIU thrice weekly s.c.) [67]. In a similar trial that utilized the same dose and schedule of 5FU, but IFN at a dose of 10 MIU s.c. thrice weekly, the response rate was 30% for 54 patients who received 5FU alone and only 19% for 52 patients who received 5FU plus IFN [39]. The lower response rate was probably due to patients withdrawing from the trial because of toxicity. Patients who received IFN experienced more toxicity, including four toxic deaths, leucopenia, lymphopenia, depression, and alopecia. Subsequent trials combined 5FU, folinic acid, and IFN-α, but the response rates were no higher than the doublets [32, 44, 69]. Randomized trials confirmed that IFN-α adds toxicity, but not benefit, to 5FU and folinic acid. In a trial that included 204 untreated patients, there was no difference in response rate was (23% to 24%) or survival (medians 11 to 12 months) [10]. The addition of IFN was associated with more toxicity, especially fever, diarrhea, and mucositis. In another trial 102

trial in which 1,703 colon cancer patients were randomized to 5FU plus levamisole or 5FU plus leucovorin plus levamisole, there was no difference in survival, but the three drug regimen was more toxic [13].

Thymosin Fraction 5 The importance of the thymus gland and thymic proteins for normal T-lymphoycte cellular immunity has been recognized for many decades. One of the preparations of interest was a crude lysate called thymosin fraction 5. As summarized in Table 1, subsequent dose-exploring and phase II trials failed to confirm significant activity in lung cancer or immune enhancing activity for thymosin fraction 5 [14, 15].

Interferons As shown in Table 1, single agent alpha-interferon (IFNα) rarely produces objective tumor responses in patients with metastatic colorectal cancer [4, 62]. In three phase II trials there were no objective responses observed with either lymphoblastoid interferon or alpha interferon [6, 9, 54]. However, there is laboratory evidence that IFN-α can enhance the antitumor activity of 5-FU by a mechanism which is different from that of leucovorin [61, 89]. An early clinical trial which combined 5-FU with IFN-α for the treatment of advanced colorectal carcinoma claimed a 75% objective response rate among patients who had not been treated previously with chemotherapy,

Table 1. Single-agent activity of various biologicals in patients with metastatic colon cancer Modality class

Biological agent

Reference

Patients

Response rate (%)

Non specific Cytokine Cytokine Cytokine Cytokine Cell therapy Cell therapy Cell therapy Cell therapy Cell therapy Mab Mab Mab Mab Mab Mab

Thymosin fraction 5 Lymphoblastoid Interferon Interferon-α Interferon-α Interleukin-2 (IL-2) IL-2 + LAK IL-2 + LAK IL-2 + TIL IL-2 + TIL CD4+ T cells Anti-CEA Edrocolomab Edrocolomab Edrocolomab Edrocolomab Bevacizumab

Dillman [6] [54] [9] [73, 74] [73, 74] [17] [64] [20] [19] [16]

12 19 18 36 22 38 35 8 8 15 30 40 20 25 52 234

0 0 0 0 0 18 0 0 0 7 0 0 5 4 2 3

Mab = monoclonal antibody LAK = lymphokine activated killer cells TIL = tumor infiltrating lymphocytes CEA = carcinoembryonic antigen

[24] [52] [57] [28]

Robert O. Dillman patients were randomized to receive 5FU and leucovorin with or without IFN; there was no difference in response rates (8% and 10%) but time to progression and overall survival both favored the group that did not received IFN (p = 0.002) [50]. Thus, clinical trials showed that 5FU plus IFN-α was no better than 5FU plus leucovorin, but more toxic, and that there was no advantage for the addition of IFN-α to 5FU plus leucovorin in the treatment of metastatic colorectal cancer. Several randomized trials have established that there is no role for IFN-α in the adjuvant treatment of highrisk colon cancer. In a Greek trial that included stage II and III colon cancer 139 patients received 5FU plus folinic acid and 141 received the same therapy plus IFN (3 MU s.c. thrice weekly) [23]. After a median follow up of 4 years, there was no difference in disease free survival or overall survival. In an Italian trial in which 322 patients with stage II or III colon cancer were randomized to infusional 5FU plus leucovorin with or without IFN (5 MU s.c. thrice weekly), there was no difference in either disease-free survival or overall survival but toxicity was greater in the IFN-arm because of flu-like syndrome [26]. In a three-arm German trial, 813 patients with stage II and 750 with stage III colon cancer were randomized to 5FU plus levamisole alone or with leucovorin, or with IFN [81]. The 4-year survival rate was highest in the arm that included leucovorin (78%), but was no better in the IFN-arm than 5FU plus levamisole alone (both 66%). In a four-arm trial in which 598 patients were randomized to receive 1 year of weekly 5FU, or 5-FU plus levamisole, or 5-FU plus IFN-α or all three agents, there was increased toxicity in the IFNarms but no effect on survival [77]. The National Surgical Adjuvant Breast and Bowel Project (NSABP) group randomized 2,176 patients with stage II or III colon cancer to receive 5FU plus leucovorin with or without IFN [97]. After 4.5 years of follow up there was no difference in disease free survival (69–70%) or overall survival (80–81%), but there was more toxicity associated with IFN. In one small randomized trial 99 patients with stage II, III, or IV colon cancer were randomized to receive IFN-gamma 0.2 mg s.c. daily for 6 months or observation after surgical resection of their disease [96]. Despite evidence that IFN-γ induced immune response, after a median follow-up of almost 5 years, disease free survival was worse for patients who received IFN-γ (p = 0.03) and they had a trend to worse survival (p = 0.12).

Interleukin-2 There is only limited clinical trial data with interleukin-2 (IL-2) in colorectal cancer, and most of the experience

661 has been in combination with adoptive cell therapy or in combination with other biologicals. High-dose bolus IL-2 yielded no objective responses in 22 patients [73, 74]. The response rate for high-dose continuous infusion IL-2 and tumor necrosis factor (TNF-α) was 0/16 [18]. The response rate for high-dose continuous infusion IL-2 and IFN-α was 1/10 [65].

Adoptive Cell Therapy There has been only limited exploration of cell-based therapies for colon cancer. As described in detail elsewhere in this book, lymphokine activated killer [LAK] cells are derived by taking peripheral blood lymphocytes (PBL) and stimulating them in vitro with IL-2 while tumor infiltrating lymphocytes (TIL) are produced by incubating tumor samples with IL-2. For patients with metstatic colon cancer, LAK administered with high dose bolus IL-2 produced responses in 7/38 (18%) patients [73, 74] AK cells infused with high-dose continuous infusion IL-2 (civ IL-2) produced no responses in 35 patients [18]. There were no responses among 16 patients treated with TIL, eight of whom received high doses of TIL plus civ IL-2, and eight who received variable doses of TIL with other schedules of IL-2 [20, 64]. Autolymphocyte therapy and autologous activated lymphocytes were used to describe autologous lymphocyte preparations that were derived from peripheral blood and stimulated with anti-CD3 monoclonal antibodies in vitro to produce an auto-cytokine preparation that was then used for incubation with additional peripheral blood lymphocytes [19, 66]. The autolymphokine preparation contained significant amounts of TNF-α, IL-1β, interferon-γ, and IL-6, but no IL-2. Culturing peripheral blood lymphocytes in this media resulted in enrichment of CD3+ cells, but decreased CD3+/CD56+ cells (NK), decreased CD4+/ CD45RA(2H4) suppressor T cells, and increased CD8+/ HLA-Dr+, and CD4+/CD29(4B4)/HLA-DR+ helper T cells. There was a slight decrease in NK cytolytic activity in Cr51 assays compared to the mononuclear cells obtained prior to incubation in ALK. These cells, which lacked cytolytic activity, were infused in doses of about 109 cells in patients who were receiving high doses of cimetidine to inhibit suppressor or negative regulatory T cells. A response rate of 1/15 was observed with such a product in the setting of metastatic colon cancer [19].

Monoclonal Antibodies Early trials with murine anti-CEA monoclonal antibodies (Mabs) were not associated with clinical responses [16]. However as described in detail elsewhere in this book,

662 there are now three monoclonal antibody products that are commercially available with marketing indications for colon cancer. These include the anti-VEGF Mab bevacizumab, the chimeric anti-EGFR Mab cetuximab, and the human anti-EGFR Mab panitumumab. The murine anti-Epcam Mab edrocolomab was approved in Germany for the treatment of colon cancer several years earlier.

Edrocolomab (17-1A, PANOREX®) Edrecolomab is a murine IgG2a now known to react with the 37–40 kd glycoprotein epithelial cell adhesion molecule (epcam) that is expressed on various adenocarcinomas and on normal epithelial tissues [31, 38]. Single injections of edrecolomab at doses of 15–1,000 mg were well tolerated, but 50% of patients developed HAMA after a single injection [22, 78, 79] summarized in Table 1, as a single agent, edrocolomab exhibited minimal anti-tumor effects against colon cancer. There were no objective responses among 40 patients who received single infusions of 15–1,000 mg of 17-1A during phase I trials in patients with various gastrointestinal adenocarcinomas [78, 79]. In a phase II trial of 20 patients who received 200–850 mg of 17-1A, there was one response in a patient with recurrent rectal cancer [24]. In a trial of 25 patients who received one to four doses of 400 mg of 17-1A, one patient had a complete response [52]. Subsequently, this group treated eight patients with a 17-1A chimeric antibody with a human IgG4 subclass heavy chain but there were no responders [53]. Mellstedt and colleagues reported one objective response among 52 patients who received 17-1A by a variety of doses and schedules [24, 57]. An apparent benefit for adjuvant edrocolomab was demonstrated in a multicenter randomized German trial in patients with resected stage III colorectal cancer [71]. There were 90 patients randomized to observation, and 99 to a post-operative regimen of edrecolomab 500 mg i.v., then 100 mg i.v. once a month for 4 months. After a median follow up of 5 years, there was superior disease-free and overall survival for the Mab-treated group. This included a 30% reduction in death rate and 27% reduction in tumor recurrence, which was similar to results reported in U.S. trials of 5-fluououracil and levamisole that had led to acceptance of 5-FU based chemotherapy in the adjuvant treatment of colon cancer. After a median follow up of 7 years, the apparent benefits of edrecolomab persisted with a 23% reduction in recurrence and 32% reduction of death in the antibody group [72]. On the basis of this trial, edrecolomab was approved for the treatment of Dukes C colorectal cancer in Germany in 1995. Unfortunately these promising results were not confirmed by four additional phase III trials. North American

Biological therapy of colon cancer trials compared 5-FU + levamisole, or 5-FU + leucovorin to the same agents plus edrecolomab in Dukes C colon cancer using the same dose and schedule used in the German trial. European trials compared 17-1A antibody alone, to 5-FU + eucovorin, and to 5-FU + leucovorin + 17-1A. Edrecolomab was also studied in combination with chemotherapy and radiotherapy in Dukes B and C rectal cancer. Results of the large randomized European adjuvant trial did not demonstrate a benefit for the use of edrecolomab as adjuvant therapy [70]. There were 2,761 patients randomized in the threearm trial. The major toxicities observed in the edrecolomab-alone arm were diarrhea in 32%, although severe or life-threatening diarrhea was noted in only 2%. Results in the edrecolomab-alone arm were inferior to the 5FU-containing arms (p < 0.05), and there was no evidence of better results when edrocolomab was combined with 5FU (p = 0.53). Three-year survival rates were 76% for 5FU plus leuovorin, 75% for the antibody plus chemotherapy combination, and 70% for the antibody alone. The differences in 3-year event free survival also revealed inferior results for antibody alone, and no advantage for the chemotherapy plus antibody combination. In another trial 377 patients with stage 2 colon cancer, stratified by whether the primary tumor was T3 or T4, were randomized post-operatively to treatment with 900 mg edrecolomab or observation [35]. The study was stopped because of discontinuation of drug supply in Germany after negative results from other trials became public. After a median follow-up of 3.5 years there was no difference in overall survival or diseasefree survival. Several trials have explored edrecolomab in combination with other biologicals with or without chemotherapy. Weiner et al. gave 150 mg of 17-1A on days 2 to 4 in combination with 1.0 MIU/m2 of IFN-γ on days 1 to 4, to 19 colorectal cancer patients, but no antitumor responses were noted [95]. In a second trial 27 patients with colon or pancreatic cancer were given IFN-γ at doses up to 40 MIU/day for 4 days followed by 400 mg 17-1A on day 5 [94]. No objective tumor responses were seen and the authors concluded that the low dose of IFN-γ was as effective as higher doses. Saleh et al. treated 15 colorectal cancer patients with 0.1 mg/m2 IFN-γ on days 1 to 15 and 400 mg of 17-1A at a dose of 400 mg on days 5, 7, 9 and 22 [75]. No significant objective tumor responses were described.

Bevacizumab (Avastin®) Bevacizumab is a humanized Mab that binds to the VEGF ligand. As a single agent bevacizumab rarely

Robert O. Dillman produced objective responses in a three-arm randomized trial in patients with metastatic colorectal cancer [28]. However, a three-arm randomized phase II trial in patients who had untreated metastatic colorectal cancer suggested that bevacizumab enhanced 5-FU based chemotherapy [46]. The 144 patients were randomized to fluorouracil and leucovorin (both at 500 mg/m2) as the control arm, or to the same therapy plus bevacizumab q 2 weeks at either 5 mg/kg or 10 mg/kg. 5FU and leucovorin were given weekly for the first 6 weeks of each 8-week cycle. Better results were seen with the addition of bevacizumab, and there appeared to be an advantage for the lower dose. In the pivotal trial for regulatory approval, 923 patients with metastatic colorectal cancer were randomized to receive irinotecan, 5-FU and leucovorin (IFL) and bevacizumab (IFL-BV), IFL and placebo (control), or FL with bevacizumab (FL-BV) at a dose of 5 mg/kg every 2 weeks [42, 43] the first phase of the trial, 313 patients were randomly assigned to these three arms, then; after a planned initial analysis, the FL-BV arm was discontinued after enrollment of 110 patients, not because of inferior results, but because IFL had become the standard control arm based on other trials in metastatic colorectal cancer. IFL-BV was superior to IFL-placebo for the 813 patients randomized to treatment with one of these arms, in terms of response rate (p = 0.004), PFS (HR 0.54, p < 0.001), and OS (HR 0.66, p < 0.001) [42]. The IFL-BV arm was associated with more grade 3 or 4 hypertension (11% versus 2%). The FL-BV arm also was superior to the IFL-placebo arm [43]. In a trial of initial therapy for patients with metastatic colorectal cancer who were not considered optimal candidates for first-line irinotecan treatment, 209 patients were randomized to receive FL + becacizumab at 5 mg/kg or FL + placebo [48]. The addition of bevacizumab resulted in a better PFS (HR = 0.50, p < 0.001), higher response rate (p = 0.055), and a trend toward better survival (HR = 0.79, p = 0.16). Hypertension was more frequent with bevacizumab. In a three-arm trial 829 patients whose cancers had recurred after IFL therapy were randomized to receive oxaliplatin, fluorouracil, and leucovorin (FOLFOX4) with bevacizumab or to FOLFOX4 alone, or to bevacizumab alone [28]. In this trial bevacizumab was given at 10 mg/kg every 2 weeks. As noted earlier, bevacizumab showed little activity as a single agent, but FOLFOX4 plus bevacizumab was superior to FOLFOX alone for all key endpoints, including OS (HR 0.75, p = 0.001), PFS (HR 0.61, p < 0.0001) and response rate (p < 0.0001). A metanalysis was performed using the raw data from three randomized trials that included 5-FU plus

663 leucovorin and bevacizumab (FL-BV) as initial treatment for patients with metastatic colorectal cancer [47]. For the combined data, FL-BV (n = 249) was superior to FL (n = 241) in terms of response rate (34% versus 24%, p = 0.019), PFS (9 versus 6 months, HR = 0.63, p < 0.001), and OS (18 versus 15 months, HR 0.74, p = 0.008). In contrast to these excellent results in previously untreated patients, in an expanded access trial 350 patients with metastatic colorectal cancer, who had relapsed after or progressed during both irinotecan and oxaloplatin based therapy, the response rate was only 4% in the first 100 patients enrolled by investigator analysis, and only 1% based on blinded central review [7]. A response rate of 49% was observed among 81 evaluable patients with untreated advanced colorectal cancer who were treated with IFL and bevacizumab [27]. The doses of irinotecan and 5FU both had to be reduced by 20% to 25% because of vomiting, diarrhea and neutropenia. Bleeding occurred in 37 patients (46%) and nine patients (11%) had grade 3 or 4 thromboembolic events. In another trial FOLFOX4 and bevacizumab was used to treat 53 patients with previously untreated metastatic colorectal cancer [21]. The response rate was 68%. Hemorrhage was not a problem in this trial, and only one patient had a severe thromboembolic event.

Cetuximab (Erbitux®) Cetuximab is a chimeric Mab that binds to EGFR. Clinical trials with cetuximab used an initial loading dose of 400 mg/m2 i.v. over 2 h, then 250 mg/m2 i.v. over 1 h weekly. Saltz et al. treated 57 patients whose metstatic cancer had not responded to prior irinotecan alone or as part of combination chemotherapy [76]. Patients were required to have EGFR demonstrated by immunohistochemistry on formalin-fixed paraffin-embedded tumor tissue, but it is now known that quantitative expression of EGFR is not predictive of response to cetuximab treatment. The most common adverse events were an acne-like skin rash, predominantly on the face and upper chest (86%) and the constellation of asthenia, fatigue, malaise, or lethargy (56%). Three patients were reported to have had a grade 3 allergic reaction; two were withdrawn. Lenz et al. treated reported a response rate of 12% for 346 patients whose metastatic cancer was considered refractory to fluoropyrimidines, irinotecan and oxaliplatin [51]. EGFR positivity by IHC was an eligibility requirement, but degree of positivity did not correlate with clinical benefit. The most prevalent toxicity was the acneiform rash which was observed in 83% of patients, and which was predictive of clinical benefit.

664 In a randomized trial, 572 patients who had progressed despite 5FU, irinotecan, and oxaloplatin, were either observed or received standard dose cetuximab [45]. The objective response rate was only 8%, but PFS and OS were better in the cetuximab arm. Skin rash, infection and hypomagnesemia were more common in the cetuximab arm. Eleven patients discontinued cetuximab because of infusion reactions. In the pivotal trial Cunningham et al. randomized 329 patients with colorectal cancer that had progressed during or within 3 months after discontinuation of irinotecan, to receive either, irinotecan plus cetuximab, or cetuximab alone, using a 2:1 randomization [11]. Protocol prescribed irinotecan was to be the same as used prestudy. The response rate was higher for the combination therapy (23% versus 11%, p = 0.007), as was PFS (4.0 months versus 1.5 months, p < 0.001). Gebbia et al. reported a response rate of 20% for cetuximab plus irinotecan in 60 patients who had received at least two prior therapies, and whose metastatic cancer was considered refractory to oxaliplatin and irinotecan [25]. Standard cetuximab dosing was combined with irinotecan 120 mg/m2 weekly for 4 out of 6 weeks. Responses were not predicted by EGFR expression. Other than the acneiform skin rash that was severe in 13% of patients, the main grade 3 or 4 toxicities were attributable to the chemotherapy. Vincenzi et al. reported a 25% response rate for cetuximab and irinotecan in 55 patients whose metastatic cancer was considered refractory to oxaliplatin and irinotecan [88]. Standard cetuximab dosing was combined with irinotecan 90 mg/m2 weekly. Skin toxicity was observed in 89% of patients. The most common grade 3 to 4 adverse events were dermatologic (33%), diarrhea (16%), fatigue (13%) and stomatitis (7%). Fever was noted in 25%, typically in association with the first infusion of cetuximab, but no allergic reactions were recorded. More recently cetuximab has been combined with modern combination therapies such as FOLFOX and FOLFIRI as the initial treatment of patients with metastatic colorectal cancer. Tabernero et al. used FOLFOX and cetuximab as the initial treatment for 43 patients with metastatic colorectal cancer [83]. Cetuximab was given day 1 at a dose of 400 mg/m2 during week 1, and then 250 mg/m2 weekly thereafter. Treatment was well-tolerated and the response rate was 72%. In a randomized trial of 337 patients, a preliminary report showed that cetuximab plus FOLFOX was superior to FOLFOX alone in terms of response rate (46% versus 36% p = 0.064) [3]. In a randomized trial of 1,198 patients, an initial report showed that cetuximab plus FOLFIRI was superior to FOLFIRI alone in terms of response rate (47% versus 39%, p = 0.004) and PFS (8.9 versus 8.0 months p = 0.048) [85].

Biological therapy of colon cancer

Panitumumab (Vectibix®) Panitumumab is a human Mab that reacts with EGFR. A pivotal trial for regulatory approval compared panitumumab at 6 mg/kg i.v. every 2 weeks to best supportive care in patients with metastatic colorectal cancer whose disease had progressed during or after standard therapy with fluoropyrimidine-, oxaliplatin-, and irinotecancontaining chemotherapy regimens [29, 30, 84, 86]. All patients had epidermal growth factor receptor (EGFR)expressing tumors. Panitumumab produced a 10% objective response rate and a longer progression free survival compared to supportive care alone. There was no difference in survival, but approximately 75% of patients in the supportive care alone arm crossed over to receive panitumumab after disease progression. Quality of life was also better in the patients who received panitumumab [80]. For 176 patients who progressed on the supportive care arm, and then subsequently did receive panitumumab, 12% had an objective response and two patients had complete responses [86]. Hecht et al. treated 148 patients whose metastatic colorectal cancer had progressed on chemotherapy that included a fluoropyrimidine and irinotecan or oxaliplatin, or both [37]. Panitumumab was given i.v. at a dose of 2.5 mg/kg weekly for 8 of each 9 weeks until disease progression or excessive toxicity. Skin toxicity occurred in 95% and 5% were grade 3 or 4. Four patients discontinued therapy because of toxicity and one patient had an infusion reaction but was able to resume treatment. EGFR of at least 1+ by IHC was an eligibility requirement. There was no difference in response for 105 patients who were judged as having high EGFR by IHC compared to 43 patients who were characterized as having a low EGFR. Berlin et al. tested the combination of irinotecan, 5-FU and leucovorin, and panitumumab as initial therapy in patients with metastatic colorectal cancer [2]. This was a two-part, multicenter, phase II study of panitumumab 2.5 mg/kg weekly with bolus 5-FU (IFL) in the first part of the trial, and infusional 5-FU (FOLFIRI) in the second part. Grade 3 to 4 diarrhea occurred in 11 patients (58%) in part 1 and six patients (25%) in part 2. All patients had dermatologic toxicity, but none was grade 4. The authors concluded that panitumumab + FOLFIRI was better tolerated than panitumumab + IFL.

Vaccines A number of trials were carried out with a vaccine consisting of about 10 million irradiated autologous cells obtained directly from fresh tumor, admixed with about 10 million bacillus Calmette-Guérin (BCG) organisms and injected i.d. weekly for 2 weeks, and then an additional injection

Robert O. Dillman of irradiated tumor cells during the third week. Initial trials had shown that injections of such a product were well-tolerated, associated with local delayed type hypersensitivity reactions to autologous tumor, and isolation of human monoclonal antibodies from circulating lymphocytes [36, 40]. In a European phase III trial 254 patients with stage II or stage III colon cancer were randomized to observation or active specific immunotherapy with this preparation beginning 4 weeks after surgery [87]. At a median follow-up of more than 5 years, there was a 44% reduction in the risk of recurrence in the treatment arm (p = 0.023). In the subset analysis there was no benefit for patients with stage III disease, but there was a great benefit in terms of decrease risk of recurrence and death in the vaccine arm. In a U.S. phase III trial 297 patients with stage II colon cancer, and 115 with stage III colon cancer, were randomized to observation or to the autologous active specific immunotherapy starting 1 month after surgery [34]. After a median follow up of more than 7.5 years, there was no difference in outcomes between the two treatments based on an intent-to-treat analysis, although there were substantial problems in compliance with the injection schedule. This product was not granted regulatory approval. In addition to the inconclusive trials, there were issues regarding sterility of such a product derived from fresh colon tumor, and the variability of the product in terms of tumor cells versus stromal and immune cells in fresh tumor. Another vaccine that has been explored in colon cancer consists of a non-replicating canarypoxvirus (ALVAC) engineered to express carcinoembryonic antigen (CEA) and the B7.1 costimulatory molecule. In a phase I trial eight patients were treated with the vaccine [55]. The investigators were able to isolate cytotoxic T cells that could lyse allogeneic and autologous tumor cells in a MHC-restricted manner [99]. In a second phase I trial involving patients with CEA-expressing adenocarcinomas, three cohorts of six patients were treated with increasing doses of an ALVAC-CEA-B7.1 vaccine at doses of 45 million, 450 million, and 0.45 billion plaqueforming units delivered i.m. monthly for 3 months [41, 56]. In this trial there again was evidence of induction of specific T cell immune reactivity to CEA.

Summary The history of immunotherapy in colon cancer is noteworthy because of the brief periods of success enjoyed in the adjuvant setting by the BCG-tumor cell vaccine, the non-specific immune stimulator levamisole in combination with 5FU, and the anti-epcam murine Mab

665 edrocolomab. All three of these products had no or little activity in the advanced disease setting, and all eventually fell out of favor in the adjuvant setting because of results from randomized trials. Combinations of interferon and 5FU appeared encouraging in patients with advanced disease, but eventually were found to be wanting in both the metastatic and adjuvant settings based on results of large randomized trials. Recently, despite limited activity as single agents in the advanced setting, two monoclonal antibodies have gained approval based on their impressive activity in combination with chemotherapy in patients with metastatic disease, including enhanced survival. Bevacizumab and cetuximab are now standard components of systemic treatment regimens, but only in combination with chemotherapy.

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668 82. Stevenson HC, Green I, Hamilton JM, et al. Levamisole: known effects on the immune system, clinical results, and future applications to the treatment of cancer. J Clin Oncol 1991;9:2052–66. 83. Tabernero J, Van Cutsem E, Diaz-Rubio E, et al. Phase II trial of cetuximab in combination with fluorouracil, leucovorin, and oxaliplatin in the first-line treatment of metastatic colorectal cancer. J Clin Oncol 2007;25:5225–32. 84. Van Cutsem E, Peeters M, Siena S, et al. Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer. J Clin Oncol 2007a;25:1658–64. 85. Van Cutsem E, Nowacki M, Lang I, et al. Randomized phase III study of irinotecan and 5-FU-FA with or without cetuximab in the first-line treatment of patients with metastatic colorectal cancer: the CRYSTAL trial. Proc Am Soc Clin Oncol 2007b;25:164s [abstract 4000]. 86. Van Cutsem E, Siena S, Humblet Y, et al. An open-label, singlearm study assessing safety and efficacy of panitumumab in patients with metastatic colorectal cancer refractory to standard chemotherapy. Ann Oncol 2008;19:92–8. 87. Vermorken JB, Claessen AM, van Tinteren H, et al. Active specific immunotherapy for stage II and stage III human colon cancer: a randomised trial. Lancet 1999;353:345–50. 88. Vincenzi B, Santini D, Rabitti C, et al. Cetuximab and irinotecan as third-line therapy in advanced colorectal cancer patients: a single centre phase II trial. Br J Cancer 2006;94:792–7. 89. Wadler S, Wiernik PH. Clinical update on the role of fluorouracil and recombinant interferon alfa-2a in the treatment of colorectal carcinoma. Semin Oncol 1990; 17:16–21 90. Wadler S, Schwartz EL, Goldman M, et al. Fluorouracil and recombinant alfa-2a-interferon: an active regimen against advanced colorectal carcinoma. J Clin Oncol 1989; 7:1769–75. 91. Wadler S, Wersto R, Weinberg V, et al. Interaction of fluorouracil and interferon in human colon cancer cell lines: cytotoxic and cytokinetic effects. Cancer Res 1990; 50: 5735–9.

Biological therapy of colon cancer 92. Wadler S, Lembersky B, Atkins M, et al. Phase II trial of fluorouracil and recombinant interferon alfa-2a in patients with advanced colorectal carcinoma: an Eastern Cooperative Oncology Group study. J Clin Oncol 1991;9:1806–10. 93. Weh HJ, Platz D, Braumann D. Treatment of metastatic colorectal carcinoma with a combination of fluorouracil and recombinant interferon alfa-2b: preliminary data of a phase II study. Semin Oncol 2003; 19(Suppl. 3):180–4. 94. Weiner LM, Steplewski Z, Koprowski H, et al. Biologic effects of gamma interferon pre-treatment followed by monoclonal antibody 17–1A administration in patients with gastrointestinal carcinoma. Hybridoma 1986;5 Suppl 1:S65–77. 95. Weiner LM, Moldofsky PJ, Gatenby RA, et al. Antibody delivery and effector cell activation in a phase II trial of recombinant gamma-interferon and the murine monoclonal antibody CO17– 1A in advanced colorectal carcinoma. Cancer Res 1988;48: 2568–73. 96. Wiesenfeld M, O’Connell MJ, Wieand HS, et al. Controlled clinical trial of interferon-gamma as postoperative surgical adjuvant therapy for colon cancer. J Clin Oncol 1995;13: 2324–9. 97. Wolmark N, Bryant J, Smith R, et al. Adjuvant 5-fluorouracil and leucovorin with or without interferon alfa-2a in colon carcinoma: National Surgical Adjuvant Breast and Bowel Project protocol C-05. J Natl Cancer Inst 1998;90:1810–6. 98. Wolmark N, Rockette H, Mamounas E, et al. Clinical trial to assess the relative efficacy of fluorouracil and leucovorin, fluorouracil and levamisole, and fluorouracil, leucovorin, and levamisole in patients with Dukes’ B and C carcinoma of the colon: results from National Surgical Adjuvant Breast and Bowel Project C-04. J Clin Oncol 1999;17:3553–9. 99. Zhu MZ, Marshall J, Cole D, et al. Specific cytolytic T-cell responses to human CEA from patients immunized with recombinant avipox-CEA vaccine. Clin Cancer Res 2000;6:24–33.

21.5 Biological therapy of breast cancer ROBERT O. DILLMAN

Breast Cancer Several studies indicate that patients with carcinoma of the breast remain reasonably immune competent throughout the natural history of their disease [45, 91]. Anti-tumor immunity is demonstrable in patients by a number of techniques. An in vitro immune reaction to tumor-associated antigens was demonstrated using the leukocyte migration inhibition (LMI) and the leukocyte adherence inhibition (LAI) assays, using as stimulants tumor cell extracts, soluble membrane extracts of MCF-7 cells (a breast cancer cell line), and soluble membrane extracts of biopsy-derived tumor tissue [15, 32, 75]. Several of these studies demonstrated blocking factors, presumably immune complexes, in the serum [83]. The T-antigen, a precursor of the M and N blood group antigens whose expression is masked on normal tissue, is expressed on nearly all breast carcinoma tissue. Patients also mount a strong cellular immune reaction to this antigen as demonstrated by both delayed hypersensitivity and in vitro LMI. The demonstration of delayed hypersensitivity to tumor-associated antigens has not been particularly useful clinically. However 85% of breast cancer patients had such a reaction to a crude membrane extract prepared from cultured MCF-7 breast tumor cells previously infected with vesicular stomatitis virus [3].

Non-specific Immune Stimulation Because of the prevalence of breast cancer, in the 1970s many studies were carried out with various nonspecific immune stimulating agents including BCG, levamisole, Corynbacterium parvum, poly A:U, and cis-retinoic acid. As a generalization, these agents failed to demonstrate significant benefit.

Bacillus Calmette Guerre (BCG) In a European trial 254 patients with surgically resected breast cancer (stages T1–3a/N0–1/M0) were randomized to either surgery alone or surgery plus adjuvant chemotherapy with chlorambucil, methotrexate and 5FU with BCG [78]. After 10 years of follow up, the treatment arm was associated with better relapse and overall

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

survival. However, because of its design, this trial did not address whether BCG made any contribution to these results. U.S. trials of adjuvant cyclophosphamide, doxorubicin, and 5-fluorouracil, with or without BCG, in patients with stage II or III breast cancer [12], or stage IV breast cancer [52]. A randomized trial of BCG after radiation therapy for locally advanced breast cancer also found no advantage for BCG [79]. A methanol-extracted residue (MER) of BCG was tested in a trial in which 395 evaluable patients with metastatic breast cancer were randomized to one of three different chemotherapy combinations with or without MER [1]. MER was injected at five sites at a dose of 100 μg or at the lowest tenfold dilution that produced a 1-cm indurated lesion. After adjustments for chemotherapy regimen, collectively response rates were inferior in the combined chemoimmunotherapy arms (38% vs. 52%, p = 0.02). MER was associated with significant toxicity including painful ulcers and fevers, but without response or survival benefit.

Corynbacterium Parvum NSABP conducted a trial in which breast cancer patients were randomized to receive l-phenylalanine mustard (L-PAM) and 5FU (PF) with or without C parvum [30]. After 8 years of follow-up there was no improvement in disease free survival (DFS) or overall survival (OS), and the trend was actually for inferiority in the immunomodulating arm. There was also more toxicity, especially fever and chills associated with C parvum administration.

Pseudomonas Antigens In a trial for metastatic breast cancer patients, 133 previously untreated women were treated with combination chemotherapy and then randomized to no further treatment or nonspecific immunotherapy with a heptavalent pseudomonas vaccine [38]. Women with estrogen receptor positive tumors also received tamoxifen after completion of the chemotherapy. The addition of the pseudomonas vaccine failed to increase the response rate, duration of response, or survival.

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Levamisole The antihelminth levamisole exhibited limited benefit as a single-agent in patients with metastatic breast cancer. The agent was generally well-tolerated although granulocytopenia and agranulocytosis were observed in some patients. Most trials focused on utilizing the agent as an adjunctive therapy. In an early small randomized trial in patients with inoperable stage III breast cancer who had been treated with radiation therapy, there was a better median DFS (25 vs. 9 months) and OS at 30 months (90% vs. 35%) for 20 patients who received adjuvant levamisole compared to 23 patient who did not [72]. In an adjuvant study conducted before adjuvant chemotherapy and/or hormonal therapy had been established as standard therapy, 72 post-surgery stage II breast cancer patients were randomized to receive levamisole or placebo [43]. After 5 years of follow up, there was a trend for survival advantage in the levamisole arm, primarily because of an apparent benefit in post-menopausal patients. During 1976 to 1978 101 patients with advanced breast cancer were randomized to receive doxorubicin, vincristine and cyclophosphamide with either placebo or levamisole 2.5 mg/kg given on 2 consecutive days every week except on the days chemotherapy was given [44]. There was a higher response rate in patients treated with levamisole (63% vs. 47%) and after 10 years of follow up, they had experienced longer survival. In another trial conducted in metastatic breast cancer, 97 women were randomized to receive cyclophosphamide, doxorubicin, and 5-fluorouracil chemotherapy with placebo or levamisole, 2.5 mg/kg, 2 days of each week [16]. The patient population was limited to those with only bone metastases or local chest wall recurrence. There was no significant difference in response rate or survival.

Poly A:U Poly A:U, an interferon inducer and stimulator of natural killer cytotoxicity, has been evaluated in a randomized adjuvant study (not incorporating chemotherapy) in patients with stage II carcinoma of the breast, all of whom had previously undergone mastectomy [42]. Patients receiving poly A:U had a better overall survival than those not receiving this polynucleotide. At the time this benefit was comparable to other studies using adjuvant chemotherapy in breast cancer. Benefit was essentially limited to patients with one to three involved lymph nodes and could be correlated with natural killer cytotoxicity augmentation and 2′,5′-A synthetase production (an indicator of interferon induction).

Biological therapy of breast cancer

Immunoactivation/Absorption/ Ultrafiltration Staphylococcal protein A is a polypeptide with a molecular weight of 42,000 that has a high affinity for the Fc portion of mammalian immunoglobulin (IgG). Preclincal studies demonstrated therapeutic efficacy in a spontaneous canine mammary carcinoma 2and a chemically induced mammary carcinoma of rats) following the exposure of plasma to staphylococcal protein A [37, 70, 86]. It was postulated that the removal of immunosuppressive immune complexes was enabling an endogenous anti-tumor response. In a highly publicized pilot study, four of five patients with advanced breast cancer exhibited significant anti-tumor effects following repeated exposure of plasma to protein A imbedded in a colloidal charcoal mixture [87]. Unfortunately several attempts to confirm the anti-tumor effects results were unsuccessful [29, 60]. It was eventually determined that the antitumor effects were the result of elution of the Staph A protein and possibly other enterotoxins off the column [59, 85]. In the blood stream this protein acted as a “super antigen”, and induced a cytokine cascade associated with fever, chills, hypotension, and other systemic symptoms in patients. The Prosorba® immunoadsorbent column consisting of Staphylococcal aureus protein A bound covalently to an inert silica matrix to remove immune complexes, received regulatory approval for the treatment of immune-mediated thrombocytopenia (IMRE Corporation, Seattle, WA). In one trial that enrolled 114 patients with various tumor types, objective partial responses were reported for 5/22 patients with measurable metastatic breast cancer [51]. The final commercial product appeared to overcome the problems with elution of the protein A off the column, but this also appeared to remove clinical activity since there was no evidence of anti-tumor effects in another clinical trial conducted in 16 patients with metastatic breast cancer [27]. A related approach, utilized dialysis filters to ultrafiltrate plasma to remove suppressive factors or induce immune activation, which appeared to induce tumor regression [47]. As was the case with the Staph A columns, the patients who had tumor responses were the same ones who developed symptoms consistent with a cytokine-release syndrome. An effort to confirm the benefits of this approach by other investigators was aborted by the proponents of the procedure after no responses were seen in seven patients (R.O. Dillman, personal observation 1990). The clinical reactions experienced by these patients suggested that impurities eluted off the dialysis filters were responsible for the severe immune

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reactions that were producing anti-tumor effects in some patients.

Cytokines Interferon When clinical trials with interferon began, there was great optimism that these natural proteins would be clinically useful in most malignancies [7]. As summarized in Table 1, several early trials suggested interferonalpha (IFN-α) had significant anti-tumor activity against breast cancer. Early studies investigating leukocytederived IFN-α reported response rates of 20% to 40% [8, 35]. However, a subsequent study using human lymphoblastoid interferon documented responses in only 1/29 evaluable patients [46]. A trial involving 32 women with recurrent loco-regional breast cancer randomized patients to observation or recombinant human IFN-α at a dose of 3 MU s.c. daily for 1 year after local treatment [28]. There were no differences in the rates of new or recurrent breast cancer, but there was a high incidence of side effects. Homogenates of breast cancer cells exposed to interferon express increased levels of estrogen receptor protein. In a trial for postmenopausal patients with advanced breast cancer that was not known to be hormone receptor negative, 99 women were randomized to receive tamoxifen alone, or with IFN-α2a 3 MU i.m. thrice weekly, or daily oral 13-cis-retinoic acid [19]. Response rates did not differ significantly, and ranged from 38% to 44%. After 8 years median followup, there was no significant difference in overall survival. In terms of trends, the interferon arm had the

lowest response rate and lowest median survival, perhaps because more patients discontinued treatment because of the various toxicities that accompanied administration of interferon. Since there were over 12 IFN-α molecules in the human-derived leukocyte interferon preparation, and at least eight in the lymphoblastoid product, but only one in each recombinant IFN-α product, it is possible that other molecules present in the natural products are important for anti-tumor effect. However, an analysis of many reported studies suggests that that IFN-α has negligible activity in breast cancer [61].

Interleukin-2 Because of the recognized efficacy of chemotherapy and hormonal therapy, and the substantial toxicity of IL-2, few patients with breast cancer were included in early clinical trials of IL-2 [24, 74]. As shown in Table 1, no anti-tumor responses were seen with these high dose regimens. Lower doses of IL-2 have been explored in combination with other therapy. Following high-dose chemotherapy with autologous peripheral blood stem cell rescue, 72 women with high-risk stage II or III breast cancer were randomized to receive either a low dose of IL-2 (1 MU/m2) s.c. daily for 28 days or combined cyclosporine A at 1.25 mg/ kg i.v. daily from day 0 to +28 and interferon gamma 0.025 mg/m2 s.c. every 2 days from day +7 to +28 [88]. Both treatments were well tolerated. At a median followup of 67 months, there were no significant differences in progression free or overall survival. The combination of high dose continuous infusion IL-2+ IFN-α yielded an objective response in 4/23 patients [62].

Table 1. Single-agent activity of various biologicals in patients with metastatic breast cancer Modality class

Biological agent

Reference

Patients

Response rate

Non specific Non-specific Non specific Cytokine Cytokine Cytokine Cytokine Cytokine Mab Mab Mab Mab Mab Mab Retinoid

Staph A protein column Staph A protein column Staph A protein column Interferon-α Interferon-α Lymphoblastoid Interferon Interleukin-2 ± LAK Interleukin-2 ± LAK Trastuzumab Trastuzumab Trastuzumab Trastuzumab Trastuzumab Bevacizumab Bexarotene

Terman 1981 Messerschmidt 1988 Fennelly 1995 Gutterman 1980 Borden 1982 Laszlo 1986 Dillman 1993 Rosenberg 1989 Burris 2004 Vogel 2002 Baselga 2005 Cobleigh 1999 Baselga 1996 Cobleigh Esteva-2003

5 22 16 16 23 29 7 6 52 114 105 222 43 75 95

80% 23% 0% 44% 22% 3% 0% 0% 33% 26% 19% 15% 12% 9% 6%

Mab = monoclonal antibody. LAK = lymphokine activated killer cells.

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Retinoids Cis-retinoic Acid In a trial for postmenopausal women with advanced breast cancer that was not known to be hormone receptor negative, 99 patients were randomized to receive tamoxifen alone, or with s.c. IFN-α with 13-cis-retinoic acid 1 mg/kg p.o. daily [19]. After 8 years median follow-up, response rates and overall survival were similar for the tamoxifen control arm and the retinoid containing arm.

Bexarotene Bexarotene is a retinoid X receptor-selective retinoid that exhibited anti-tumor activity against breast cancer in preclinical testing. A multicenter phase II trial randomized 145 women with metastatic breast cancer to oral bexarotene at a doses of 200 or 500 mg/m2 per day or placebo [26]. A response rate of 6% was observed in 48 tamoxifen-refractory patients and in 47 chemotherapyrefractory patients, and 3% in 51 patients with tamoxifen-resistant disease. Hypertriglyceridemia is noted in 84% of patients. Other common drug-related adverse events were dry skin (34%), asthenia (30%), and headache (27%).

Monoclonal Antibodies Trastuzumab As summarized in Table 1, an extensive clinical experience has confirmed the anti-tumor activity of the antiHer2 humanized Mab trastuzumab in patients whose breast cancers over express Her2. Trastuzumab is now standard therapy for the treatment of patients with highrisk or advanced breast cancer. Various clinical trials have confirmed the efficacy of trastuzumab, especially in combination with chemotherapy, for the treatment of Her-2 positive breast cancer in patients with recurrent metastatic breast cancer, as the initial treatment for metastatic breast cancer, and as adjuvant therapy. At this time most physicians continue trastuzumab maintenance indefinitely or until disease progression because the Her2 receptor is continually being produced by some malignant cells. As shown in Table 1, as a single agent, trastuzumab alone produced objective response rates of 12–33% in patients with Her2-positive metastatic breast cancer that had relapsed after chemotherapy eHer2 [5, 6, 9, 20, 90]. In a phase II trial in 46 patients with metastatic breast cancer in whom at least 25% of tumor cells expressed Her2 by immunohistochemistry (IHC), a schedule of

Biological therapy of breast cancer 250 mg i.v. followed by weekly doses of 100 mg for 9 additional weeks yielded five responses among 43 evaluable patients [5]. The largest trial of trastuzumab as a single-agent was conducted in 222 patients with Her2positive metastatic breast cancer that had recurred after chemotherapy [20]. Trastuzumab alone was given at an initial dose of 4mg/kg followed by weekly doses of 2 mg/kg. There were eight complete and 26 partial responses for a response rate of 15% with a median duration of response of 13 months. Burris et al. gave trastuzumab at twice the standard doses by the weekly schedule as initial treatment for 8 weeks for patients with Her2-positive metastatic breast cancer who had never received chemotherapy [9]. They reported a response rate of 33% among 52 evaluable patients in a trial that enrolled 61 patients. This response rate is the highest reported for single-agent trastuzumab. However, in a randomized phase II trial conducted by Vogel et al., there was no suggestion that this higher dose produced a higher response rate [90]. In this trial there was no significant difference in results for an 8 mg/kg loading dose and 4 mg/kg per week maintenance versus the standard 4 mg/kg loading dose and 2 mg/kg per week maintenance. The failure to demonstrate any difference between these doses is not surprising in view of the sustained serum levels of trastuzumab that were achieved at the lower dose. In this trial trastuzumab alone as initial treatment for metastatic breast cancer resulted in seven complete and 23 partial responses among 114 patients (26%), but the response rates were 35% for patients whose tumors were IHC3+ compared to 0% for tumors that were IHC2+, and 34% among patients whose tumors were FISH+ compared to 7% for tumors that were FISH-. Baselga et al. tested an every 3-week schedule of trastuzumab delivery as initial therapy for 105 patients with Her2-positive metastatic breast cancer [6]. They observed a response rate was 19%, but it was 34% for patients whose tumors were IHC3+ and/or FISH+ for Her2. Numerous phase II trials have been reported in which trastuzumab was combined with single agent chemotherapy for treatment of patients with Her2-positive breast cancer. These doublet combinations were associated with response rates between 25% and 75% with median PFS of 6 to 12 months. Response rates of 50% or greater were reported in 17 of the 20 trials, and appeared to be much higher than observed with chemotherapy alone. Many of these reports found higher response rates in the subsets of patients who had 3+ IHC Her2 expression by IHC and/or overexpression by FISH, as opposed to 2+ IHC Her2 or negative FISH. The addition of trastuzumab was not associated with

Robert O. Dillman any additive toxicities, other than cardiac disease in association with anthracyclines. Most of the single agents used in these trials were the taxanes paclitaxel or docetaxel, or the vinca vinorelbine. In phase II trials with 23 to 40 patients, various schedules of docetaxel were associated with response rates ranging from 50% to 70% depending on prior treatment [2, 26, 57, 76, 84]. In phase II trials with 25 to 88 patients, various schedules of paclitaxel and trastuzumab produced response rates ranging from 36% to 62% depending on prior treatment [31, 34, 48, 71, 77]. In phase II trials with 30 to 62 patients, various schedules of vinorelbine and trastuzumab produced response rates ranging from 50% to 78% depending on prior treatment [4, 10, 11, 17, 24, 40, 64]. There was a 38% response rate for gemcitabine and trastuzumab among 61 patients who had previously been treated with a taxane and anthracycline [63]. Liposomal doxubicin and trastuzumab produced a 52% rate in 29 patients who had received little or no prior chemotherapy [18]. A response rate of 24% was observed for cisplatin and trastuzumab in 37 patients who had received multiple prior chemotherapy regimens [66]. Many phase II clinical trials have used combination chemotherapy plus trastuzumab for treatment of patients with Her2-positive breast cancer. Most of these trials utilized chemotherapy combinations of taxanes and platinum agents. Combination chemotherapy has consistently produced higher response rates and longer progression free survival in clinical trials in patients with metastatic breast cancer, although survival advantages have been harder to establish. Phase II trials of combination chemotherapy plus trastuzumab appear to produce slightly higher response rates (40% to 92%) than were seen with single agents plus trastuzumab, and the PFS was somewhat longer (range 6–16 months). Docetaxel, carboplatin and trastuzumab yielded a response rate of 58% in 59 patients with metastatic breast cancer, 15% of whom had received prior taxane therapy in the adjuvant setting [67]. Docetaxel, cisplatin and trastuzumab produced response rate of 79% in 62 previously untreated patients [67]. Docetaxel, epirubicin and trastuzumab produced a response rate of 67% as initial therapy for 45 patients, but the use of an anthracycline was associated with an unacceptable rate of cardiac toxicity [89]. In a randomized trial, trastuzumab and paclitaxel with or without carboplatin were compared as first-line therapy for 196 women with HER-2-overexpressing (2+ or 3+ by IHC) metastatic breast cancer [71]. The addition of carboplatin produced a higher response rate 52% versus 36% (p = 0.04), and longer median PFS (11 vs. 7 months,

673 hazard ratio 0.66, p = 0.03). In consecutive phase II trials, weekly paclitaxel and carboplatin plus trastuzumab appeared superior to an every 3-week schedule of the same agents with response rates of 81% versus 65%, median PFS 14 versus 10 months, and median OS 3.2 versus 2.3 years, and was associated with less hematologic toxicity [68]. In 40 patients who had received no prior chemotherapy the combination of paclitaxel, gemcitabine and trastuzumab produced a response rate of 52% [31]. The same combination had a response rate of 92% in 13 previously untreated patients [54]. Gemcitabine, cisplatin and trasutuzumab produced a response rate of 40% in 20 patients, but most had previously received both a taxane and anthracycline [82]. Gemcitabine, vinorelbine and trastuzumab as second-line therapy produced a 50% response rate in 30 patients [58]. Randomized trials have established the superiority of combining trastuzumab with chemotherapy compared to chemotherapy alone. One of the pivotal trials that led to regulatory approval of trastuzumab compared trastuzumab plus chemotherapy to chemotherapy alone in 469 patients with metastatic disease who had not received prior chemotherapy for metastatic disease [80]. All patients had tumors that overexpressed Her2, which was defined as 2+ or 3+ by IHC on a scale of 0–3. An initial 4 mg/kg trastuzumab dose was followed by 2 mg/kg weekly. The first dose of Mab was infused i.v. over 90 min, but in the absence of significant infusion related toxicity, subsequent doses were infused i.v. over 30 min. Patients who had not received an anthracycline previously were randomized to receive trastuzumab alone or with cyclophosphamide 600 mg/m2 i.v. and doxorubicin 60 mg/m2 or epirubicin (AC) i.v. every 3 weeks for six cycles. Patients who had received adjuvant chemotherapy that included an anthracycline were randomized to receive paclitaxel 175 mg/m2 i.v. over 3 h alone every 3 weeks, or with trastuzumab. The combination of chemotherapy plus trastuzumab was superior for key end points including: response rate (50% vs. 32%, p < 0.0001), duration of response (9.1 vs. 6.1 months), PFS (median 7.4 vs. 4.6 months, p < 0.001), and OS (death at 1 year 22% vs. 33%, p = 0.008; median survival 25 vs. 20 months, p = 0.046) with a 20% risk reduction of death. The differences were most striking for paclitaxel plus trastuzumab versus paclitaxel alone. The median OS was 22 months for paclitaxel plus trastuzumab versus 18 months for paclitaxel alone (p = 0.17) and 27 months for AC plus trastuzumab versus 21 months for AC alone (p = 0.16). In another randomized trial, docetaxel plus trastuzumab was superior to docetaxel alone in patients with metastatic breast cancer who had received no prior

674 chemotherapy [50]. Docetaxel was given every 3 weeks and trastuzumab was given weekly. The addition of trastuzumab roughly doubled the response rate and PFS, and yielded a better OS 31 versus 23 months (p = 0.032). Three large randomized trials have confirmed the expected advantage of combining trastuzumab with chemotherapy in the adjuvant treatment of patients with Her2 positive breast cancer [41, 69, 73, 81]. Two U.S. cooperative group trials were combined for one preliminary analysis [73]. The National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31 compared doxorubicin and cyclophosphamide followed by paclitaxel every 3 weeks with the same regimen plus 52 weeks of trastuzumab beginning with the first dose of paclitaxel. The North Central Cancer Treatment Group (NCCTG) trial N9831 compared three regimens: doxorubicin and cyclophosphamide followed by weekly paclitaxel, the same regimen plus 52 weeks of trastuzumab initiated concomitantly with paclitaxel, and the same chemotherapy regimen followed by 52 weeks of trastuzumab after paclitaxel. For an early analysis at a median follow up of 2 years, the two similar arms from each trial were combined and compared: doxorubicin plus cyclophosphamide followed by paclitaxel with or without trastuzumab concurrent with paclitaxel [73]. At a median follow up of 3 years, DFS was superior in the there trastuzumab arm (87% vs. 75%, p < 0.0001) with a 52% reduction in the risk of disease recurrence. The 3-year OS also favored the use of trastuzumab (94% vs. 92%, p = 0.015). Because of the use of anthracyclines in this trial, patients have been closely monitored for cardiac toxicity, which has been higher in the trastuzumab arms. In the NSABP B-31 trial 16% of women discontinued trastuzumab therapy due to clinical evidence of myocardial dysfunction or significant decline in left ventricular function. An international, multicenter trial (HERA) randomized more than 5,000 women with HER2-positive node positive or high-risk node negative breast cancer, compared 1 or 2 years of trastuzumab given every 3 weeks with observation after completion of locoregional therapy and at least four cycles of neoadjuvant or adjuvant chemotherapy (Piccart-Gebhart et al. 2007) [81]. Initial reports after 2 years of follow up indicated that 1-year of trastuzumab conveys an advantage over observation with a 46% reduction in disease recurrence (p < 0.0001) (Piccart-Gebhart et al. 2007), and a 34% reduction in death (p < 0.0001) [81]. In a European trial 1,010 women with node-positive or high risk node-negative breast cancer were randomized to adjuvant therapy that consisted of three cycles of either docetaxel or vinorelbine, followed by three cycles

Biological therapy of breast cancer of fluorouracil, epirubicin, and cyclophosphamide in both groups, with a secondary post-chemotherapy randomization for the 232 patients whose tumors were Her2positive to either observation or trastuzumab for 9 weeks [41]. The 3-year DFS was better for patients who received trastuzumab (91% vs. 86%, p = 0.005) with a 42% reduction in the risk of recurrence. Based on Her2 biology, it is probable that there is more of an advantage to give trastuzumab during, rather than only after chemotherapy, but this has not yet been established in trials. It is also probable that there is an advantage for giving trastuzumab indefinitely, even if it has already been given with chemotherapy, but this also has not been proven. Finally, completed trials have attempted to address whether it is better to give adjuvant trastuzumab for 2 years or 1 year, but it is probable that it is better to give trastuzumab indefinitely if there is reason to believe that the breast cancer has not been completely eliminated. Trastuzumab is also being combined with chemotherapy as part of neo-adjuvant therapies with pathologic complete response rates ranging from 13% to 65% [11, 13, 14, 22, 39, 56]. After 3 weeks of neoadjuvant treatment with trastuzumab alone, 23% of patients had a partial response before going on to receive 12 more weeks of trastuzumab combined with paclitaxel before surgery [56]. One randomized trial, which was designed to accrue 164 patients, was closed early because of the marked superiority of the trastuzumab-containing arm [13]. Chemotherapy consisted of four cycles of paclitaxel followed by four cycles of Z5FU, epirubicin, and cyclophosphamide, with weekly trastuzumab for all 24 weeks. After 3 years of follow up, there had been no recurrences in the patients who received neoadjuvant therapy, and they have a better PFS (p = 0.041) [14]. About half of Her2-positive patients are hormone receptor positive which has led to interest in the interaction “cross-talk” between Her2 and hormone receptors which appears to increase resistance to hormonal therapies. There is a recent report of a 26% response rate for 31 evaluable patients who were treated with the aromatase inhibitor letrozole in combination with weekly trastuzumab [49]. Her2 positivity was confirmed for 82% (IHC3+ and/or FISH+) and 82% had previously been treated with tamoxifen. The median PFS was 6 months. The anti-vascular endothelial growth factor Mab bevacizumab has been approved for the treatment of breast cancer in combination with chemotherapy. As shown in Table 1, bevacizumab had demonstrable, but limited activity as a single agent in breast cancer [21]. In that phase I/II trial, cohorts of patients received 3, 10 or 20 mg/kg of bevacizumab i.v. every other week. However,

Robert O. Dillman as a third-line treatment for patients with metastatic breast cancer, the addition of bevacizumab to capecitabine produced a higher response rate compared to capecitabine alone (19% vs. 9%, p = 0.001), although it did not increase DFS or OS [55]. In subsequent trial the addition of bevacizumab to paclitaxel as initial therapy for metastatic disease, produced higher response rates (37% vs. 21%) and doubled the PFS from 6 to 12 months compared to paclitaxel alone [53]. Many other antibody products are in development, but none have been approved by regulatory authorities for standard treatment of breast cancer. These are described elsewhere in this text in other chapters that cover monoclonal antibodies, radioimmunotherapy and immunotoxins.

Vaccines Because of the importance of HER-2 as a target, a vaccine was formulated with peptides 15–18 amino acids in length derived from the HER-2/neu domains and admixed with GM-CSF as an adjuvant [25]. Patients with breast or ovarian cancer underwent i.d. immunization once a month for a total of two to six immunizations. All eight patients immunized with HER-2/neu peptides developed HER-2/neu peptide-specific T-cell responses and six also developed HER-2/neu protein-specific responses. BA7072 is a recombinant fusion protein consisting of sequences from intracellular and extracellular domains of HER-2 linked to GM-CSF. Lapuleucel-T (APC8024) is product produced by Dendreon Inc. that consists of patient-specific peripheral-blood mononuclear cells obtained by leukapheresis and then activated in vitro with BA7072. In an exploratory trial, 18 patients with HER-2 positive breast cancer underwent leukapheresis and subsequent lapuleucel-T infusion 2 days later every 2 weeks for at least three doses [65]. Laboratory assays demonstrated cellular immune responses specific for BA7072 and HER-2 sequences. One patient had a partial response that persisted for 6 months. MUC1 is a tumor-associated antigen that is expressed on many adenocarcinomas including breast cancer. Sialyl-Tn is a carbohydrate associated with MUC1. Theratope® is a synthetic Sialyl-Tn antigen produced by Biomira, Inc. Most of the clinical trials with this product have been conducted in breast cancer patients [36]. A related formulation combined clustered sTn conjugated to keyhole limpit hemocyanin (KLH) (sTn(c)-KLH) combined with the adjuvant QS-21. sTn(c)-KLH was tested in 27 patients with high risk breast cancer who received 1, 3, 10, or 30 μg. Immunizations consisted of

675 sTn(c)-KLH conjugate containing 30, 10, 3, or 1 μg of Tn(c) plus 100 micrograms of QS-21 [33]. All patients developed significant IgM and IgG antibody titers against sTn(c).

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677 65. Park JW, Melisko ME, Esserman LJ, et al. Treatment with autologous antigen-presenting cells activated with the HER-2 based antigen Lapuleucel-T: results of a phase I study in immunologic and clinical activity in HER-2 overexpressing breast cancer. J Clin Oncol 2007; 25:3680–3687. 66. Pegram MD, Lipton A, Hayes DF, et al. Phase II study of receptorenhanced chemosensitivity using recombinant humanized antip185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 1998; 16:2659–2671. 67. Pegram MD, Pienkowski T, Northfelt DW, et al. Results of two open-label, multicenter phase II studies of docetaxel, platinum salts, and trastuzumab in HER2-positive advanced breast cancer. J Natl Cancer Inst 2004; 96:759–769. 68. Perez Ea, Suman VJ, Rowland KM, et al. Two concurrent phase II trials of paclitaxel/carboplatin/trastuzumab (weekly or every-3week schedule) as first-line therapy in women with HER2overexpressing metastatic breast cancer: NCCTG study 983252. Clin Breast Cancer 2005; 6:425–432. 69. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005; 353:1659–1672. 70. Ray PK, Raychaudhuri S, Allen P. Mechanism of regression of mammary adenocarcinomas in rats following plasma adsorption over protein A-containing Staphylococcus aureus. Cancer Res 1982; 42:4970–4974. 71. Robert N, Leyland-Jones B, Asmar L, et al. Randomized phase III study of trastuzumab, paclitaxel, and carboplatin compared with trastuzumab and paclitaxel in women with HER-2-overexpressing metastatic breast cancer. J Clin Oncol 2006; 24:2786–2792. 72. Rojas AF, Feierstein JN, Mickiewicz E, et al. Levamisole in advanced human breast cancer. Lancet 1976; 1:211–215. 73. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005; 353:1673–1684. 74. Rosenberg SA, Lotze MT, Yang JC, et al. Experience with the use of high-dose interleukin-2 in the treatment of 652 patients. Ann Surg 1989; 210:474–485. 75. Rudczynski AB, Dyer CA, Mortensen RF. Detection of cell-mediated immune reactivity of breast cancer patients by the leukocyte adherence inhibition response to MCF-7 extracts. Cancer Res 1978; 38:3590–3594. 76. Sato N, Sano M, Tabei T, et al. Combination docetaxel and trastuzumab treatment for patients with HER-2-overexpressing metastatic breast cancer: a multicenter, phase-II study. Breast Cancer 2006; 13:166–171. 77. Seidman AD, Fornier MN, Esteva FJ, et al. Weekly trastuzumab and paclitaxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification. J Clin Oncol 2001; 19:2587–2595. 78. Senn HJ, Barett-Mahler AR, Jungi WF, et al. Adjuvant chemoimmunotherapy with Leukeran, Methotrexate and 5FU (LMF) + BCG in node-negative and node-positive breast cancer patients: 10 year results. Eur J Cancer Clin Oncol 1989; 25:513–525. 79. Serrou B, Sancho-Garnier H, Cappelaere P. Inefficacy of post radiotherapeutic BCG immunotherapy in T3-T4 breast cancer patients: a randomized trial. In: Terry WD, Rosenberg SA, eds. Immunotherapy of human cancer. New York: Elsevier, 1982: 195–198. 80. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001; 344:783–792.

678 81. Smith I, Procter M, Gelber RD, et al. 2-year follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer: a randomised controlled trial. Lancet 2007; 369:29–36. 82. Stemmler HJ, Kahlert S, Brudler O, et al. High efficacy of gemcitabine and cisplatin plus trastuzumab in patients with HER2overexpressing metastatic breast cancer: a phase II study. Clin Oncol 2005; 17:630–635. 83. Tanaka F, Yonemoto RH, Waldman SR. Blocking factors in sera of breast cancer patients. Cancer 1979; 43:838–847. 84. Tedesco KL, Thor AD, Johnson DH, et al. Docetaxel combined with trastuzumab is an active regimen in HER-2 3 + overexpressing and fluorescent in situ hybridization-positive metastatic breast cancer: a multi-institutional phase II trial. J Clin Oncol 2004; 22:1071–1077. 85. Terman DS. Protein A and staphylococcal products in neoplastic disease. Crit Rev Oncol Hematol 1985; 4(2):103–124. 86. Terman DS, Yamamoto T, Mattioli M, et al. Extensive necrosis of spontaneous canine mammary adenocarcinoma after extracorporeal perfusion over Staphylococcus aureus Cowans I. Description of acute tumoricidal response: morphologic, histologic,

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immunohistochemical, immunologic, and serologic findings. J Immunol 1980; 124:795–805. Terman DS, Young JB, Shearer WT, et al. Preliminary observations of the effects on breast adenocarcinoma of plasma perfused over immobilized protein A. N Engl J Med 1981; 305:1195–1200. Vahdat LT, Cohen DJ, Zipin D, et al. Randomized trial of low-dose interleukin-2 vs cyclosporine A and interferon-gamma after highdose chemotherapy with peripheral blood progenitor support in women with high-risk primary breast cancer. Bone Marrow Transplant 2007; 40:267–272. Venturini M, Bighin C, Monfardini S, et al. Multicenter phase II study of trastuzumab in combination with epirubicin and docetaxel as first-line treatment for HER2-overexpressing metastatic breast cancer. Breast Cancer Res Treat 2006; 95:45–53. Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2overexpressing metastatic breast cancer. J Clin Oncol 2002; 20:719–726. Weese J, Oldham RK, Tormey DC. Immunologic monitoring in carcinoma of the breast. Surg Gynecol Obstet 1997; 145:208–218.

21.6 Biological therapy of lung cancer ROBERT O. DILLMAN

Non-specific Immunostimulants Bacillus Calmette Guerin (BCG) Bacillus Calmette Guerin (BCG) was the first biotherapy tested in lung cancer in the modern era. Early randomized trials suggested that various forms or extracts of BCG could induce immune responses in patients with lung cancer [88]. However, after the reporting of numerous randomized trials that explored intrapleural, cutaneous, and intralesional BCG, this approach was abandoned. Intrapleural BCG: Following an initial encouraging report based on 40 patients, after 4 years of follow up of 169 patients with stage I lung cancer, there appeared to be a survival advantage for intrapleural BCG following surgery [68, 69]. However, other small randomized trials of intrapleural BCG involving 52, 92 and 118 patients respectively, found no difference in survival and documented significant toxicity [56, 61, 89]. A large U.S. randomized trial of 425 patients also failed to confirm a survival advantage for intrapleural BCG in patients with stage I lung cancer [72]. A large European randomized trial of 407 patients also found no advantage for intrapleural BCG, and noted a shortened survival in patients who received BCG and underwent pneumonectomy [58]. Intratumoral BCG: A randomized trial in 88 patients with lung cancer examined the effects of pre-operative intratumoral treatment with BCG, and found no survival advantage [64]. Cutaneous BCG: A report of 48 patients suggested an increased progression free survival (PFS) for s.c. BCG in lung cancer patients treated with radiotherapy [80]. After 2 years of follow up, a randomized trial of 500 patients suggested a slight, but statistically insignificant, survival advantage for patients who received i.d. BCG post-operatively [23]. In a randomized trial of 103 patients with advanced or metastatic cancer, a methanol extracted residue (MER) of BCG provided no response or survival benefit beyond chemotherapy with CCNU, methotrexate, and doxorubicin [85]. In a three-arm trial in 92 surgically resected patients, investigators observed the same 37% 5-year survival rate in all three-arms that

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

included two different types of cutaneous BCG therapy or no immuotherapy [70]. SRL172, a preparation of heat-killed Mycobacterium vaccae, was combined with chemotherapy to treat patients with inoperable non-small cell lung cancer (NSCLC) or mesothelioma [75]. In a small study, 28 patients were randomized to receive chemotherapy alone or with monthly i.d. injections of SRL172. The response rate was higher for the SRL172 arm (54% versus 33%), as was median survival (9.7 months versus 7.5 months) and 1-year survival (42% versus 18%). SRL172 produced mild inflammation at the injection site. In 298 patients with small cell lung cancer (SCLC) a randomized trial of induction chemotherapy and radiation therapy with or without concurrent BCG by scarification or found no beneficial effect on response rate, duration of response, or survival [67]. A second study in 102 patients SCLC found no advantage for the MER form of BCG [34]. A third randomized trial in limited stage SCLC compared MER to observation after four cycles of chemotherapy and again there was no difference in outcome in terms of maximum response or survival [66].

Corynebacterium Parvum Another non-specific immunostimulant form of biotherapy that was studied in lung cancer is Corynebacterium parvum (C parvum). There was no difference in response rate in a small randomized trial of 49 NSCLC patients who received C parvum in combination with ifosphomide and doxorubicin chemotherapy compared to those who received chemotherapy alone [33]. A 49 patient randomized trial of i.v. C parvum as an adjuvant to surgery also found no advantage for this approach [111]. A randomized trial in 79 patients failed to confirm a positive effect on response rate or PFS in patients who received chemotherapy with doxorubicin, cyclophosphamide and vincristine, and methotrexate with or without C-parvum [96]. A large European trial randomized 286 evaluable patients with stage I or II non-small cell lung cancer to no further treatment or adjuvant therapy with a combination of intrapleural and intravenous C parvum [57]. There was no difference in outcome.

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Levamisole Levamisole alone: The antihelminth levamisole has also been studied in lung cancer. A randomized trial in which 211 post-operative patients were either observed or treated with levamisole found no difference in survival [1]. In a randomized trial of 318 patients, there was a 15% increase risk of death associated with a regimen of pre and post operative levamisole compared to a placebo group, primarily because of post surgical respiratory failure [2]. Levamisole and chemotherapy: A Veterans Administration trial randomized 381 patients with metastatic lung cancer to cyclophosphamide and CCNU alone or with levamisole [20]. After statistical adjustments, levamisole had a statistically significant negative effect on survival. Levamisole and radiation therapy: In a four-arm trial in 107 patients with squamous cell NSCLC, there was no advantage for levamisole or levamisole plus doxorubicin over radiation therapy alone [110]. The Southeastern Cancer Study Group randomized 251 patients with inoperable NSCLC to radiation therapy alone or with levamisole [46]. The trends for response rate and local control favored radiation alone; survival was the same in both arms. The Radiation Therapy Oncology Group (RTOG) conducted a similar trial in 285 patients with inoperable stage I or II or unresectable T3 N0 or N1 NSCLC [79]. Patients received radiation therapy and were randomized to either placebo or twice weekly levamisole for 2 years. Levamisole failed improve PFS or OS. RTOG also found no difference in a study of 74 patients with resected stage II–III nonsmall cell lung cancer with positive nodes randomized to post-operative thoracic irradiation plus either placebo or levamisole [31]. BCG and/or C parvum and/or levamisole: Some randomized trials explored C parvum and/or levamisole and/or BCG simultaneously. In one trial, 76 patients with stage III lung cancer were randomized to chemotherapy alone (cisplatin, doxorubicin, cyclophosphamide and vincristine) chemotherapy plus BCG, or chemotherapy plus C-parvum. Survival favored the group that received C-parvum (p = 0.05) [12]. A randomized trial in 100 patients with resectable lung cancer showed no difference in post-operative survival among intrapleural BCG, BCG plus C-Parvum, or placebo [112]. A total of 109 patients with advanced lung cancer of various cell types, were randomized to MACC chemotherapy alone (methotrexate, doxorubicin cyclophosphamide, and CCNU), or with oral levamisole or with s.c. C parvum. There were no differences among the

Biological therapy of lung cancer three treatment groups in terms of response rate or survival [16]. Another trial randomized 141 patients with resected stage II and III adenocarcinoma and large-cell undifferentiated carcinoma to receive postoperative chemotherapy (cyclophosphamide, cisplatin and doxorubicin) or immunotherapy (intrapleural BCG and oral levamisole) [32]. Survival was better for the patients who received chemotherapy.

OK-432 OK-432 (Picibanil) is a lyophilized preparation of penicillin-treated and heat killed Streptococcus pyogenes that has been studied for many years as an immunotherapy in Japan. OK-432 has also been explored as treatment for malignant ascites and malignant pleural effusions, and also as inhalational therapy for bronchoalveolar cell carcinoma [74, 113]. A prospective randomized study in 93 patients with primary lung cancer failed to demonstrate an immunological or survival benefit from adjuvant intrapleural OK-432 immunotherapy after complete resection of the primary lung tumor [49]. A recent three-arm randomized trial compared intrapleural OK-432, intrapleural bleomycin, or systemic cisplatin plus etoposide in 102 patients with NSCLC who had malignant pleural effusions [114]. Pleural PFS and OS both favored the OK-432 arm, but the differences were not statistically different. Intrapleural OK-432 as also been given in combination with chemotherapy as adjuvant treatment for patients with resected non-small-cell lung cancer. A recent meta-analysis was conducted based on data from 1,520 such patients enrolled in 11 randomized clinical trials, all of which were started before 1991 in which standard chemotherapy was compared with the chemotherapy plus OK-432 [92]. The 5-year survival rate for all eligible patients in the 11 trials was 51% in the immunochemotherapy group versus 44% in the chemotherapy group (hazard ratio 0.70, p = 0.001). Because, only four of these trials actually utilized a centralized randomization, an analysis of that subset of trials was analyzed and also showed longer survival time for the OK-432 plus chemotherapy group (odds ratio = 0.66, p = 0.049). Confirmatory trials have not been performed in the United States or Europe.

Thymic Hormones A number of different thymic hormones, including crude preparations such as Thymosin fraction V, and purified agents such as thymosin alpha-1, have significant effects on T cells and cell mediated immunity that led to investigation of their immunopotientiation in lung cancer [55].

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681

In a small randomized trial in SCCL, thymosin fraction V plus chemotherapy did not increase the response rate compared to chemotherapy alone, but was associated with a significant prolongation of survival [19]. Unfortunately, a larger study of 91 patients found no advantage for the addition of thymosin fraction 5 over chemotherapy alone in terms of response rate or survival [97]. A randomized trial involving 105 NSCLC patients found a higher response rate for patients who received VAP (vindesine, doxorubicin, cisplatin) alone compared to patients who received VAP plus thymosin fraction V, and there was a statistically insignificant trend for better survival for chemotherapy alone [8]. As summarized in Table 1, dose-exploring and phase II trials failed to confirm significant anti-tumor effects or enhancing activity for thymosin alpha-1 in NSCLC [21, 22]n a phase II trial in patients with advanced NSCLC, the combination of cisplatin and etoposide chemotherapy combined with thymosin-alpha 1 and low-dose IFN-α2a produced a response rate of 43% (24/56) and a median survival of 12.6 months [25]. In a small randomized trial involving 22 patients with advanced NSCLC, following ifosfamide chemotherapy half of patients received the combination of thymosin alpha 1 with interferon alpha (IFN-α) [93]. The chemoimmunotherapy cohort had a higher response rate (33% versus 10%) and longer PFS as well as less suppression of T cell counts and less hematologic toxicity.

Interferon alpha Non Small Cell Lung Cancer: In a phase II trial in 38 patients with measurable NSCLC, human leukocyte interferon, which is predominantly interferon-alpha, produced no objective responses [95] Preclinical and preliminary clinical data suggested IFN-α potentiates the cytotoxic activity of several chemotherapy agents against NSCLC which provided the rationale for several trials of combination therapy in NSCLC. In one trial 182 patients were randomized to receive either cisplatinepidoxorubicin-cyclophosphamide (CEP) alone or with i.m. IFN-α [3]. The response rate and toxicity were statistically higher in the combination therapy arm, but there was no difference in OS.. In a phase II trial the 3/41 (7%) response rate and median survival of only 6 months for patients treated with carboplatin and IFN-α was considered disappointing [62]. In a randomized phase II trial a combination of ifosfamide, platinum and IFN-α produced a response rate that similar to the same chemotherapy alone, but was associated with much greater neutropenia [82]. In a small pilot study the combination of 5FU, leucovorin, and IFN-α resulted in responses in 7/18 (39%) of patients with metastatic NSCLC, but this same response rate has been reported for 5FU chemotherapy alone in patients with adenocarcinoma for the lung [82]. A phase II study in 100 previously untreated patients with unresectable,

Table 1. Single-agent activity of various biologicals in patients with metastatic lung cancer Modality class

Biological agent

Lead author

Patients

Response rate (%)

Non-specific Non-specific Cytokine Cytokine Cytokine Cyokines Cytokines Cytokines Cytokine Cell therapy Immunotoxin Mab Mab Mab Mab Mab Vaccine

Thymosin alpha 1 All-trans retinoic acid Interferon-α (IFN) Interferon-α + isotretinoin Interleukin-2 (IL-2) IL-2 ± IFN-β IL-2 + IFN-α IL-2 + IFN-α IL-4 IL-2 + LAK Ricin A-Anti-CD56 Anti-Epcam Anti-GRP Cetuximab Trastuzumab Trastuzumab Belagenpumatucel-L

[22] [104b] [95] [5] [4] [103] [76] [35] [109] [9] [59] [24] [40] [29] [18] [48] [73]

10 28 38 17 11 76 7 11 55 15 21 6 13 66 24 13 61

0 7 0 0 0 3 0 0 2 6 4 0 8 5 1 0 15

Mab = monoclonal antibody LAK = lymphokine activated killer cells Epcam = epithelial cell adhesion molecule GRP = gastrin releasing peptide

682 measurable or evaluable stage III or IV NSCLC examined the combination of IFN-α2a and cisplatin in NSCLC [39]. After an initial planned 6 months of therapy, responding patients could receive maintenance IFN-α for up to 6 more months. The overall response rate was 33% among 84 evaluable patients with a median survival of 6.4 months. As would be expected, better results were achieved in stage III than IV patients. In another phase II trial 35 patients received MVP chemotherapy (mitomycin C, vindesine or vinblastine, cisplatin) plus IFN-α2a-IFN, 3 MIU during the first week of each 28 day cycle of therapy [99]. There was a tumor response rate of 51% (18/35), median PFS of 6 months, and median OS of 9.5 months. Small Cell Lung Cancer: Following induction chemotherapy with cisplatin, doxorubicin, cyclophosphamide (CAP) 237 SCLC patients were randomized to receive no further treatment or IFN-α as maintenance therapy [65]. Although there were more long term survivors among the subset of patients with limited stage disease who received IFN-α, there was no difference in OS. In a trial involving 215 patients with limited stage SCLC, 171 responded to induction chemoradiotherapy and 140 were randomized to receive maintenance IFN-α or no further treatment [41]. The IFN-α afforded no improvement in duration of response or survival, and was tolerated poorly. In a trial that included patients with both limited and extensive stage SCLC, 90 previously untreated patients were randomly assigned to receive Ifosfamide, Carboplatin, and Etoposide (ICE) alone or in combination with twice weekly low dose s.c. IFN-α [115]. There was no significant difference in overall response rates, but toxicity, complete responses and survival were higher in the IFN-α arm (p < 0.05). Most of the impact was in patients who had limited stage disease. In a similar trial with different induction chemotherapy 85 patients were randomized to chemotherapy alone or concurrent with IFN [81]. The IFN-α arm was associated with higher rates of complete (30% versus 15%) and partial remission (42% versus 29%), and longer survival (p < 0.02). In patients with extensive stage SCLC, a single arm phase II trial that included IFN-α in both the induction and maintenance therapy did not suggest results that were better than the historical experience with the etoposide-cisplatin chemotherapy alone [42]. A three-arm trial in 219 patients with any stage of SCLC randomized patients to cisplatin plus etoposide alone, or to low doses of natural or recombinant IFN [90]. Hematologic toxicity was greater in the interferon arms and there was no difference in survival. Radiation Therapy and Interferon Alpha: In vitro studies suggest that IFN-α may be a radiosensitizer for

Biological therapy of lung cancer both NSCLC and SCLC, but this has been explored in only a limited manner clinically. In one study 20 patients with inoperable NSCLC, were randomly assigned to receive either hyperfractionation radiotherapy alone or concurrently with IFN-α 3MU i.m. and 1.5 MU inhaled via a dosimeter-equipped jet nebulizer 30 min before each radiotherapy session [60]. Combined treatment with radiotherapy was feasible but impractical. There was no difference in response rate and some early deaths occurred in the combination arm that could have been treatment related. In a phase I study, escalating doses of IFN-α2a were combined with three different schedules of cisplatin and twice-daily radiotherapy [108]. IFN-α was injected s.c. 2 h before the first daily fraction of radiation. The objective response rate was 46% (11/24). The Radiation Therapy Oncology Group (RTOG) conducted a trial in 123 patients with stage III NSCLC in which patients were randomized to 50 Gy RT over 6 weeks alone or with IFN-α 16 MIU by i.v. bolus every other day thrice weekly on weeks 1, 3, and 5 during radiation therapy [14]. Because of toxicity, only 76% completed IFN-α, and only 82% completed radiotherapy compared to 94% in the control arm. Median survival and 1-year survival rates were similar, respectively 9.5 to 10.3 months and 42% to 44%.

Retinoids Prevention Trials It had been proposed that analogs of vitamin A such as arytenoids and retinoids might help prevent lung cancer and heart disease. The effects of a combination of 30 mg of beta carotene per day and 25,000 IU of retinol (vitamin A) in the form of retinyl palmitate were explored in a multicenter, randomized, double-blind, placebo-controlled primary prevention trial involving 18,314 smokers, former smokers, and workers exposed to asbestos [77]. There were 388 new cases of lung cancer diagnosed during a mean follow-up of 4 years. Surprisingly, the vitamin-treated group had a higher relative risk 1.28 (p = 0.02) of developing lung cancer compared with the placebo group. There were no differences in the risks of other types of cancer, but there was also a higher risk of cardiac related mortality in the retinoid treatment group. The investigators concluded that the combination of beta carotene and vitamin A had no benefit and may have had an adverse effect on the incidence of lung cancer and on the risk of death from lung cancer, cardiovascular disease, and for these reasons the study was stopped early.

Robert O. Dillman Beta carotene (50 mg on alternate days) alone was tested in a similar randomized, double-blind, placebocontrolled trial involving some 22,071 male physicians, about half of whom were current or former smokers [30]. After 12 years of follow up 170 cases of lung cancer had been diagnosed. There were no differences in the overall incidence of lung cancer, overall malignancy or cardiovascular disease, or in overall mortality. National Cancer Institute (NCI) Intergroup phase III trial (NCI #I91-0001) tested the hypothesis that retinoid chemoprevention would decrease recurrences or second primary tumors in the setting of stage I NSCLC [52]. The 1,166 patients had pathologic stage I NSCLC and were 6 weeks to 3 years post potentially curative surgery without other adjuvant therapy. They were randomized to received placebo or 13-cisretinoic acid (isotretinoin, CRA, Accutane®) at a dose of 30 mg/day for 3 years. After a median follow-up of 3.5 years, there were no differences between the placebo and isotretinoin arms, but there was more mucocutaneous toxicity and noncompliance in the isotretinoin arm. Secondary multivariate and subset analyses suggested that isotretinoin was harmful in current smokers, but beneficial in those who had never smoked. Squamous metaplasia is frequently observed in bronchial biopsy samples from chronic smokers. In a small randomized trial the synthetic retinoid etretinate was evaluated to see if could reverse such squamous metaplasia [50]. Following bronchoscopy 86 individuals with significant dysplasia and/or metaplasia were randomized to receive either 1 mg/kg isotretinoin or placebo daily for 6 months, 69 were reevaluated at the completion of treatment. A reduction in the metaplasia was noted in more than 50% of subjects in each group, but no significant change in metaplasia was found among those who continued to smoke. In an exploratory trial, the frequency of abnormalities in the expression of retinoic acid receptor-beta (RARbeta) in bronchial cells and the ability of CRA to correct such abnormalities, were examined in 188 smokers who underwent bronchoscopy [7]. Bronchial brushing samples were obtained for cytology analysis and for molecular analysis. Forty-four individuals with diminished RARbeta expression were randomized to receive a placebo or CRA 30 mg p.o. daily for 6 months. Only 27 patients completed treatment and 18 underwent repeat bronchoscopy. There was no difference between the results of RARbeta expression before and after treatment in the placebo group (p = 0.43), but there was upregulation of RARbeta expression in the CRA group (p = 0.001). The authors felt that these results supported undertaking a phase III chemoprevention trial of CRA treatment for lung cancer. In another trial 307 post operative patients with stage I NSCLC were

683 randomized to no treatment or retinol palmitate (300,000 IU p.o. daily for 12 months) [78]. After 46 months there were fewer new tumors in the retinol group (p = 0.045) but few recurrences had taken place.

Cis-retinoic Acid and Interferon The combination of IFN-α plus CRA, which had produced high objective response rates in squamous cell cancers of the skin and cervix, produced objective responses in only 1/21 patients with squamous cell cancer of the lung, and that response was in a patient who only had regionally advanced disease [86]. A three-arm randomized phase II multi-center study of 85 patients with SCLC revealed no differences in survival among the three arms, one of which included IFN-α during induction, and IFN plus CRA as maintenance therapy [91].

Cis-retinoic Acid and Chemotherapy CRA has also been combined with chemotherapy. Carboplatin, vindesine and 5-fluorouracil/leucovorin were combined CRA 1 mg/kg orally in 28 patients with advanced, measurable NSCLC [84]. Toxicity was manageable. The response rate was 39% (11/28) with a median survival of 9.7 months for the whole group.

Trans-retinoic Acid and Interferon The combination of all trans-retinoic acid (ATRA) and IFN-α was tested in patients with various types of nonsmall cell lung cancer that were unresectable, locally advanced, or metastatic [6]. The authors reported an objective response rate of 21% (6/29) with several responses lasting more than a year in duration. ATRA alone produced two objective response in 28 patients with NSCLC [104b].

Transretinoic Acid and Chemotherapy In a phase II study 38 patients with advanced squamous cell NSCL were treated with a combination of low-dose ATRA (40 mg/m2/day), IFN-α (6 MU/day) and monthly cisplatin (40 mg/m2) for 12 weeks [27]. There was a response rate of 21% but only 16% of patients were able to complete the planned treatment. Two large randomized trials were conducted in which patients with pleural effusions or distant metastatic NSCLC were randomized to receive standard combination chemotherapy with placebo or bexarotene capsules (Targretin®) at a dose of 400 mg/m2 p.o. daily [106]. In both trials the 2-year survival rate was 16% for chemotherapy alone, and

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12% to 13% in the chemotherapy plus bexarotene arms; so the studies were conclusively negative in terms of any benefit for this retinoid [13, 36].

Interleukin-2 Alone, in Combination, or with Adoptive Cellular Therapy

Interferon Gamma

IL-2 in NSCLC

Randomized trials have failed to support a benefit for IFN-gamma in lung cancer, even in the setting of minimal residual disease.

Eleven subjects with Stage III–IV non small cell lung cancer were treated with continuous i.v. IL-2 at a dose of 18 MIU/m2/day from day 1 to day 13 with a rest on day 7 [4]. There were no tumor responses, but immune effects were noted. In an Italian study, 20 patients with NSCLC received the pineal hormone melatonin orally at a dose of 10 mg day-1 each evening starting 1 week before starting s.c. IL-2 at a dose of 3 MIU/m2 every 12 h for 5 days/week for 4 weeks [53]. The authors reported partial responses in 4/20 (20%), with little toxicity. In a subsequent trial, the same group conducted a randomized trial in 60 patients with locally advanced n = 8) or metastatic NSCLC (n = 52) who received either lowdose s.c. IL-2 plus melatonin or cisplatin plus etoposide chemotherapy [54]. Similar response rates (all partial) were observed (7/29 patients treated with biotherapy and 6/31 in patients receiving biotherapy, but the survival rate at 1 year actually favored the biotherapy group (45% versus 19%, p = 0.02). This study is of interest because it suggests that a relative non toxic immunotherapy regimen may be as efficacious as standard chemotherapy in the treatment of advanced NSCLC. In a phase II trial, 76 patients with stage IV NSCLC were randomized to receive either IL-2 alone 18 MIU/ m2 i.v. 3 days weekly or IL-2 plus i.v. IFN-β (6 MU/m2), both given thrice weekly [103]. Objective responses were observed in only 3/76 (4%) patients; grade 4 toxicity was about 10% in each arm. Continuous infusion IL-2 (18 MIU/m2/day for 5 days) combined with IFN-α (5 MU s.c. every other day during IL-2) produced no responses in seven patients with NSCLC [76]. In another trial of continuous i.v. IL-2 (18 MIU/m2/day for 3 days) and IFNα (5 MU/m2/day i.m. for 3 days) produced no responses in 11 patients [35]. IL-2 at a dose of 3 MU/m2 s.c. twice daily, and IFN-α at a dose of 3 MU once daily, 5 days a week was given as a consolidation treatment to patients with NSCLC cancer who had responded to chemotherapy, but no benefit was observed in a study of 52 patients in which many patients dropped out because of toxicity [105].

Non Small Cell Lung Cancer In a small randomized phase II trial, 37 patients with inoperable NSCLC were randomized to receive either two cycles of etoposide and cisplatin chemotherapy or 6 weeks of thrice weekly IFN-β (30 MU) plus IFN-γ (200 μg) followed by two cycles of chemotherapy [98]. There was more hematologic toxicity during chemotherapy on the combined modality arm (p = 0.02), but no difference in response rates (11–17%) or survival.

Small Cell Lung Cancer Of 71 patients with extensive stage SCLC, 41 had a complete or partial response after four cycles of PACE (cisplatin, doxorubicin, cyclophosphamide, and etoposide) and then were treated with INF-γ 0.2 mg s.c. daily until grade IV toxicities or disease progression occurred [11]. There was an increased tumor regression in only 2/30 (7%) patients who had a partial response at the end of chemotherapy, and no suggestion of a survival benefit. Based on this the authors concluded that INF-γ was not active against SCLC. In a phase III trial, 100 patients in complete remission following treatment with six cycles of combination chemotherapy, thoracic radiotherapy, and prophylactic cranial irradiation (PCI), were randomized to observation or IFN-γ at a dose of 4 MU s.c. per day for 6 months [37]. Significant toxicity including chills, myalgia, lethargy, and alteration of mood-personality were observed in the IFN- γ group. There was no difference in progression free survival or overall survival, although the trends favored the group who did not receive IFN-γ. The study had sufficient power to exclude a 33% improvement in survival (P = 0.04) for the IFN-γ. In another phase III trial, 127 of 177 patients with SCLC who were in complete or nearly-complete response following chemotherapy with or without thoracic radiotherapy, were randomized to observation or to receive either IFN-γ 4 MU (0.2 mg) sc every other day for 4 months or observation [107]. There was no difference in progression free or overall survival.

IL-2 in SCLC In a phase II trial, 24 of 50 patients with extensive SCLC who had not achieved complete remission with

Robert O. Dillman PACE (cisplatin, doxorubicin, cyclophosphamide, and etoposide) chemotherapy, were treated with IL-2 by 4-day continuous i.v. infusion of 4.5 MIU/m2 per day for up to 8 weeks [17]. Four patients improved to a complete response and one improved from stable disease to a partial response for an improvement rate of 21% (5/21), but the treatment was quite toxic, and only five patients completed the planned 8 weeks of treatment. These radiographic improvements in response may have been because of faster resolution of dead tumor cells by activated macrophages rather than a cytotoxic anti-tumor effect.

IL-2 and LAK A phase II study of interleukin-2 (IL-2) at a fixed dose of 6 MU/m2 per day as a 24 h continuous intravenous infusion (CIV) with lymphokine-activated killer (LAK) cells yielded one near complete response that lasted 18 months [9]. Two Japanese trials have suggested that IL-2 plus LAK may be of value in NSCLC. A randomized trial of immunotherapy with IL-2 plus LAK cell was conducted in 105 patients after noncurative resection of primary lung cancer [43]. All patients received standard chemotherapy and/or radiotherapy with or without the addition of the immunotherapy. The 7-year survival rate was greater in the immunotherapy group than in the control group (39% versus 13%, p < 0.01). The same investigators conducted a randomized prospective study of post surgical adjuvant immunotherapy using IL-2 and LAK cells in 82 patients who had undergone a curative resection of primary lung cancer [44]. The three arms included observation alone, adjuvant chemotherapy (cisplatin, vindesine, and mitomycin C) and adjuvant chemotherapy for two cycles followed by IL-2 plus LAK. Chemotherapy plus immunotherapy produced a 5-year survival rate of 50% compared to 33% for observation and 30% for chemotherapy alone (p = 0.03).

IL-2 and TIL In a Phase I clinical-trial retroviral-mediated IL-2 genes were transferred to tumor infiltrating lymphocytes (TIL) that were infused into ten lung cancer patients with refractory pleural effusions [101]. Autologous TIL were exposed to the retroviral plasmid pL(IL-2)SN containing the human IL-2 gene with about 10 to 16 billion IL-2-transfected TIL cells infused into the chest cavity of each patient. Pleural effusions did not re-accumulate for at least 4 weeks in six of ten patients. In a randomized study, TIL cultures were successfully established for 113 of 131 patients, 13 stage II, 42 IIIA,

685 and 32 IIIB NSCLC who were randomized to receive s.c. IL-2 with TIL with either chemotherapy or radiation for stage III A disease, or to observation alone in patients with stage II disease [83]. The radiation therapy consisted of 60 Gy and the chemotherapy of cisplatin and vinblastine followed by radiation therapy. The radiation/chemotherapy was not given until 2–3 months after the administration of IL-2 and TIL. Better survival was seen for the patients who received IL-2 plus TIL in stage IIIA (median survival 22 months versus 9 months, p = 0.06) and stage III B (median survival 24 months versus 7 months, p < 0.01). There was no difference in survival for patients with stage II disease. There has been no effort to replicate this study in the United States. One criticism of this study is that the median survival rates for this chemotherapy followed by radiation therapy has been 13–14 months in U.S. trials, and the analysis was based on treatment rather than intent to treat.

Interleukin-4 In a randomized phase II trial, 63 patients with advanced NSCLC, 44 of whom had received prior combination chemotherapy were randomized to receive one of two different dose of Interleukin-4 (IL-4) (0.25 μg/kg and 1.0 μg/kg) given s.c. thrice weekly [109]. Common side effects were fatigue and fever. Only one of 55 evaluable patients had an objective tumor response. A subsequent randomized three-arm placebo-controlled phase III trial of chemotherapy alone or with one of two different dose of IL-4 showed no difference in response rate or survival (unpublished results).

Amifostine (Ethyol™) Amifostine is an analog of cysteamine that selectively protects normal tissues of various organ systems against the toxic effects of various cytotoxic drugs and radiation. Because of this it has been suggested that amifostine be given concurrently with chemotherapy and radiation therapy in the treatment of lung cancer in an effort to decrease toxicity [102, 15]. It was originally named Walter Reed 2721, a reflection of its origin from a classified military research program that was trying to develop agents that would protect personnel from the lethal effects of radiation. Amifostine is actually a prodrug that is converted by tissue alkaline phosphatase to WR-1065, a free thiol with a sulfhydryl group that binds to oxygen-free radicals generated by radiation and chemotherapy. It may also upregulate p53 as an additional

686 mechanism of chemoresistance. It acts a broad-spectrum cytoprotective agent that seems to protect virtually all normal tissues except perhaps the brain. The alkaline phosphatase enzyme that converts WR-2721 to WR-1065 is expressed in much greater amounts in normal blood vessels and normal tissues than in neoplastic vasculature or membranes of malignant cells. Numerous clinical trials have shown that amifostine decreases the toxic effects of chemotherapy and radiation on normal tissues without any diminution in clinical efficacy as manifest by equivalent response rates and survival. Cytoprotective effects without decreased anti-tumor efficacy were demonstrated for patients with NSCLC who were receiving carboplatin [10, 51]. A small randomized trial in 62 patients suggested a better survival for patients with stage II and III NSCLC who received chemoradiotherapy with amifostine compared to chemoradiotherapy alone [45]. However, a larger national trial in 242 patients stage II to IIIA/B non-small-cell lung cancer failed to confirm any benefit for amifostine to carboplatin/paclitaxel chemotherapy [71].

Monoclonal Antibodies The murine anti-Epcam monoclonal antibody (Mab) KS1/4 and KS1/4-methotrexate immunoconjugate were administered to patients with Stage IIIB or IV NSCLC. Six patients received KS1/4 alone and five patients received KS1/4-methotrexate conjugate [24]. Mild to moderate side effects including fever, chills, anorexia, nausea, vomiting, diarrhea, anemia, and brief transaminasemia were seen in both groups. There was one possible clinical response in a patient treated with unconjugated antibody. Results were not better with a KS1/4 methotrexate immunoconjugate, but the latter was associated with greater GI toxicity because of reactivity with antigen on normal small bowel. An immunotoxin consisting of murine Mab N901, that binds to the CD56 (neural cell adhesion molecule [NCAM]) antigen found on cells of neuroendocrine origin, and blocked ricin was tested in a phase I trial in 21 patients with SCLC [59]. Successive cohorts of at least three patients were treated at doses from 5 to 40 μg/kg/ day for 7 days. The dose-limiting toxicity was a capillary leak syndrome that occurred in two of three patients at the highest dose. No patient developed clinically significant neuropathy. One patient achieved a partial response. The murine Mab 2A11 binds to gastrin-releasing peptide (GRP), which binds to receptors and stimulates growth of SCLC cells [40]. 2A11 at a dose of 250 mg/m2 over 1 h thrice weekly for 4 weeks was given to 13 previously

Biological therapy of lung cancer treated SCLC patients. One patient had complete resolution of radiographically detectable tumor that lasted 4 months. Four patients (33%) had stable disease. No toxic reactions were observed. The anti-VEGF Mab bevacizumab (Avastin®) is now part of standard therapy for patients with non-squamous NSCLC. In a three-arm randomized phase II trial 99 previously untreated NSCLC patients were randomized to treatment every 3 weeks with paclitaxel 200 mg/m2 and carboplatin (AUC = 6) (PC) alone or in combination with bevacizumab at 15 or 7.5 mg/kg [38]. The response rate and PFS were higher for the 15 mg/kg bevacizumab arm compared to PC alone with a trend toward better survival. Bleeding was the most significant complication associated with bevacizumab and included minor mucocutaneous hemorrhage, and major hemoptysis associated with centrally located squamous cell cancers accompanied by tumor necrosis and cavitation. In a large two-arm randomized trial, 878 patients with previously untreated non-squamous NSCLC were randomized to PC + bevacizumab (15 mg/kg) or PC alone on a 3 week schedule [94]. The bevacizumab arm was associated with a higher response rate (p < 0.001), better OS (HR = 0.79, p = 0.003), and PFS (HR 0.66, p < 0.001). Even though patients with squamous cell histology were not enrolled in this trial, there were still five hemorrhagic deaths in the bevacizumab arm (1.2%) and significant bleeding was more frequent in the bevacizumab arm (4.4% versus 0.7%, p < 0.001). In a large European trial 1,043 patients with previously untreated non-squamous NSCLC were randomized to gemcitabine plus cisplatin with bevacizumab at 10 mg/kg or 5 mg/kg or placebo [63]. Results favored the bevacizumab arms, although the benefit was not as impressive as in the U.S. trial with PC. As shown in Table 1, the anti-EGFR Mab cetuximab (Erbitux®) also enhances the effects of chemotherapy in NSCLC. In NSCLC Hanna et al. observed a response rate of only 5% for cetuximab alone in 66 patients with metastatic disease who had progressed after previous systemic therapy [29]. Other studies have combined cetuximab with chemotherapy. Thienelt et al. reported a 26% response rate in 31 previously untreated patients who received paclitaxel, carboplatin, and cetuximab [104a]. Robert et al. reported a 29% response rate in 35 patients who were treated with gemcitabine, carboplatin and cetuximab [87]. Some patients with NSCLC have tumors that express Her2, the target of trastuzumab (Herceptin®). In one study only 24/209 (11%) patients had Her2-positive tumors by immunothistochemistry (IHC), and only 1/24 (4%) of them had a response [18]. In another NSCLC

Robert O. Dillman trial 13/69 had Her2-positive tumors by IHC, and 0/13 responded to trastuzumab [48]. Trials of chemotherapy and trastuzumab were also compromised by the low proportion of patients with HER2-positive tumors. For example, only 16% to 21% of patients in three NSCLC trials had HER2-positive tumors, and in two of the trials less than 5% of patients had tumors that were Her2 3 + by IHC [26, 47, 116]. Gatzemeier et al. randomized 101 patients with Her2-positive NSCLC to trastuzumab plus gemcitabine and cisplatin or the chemotherapy alone, and found no difference in response rate (36% versus 41%) or PFS (6.1 versus 7.0 months), but only six patients had tumors that were 3 + Her2 by IHC, and five of them did have an objective response to treatment [26]. Zinner et al. observed a 38% response rate in 21 patients treated with gemcitabine and cisplatin [116]. Krug et al. observed a 28% response rate among 64 patients including 7/30 in patients treated with docetaxel plus trastuzumab and 11/34 in the patients treated with paclitaxel plus trastuzumab [47]. Response rates did not appear to be higher than anticipated response rates for chemotherapy alone, but to determine the true contribution of trastuzumab would require randomized trials. Collectively these studies have been disappointing. This does not appear to be a fruitful area for further investigation given that so few NSCLC express Her2.

Vaccines There is great interest in the potential role of vaccines in the adjuvant setting of lung cancer, but no products are commercially available. In a randomized trial in which 95 post operative NSCLC received no additional treatment or an i.d. dose of an autologous irradiated suspension of fresh tumour cells combined with a small dose of C parvum, there was no difference in outcome [100]. A vaccine consisting of an anti-idiotype antibody BEC2, a mouse Mab that mimics the structure of the disialoganglioside GD3, was administered with BCG to 15 SCLC patients following primary therapy with chemotherapy with or without radiation therapy [28]. All patients developed anti-BEC2 antibodies and five were found to have antibodies against GD3. A Phase III trial is being conducted to evaluate BEC2 plus BCG as adjuvant therapy after chemotherapy and irradiation. The most encouraging vaccine in lung cancer at the moment is belagenpumatucel-L, a transforming growth factor beta-2 (TGF-β) antisense gene-modified allogeneic tumor cell vaccine designed to inhibit secretion of TGF-β at the site of immunization. In a randomized phase II trial, 75 NSCLC patients with stage II, III, or

687 low tumor burden stage IV NSCLC were randomized to receive 12.5, 25, or 50 million cells per injection on a monthly or every other month schedule to a maximum of 16 injections [73]. There were no significant adverse events associated with the treatment. There was a doserelated survival benefit in patients who received the two higher doses who had a 2-year survival rate of 52% compared to 20% for patients in the lowest dose cohort (p = 0.007). There was response rate of 15% among 61 patients who had stage IIIB or IV disease, although this interpretation was confounded by previous therapy. Belagenpumatucel-L is being tested further in a pivotal randomized phase III trial.

Summary As of 2008, the anti-VEGF Mab bevacizumab is the only biotherapeutic that has a ever received a commercial marketing indication in lung cancer. None of the cytokine or cell therapy approaches are considered promising at this time, but other Mabs are still being tested. Some of the results with adoptive cell therapy are provocative, but have not been replicated. IFN-α adds little if any benefit after chemotherapy, and adds to toxicicty when administered with chemotherapy. Doses of IL-2 that are effective in other malignancies are too toxic to administer to patients with significant underlying lung disease. The results with retinoids have been disappointing. Randomized trials will determine whether any of the current vaccine approaches are worthwhile.

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21.7 Biological therapy of B and T cell lymphoproliferative disorders ROBERT O. DILLMAN

Biotherapy has consistently exhibited significant activity in lymphoid malignancies. The first report of a complete remission with a monoclonal antibody in 1982 was in a patient with follicular lymphoma. Interferon alpha (IFNα) became the first biological approved for the treatment of malignancy in 1986, because of its activity in hairy cell leukemia, and for many years was used in the treatment of lymphoma. The first monoclonal antibody approved for the treatment of a malignancy was the antiCD20 chimeric Mab rituximab, which has become a block-bluster drug since its approval in 1997. The first immunotoxin to gain regulatory approval, denileukin diftitox was approved based on activity in T cell lymphoma. The anti-CD52 humanized Mab alemtuzumab was approved based on its activity in CLL. The first two radiolabeled antibodies approved for cancer treatment were the anti-CD20 products Y-90 ibritumomab tiuxetan and I-131 tositumomab which are very active in the treatment of B cell lymphoma. Finally, it is widely anticipated that perhaps one or more of the patient-specific vaccines consisting of idiotype protein may become the first tumor specific vaccines to receive regulatory approval as treatment for follicular lymphoma.

Hairy Cell Leukemia This section starts with this relatively uncommon B cell lymphoproliferative disorder because of its significance in the validation of biotherapy. In 1986, IFN-α became the first biological to achieve regulatory approval based on its activity in hairy cell leukemia (HCL) [66, 75, 86, 164]. In one report 75% of 64 patients with HCL, 61 of whom had undergone prior splenectomy, who were treated with 2 MU/m2 IFN-α2b s.c. thrice weekly demonstrated an objective response [75]. In another report 78% of 212 patients with HCL, 166 of whom had undergone prior splenectomy, who were treated with 2 MU/ m2 s.c. IFN-α2b s.c. thrice weekly had an objective response [164]. These responses proved to be durable with 28% in continuous remission beyond 6 years and 83% alive beyond 6 years [141]. A small randomized

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

trial confirmed that IFN-α was superior to splenectomy as initial treatment of the disease [155]. However, in 1991 the purine analog deoxycoformycin (pentostatin) gained regulatory approval for the treatment of HCL and displaced IFN-α as the treatment of choice because was it active in interferon-refractory disease [67, 81] less toxic, and superior to IFN-α when compared directly in randomized trials in previously untreated patients [77]. In 1993 another purine analog 2-chlorodeoxyadenosine (cladribine), which like pentostatin was also associated with durable responses in over 90% of previously untreated patients, was also approved for the treatment of HCL [17, 136]. The circulating lymphocytes of HCL typically express very high levels of CD20. The anti-CD20 chimeric Mab rituximab has been used to treat relapsed HCL using both the 4 and 8 week treatment schedules of 375 mg/m2 per week. The 4-week schedule produced responses in 5/10 patients in one study [103], but only in 6/24 patients who had all relapsed after cladbribine [126]. The 8-week schedule produced responses in 8/15 relapsed patients [163].

B Cell Lymphoma Interferon In early trials single agent activity for IFN-α was demonstrated in the lymphomas with response rates near 50% in indolent lymphomas, and as high as 20% in more aggressive lymphomas [185, 190, 192]. Numerous trials focused on the role of IFN-α in combination with chemotherapy or as maintenance therapy following induction chemotherapy as summarized in Table 1. In a U.S. multicenter trial 249 patients with B cell lymphoma were randomized to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (COPA) with or without IFN-α [156, 157]. There was better progression-free survival (PFS) and overall survival (OS) in the IFN-α arm, but there was also more toxicity. In a French trial 268 patients with B cell lymphoma were randomized to combination chemotherapy with cyclophosphamide, doxorubicin, vindesine,

693

694

Biological therapy of B and T cell lymphoproliferative disorders

Table 1. Randomized trials of interferon-alpha in the treatment of B-cell lymphoma. Author

Year

Ref.

# of Pts

Induction

Maintenance

Results

Smalley et al. Peterson et al. Solal-Celigny et al. Aviles et al. Hagenbeek et al. Unterhalt et al. Fisher et al. Rohatiner et al. Neri et al.

1992, 2001 1993 1997 1993 1994 1998 1996 2000 2001 2001

NEJM [156,157] Leukemia ASCO [133,191] NEJM [158] Leuk Lymphoma [2] J Clin Oncol [69] Leukemia [168] J Clin Oncol [49] Br J Cancer [151] J Hematother [125] Stem Cell Res

249 581 268 384 347 498 571 204 151

COPA ± IFN CTX ± IFN CHVP ± IFN CEOP-Bleo CVP + RT to bulky PmM or CVP CHOP & ProMACE CLB ± IFN MXT/Leu CEOP CVP

None ± IFN for CR CHVP ± IFN ± IFN for CR ± IFN for OR/SD ± IFN for CR ± IFN 1 yr for OR ± IFN for OR ± IFN 1 yr

↑PFS ↑OS NSD ↑PFS ↑OS ↑EFS ↑OS ↑PFS ↑DFS NSD NSD ↑DFS ↑OS

CTX = cyclophosphamide CLB = chlorambucil COPA = cyclophosphamide, vincristine, prednisone, doxorubicin CHOP = cyclophosphamide, vincristine, doxorubicin,prednisone CHVP = cyclophosphamide, doxorubicin, vindesine, prednisone CEOP-Bleo = cyclophosphamide, epirubicin, vincristine, prednisone, bleomycin CVP = cyclophosphamide, vincristine, prednisone ProMACE = procarbazine, methotrexate, doxorubicin, cyclophosphamide, etoposide PmM = prednimustine and mitoxantrone MXT/Leu = mitoxantrone, chlorambucil CR = complete response OR = objective response (partial or complete response) PFS = progression free survival EFS = event free survival (neither death nor disease progression) DFS = disease free survival OS = overall survival

prednisone (CHVP) with or without IFN, and the same treatment was continued as maintenance treatment in responders [158]. This trial also demonstrated improvement in PFS and OS. It should be noted that these two randomized trials were not limited to patients with indolent lymphoma. The US trial included patients with high-risk follicular lymphoma as well as patients with intermediate grade non-follicular lymphoma. The French trial was conducted in patients with high-risk follicular lymphoma defined by tumor mass >7 cm, B-symptoms, or multiple extranodal sites of disease. CALBG randomized 581 indolent lymphoma patients to cyclophosphamide with or without IFN-α, with a second randomization to observation or IFN-α for patients who had complete remissions [133, 191]. There was no difference based on the initial therapy, but there was some trend toward better PFS for the patients who achieved a complete response (CR) and subsequently received maintenance IFN-α. A German trial randomized indolent lymphoma patients who had achieved a CR with chemotherapy alone to IFN-α or observation [168]. Maintenance IFN-α was associated with longer disease free survival. A European cooperative group trial randomized indolent lymphoma patients whose disease had

no progressed during chemotherapy, to observation or IFN-α [69]. IFN-α maintenance therapy was associated with longer PFS. In another 151 indolent lymphoma patients were randomized to receive IFN-α or observation after chemotherapy [125]. Again there was improvement in PFS for patients who experienced a CR, and improved OS for all patients. In a Spanish trial 384 patients with follicular lymphoma who were in complete remission after six cycles of cyclophosphamide, epirubicin, vincristine, prednisone and bleomycin (CEOP-Bleo) chemotherapy, were randomized to observation or IFN-α maintenance [4]. At a median follow up of almost 10 years, the IFN-α arm had a better event free survival (64% versus 35%, p < .01) and OS (81% versus 57%, p < .001). In contrast to these results, in a trial in which 204 patients were randomized to chlorambucil with or without IFN-α, and the responders were randomized to observation or IFNα, there was no difference in outcome [150]. In a U.S. cooperative group trial there was no survival difference for 571 patients with low-grade stage III or IV lymphomas who were randomized to CHOP and PROMACE induction therapy with or without maintenance IFN-α, and subsequently 268 responders were randomized to observation or 1 year of IFN-α [49].

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A meta-analysis focused on ten randomized trials in which IFN-α was tested as part of initial therapy in 2005 patients with lymphoma [151]. In seven of these trials IFN-α was used as part of initial therapy, and in seven IFN-α was used as maintenance therapy. In the trials in which IFN-α was combined with chemotherapy, there was no increase in response rates compared to chemotherapy alone. However, the use of IFN-α either with or after chemotherapy was associated with better survival. The best results were obtained in four trials in which the intensity of IFN-α therapy was greater than 36 MU per month (p = .0004). Better survival was also noted in the five trials in which doxorubicin or mtioxantrone were used in the combination chemotherapy (p = .0008). Based on these trials, prior to the introduction of rituximab, it appeared that IFN-α added nothing to chemotherapy in the initial treatment of patients with lowrisk indolent lymphoma, but the use of maintenance IFN-α in responding patients was associated with prolonged PFS, but not OS. The converse appeared to be true in high-risk, low-grade lymphoma. In that disease setting it appeared that concurrent administration of IFN-α with “CHOP-like” regimens was associated with a progression free and perhaps overall survival advantage, but maintenance IFN-α did not appear to add benefit in that setting. Two additional trials have been completed in lymphoma patients who had responded to induction therapy, and then were randomized to consolidation with either intensive chemotherapy and stem cell rescue, or IFN-α. In patients with mantle cell lymphoma who were in CR after chemotherapy the median PFS was much better in the transplant arm than the IFN-α arm (39 versus 17 months, p = .011) [40]. Similarly, in 244 patients with follicular lymphoma who were in remission following chemotherapy, the 5-year PFS was twice as high in the

transplant arm than the IFN-α maintenance arm (65% versus 33%, p < .0001) [105]. More recently the inferiority of IFN-α compared to other therapies was confirmed in another trial that compared rituximab plus CHOP to CHOP as induction therapy in patients with follicular lymphoma, and then randomized responders to high-dose chemotherapy or maintenance IFN-α [72]. Survival was similar for RCHOP-transplant, RCHOPIFN-α, and CHOP-transplant, all of which were superior to the CHOP-IFN-α arm. This trial seems to have solidified chemotherapy plus rituximab as the current standard initial therapy for patients with follicular lymphoma and suggests there is no general benefit for the addition of IFN-α or high dose chemotherapy for patients who respond to RCHOP. Thus, controversies regarding the role of IFN-α in B-cell lymphoma have been muted by the introduction of rituximab, which as a single agent had much higher response rates and a much milder toxicity profile than interferon.

Interleukin-2 The published experience in lymphoma with interleukin-2 (IL-2) alone, or with adoptive cellular therapy, is summarized in Table 2. In three small pilot studies, IL-2 + lymphokine activated killer (LAK) cells were associated with response rates of 0%, 8%, and 50% [6, 113, 172]. Five studies of IL-2 alone using high-dose bolus, low-dose bolus, or intermediate-dose continuous infusion intravenous [CIV] IL-2 were associated with response rates of 0%, 4%, 20% and 22% [1, 41, 65, 172]. Most of these studies had a mix of all types of lymphoma patients. Interestingly, in one French trial the response rate for intermediate-dose CIV IL-2 was 22% for intermediate lymphomas compared to only 4% in indolent lymphoma [65]. In a cooperative group clinical

Table 2. Interleukin-2-based treatment of B-cell lymphoma Lead author

Year

Ref.

Regimen

# Pts

Response rate

Allison Margolin Bernstein Weber Weber Duggan Gisselbrecht Gisselbrecht

1989 1991 1991 1992 1992 1992 1994 1994

J Clin Oncol [1] J Immunother [113] J Immunother [6] J Clin Oncol [172] J Clin Oncol [172] J Immunother [41] Blood [65] Blood [65]

LD bolus HD bolus & hybrid bolus/CIV + LAK ID CIV + LAK HD bolus + LAK HD bolus LD bolus HD CIV for low-grade HD CIV for intermediate-grade

9 15 12 8 11 20 24 23

22% 0% 8% 50% 0% 20% 4% 22%

HD = high dose ID = intermediate dose CIV = continuous intravenous LAK = lymphokine activated killer cells IFN = interferon

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trial patients were randomized to a combination of lowdose bolus IL-2 with or without IFN-β [41]. Toxicity was substantial, and there appeared to be no advantage of the addition of IFN-β since the response rates were 4/20 for IL-2 alone and 3/21 for IL-2 plus IFN-β. Because of the success of rituximab in lymphoma, and the toxicity of higher dose IL-2 regimens, outpatient s.c. IL-2 was combined with rituximab for the treatment of relapsed follicular lymphoma with reported response rates of 10% in a trial of 54 patients [95], 26% in a trial of 34 patients [186], and 55% in a trial of 20 patients [61]. An industry sponsored randomized phase 2 trial of 8 weeks of s.c. IL-2 with 4 weeks of standard rituximab for patients who had relapsed after prior chemotherapy was terminated because of poor accrual. Because of the toxicity associated with high dose schedules of IL-2, the limited single agent activity, and the emergence of other agents for the treatment of lymphoma, there has been little interest in pursuing trials with IL-2 in recent years in the lymphomas other than as low doses for immune reconstitution purposes following myelosuppressive therapy, such as in the transplant setting.

Monoclonal Antibodies In November 1997 Rituximab [Rituxan®] became the first monoclonal antibody (Mab) approved with a marketing indication for a malignant disease. Rituximab has revolutionized the treatment of B cell lymphoproliferative disorders and is now standard therapy. The clinical trial experience with rituximab is summarized in Table 3. In nine trials of relapsed, previously treated follicular lymphomas rituximab produced response rates ranging from 46% to 76% [3, 8, 15, 28, 54, 64, 116, 137, 178]. Responses were evident within 2 months in most patients, and persisted for over a year from the date of first treatment. Response rates of over 40% were also noted in patients with bulky B-cell lymphoma [31] and patients who

relapsed after a previous response to rituximab [29, 85]. In four trials in previously untreated follicular lymphomas rituximab produced response rates ranging from 67% to 78% [21, 64, 78, 179]. In small lymphocytic lymphoma three trials in previously treated patients yielded response rates of only 14% [54, 116, 137], but the response rate was 65% in such patients who had previously received chemotherapy and who received maintenance rituximab [78]. In marginal zone lymphoma response rates were 85% in previously untreated patients with extranodal MALT disease compared only 45% in such patients who had relapsed after prior chemotherapy [22], and 77% in patients who had relapsed with gastric MALT [114]. In lymphoplasmacytic lymphoma, four trials in previously treated patients reported response rates ranging from 16% to 50% [37, 63, 166] while three trials with untreated patients reported response rates ranging from 35% to 65% with the best results obtained with extended rather than only 4 weeks of therapy [37, 63, 167]. In the limited experience reported for single agent rituximab in mantle cell lymphoma responses were about 30% regardless of whether patients were previously untreated or had relapsed after prior therapy [19, 54]. There are only two published reports regarding single-agent rituximab in large B cell lymphoma, both of which were conducted in patients who had relapsed after prior therapy, and both recorded a response rate of 37% [19, 165]. In practice rituximab is almost always used in combination with chemotherapy in aggressive lymphomas, but is often used alone as initial therapy of patients with follicular lymphoma or marginal zone lymphomas. Rituximab has been safely combined or sequenced with various types of chemotherapy with numerous reports of high response rates and durable remissions. These are summarized in Chapter 10 of this text. In large randomized trials rituximab plus chemotherapy has consistently produced superior outcomes compared to the same chemotherapy alone. In patients with previously

Table 3. Response rates for rituximab as a single agent Lymphoma histology

Setting

# trials

# patients

Response range

Median response rate

Follicular lymphoma Follicular lymphoma Small lymphocytic Small lymphocytic Marginal zone Marginal zone Lymphoplasmacytic Lymphoplasmacytic Mantle Cell Mantle Cell Large B cell

Untreated Relapsed/refractory Untreated Relapsed/refractory Untreated Relapsed/refractory Untreated Relapsed/refractory Untreated Relapsed/refractory Relapsed/refractory

4 9 1 3 1 2 4 3 1 2 2

180 547 23 68 24 37 78 92 34 54 87

67–78% 46–76% 65% 13–14% 87% 45–77% 35–65% 16–50% 29% 30–33% 37%

73% 58% 65% 14% 87% 61% 50% 33% 29% 32% 37%

Robert O. Dillman untreated follicular lymphomas response rates were higher for RCHOP versus CHOP [72], RCVP versus CVP [112], and RMCP versus MCP [71]. In previously untreated indolent lymphoma response rates were higher for RFND versus FND [117]. In previously untreated lymphoplasmacytoid lymphomas response rates were higher for RCHOP versus CHOP [11]. In relapsed follicular lymphoma response rates were higher for RCHOP versus CHOP [169] and for RFCM versus FCM [56]. In relapsed mantle cell there was a higher response rate for RFCM versus FCM [55]. In the trials in which it is an endpoint that has been assessed, PFS has also been longer in those patients who received rituximab with their chemotherapy [71, 112, 169]. Survival advantages have not been established in any of these trials, and probably will not be because of the clinical benefit associated with second and third-line therapies in these diseases. In patients with large B cell lymphoma the addition of rituximab has not only typically increased response rates and complete response rates, but more importantly, it has also produced longer progression free and overall survival and is probably curative therapy for more than half of all patients. All four large randomized trials in which RCHOP was compared to CHOP as initial therapy for patients with large B cell lymphoma, RCHOP has been associated with a superior PFS and OS with a substantial reduction in the risk of death [20, 47, 68, 134, 135]. In the one relatively small randomized trial in mantle cell that has been published, RCHOP was superior to CHOP in terms of response rate (94% versus 75%) complete response rate (34% versus 7%), with a 50% longer median PFS (21 versus 14 months), but with no difference in survival [106]. As discussed in Chapter 10, the role of maintenance rituximab remains controversial. In indolent lymphomas four randomized trials utilizing a variety different maintenance schedules, have shown that administration of additional doses or courses of rituximab clearly prolongs progression free survival after initial treatment with rituximab alone in untreated patients [64], after rituximab alone in patients who have relapsed after prior chemotherapy [64, 70], and after chemotherapy and rituximab in patients who had relapsed after prior chemotherapy [56, 169]. However, none of these trials have established a survival benefit. The only published randomized trial that has tried to address the issue of duration of benefit from rituximab (or time to becoming rituximab refractory, is of uncertain relevance because it was conducted in patients who had relapsed after chemotherapy, and they were then treated with rituximab alone. However, the additional rituximab therapy was associated with a higher response rate 52% versus 35%,

697 complete response rate (27% versus 4%), was a striking difference in the time to disease progression, medians of 31 months versus 8 months (p = .007), but no difference in time to become “rituximab refractory” which was about 30 months in both arms, nor in overall survival at a median follow up of 42 months [70]. In large B cell lymphoma the one randomized trial that addressed this issue found there was no advantage in terms of PFS or OS derived by giving four weekly doses of rituximab every 6 months for 2 years after responding to RCHOP induction chemotherapy [68]. In patients with relapsed mantle cell lymphoma who responded to RFCM as second line therapy, compared to observation there was also prolongation of PFS for patients who received two additional 4 week courses of rituximab 3 and 9 months after completing RFCM [56]. There is no specific data regarding this issue in small lymphocytic lymphoma, however, one would expect that any sort of additional rituximab would prolong PFS based on the higher response rates achieved with prolonged rituximab in that disorder, and the results in follicular lymphoma. Two radiolabeled murine Mabs have been approved for the treatment of B cell lymphoma. These are both anti-CD20 antibodies: Y-90 ibritumomab tiuxetan (Zevalin®) which was approved in February 2002, and I-131 tositumomab (Bexxar®) which was approved in June 2003 [32]. In both products the original mouse antibodies ibritumomab tositumomab were kept as murine proteins in order to decrease half-life in the circulation to reduce non-specific total body irradiation. Ibritumomab is the same antibody that was modified to create the chimeric antibody rituximab; therefore, it has exactly the same binding to CD20 as rituximab. The clinical trial results for these two products are summarized in Table 4. As expected, in phase I trials the dose limiting toxicities of these agents were cytopenias secondary to bone marrow suppression with nadirs 5 to 8 weeks after treatment, but both produced objective response rates of 65–70% with durabilities of 9 to 12 months [87, 176]. Both of these products have produced response rates of about 40% with durabilities of about 1-year in patients with transformed B-cell lymphomas that had been heavily treated with prior chemotherapy. Both products have produced response rates of about 80% with a median duration of response of 11 to 12 months in patients with follicular lymphoma who had failed prior chemotherapy, but were not refractory to Rituximab [27, 89, 178]. Both products produced higher response rates that an unconjugated Mab in randomized trials, as compared to tositumomab for the I-131 product, and to rituximab for the Y-90 product [30, 178]. Both products produced response rates in over 70% of patients

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Table 4. Radioimmunotherapy of B cell lymphoma: radiolabeled anti-CD20 antibodies Dose Limiting Toxicity Therapeutic Dose Phase I response rate Response rate in relapsed follicular lymphoma Response rate in rituximab refractory lymphoma Response rate in transformed lymphoma RIT versus unlabeled Moab

131-I tositumomab (Bexxar)

90-Y-ibritumomab tiuxetan (Zevalin)

Bone marrow suppression 75 cGy total body dose 71% 81%

Bone marrow suppression 0.4 mCi/kg (max 32 mCi) 67% 86%

70%

74%

39% 67% versus 28% (tositumomab)

56% 80% versus 56% (rituximab)

with indolent B-cell lymphomas that had become refractory to Rituximab, defined as either no response or progression with 6 months after a response [82, 177]. Long term follow up has confirmed the remissions in these various settings are usually quite durable [50, 76, 175, 180]. In a young cohort (median age 49 years) of 76 patients with previously untreated follicular lymphoma, treatment with I-131 tositumomab produced a response rate of 95% with a 75% complete response rate, and 60% molecular complete response rate [88]. At a median follow-up of greater than 5 years, the 5-year progression-free survival rate was 59%, and the median progression-free survival was projected to be 6.1 years. Seventy percent of the 57 complete responders were still in remission 4 to 8 years following therapy. Despite these tremendous results, these two radiolabeled products have struggled in the marketplace because of the reluctance of medical oncologists to refer their patients for this treatment. One reason for this is the limited numbers of sites able and willing to provide therapy, so that often patients have to travel long distances seeking such treatment. The treatment itself requires some complex coordinated planning. Many hospitals have kept these products off their formularies because of the high single cost of the radioisotope and the financial risks associated with fixed payments. Since these treatments have to be delivered by physicians with radiation credentials, medical oncologists do not share in the profit margin associated with such treatment in the way that they do with the administration of chemotherapy and other intravenous products. Because there are so many systemic agents available for lymphoma, physicians can easily rationalize that they should try all other therapies before considering referral for radioimmunotherapy. Despite the high response rates, to date there are no trials directly comparing radioimmunotherapy plus rituximab to chemotherapy plus rituximab, although it is likely efficacy would be similar and quality of life superior in the radioimmunotherapy arm. At this time it appears that the most acceptable application

of these products will be as consolidation treatment after induction therapy [107, 138, 183], or as part of marrow ablative therapy and hematopoietic stem cell rescue [121, 124, 170, 171].

Immunotoxins Denileukin diftitox, an immunotoxin consisting of IL-2 fused to diphtheria toxin, is approved for treatment of CD-25 expressing cutaneous T-cell lymphomas (CTCL). The IL-2 receptor is also expressed on many B cell malignancies; so denileukin diftitox has been tested in patients with relapsed indolent lymphoma. In 45 patients with progressive B cell lymphoma, all of whom had been treated with rituximab in the past, there was a 24% response rate with a median duration of 7 months [24]. A second phase II trial was aborted because of slow accrual after 35 of a planned 77 patients had been enrolled [99]. Objective responses were observed in 3/29 evaluable patients, 1/21 with follicular lymphoma and 2/8 with small lymphocytic lymphoma. In both of these trials response rates were similar regardless of whether the lymphoma tested positive or negative for the IL-2 receptor. The combination of denileukin diftitox and rituximab produced a response rate of 32% in 38 evaluable patients, 30 (80%) were rituximab-refractory [25]. The overall response rate (ORR) was 32% and median duration of response was 8 months. Of note 6/11 patients with rituximab-refractory follicular lymphoma had an objective response.

Vaccines As of early 2008 there were no vaccines approved for use in any B cell malignancy, although three products, MyVaxID, FavID, and BiovaxID were being evaluated in pivotal regulatory trials [51, 145, 184]. Initial investigation established that vaccination with an individual patient’s idiotype protein could induce an endogenous

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idiotype response [100]. In a trial of 41 patients with indolent B cell lymphoma, vaccination with an idiotypekeyhole limpet hemocyanin (KLH) product produced anti-idiotype responses in 49% of patients [83]. For the subset of 32 patients who were in their first remission, PFS was much longer in those patients who exhibited an immune response compared to those who did not (7.9 versus 1.3 years, p = .0001). The vast majority of patients in these trials had follicular lymphoma, in part because of the inherent difficulty in isolating the idiotype from other B cell malignancies [149]. In a trial of 33 consecutive patients with relapsed follicular lymphoma, who had relapsed after an initial complete response and were induced into a second remission with CHOP, an idiotype vaccine was able to be prepared for 83% [74]. Overall 80% of the 25 treated patients exhibited some sort of a cellular idiotype-specific response. All 20 responders had a longer disease free interval after their second remission than their first. A cellular idiotypespecific response was detected in 18/25 (72%) while a humoral idiotype-specific response was demonstrated in 13 patients (52%). In another trial of an idiotype-KLH product co-administered with GM-CSF, objective tumor responses were noted in 4/32 previously treated indolent lymphoma patients [144]. Encouraging results such as these have attracted commercial interest, and at present there are at least anti-idiotype vaccine products that have entered into phase III trials, all of which are being conducted in patients with follicular lymphoma. All three utilize the idiotype paraprotein of the malignant B cell clone as the immunogen to produce an endogenous anti-idiotype response as summarized in Table 5. It has been announced that the Genitope trial did not meet the necessary regulatory endpoints, but those patients who were able to mount an anti-idiotype response did have a

longer PFS. It is possible the dendritic cells pulsed with idiotype protein may be a more powerful vaccination approach [147, 193].

T Cell Lymphoproliferative Malignancies Even though T cell malignancies account for less than 10% of the malignant lymphoproliferative disorders, they have been the object of many trials of biological therapy. Most of the trials or biologics in T cell lymphoma have been in mycosis fungoides cutaneous T cell lymphoma (CTCL) with predominantly patients who were in the Sezary syndrome stage of disease which features a characteristic desquamative rash and circulating malignant T cells. The single-agent activities of different biologicals in the T cell lymphoproliferative diseases are summarized in Table 6.

Interferon As a single agent interferon alpha produced response rates ranging from 40% to 60% in patients with CTLC with most of the responses observed in patients with mycosis fugoides [10, 39, 131]. Many trials were conducted with IFN-α in combination with other therapeutic modalities. A response rate of 83% was reported for the combination of IFN-α and psolaren with ultraviolet light A (PUVA) and (IFN-α2a) in 63 symptomatic early-stage CTCL patients, with most of the responses being CRs [18]. In another trial of IFN-α and PUVA in 25 patients with early-stage CTCL, 91% of patients had an objective response with 76% achieving a CR [152]. In two trials

Table 5. Anti-idiotype vaccines in follicular lymphoma Vaccine type Production Method Disease Setting Induction therapy Eligibility Accrual

MyVaxID (Genitope)

FavID (Favrille)

BiovaxID (Biovest)

Id-KLH/GM-CSF Recombinant DNA Untreated CVP CR or PR Finished

Id-KLH/GM-CSF Recombinant DNA Untreated or Relapsed Rituximab CR or PR or SD Finished

Id-KLH/GM-CSF Hybridoma rescue Untreated PACE or RCHOP CR only In progress

Id = idiotype KLH = keyhole limpet hemocyanin GM-CSF = granulocyte-macrophage colony stimulating factor CVP = cyclophosphamide, vincristine, prednisone PACE = platinum, doxorubicin, cyclophosphamide, and etoposide RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone CR = complete response PR = partial response

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Biological therapy of B and T cell lymphoproliferative disorders Table 6. Biotherapy of T cell lymphoma with single agent biologicals Modality Class

Biological agent

Reference

Disease

Patients

Response rate

Cytokine Cytokine Cytokine Cytokine Cytokine Immunotoxin Immunotoxin Immunotoxin Mab Mab Mab Mab Mab Mab Mab Mab Retinoid Retinoid Retinoid Retinoid Retinoid

Interferon-α Interferon-α Interferon-α Interleukin-2 Interleukin-2 Denileukin diftitox Denileukin diftitox Denileukin diftitox antiCD4 antiCD5 Y90 – antiCD5 Alemtuzumab Alemtuzumab Alemtuzumab Alemtuzumab Alemtuzumab Isotretinoin Isotretinoin Isotretinoin Etretinate Bexarotene

[10] [131] [39] [65] [140] [130] [104, 153] [25] [97] [35] [60] [108] [45] [5] [110] [93] [94] [120] [16] [120] [42]

CTCL CTCL CTCL CTCL CTCL CTCL CTCL PTCL CTCL CTCL CTCL PTCL PTCL CTCL CTCL CTCL CTCL CTCL PTCL CTCL CTCL

20 22 45 7 22 71 35 27 8 10 8 8 14 14 22 8 25 39 12 29 94

45% 59% 38% 71% 18% 30% 37% 48% 62% 40% 38% 50% 36% 86% 55% 38% 44% 59% 50% 67% 49%

CTCL = cutaneous T cell lymphoma PTCL = peripheral T cell lymphoma

s.c. IFN-α was combined with extracorporeal photochemotherapy (photopheresis) using oral 8-methoxypsoralen as the photosensitizer. A response rate of 90% was observed in 39 patients, most of whom had early-stage CTCL, with 24 achieving a CR [98]. In the second trial, which was limited to 14 patients with stage II CTCL, a response rate of only 50% was observed [181]. IFN-α has combined with a variety of retinoids to treat patients with T cell lymphomas. In an early report all seven patients treated with IFN-α and etretinate had a response [161]. In another small study there was an 83% CR rate in 12 patients with CTCL who received IFN-α and acitretin [2]. In a more complex trial 45 patients, 13 of whom had stage I Sezary syndrome and the other 32 mycosis fungoides, were treated with 3 months of daily s.c. IFN-α alone, and responders then continued to received thrice weekly s.c. IFN-α, while non-responders were treated with thrice weekly s.c. IFN-α plus etretinate [39]. After 1 year 28 patients (62%) were in remission. IFN-α alone produced a response in 17 patients (38%). Among the 28 patients who had not responded after 3 months of IFN-α, 11 (39%) eventually achieved a response after changing to IFN-α plus etretinate. The combination of s.c. IFN-α and the oral synthetic retinoid bexarotene produced a response rate of 39% among 18 CTCL patients, which appeared no better than either agent used alone [160]. A response rate of 31% was noted for 17 patients with

anthracycline-resistant peripheral T-cell lymphoma (PTCL) who were treated with thrice weekly s.c. IFNα2a and daily oral 13-cis retinoic acid (isotretinoin) [84]. A response rate of 100% was reported for 28 patients with Sezary syndrome who were treated with extracorporeal photochemotherapy and two or more of various biologicals including IFN-α, retinoids, and GM-CSF [146]. One small randomized trial compared thrice weekly 9 MU IFNα-2a plus PUVA to 9 MU IFNα-2a plus acitretin in 98 patients with stage I and II CTCL, only 82 of whom were considered evaluable for response [159]. IFNα-2a plus PUVA was not only less toxic, but was associated with more rapid response, and a complete response rate of 70% compared to only 38% for IFNα-2a plus acitretin. IFNα has also been combined with chemotherapy in the treatment of patients with T cell lymphoma. The purine analogs fludarabine and pentostatin have substantial single-agent activity in CTCL. A response rate of 41% was observed in a trial of 41 CTCL patients, only six of whom had not received prior therapy, and 29 of whom had not responded to prior therapy, who were treated with pentostatin 4 mg/m2 i.v. days 1 through 3 and IFNα 10 MU/m2 i.m. on day 22, and 50 MU/m2 i.m. 23 through 26 [58]. A response rate of 51% was observed in 35 patients, 10 of whom had received pentostatin previously and 21 of whom had not responded to prior

Robert O. Dillman therapy, who were treated with fludarabine 25 mg/m2 i.v. days 1 to 5 every 28 days with IFNα 5 MU/m2 s.c. thrice weekly for up to eight cycles [59].

Interleukin-2 There has been limited experience with IL-2 in the T cell malignancies. In one trial that utilized continuous i.v. infusion of relatively high doses of IL-2, responses were noted in 5/7 patients [65]. In a recent trial of s.c. IL-2 a response rate of only 18% was reported for 22 heavily pretreated CTCL patients [140].

Retinoids The vitamin analogs collectively known as the retinoids, have been shown to modulate proliferation and differentiation in premalignant and malignant cells, and also have immunologic effects. The retinoid response is mediated by nuclear receptors, which have been characterized as retinoic acid receptors (RARs) and retinoid “X” receptors (RXRs). Because of the dramatic effects of retinoids in skin disorders including acne and aging of the skin, there has been intense investigation of retinoids in the treatment of CTCL, especially in early stage mycosis fungoides and Sezary syndrome, and also in PTCL. As summarized in Table 6, as single-agents retinoids have produced response rates ranging from 50% to 70% in CTCL, depending on the product and the specific CTCL patient population [42, 43, 94, 120], and 50% in PTCL [16]. Substantial anti-tumor activity was demonstrated with cis-retinoic acid (isotretinoin) and related retinoids, but none of these agents were ever submitted for regulatory approval for marketing these agents in T cell malignancies. The retinoid X receptor-selective agent LGD1069 was given orally by mouth to 52 patients in an dose escalation trial in which some tumor response were observed at higher doses [119]. This product became bexarotene (Targretin capsules; Ligand Pharmaceuticals Incorporated, San Diego, Calif), which received regulatory approval based on the activity of this agent in early-stage cutaneous T-cell lymphoma at doses of 300 mg/m2 per day [42, 43]. Hypertriglyceridemia and hypothyroidism were the major adverse events noted. High response rates have also been observed with bexarotene gel [9, 139]. Interestingly, a single institution retrospective comparison of all-trans retinoic acid (RAR-specific) and bexarotene (RXR-specific) in patients with relapsed mycosis fungoides/Sézary syndrome detected no substantial differences between the products in terms of duration of disease control, although the response rate was higher for bexarotene (21% versus 12%) [140]. The combination of bexarotene and immunotoxin denileu-

701 kin diftitox yielded a response rate of 67% among 14 patients with relapsed or refractory CTCL [57].

Immunotoxins Denileukin diftitox (Ontak™) is a genetically engineered fusion protein that combines the active chain of diphtheria toxin to the cytokine IL-2 which binds to the CD25 receptor full-length sequence for interleukin-2 (IL-2). This immunotoxin has therapeutic potential for any malignancy, including B cell lymphomas and Hodgkin’s disease, in which there is over-expression of CD25, or more specifically, the subunit of the IL-2 receptor. Denileukin diftitox was approved by the US FDA based on its activity in cutaneous T cell lymphoma where response rates 30% to 40% were achieved in trials [104, 130, 153]. Response rates in peripheral T cell lymphomas may be even higher [26] This product may be useful in any CD25 overexpressing malignancy, which includes some cases of B-cell lymphoma and Hodgkin’s disease in addition to most T cell malignancies.

Monoclonal Antibodies The humanized anti-CD52 Mab Alemtuzumab (Campath®) was approved in May 2001 based on data submitted for the treatment of patients with CLL that had recurred or been refractory to the purine analog fludarabine. As shown in Table 6, alemtuzumab has exhibited significant activity as a single agent in both CTCL with where response rates ranged from 38% to 86% [5, 45, 110], and in PTCL where response rates ranged from 36% to 50% in two small studies [93, 108]. Alemtuzumab has also been combined with chemotherapy in the treatment of PTCL with very encouraging results. In an Italian trial the combination of CHOP chemotherapy and s.c. alemtuzumab yielded a response rate of 75% in 24 patients with 17 of the 18 responses recorded as CRs [62]. In a Korean trial in 20 PTCL patients the combination of CHOP chemotherapy and i.v. alemtuzumab was associated with a response rate of 80% with 13 of the 16 responses being CRs [96].

Vaccines A vaccine consisting of dendritic cells loaded with the lysate from CTCL cells and KLH was given as weekly intranodal injections in ten patients with CTCL [111]. Tumor-specific delayed-type hypersensitivity (DTH) reactions to tumor lysate were observed in 3/8 patients so tested. There was a 50% objective response rate including one CR and four PRs with all of the response occurring in patients who had a low tumor burden.

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Summary Although T cell malignancies are relatively rare, they have been the target of many biological therapies. Interferonalpha, the retinoid bexarotene, the immunotoxin denileukin diftitox, and the Mab alemtuzumab are all routinely used in the treatment of these malignancies both as single agents, and increasingly in combination therapies.

Chronic Lymphocytic Leukemia Interferon Early U.S. trials using leukocyte-derived interferon and recombinant interferon in small numbers of patients with chronic lymphocytic leukemia (CLL) were associated with non-durable response rates of 11% to 15% [52]. However a Greek trial reported objective responses in 10/26 (38%) [7], and a British trial reported responses in 8/18 (44%), although all were transient [118]. Other trials focused on IFN-α as maintenance therapy following chemotherapy. A small randomized trial of Italian 45 patients showed a significantly longer duration of response and fewer infections in patients who received IFN-α maintenance therapy [46]. However investigators who conducted a single-arm U.S. trial of maintenance therapy in 31 patients who had responded to fludarabine, concluded that adding IFN-α did not enhance the degree of chemotherapy-induced remission, and that the time to progression was similar to a historical control group [127]. In a German trial in which 44 high-risk patients were randomized to observation or IFN-α, there was no difference in PFS or OS [102]. In the largest trial of IFN-α in CLL, 133 previously untreated patients were randomized to receive fludarabine and prednisone with or without interferon, and then 78 responders were randomized to observation versus maintenance interferon [115]. The initial response rates were similar (84% and 86%) suggesting that IFN-α added nothing over chemotherapy alone. There was a longer response duration in patients in patients who received maintenance therapy, however, this difference could be explained by an imbalance in the distribution of patients who were in CR at that the time they entered the maintenance phase of the study. Although these were all relatively small studies by the standards of modern clinical trial design, the failure of IFN-α to consistently achieve durable disease control, and the toxicity associated with IFN-α, dampened enthusiasm for larger studies. Furthermore, the high durable response rates achieved with combinations of rituximab with fludarabine, or

rituximab with cyclophosphamide and a purine analog, quickly attracted the interest of investigators, and enthusiasm for pursuing IFN-α in CLL waned.

Monoclonal Antibodies A summary of clinical trials of Mab in CLL are shown in Table 7. Some of the earliest trials with Mabs were conducted in patients with CLL. The anti-CD5 murine Mab T101 bound to CLL cells and decreased circulating lymphocyte numbers, but at the doses given, and with the murine construct, was not able to induce sustained responses [33–35, 53]. Trials were never carried out at higher doses or with humanized constructs. In May 2001 the anti-CD52 monoclonal antibody alemtuzumab (Campath) became the second Mab approved by the U.S. FDA for a hematologic malignancy when it was granted regulatory approval based on its activity in patients with CLL whose disease had recurred or been refractory to the purine analog fludarabine [91]. Trials conducted in the fludarabine resistant or refractory setting were associated with response rates ranging from 33% to 55% with a median of 40% [23, 91, 123, 132, 142, 143]. In 115 patients who had progressive disease after multiple course of prior chemotherapy, the response was still 23% [48] In the untreated setting alemtuzumab produced responses of 83% to 87% when given by the i.v. or s.c. route [109, 187]. In a European registration trial 297 previously untreated CLL patients were randomized to oral chlorambucil 40 mg/m2 monthly versus standard i.v. alemtuzumab [80]. The Mab produced superior response rate, 83% versus 55% (p < .0001) and Table 7. Single-agent activity of monoclonal antibodies in CLL Antibody

Reference

Setting

Patients Response rate

Anti-CD5 Anti-CD5 Alemtuzumab Alemtuzumab Alemtuzumab Alemtuzumab Alemtuzumab Alemtuzumab Alemtuzumab Alemtuzumab Alemtuzumab Rituximab Rituximab Rituximab Rituximab Rituximab Rituximab Rituximab

[35] [53] [109] [80] [123] [23] [142] [132] [91] [143] [48] [162] [78] [79] [12] [128] [188] [73]

Relapsed Relapsed Untreated Untreated Relapsed Relapsed Relapsed Relapsed Relapsed Relapsed Relapsed Untreated Untreated Untreated Relapsed Relapsed Relapsed Relapsed

10 13 38 149 91 16 136 29 93 24 115 19 26 43 33 40 23 28

0% 0% 87% 83% 55% 50% 40% 38% 33% 33% 23% 90% 70% 58% 45% 36% 35% 25%

Robert O. Dillman PFS which led to a marketing indication as initial therapy, even though in the U.S. purine-analog based therapy is considered the treatment of choice. Fludarabine and alemtuzumab combination therapy as the initial treatment of patients with CLL produced responses in 83% of 36 patients, with 11 of the 30 responses being CR [44]. Combining GM-CSF with alemtuzumab in 14 patients with relapsed disease did not appear to increase the response rate (36%) or decrease the risk of infection [189]. Because of the additive toxicity and immuosuppression associated with combining alemtuzumab with chemotherapy, most of the recent emphasis has been on using alemtuzumab after chemotherapy in an effort to eradicate minimal residual disease in responding CLL patients. Thrice weekly doses of 10 to 30 mg have been administered i.v. or s.c. for 4 to 12 weeks as a consolidative therapy [122, 129, 173]. These studies have shown that consolidation with alemtuzumab does increase clinical, phenotypic, and molecular complete response rates, but there is an increased risk of opportunistic infections, even if anti-viral, anti-bacterial, and anti-Pneumocystis prophylaxis is used. Although rituximab does not have regulatory approval for marketing as a treatment for CLL, it is widely used in combination with chemotherapy. As shown in Table 7, in patients with previously treated, relapsed CLL, as a single agent rituximab was associated with response rates ranging from 25% to 45% with a median of 35%. over a wide range of doses and by various schedules of administration [12, 73, 128, 188]. Response rates were much higher in patients who had not been previously treated with a range from 58% to 90% with the highest rate occurring in patients with early stage disease who were considered to be high risk. [78, 79, 162]. The popularity of rituximab in CLL is due to the ability to combine it with chemotherapy with no apparent increase in toxicity. Rituximab combined with purine analogs has produced response rates of about 90% in untreated patients treated with rituximab and fludarabine [12, 154], and 33% in previously treated patients who received pentostatin plus rituximab [38]. The combinations of purine analogs plus cyclophosphamide plus rituximab have been associated with very high response rates and durable complete remissions in previously untreated patients. In single institution trials the combination of fludarabine, cyclophosphamide, and rituximab (FCR) has produced response rates of 77% in the relapsed setting [174], and 95% as initial therapy [92]. The combination of pentostatin, cyclophosphamide, and rituximab (PCR) has produced response rates of 75% to 90% in previously untreated patients [36, 90, 101], but only 33% in previously treated elderly patients in the

703 community setting [36]. In a trial of young patients with relapsed CLL, cladribine, cyclophosphamide, and rituximab (CCR) produced a response rates of 58% [148]. In one randomized phase II trial, concurrent fludarabine plus rituximab was compared to the sequence of fludarabine followed by rituximab in 104 patients [13]. The response rate was higher for concurrent therapy (90% versus 77%) as was the complete response rate (47% vs 28%). A retrospective comparison of these patients to a similar population of patients treated with fludarabine alone at the same dose in an earlier randomized trial suggested that patients treated with the combination of fludarabine plus rituximab had much better outcomes, including a higher response rate (84% versus 63%), higher complete response rate (38% versus 20%) better progression free survival after 2 years (67% versus 45%), and better overall survival at 2 years (93% versus 81%) [14].

Summary Biotherapy now plays a crucial and ever increasing role in the management of both B-cell and T cell lymphoproliferative malignancies. Because of its ease of delivery, anti-tumor activity and relatively low toxicity rate, rituximab has become the most important single agent in the treatment of indolent B cell lymphoma., and combined with chemotherapy, has become the standard of treatment for more aggressive B cell lymphomas. Rituximab alone or in combination with chemotherapy has become the initial treatment of choice for indolent lymphomas, and CHOP + Rituximab has become the treatment of choice for large B cell lymphoma. Despite evidence-based data supporting the use of interferon-α in lymphoma, because of its toxicity and need for chronic treatment, this biological never gained widespread acceptance in the treatment of lymphoma in the U.S. IL-2 was never an important agent in these disorders because of the need for hospitalization and toxicity associated with higher doses, and the lack of clinical activity at lower doses. Two biological agents, the immunotoxin denileukin diftitox and the retinoid bexarotene were both received regulatory approval based on studies conducted in cutaneous T cell lymphoma. The use of denileukin difitox is limited by its immunogenicity. The antiCD52 Mab alemtuzumab is being used increasingly in combination with chemotherapy for the T cell malignancies, but its immune inhibition and toxicity profile make it a less attractive option in B cell lymphoma because of the availability of rituximab. Vaccine approaches are interesting and promising, but at this time there is no commercial product.

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21.8 Biological therapy of multiple myeloma ROBERT K. OLDHAM

Interferon Several studies have demonstrated response rates of about 20% in patients with refractory multiple myeloma treated with recombinant alpha-interferon [1, 3, 7]. An attempt to augment this response experience by using high dose induction schedules, often with the addition of Prednisone, did not result in enhanced response [1]. Anecdotal observations have suggested possible synergism between the interferons and cytotoxic drugs [2, 5]. Because of response rates of 50% in untreated patients [8], studies were done to evaluate the combination of interferon with standard chemotherapeutic regimens as initial therapy for multiple myeloma. Preliminary interpretation suggested that the duration of initial response may be extended with the addition of alpha-interferon [6]. Follow-up studies were not confirmatory. The availability of very active new agents such as Velcade has precluded further studies with Interferon combinations. In addition, thalidomide and reilamide, with their immunomodulating capacities, are active in multiple myeloma [9]. Given the high degree of activity both with chemotherapy and immunomodulators, as well as the use of autologous bone marrow transplants, Interferon is now little studied in multiple myeloma [4, 9]. With the advent of Velcade and revlamide as targeted therapy for multiple myeloma [4, 9], there has been little subsequent activity with other forms of biotherapy in this disorder. However, given the activity of revlamide as an immunomodular, we may soon see other drugs working through the immune system to treat this disseminated disease.

Antibodies Plasma cells produce myeloma protein (antibody), giving ready availability of a secreted protein target for antibody induction. However, this very characteristic makes it unlikely that antibody therapy will be useful in myeloma, at least not with the myeloma protein as target, since the therapeutic antibody would be totally absorbed in the vascular and extracellular pool. Anti-CEA studies in colon cancer suggest this is not a rational approach to follow, but targeting myeloma cell membrane proteins

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(nonsecreted) could be attempted. No major clinical studies of antibody against other plasma cell membrane targets have been reported. CD20 is rarely expressed on plasma cells, but has been reported to be present on malignant cells from up to 20% of patients with multiple myeloma [11]. Rituximab has rarely produced objective responses in multiple myeloma even in patients whose plasma cells were felt to overexpress CD20. Using the standard dosing of 375 mg/m2, Treon et al saw objective responses in only 1/19 patients [11], and Hussein et al reported responses only 2/21 patients prior to starting chemotherapy five weeks later [10]. There has been a much greater unpublished experience in the community practice of medicine with anecdotal reports of occasional excellent responses in refractory myeloma patients whose plasma cell overexpressed CD20. Another strategy is to use rituximab as an adjuvant treatment after an intial response to chemotherapy, in an effort to suppress the B cell clone that leads to the malignant plasma cells. Based on this same rationale, rituximab may be more active against smoldering myeloma than myeloma with a high proliferative index.

References 1. Case DC, Jr., Sonneborn HL, Paul SD et al. Phase II study of rDNA alpha-2 interferon (INTRON A) in patients with multiple myeloma utilizing an escalating induction phase. Cancer Treat Rep 1986; 70:1251–1254. 2. Clark RH, Dimitrov NV, Axelson JA, and Charmella LJ. Leukocyte interferon as a biological response modifier in lymphoproliferative diseases resistant to standard therapy. Blood 1983; 62:188a (abstract). 3. Cooper MR. Interferons in the treatment of multiple myeloma. Semin Oncol 1986; 13:13–20. 4. Dimopoulos M, Spencer A, Attal M, et al. Lenalidomide plus dexamethasone for relapsed or refractory multiple myeloma. N Engl Med 2007;357:2123–2132. 5. Ferraresi R. Enhanced response to chemotherapy after treatment with DNA alpha-2 interferon. Am Soc Hematol 1983; 62:212a. 6. Mandell F, Tribalto M. Recombinant alpha-2b interferon as maintenance therapy in responding multiple myeloma patients. Blood 1987; 70:247a, 318. 7. Ohno R, Kimura K. Treatment of multiple myeloma with recombinant interferon alfa-2a. Cancer 1986; 57:1685–1688.

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712 8. Quesada JR, Alexanian R, Hawkins M et al. Treatment of multiple myeloma with recombinant alpha-interferon. Blood 1986; 67:275–278. 9. Weber DM, Chen C, Niesbizky R, et al. Lenalidomide plus dexamethasone for relapsed multiple myeloma in North America. N Engl J Med 2007;357:2133–2142.

Biological therapy of multiple myeloma 10. Hussein MA, Karam MA, McLain DA, et al. Biologic and clinical evaluation of rituxan in the management of newly diagnosed multiple myeloma patients. Blood 1999;94:313a [abstract 1400]. 11. Treon SP, Pilarski LM, Belch AR, et al. CD20-directed serotherapy in patients with multiple myeloma: biologic considerations and therapeutic applications. J Immunother. 2002;25:72-81.

21.9 Biological therapy of squamous cell cancers of the head and neck ROBERT O. DILLMAN

Introduction This category includes squamous cell cancers that originate in the mouth, oropharynx, nasopharynx, and larynx. Many of the cancers are associated with smoking and other forms of tobacco abuse that are local irritants and mutants, and some are caused by human papilloma virus, and other viruses. Primary therapy typically includes surgery and/or radiation therapy, and increasingly chemotherapy as part of organ sparing approaches that minimize the morbidity associated with extensive surgery in this area.

Non-specific Immune Stimulants Bacillus Calmette Guerin (BCG) There were no trials that tested single-agent activity of BCG, but several trials addressed the issue of whether BCG augmented methotrexate or other chemotherapy regimens in the treatment of advanced squamous cell cancers of the head and neck. In a large, non-randomized phase II trial, 100 patients with advanced, recurrent, or metastatic squamous cell carcinoma of the head and neck were treated with the combination of chemotherapy and BCG [49]. The objective response rate was 35%, and the median duration of response was 17 weeks, both of which were considered similar to historical results obtained with chemotherapy alone. In a small randomized phase II trial, 38 patients with advanced, inoperable squamous cell carcinoma of the head and neck were randomized to receive methotrexate alone or with BCG [35]. The response rates, 3/19 in the methotrexate alone arm and 4/19 for methotrexate plus BCG, were similar as was the duration of response and survival. In another small randomized phase II trial, 23 patients with advanced recurrent head and neck carcinoma were randomized to receive either high dose methotrexate with calcium leucovorin rescue (HDMTX) (n = 12) or HDMTX in combination with BCG (n = 11) [6]. There were three objective tumor responses of similar brief duration in both groups. Although this trial was too small to be conclusive, it cast doubt on whether BCG added anything to HDMTX chemotherapy for such

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

patients. In another randomized prospective study of patients with advanced squamous cell carcinoma of the head and neck, treatment with methotrexate plus BCG was no better than methotrexate alone in terms of response rate or survival [62]. There were two small trials with relatively positive results. One was another small randomized phase II trial, in which 34 patients with recurrent squamous cell cancer of the head and neck were randomized to receive the fivedrug chemotherapy regimen BACON (bleomycin, doxorbucin, CCNU, vincristine and nitrogen mustard) alone (n = 14) or with BCG by scarification (n = 20) [41]. The patients treated with BACON plus BCG experienced a longer survival (P = 0.014) than those treated with BACON alone. The other positive trial was in the neoadjuvant setting. Following neoadjuvant methotrexate and definitive local therapy, 52 patients with locally advanced squamous cell cancer were randomized to adjuvant methotrexate alone (n = 27) or with an adjuvant treatment with a BCG vaccine (n = 25) consisting of 2 to 4 million Tice strain BCG organisms given i.d. in alternating sides of the neck every 2 weeks for six doses, then every 4 weeks for nine doses [50]. At a median follow-up of about 3.5 years, 52% of the BCG vaccine-treated group remained disease free compared to only 26% of the controls. Similarly, 68% of the BCG vaccine-treated group were alive at 3.5 years compared to only 41% of the controls. Based on this trial BCG showed promise as adjuvant immunotherapy in ear, nose, and throat cancers.

Levamisole Several small randomized trials were conducted in head and neck cancer with the antihelminth levamisole. A randomized double-blind study compared levamisole (n = 31) with placebo (n = 34) as adjuvant treatment following surgery of patients with squamous cancer of the head and neck [61]. There was no difference in progression free survival (PFS) or overall survival (OS). The largest randomized trial ever conducted with levamisole in head and neck cancer patients, actually suggested a detrimental effect. After treatment of a primary

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squamous cell carcinoma of the head and neck, 134 patients were randomized to receive placebo (n = 65) or levamisole, 150 mg/day orally for 3 consecutive days every other week [33]. Patients with stage I and II disease who were treated with levamisole had a significantly higher incidence of recurrence than the placebo-treated patients (p = 0.02). Some other small studies were more encouraging. In a small randomized study of 24 patients with squamous cell cancer of the larynx or hypopharynx, 12 patients received placebo and 12 received levamisole 50 mg t.i.d. for 3 consecutive days every 2 weeks following surgery and/or radiation [30]. After 20 months of follow up, the recurrence rate was only 20% in the levamisole group compared to 50% in the control group. In another trial, following conventional radiotherapy, 82 patients with T1 or T2 N0 M0 squamous cell carcinoma of the oral cavity were randomized to receive either placebo or levamisole at a dose of 150 mg daily for 3 consecutive days every 2 weeks [34]. After a median of 3 years of follow up, 44% of patients who received levamisole were still disease-free compared to only 32% in the placebo group. There also was a faster recovery from radiation-induced leukopenia and lymphopenia in the levamisole group. In another small randomized study, 34 patients without distant metastases were randomized to observation and 31 to receive futraful and uracil (UFT) plus levamisole as adjuvant treatment for stage III and IV squamous cell carcinomas of oral cavity, oropharynx, hypopharynx and larynx following primary therapy [24]. The 5-year disease free survival rate favored the UFT-levamisole arm (57% versus 39%), but was not statistically significant (p = 0.21). There was also a trend for a lower rate of distant metastasis (10% versus 32%, p = 0.06). The contribution of levamisole beyond UFT is unclear.

Cornybacterium Parvum (C parvum) Three randomized trials failed to demonstrate any benefit for C parvum in the treatment of epidermoid cancers of the head and neck. In a small randomized trial, patients with advanced squamous cancer of the head and neck were randomized to treatment with weekly methotrexate alone or methotrexate with C parvum given s.c. weekly as bilateral doses in sites around the neck [63]. The addition of C. parvum did not improve the response rate, response duration or survival. In a randomized trial involving 57 patients with previously untreated squamous cell carcinoma of the head and neck, 29 received radiation alone and 28 patients received C. parvum administered i.v. and locally into

the tumor-bearing lymph nodes of the neck, or into the cervical node region in patients who lacked palpable lymph nodes, before and after radiation therapy [10]. After 2.5 years of follow up, the study was terminated when it became evident that there was no difference disease free survival. In a large trial 209 operable patients with squamous cell head and neck cancer were randomized to surgery alone or pre-operative intratumoral immunotherapy with C parvum followed by post-operative s.c. C parvum for 2 years [31]. Only 176 were considered fully evaluable, but there was no difference disease free or overall survival.

OK-432 The streptococcal preparation OK-432 has been extensively studied in Japan in various malignancies including epidermoid cancers of the head and neck. During 1984 to 1989 120 newly diagnosed patients of laryngeal squamous cell carcinoma were registered at ten different institutions, stratified by stages of disease, treated with radiation therapy and 5FU chemotherapy with or without surgery, with or without chemotherapy, and randomized to no further therapy or immunotherapy with OK-432 [23]. There was no apparent advantage for those patients who received OK-432. The 5-year disease free survival rate for 109 evaluable patients was 76% in the OK-432 arm and 75% in the control arm. The 5-year overall survival rate was 84% in the OK-432 arm and 78% in the control arm.

Thymic Hormones It has long been recognized that the thymus gland is important in the development of cell-mediated immunity and that many thymic hormones have stimulating affects on T lymphocytes. Clinical experiments have been performed with crude thymus extracts, isolated purified products, and synthesized peptides. Several thymic preparations have been of clinical interest, including thymosin fractions 5, thymosin-α1, prothymosin, thymulin, thymopoietin, thymostimulin (TP-1), and thymic humoral factor [46]. In vitro thymostimulin restores a number of immunologic defects noted in the monocytes and dendritic cells of patients with head and neck cancers. In a small study, 18 patients with squamous cell cancer of the head and neck were treated with thymostimulin at one of three dosages (0.5, 1.0, or 2.0 mg/ kg) prior to surgery, and then their exicised tumors were examined and results compared to those from 16 contemporary controls who had not received treatment with the thymic hormone [21]. Preoperative treatment with

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thymostimulin enhanced T-cell infiltration. Anti-tumor effects were not determined. There have been no publications of follow up studies.

Interferons

a 30% response rate in 18 evaluable patients who had an 8.5 month median survival [3]. IFN-α2b, cisplatin and 5FU were combined to treat 14 patients with previously treated squamous cell head and neck cancers [2]. Hematologic toxicity caused treatment delays in 9/14 patients, which led to early closure of the trial, but 8/14 (54%) of the patients had an objective response rate.

Interferon-α Interferon-alpha (IFN-α) was studied in several trials in squamous cell cancer of the head and neck. In a phase II trial, 71 patients with recurrent or metastatic squamous cell carcinoma of the head and neck were randomized to receive recombinant IFN-α2b at a relatively low dose (6 MU/m2 daily × three every 4 weeks) or a relatively high dose (12 MU thrice weekly) [59]. As shown in Table 1, the response rate was 5%, with a response rate of 1/32 for evaluable patients who received the lower dose, and 2/29 for those who received the higher dose. Median survival was about 6 months in both arms. Other trials combined IFN-α with radiation therapy and/or chemotherapy. In a small study, 22 patients with operable head and neck cancer were randomized to receive radiotherapy alone prior to surgery, or radiotherapy with natural leukocyte IFN-α at a dose of 6 MU i.m. daily for 4 weeks prior to surgery, and then thrice weekly for 2 months [54]. Objective responses were observed in 4/10 patients who received IFN-α and radiotherapy, compared to 2/12 among those who only underwent radiation therapy, but there was no difference in survival. The study was discontinued because of worse toxicity in the IFN-α cohort. IFN-α was combined with 5-FU and cisplatin to treat patients with recurrent or metastatic squamous cell carcinoma of the head and neck who had not received prior chemotherapy [20]. The response rate was 25% among 50 eligible patients and the median survival was 5 months. The authors concluded that IFN-α added to toxicity without conveying a benefit to the chemotherapy. In 20 patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck, the same combination of chemotherapy and IFN-α produced

Table 1. Single-agent activity of various biologicals in patients with head and neck cancer Modality class

Biological agent

Lead author

Patients

Response rate (%)

Non-specific Cytokine Cytokine

Isotretinoin Interferon-α Interleukin-2 (IL-2) Cetuximab

[25] [59] [12]

19 61 31

16 5 13

[57]

109

13

Mab

Interferon-gamma Interferon-gamma (IFN-γ) was given at a dose of 0.25 mg/m2 as a 24-h infusion i.v. weekly for 4 weeks to eight patients with locally advanced, but resectable head and neck cancer [42]. There were minimal side effects, and three patients had clinically measurable responses prior to surgery.

Interleukins Interleukin-2 Local injections of IL-2 might enhance an immune response in lymph nodes that drain head and neck cancers. In one study, 20 patients with recurrent, inoperable head and neck squamous cell carcinoma received perilymphatic injections of natural IL-2 for 10 days [11]. Irrespective of the site of the recurrence, injections were always given 1.5 cm below the insertion of the sternocleidomastoid muscle on the mastoid. Injections were performed on the ipsilateral side if the lymphatic chain was still present, or on the contralateral side if the patient had previously undergone an extensive neck dissection. Thirteen patients had brief response to such treatment, and there was no benefit from repeated treatment. The same study investigators treated 31 patients with recurrent head and neck squamous cell carcinoma with ten daily doses of 500 or 500,000 U of IL-2 as injections 1.5 cm from the insertion of the sternocleidomastoid muscle on the mastoid [12]. There were no toxic effects. Objective, but non-durable tumor responses were noted in 4/16 patients who received the 500 U dose, but the response rate was 0/15 at the higher dose. In a U.S. dose escalation trial of local therapy, IL-2 was injected perilesionally in divided doses in each of four quadrants and bilaterally into the superior jugular lymph nodes in 36 patients with unresectable squamous cell carcinoma of the head and neck [58]. The maximum tolerated dose was determined to be 2 MU/day. There were two objective responses for a response rate of 6%. In a European multicenter trial, 202 patients with advanced, but potentially resectable cancers of the oral

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Biological therapy of squamous cell cancers of the head and neck

cavity or oropharynx, were randomized to undergo surgery and radiation therapy alone or in combination with perilymphatic recombinant IL-2 [14]. A dose of 5,000 U IL-2 was injected around the ipsilateral cervical lymph node chain daily for 10 days before surgery, and after surgery the same IL-2 dose was injected into the contralateral cervical lymph node chain 5 consecutive days monthly for 1 year. Treatment was well-tolerated and did not interfere with the treatment plan. Those patients who received IL-2 had a longer disease free survival (p < 0.01) and overall survival (p < 0.03). A preparation of polyethylene glycol-modified IL-2 (PEG-IL-2) provides a slower release of IL-2 from sites of injection, and therefore a more continuous exposure to the IL-2. PEG-IL-2 was given to 19 patients with recurrent head and neck squamous cell carcinoma as intratumoral injections of 200,000 U of (PEG-IL-2) two or three times a week for 4 weeks [29]. Temporary regional swelling and redness developed in ten patients, and nine patients had systemic eosinophilia. One patient had an objective response. Several trials combined IL-2 and IFN-α. In a phase II study 11 patients with recurrent head and neck cancer were treated with continuous i.v. recombinant human IL-2 and i.m. or s.c. IFN-α2a [53]. Two patients (18%) achieved a partial response, but toxicity was substantial. In another phase II trial, a response rate of 36% was reported for 14 patients with advanced head and neck cancer, even though toxicity was such that only three patients were able to complete three cycles of therapy [45]. The degree of natural killer cell activation correlated with response. Other trials combined IL-2 with chemotherapy for the treatment of squamous cell head and neck cancer. In a non-randomized phase II study, 23 patients with advanced (stage III or IV) head and neck squamous cell carcinoma received chemotherapy with cisplatin, 5FU, and vinorelbine, or the same combination with IL-2 at a dose of 9 MIU s.c. daily from day 9 to 13 and from day 16 to 20 of every cycle [27]. The response rate was 63% for the chemotherapy alone, and 100% with the addition of IL-2. In a randomized, phase III, multicenter clinical trial, only 33 patients with advanced head and neck squamous-cell carcinoma were randomized to 5FU and cisplatin alone, or combined with s.c. IL-2 at a dose of 4.5 MIU/day on days 8–12 and 15–19 of each treatment cycle [28]. Response rates were similar between the arms, 12/17 in the chemotherapy alone arm, and 10/16 in the IL-2 containing arm. Multikine is a combination of natural interleukins derived from leukocytes. In a pilot study, 12 previously untreated patients with various head and neck cancers

were treated by peritumoral injection of this cytokine combination, in addition to oral zinc sulfate, oral indomethacin, i.v. cyclophosphamide. [15]. Four patients reportedly had an objective response. In a four-center phase I/II dose-escalation clinical trial, 54 patients with locally advanced, node-negative primary oral squamous cell carcinoma were treated with a leukocyte multikine interleukin product prior to surgical resection [51]. Paraffin-embedded tumor samples obtained at surgical resection of the residual tumor showed what the authors interpreted as increased intraepithelial T cell infiltration. Because of the trial design, it was not possible to tell whether significant anti-tumor effects occurred.

Interleukin-12 Interleukin-12 (IL-12) was injected intratumorally prior to surgery once weekly, two or three times, at either 100 or 300 ng/kg, in ten previously untreated patients with head and neck squamous cell carcinoma [55]. The injections resulted in a greater infiltration of natural killer cells and CD20+ B cells than were seen in samples from 20 patients who had not received IL-12. Because of the trial design, it was not possible to tell whether significant anti-tumor effects occurred.

Retinoids and Vitamins There is a long history of interest in the effects of vitamins and especially vitamin A analogs, for the treatment and prevention of head and neck cancers. Much of the focus has been on 13-cis-retinoic acid (isotretinoin, CRA). As summarized in Table 1, an objective response rate of 16% was observed in 19 evaluable patients who were treated with isotretinoin as part of a multi-institutional, randomized phase II trial that included 40 patients with advanced head and neck squamous cell carcinoma who were randomized to treatment with either methotrexate or isotretinoin [25]. In this trial there were no objective responses in patients treated with methotrexate chemotherapy. Because of in vitro evidence of synergy, and some evidence of single-agent activity, several small trials combined 13-cis-retinoic acid (isotretinoin, CRA) with IFN-α. A response rate of 13% was reported in a trial of 16 patients with unresectable recurrent head and neck carcinomas who were treated with CRA 40 mg p.o. daily plus IFN-α 3 MU s.c. every other day [32]. A response rate of only 5% was reported in another phase II trial of this regimen in 21 patients with recurrent squamous cell carcinoma of the head and neck [60]. In another trial, no

Robert O. Dillman responses were observed in ten patients with advanced squamous cell carcinoma of the lung and of the head and neck who were treated with high dose 13-cis-retinoic acid (2 mg/kg/day) and IFN-α [44]. Collectively, the results of these three trials were disappointing, with a cumulative recorded objective responses of only 3/47 (6%) of patients. The retinoids have produced impressive results in the prevention of squamous cell cancer of the head and neck. Patients with squamous cell cancers of the head and neck remain at high risk for both recurrent and second primary tumors after initial therapy. After completion of surgery or radiotherapy (or both), 103 patients who were disease-free after primary treatment for squamous-cell cancers of the larynx, pharynx, or oral cavity were randomized to receive 12 months of either isotretinoin (13-cis-retinoic acid) (50 to 100 mg/m2/day) or placebo [19]. There was no reduction in the rate of recurrence of the original cancer, but after a median follow-up of 32 months, only 4% of patients in the isotretinoin group had second primary tumors, as compared with 24% in the placebo group (p = 0.005). Previously high-dose isotretinoin therapy had been shown to be an effective treatment for leukoplakia, a precursor of squamous cell cancer in the mouth. In a follow up study, 70 patients with leukoplakia underwent induction therapy with a high dose of isotretinoin (1.5 mg/kg/day) for 3 months, then patients with responses or stable lesions were randomly assigned to maintenance therapy with either beta carotene (30 mg/ day) or a low dose of isotretinoin (0.5 mg/kg/day) for 9 months [26]. Of 66 evaluable patients, 55% responded to the high-dose CRA, and 59 went on to randomization between beta carotene and low-dose CRA. Of the 53 patients who could be evaluated, 92% in the low-dose isotretinoin group and 45% in the beta carotene group had sustained disease control (p < 0.001). Trials conducted more recently outside the United States failed to confirm these impressive results. During 1992 to 1995, 272 Italian patients who had undergone radical treatment for advanced (stage III and IV) squamous cell head and neck cancer were randomized to no further treatment (n = 126) or to receive 1 year of CRA at a dose of 0.5 mg/kg/day p.o. (n = 126) [52]. Because of difficulties in getting IFN-α, a third arm of CRA plus IFN-α was discontinued after enrolling only 15 patients. After 3 years of follow up, the 5-year actuarial OS rates were similar for both arms at 59% and 57%, as were DFS rates at 66% and 63%. Adverse effects were mostly grade I, and occurred in 69% of treated patients. As in other trials, adverse events included cytopenia, mucositis, conjunctivitis, cutaneous

717 toxicity, hypertriglyceridemia and hypercholesterolemia. These authors concluded that CRA was ineffective as chemoprevention in patients with radically treated HNSCC. In an Australian trial, 151 patients head and neck squamous cell carcinoma, who had undergone curative treatment, were randomized to 3 years of treatment with isotretinoin at a high dose (1.0 mg/kg/day) or a moderate dose (0.5 mg/kg/day) or placebo [36]. There was no difference in the rate of second primary cancers of the head and neck, lung, or bladder, or time to recurrence of disease. These authors concluded that the use of CRA as prophylaxis against a second cancer in head and neck cancer patients is not indicated. The largest trial conducted to date was a phase III randomized trial of 3 years of low-dose isotretinoin (30 mg/day) versus placebo in 1,190 patients with earlystage (stage I or II) squamous cell cancer of the head and neck [22]. After 4 years of follow up, it was determined that isotretinoin did not statistically significantly reduce the rate of second primary tumors or increase survival. This study reaffirmed that active smoking was the greatest risk for subsequently being diagnosed with additional smoking related cancers. Higher doses of cis-retinoic acid and interferon are associated with some toxicity which might be mitigated by concomitant administration of vitamin E; so, all three agents have combined as a chemoprevention therapy after treatment of advanced head and neck cancer. After definitive local treatment with surgery, radiotherapy, or both, 45 patients with locally advanced squamous cell head and neck cancer were treated with 13-cRA (50 mg/ m2/day orally, IFN-α 3MIU/m2 s.c. thrice weekly, and alpha-tocopherol (1,200 IU/day orally) for 12 months with 85% completing the planned therapy [48]. The 2-year disease free survival (DFS) rate was 81%, and overall survival (OS) 91%. In a similar trial CRA, IFNα2a, and vitamin E were used to treat 45 patients who had locally advanced (III or IV) squamous cell carcinoma of the head and neck that had been treated with surgical resection, radiation, or both [47]. The threedrug bioadjuvant chemopreventive treatment was continued for 12 months. The 5-year PFS was 80% and 5-year OS 81%, which the authors felt was much better than the 40% 5-year survival that is considered the historical standard. Other combinations of biologicals have been tested as chemoprevention strategies for these patients. In a large European trial, from 1988 to 1994, 2,592 patients (60% with head and neck cancer and 40% with lung cancer) were randomized to receive one of four treatment arms: retinyl palmitate (0.3 MIU daily for 1 year followed by 0.15 MIU for an additional year),

718

Biological therapy of squamous cell cancers of the head and neck

N-acetylcysteine (600 mg daily for 2 years), the combination of retinyl palmitate and N-acetylcysteine for 2 years, or no additional treatment [56]. In terms of risk, 94% had smoked tobacco at sometime in their lives and 25% continued to smoke after the cancer diagnosis. After a median follow-up of 4 years, 916 (35%) patients had experienced a recurrence, second primary tumor, or death. There was no statistically significant difference in event-free or overall survival. or event-free survival for any of the 2 × 2 analyses for the impact of retinyl palmitate or N-acetylcysteine. There was a lower incidence of second primary tumors in the no intervention arm that was not statistically significant. Retinoids have been combined with chemotherapy agents and biologicals other than IFN-α. Ifosfamide, cisplatin, and CRA (0.5 mg/kg) were given in combination to treat 52 patients with squamous cell carcinoma of the head and neck, who had either locally advanced disease, that could not be managed by surgery or radiation therapy, locoregional recurrence or distant metastases [38]. Treatment was well-tolerated. The response rate was 72%, with a median PFS of 10 months and OS of 13 months. Cisplatin, IFN-α (6 MIU/day i.m.), and CRA 1 (mg/kg/day) were given in combination for 12 weeks to 23 previously treated patients who had developed advanced or metastatic head and neck squamous cell carcinoma [16]. There were four objective responses. In a trial that enrolled 54 patients, 42 who had exhibited clinical benefit (complete or partial response, disease stability) from docetaxel, ifosfamide, and cisplatin chemotherapy as treatment for recurrent or metastatic squamous cell carcinoma of the head and neck, lowdose s.c. IL-2 and oral CRA were given as maintenance immunotherapy [39]. Median PFS was 11 months and median OS was 22 months. Progressive increases in the numbers of lymphocytes and natural killer cells were documented, as well as a decline in levels of vascular endothelial growth factor (VEGF).

with virus-modified autologous tumor cells prepared from short-term tumor cultures [18]. Delayed type hypersensitivity reaction to unmodified tumor cells was demonstrated, but anti-tumor effects could not be measured because of the trial design.

Adoptive Cell Therapy There has been only limited investigation of adoptive cell therapy in patients with head and neck cancer. In one study, irradiated autologous tumor cells were admixed with BCG and injected i.d. in six patients with advanced head and neck cancers, then vaccine-primed lymphocytes were isolated from regional lymph nodes, expanded in vitro with IL-2 and anti-CD3 Mab, then infused into patients along with i.v. IL-2 [9]. Specific cytoxicity against autologous tumor was demonstrated in four patients. There were no significant tumor responses after transfer of the activated lymphocytes. In another approach, three patients with recurrent head and neck cancer underwent leukapheresis to collect peripheral blood mononuclear cells (PBMC) which were then incubated in vitro with the bifunctional monoclonal antibody (Mab) catumaxomab, which has one arm that binds to the epithelial cell adhesion molecule (Epcam) and the other arm binds to the T cell antigen CD3 [40]. Intravenous administration of catumaxomab is complicated by the release of cytokines when the Mab binds to circulating T cells; so in this trial the strategy was to activate the T cells in vitro where the release of cytokines would take place ex vivo, then infused the cells i.v. in hopes that the catumaxomab would target the cells to tumor. Assays showed that the PBMNC indeed did release substantial amounts of interferon gamma and tumor necrosis factor alpha in vitro. Patients received escalating doses from 10,000 to 10 million cells per kilogram, with 1 million per kilogram being the maximum tolerated dose. One patient achieved a CR that lasted more than 2 years.

Vaccines The vaccines that were recently approved that prevent infection with human papilloma virus (HPV) will prevent HPV- squamous cell head and neck cancers, which account for a small subset of all head and neck cancers. In terms of the more traditional vaccine approach, there have been relatively few studies in patients with head and neck cancer. In a pilot study, following radiation therapy 20 patients with squamous cell cancer of the head and neck were preconditioned with IL-2 and subsequently vaccinated

Antibody Therapy Cetuximab has been extensively evaluated in squamous cell cancers of the head and neck. Vermorken et al. gave single-agent cetuximab at the standard dose and schedule to 109 patients who had recurrent and/or metastatic disease that progressed during platinum chemotherapy [57]. Acneiform skin rash, which occurred in 49%, was the most common toxicity There was one death due to an infusion-related allergic reaction. The 13% response

Robert O. Dillman rate was similar to that of single-agent cetuximab in colorectal cancer. Phase I trials suggested that cetuximab could be safely given with radiation therapy and might enhance therapeutic benefit [43]. Bonner et al. conducted a multinational trial comparing cetuximab plus radiotherapy to radiotherapy alone in 424 patients with locoregionally advanced disease, that showed a benefit for the addition of cetuximab, including a 26% reduction in death, with an increase in survival from 29 to 49 months (p = 0.03) [4]. There was no increase in toxicity other than the acneiform rash. Pfister et al. treated 22 patients with locoregionally advanced disease with cisplatin, cetuximab, and 70 Gy radiotherapy [37]. This trial was closed early because of several adverse events from infection and cardiac events. Grade 3 or 4 cetuximab-related toxicities included acneiform rash in 10% and hypersensitivity in 5%. With a median follow-up of 52 months, the 3-year overall survival rate is 76%, the 3-year progression-free survival rate is 56%, and the 3-year locoregional control rate is 71%. Herbst et al. treated 130 patients who did not have an objective response or relapsed within 90 days of completing two cycles of cisplatin/paclitaxel or cisplatin/ fluorouracil, with standard cetuximab and cisplatin (75 or 100 mg/m2 i.v.) every 3 weeks [17]. The most common toxicities were anemia, acneiform skin rash, leukopenia, fatigue/malaise, and nausea/vomiting. Seven patients (5%) experienced a grade 3 or 4 hypersensitivity reaction to cetuximab. Baselga et al. treated 96 patients who were refractory to cisplatin, with standard cetuximab and cisplatin at the same dose and schedule, during which progressive disease had occurred [1]. Acneiform rash was the most common toxicity. Burtness et al. randomized 117 patients who had recurrent or metastatic disease to receive cisplatin every 4 weeks with weekly cetuximab or placebo [7]. Response rate was higher with the addition of cetuximab, but PFS and OS were not improved. Survival was better for those patients who developed the skin rash. Bourhis et al. treated 53 patients who had recurrent or metastatic disease with a combination of cetuximab, cisplatin or carboplatin, and escalating doses of 5FU as initial therapy [5]. Dermatologic toxicity was the most common adverse event, but the most common grade 3 or 4 adverse events were leucopenia (38%), asthenia (25%), thrombocytopenia (15%), and vomiting (14%). Chan et al. treated 59 patients who had recurrent or metastatic nasopharyngeal cancer that had recurred after cisplatin, with cetuximab and carboplatin [8]. Six patients (10%) experienced serious treatment-related adverse events during cetuximab.

719 The humanized anti-EGFR Mab h-R3 was given at four dose levels weekly for 6 weeks in combination with radiotherapy, to 24 patients with advanced head and neck cancer [13]. In this trial there were some infusion reactions, but no dermatologic or allergic toxicities were noted. The overall survival of patients was considered encouraging.

Summary The role of biotherapy in squamous cell cancer of the head and neck is very limited at this time. Enthusiasm for retinoid therapy alone, or in combination with interferon has waned with the publication of recent randomized trials. The results associated with regional and intranodal injections are interesting, but have not created commercial interest. In the near future, monoclonal antibody combined with chemotherapy may emerge as a new standard of care for systemic disease.

References 1. Baselga J, Trigo JM, Bourhis J, et al. Phase II multicenter study of the antiepidermal growth factor receptor monoclonal antibody cetuximab in combination with platinum-based chemotherapy in patients with platinum-refractory metastatic and/or recurrent squamous cell carcinoma of the head and neck. J Clin Oncol 2005;23:5568–77. 2. Benasso M, Merlano M, Blengio F, et al. Concomitant alpha-interferon and chemotherapy in advanced squamous cell carcinoma of the head and neck. Am J Clin Oncol 1993;16:465–8. 3. Bensmaine ME, Azli N, Domenge C, et al. Phase I-II trial of recombinant interferon alpha-2b with cisplatin and 5-fluorouracil in recurrent and/or metastatic carcinoma of head and neck. Am J Clin Oncol 1996;19:249–54. 4. Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 2006;354:567–78. 5. Bourhis J, Rivera F, Mesia R, et al. Phase I/II study of cetuximab in combination with cisplatin or carboplatin and fluorouracil in patients with recurrent or metastatic squamous cell carcinoma of the head and neck. J Clin Oncol 2006;24:2866–72. 6. Buechler M, Mukherji B, Chasin W, Nathanson L. High dose methotrexate with and without BCG therapy in advanced head and neck malignancy. Cancer 1979;43:1095–100. 7. Burtness B, Goldwasser MA, Flood W, et al. Phase III randomized trial of cisplatin plus placebo compared with cisplatin plus cetuximab in metastatic/recurrent head and neck cancer: an Eastern Cooperative Oncology Group study. J Clin Oncol 2005;23:8646–54. 8. Chan AT, Hsu MM, Goh BC, et al. Multicenter, phase II study of cetuximab in combination with carboplatin in patients with recurrent or metastatic nasopharyngeal carcinoma. J Clin Oncol 2005;23: 3568–76. 9. Chang AE, Li Q, Jiang G, et al. Generation of vaccine-primed lymphocytes for the treatment of head and neck cancer. Head Neck 2003;25:198–209.

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10. Cheng VS, Suit HD, Wang CC, et al. Clinical trial of Corynebacterium parvum (intra-lymph-node and intravenous) and radiation therapy in the treatment of head and neck carcinoma. Cancer 1982;49:239–44. 11. Cortesina G, De Stefani A, Galeazzi E et al. Interleukin-2 injected around tumor-draining lymph nodes in head and neck cancer. Head Neck 1991;13:125–31. 12. Cortesina G, De Stefani A, Galeazzi E, et al. Temporary regression of recurrent squamous cell carcinoma of the head and neck is achieved with a low but not with a high dose of recombinant interleukin 2 injected perilymphatically. Br J Cancer 1994;69:572–6. 13. Crombet T, Osorio M, Cruz T, et al. Use of the humanized antiepidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy in the treatment of locally advanced head and neck cancer patients. J Clin Oncol 2004;22:1646–54. 14. De Stefani A, Forni G, Rogona R, et al. Improved survival with perilymphatic interleukin 2 in patients with resectable squamous cell carcinoma of the oral cavity and oropharynx. Cancer 2002;95:90–7. 15. Feinmesser R, Hardy B, Sadov R, et al. Report of a clinical trial in 12 patients with head and neck cancer treated intratumorally and peritumorally with multikine. Arch Otolaryngol Head Neck Surg 2003;129:874–81. 16. Gravis G, Pech-Gourgh F, Viens P, et al. Phase II study of a combination of low-dose cisplatin with 13-cis-retinoic acid and interferon-alpha in patients with advanced head and neck squamous cell carcinoma. Anticancer Drugs 1999;10:369–74. 17. Herbst RS, Arquette M, Shin DM, et al. Phase II multicenter study of the epidermal growth factor receptor antibody cetuximab and cisplatin for recurrent and refractory squamous cell carcinoma of the head and neck. J Clin Oncol 2005a;23:5578–87. 18. Herold-Mende C, Karcher J, Dyckhoff G, Schirrmacher V. Antitumor immunization of head and neck squamous cell carcinoma patients with a virus-modified autologous tumor cell vaccine. Adv Otorhinolaryngol 2005;62:173–83. 19. Hong WK, Lippman SM, Itri LM, et al. Prevention of second primary tumors with isotretinoin in squamous-cell carcinoma of the head and neck. N Engl J Med 1990;323:795–801. 20. Hussain M, Benedetti J, Smith RE, et al. Evaluation of 96-hour infusion fluorouracil plus cisplatin in combination with alpha interferon for patients with advanced squamous cell carcinoma of the head and neck: a Southwest Oncology Group study. Cancer 1995;76:1233–7. 21. Kerrebijn JD, Simons PJ, Balm AJ, et al. Thymostimulin enhancement of T-cell infiltration into head and neck squamous cell carcinoma. Head Neck 1996;18:335–42. 22. Khuri FR, Lee JJ, Lippman SM, et al. Randomized phase III trial of low-dose isotretinoin for prevention of second primary tumors in stage I and II head and neck cancer patients. J Natl Cancer Inst 2006;98:441–50. 23. Kimura T, Suzuki K, Motai H, et al. Final report of a randomized controlled study with streptococcal preparation OK-432 as a supplementary immunopotentiator for laryngeal cancer. Acta Otolaryngol Suppl 1996;525:135–41. 24. Lam P, Yuen AP, Ho CM, et al. Prospective randomized study of post-operative chemotherapy with levamisole and UFT for head and neck carcinoma. Eur J Surg Oncol 2001;27:750–3. 25. Lippman SM, Kessler JF, Al-Sarraf M, et al. Treatment of advanced squamous cell carcinoma of the head and neck with isotretinoin: a phase II randomized trial. Invest New Drugs 1988;6:51–6. 26. Lippman SM, Batsakis JG, Toth BB, et al. Comparison of lowdose isotretinoin with beta carotene to prevent oral carcinogenesis. N Engl J Med 1993;328:15–20.

27. Mantovani G, Bianchi A, Curreli L, et al. Neo-adjuvant chemotherapy +/- immunotherapy with s.c. IL 2 in advanced squamous cell carcinoma of the head and neck: a pilot study. Biotherapy 1994;8:91–8. 28. Mantovani G, Gebbia V, Airoldi M, et al. Neo-adjuvant chemoimmuno therapy of advanced squamous-cell head and neck carcinoma: a multicenter, phase III, randomized study comparing cisplatin + 5-fluorouracil (5-FU) with cisplatin + 5-FU + recombinant interleukin 2. Cancer Immunol Immunother 1998;47:149–56. 29. Mattijssen V, De Mulder PH, De Graeff A, et al. Intratumoral PEG-interleukin-2 therapy in patients with locoregionally recurrent head and neck squamous-cell carcinoma. Ann Oncol 1994;5:957–60. 30. Mussche RA, Kluyskens P. Prognosis of primarily treated localized laryngeal carcinoma ameliorated through levamisole treatment: a randomized pilot study. Oncology 1980;37:329–35. 31. Neifeld JP, Terz JJ, Kaplan AM, Lawrence W Jr. Adjuvant Corynebacterium parvum immunotherapy for squamous cell epitheliomas of the oral cavity, pharynx, and larynx. J Surg Oncol 1985;28:137–45. 32. Nikolaou AC, Fountzilas G, Daniilidis I. Treatment of unresectable recurrent head and neck carcinoma with 13-cis-retinoic acid and interferon-alpha. A phase II study. J Laryngol Otol 1996;110:857–61. 33. Olivari AJ, Glait HM, Guardo A, et al. Levamisole in squamous cell carcinoma of the head and neck. Cancer Treat Rep 1979;63:983–90. 34. Padmanabhan TK, Balaram P, Vasudevan DM. Role of levamisole immunotherapy as an adjuvant to radiotherapy in oral cancer. I. A three-year clinical follow up. Neoplasma 1987;34:627–32. 35. Papac R, Minor DR, Rudnick S, et al. Controlled trial of methotrexate and Bacillus Calmette-Guérin therapy for advanced head and neck cancer. Cancer Res 1978;38:3150–3. 36. Perry CF, Stevens M, Rabie I, et al. Chemoprevention of head and neck cancer with retinoids: a negative result. Arch Otolaryngol Head Neck Surg 2005;131:198–203. 37. Pfister DG, Su YB, Kraus DH, et al. Concurrent cetuximab, cisplatin, and concomitant boost radiotherapy for locoregionally advanced, squamous cell head and neck cancer: a pilot phase II study of a new combined-modality paradigm. J Clin Oncol 2006;24:1072–8. 38. Recchia F, Lalli A, Lombardo M, et al. Ifosfamide, cisplatin, and 13-Cis retinoic acid for patients with advanced or recurrent squamous cell carcinoma of the head and neck: a phase I-II study. Cancer 2001;92:814–21. 39. Recchia F, Candeloro G, Di Staso M, et al. Maintenance immunotherapy in recurrent or metastatic squamous cell carcinoma of the head and neck. J Immunother 2008;31:413–9. 40. Riechelmann H, Wiesneth M, Schauwecker P, et al. Adoptive therapy of head and neck squamous cell carcinoma with antibody coated immune cells: a pilot clinical trial. Cancer Immunol Immunother 2007;56:1397–406. 41. Richman SP, Livingston RB, Gutterman JU, et al. Chemotherapy versus chemoimmunotherapy of head and neck cancer: report of a randomized study. Cancer Treat Rep 1976;60:535–9. 42. Richtsmeier WJ, Koch WM, McGuire WP, et al. Phase I-II study of advanced head and neck squamous cell carcinoma patients treated with recombinant human interferon gamma. Arch Otolaryngol Head Neck Surg 1990;116:1271–7. 43. Robert F, Blumenschein G, Herbst RS, et al. Phase I/IIa study of cetuximab with gemcitabine plus carboplatin in patients with chemotherapy-naive advanced non-small-cell lung cancer. J Clin Oncol 2005;23:9089–96.

Robert O. Dillman 44. Roth AD, Abele R, Alberto P. 13-cis-retinoic acid plus interferonalpha: a phase II clinical study in squamous cell carcinoma of the lung and the head and neck. Oncology 1994;51:84–6. 45. Schantz SP, Dimery I, Lippman SM, et al. A phase II study of interleukin-2 and interferon-alpha in head and neck cancer. Invest New Drugs 1992;10:217–23. 46. Schulof RS. Thymic peptide hormones: basic properties and clinical applications in cancer. Crit Rev Oncol Hematol 1985;3:309–76. 47. Seixas-Silva JA Jr, Richards T, Khuri FR, et al. Phase 2 bioadjuvant study of interferon alfa-2a, isotretinoin, and vitamin E in locally advanced squamous cell carcinoma of the head and neck: long-term follow-up. Arch Otolaryngol Head Neck Surg 2005;131:304–7. 48. Shin DM, Khuri FR, Murphy B, et al. Combined interferon-alfa, 13-cis-retinoic acid, and alpha-tocopherol in locally advanced head and neck squamous cell carcinoma: novel bioadjuvant phase II trial. J Clin Oncol 2001;19:3010–7 49. Suen JY, Richman SP, Livingston RB, et al. Results of BCG adjuvant immunotherapy in 100 patients with epidermoid carcinoma of the head and neck. Am J Surg 1977;134:474–8. 50. Taylor SG 4th, Sisson GA, Bytell DE, Raynor WJ Jr. A randomized trial of adjuvant BCG immunotherapy in head and neck cancer. Arch Otolaryngol 1983;109:544–9. 51. Timar J, Forster-Horvath C, Lukitis J, et al. The effect of leukocyte interleukin injection (Multikine) treatment on the peritumoral and intratumoral subpopulation of mononuclear cells and on tumor epithelia: a possible new approach to augmenting sensitivity to radiation therapy and chemotherapy in oral cancer – a multicenter phase I/II clinical Trial. Laryngoscope 2003;113:2206–17. 52. Toma S, Bonelli L, Sartoris A, et al. 13-cis retinoic acid in head and neck cancer chemoprevention: results of a randomized trial from the Italian Head and Neck Chemoprevention Study Group. Oncol Rep 2004;11:1297–305. 53. Urba SG, Forastiere AA, Wolf GT, Amrein PC. Intensive recombinant interleukin-2 and alpha-interferon therapy in patients with advanced head and neck squamous carcinoma. Cancer 1993;71:2326–31. 54. Valavaara R, Kortekangas AE, Nordman E, et al. Interferon combined with irradiation in the treatment of operable head and neck carcinoma. A pilot study. Acta Oncol 1992;31:429–31.

721 55. van Herpen CM, van der Laak JA, de Vries IJ, et al. Intratumoral recombinant human interleukin-12 administration in head and neck squamous cell carcinoma patients modifies locoregional lymph node architecture and induces natural killer cell infiltration in the primary tumor. Clin Cancer Res 2005;11:1899–909. 56. van Sándwich N, Dalesio O, Pastorino U, et al. EUROSCAN, a randomized trial of vitamin A and N-acetylcysteine in patients with head and neck cancer or lung cancer. For the European Organization for Research and Treatment of Cancer Head and Neck and Lung Cancer Cooperative Groups. J Natl Cancer Inst 2000;92:977–86. 57. Vermorken JB, Trigo J, Hitt R, et al. Open-label, uncontrolled, multicenter phase II study to evaluate the efficacy and toxicity of cetuximab as a single agent in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck who failed to respond to platinum-based therapy. J Clin Oncol 2007;25:2171–7. 58. Vlock DR, Snyderman CH, Johnson JT, et al. Phase Ib trial of the effect of peritumoral and intranodal injections of interleukin-2 in patients with advanced squamous cell carcinoma of the head and neck: an Eastern Cooperative Oncology Group trial. J Immunother Emphasis Tumor Immunol 1994;15:134–9. 59. Vlock DR, Andersen J, Kalish LA, et al. Phase II trial of interferon-alpha in locally recurrent or metastatic squamous cell carcinoma of the head and neck: immunological and clinical correlates. J Immunother Emphasis Tumor Immunol 1996;19:433–42. 60. Voravud N, Lippman SM, Weber RS, et al. Phase II trial of 13-cis-retinoic acid plus interferon-alpha in recurrent head and neck cancer. Invest New Drugs 1993;11:57–60. 61. Wanebo HJ, Hilal EY, Pinsky CM, et al. Randomized trial of levamisole in patients with squamous cancer of the head and neck: a preliminary report. Cancer Treat Rep 1978;62:1663–9. 62. Woods JE, DeSanto LW, Ritts RE Jr. A controlled study of combined methotrexate, BCG, and INH therapy for squamous cell carcinoma of the head and neck. Surg Clin North Am 1977;57: 769–78. 63. Vogl SE, Schoenfeld DA, Kaplan BH, et al. Methotrexate alone or with regional subcutaneous Corynebacterium parvum in the treatment of recurrent and metastatic squamous cancer of the head and neck. Cancer 1982;50:2295–300

21.10 Biological therapy of glioblastoma ROBERT O. DILLMAN

Current Status of Therapy for Glioblastomas Glioblastoma multiforme (GBM) or glioblastoma is a collection of morphologically and genetically diverse poorly differentiated neoplasms which collectively are the most common and most primary adult brain tumor [50, 52]. More than 90% of GBMs are rapidly progressive primary tumors without clinical or histological evidence of a less malignant precursor lesion, typically appearing in the elderly. Primary GBM are genetically characterized by loss of heterozygosity 10q (70% of cases), EGFR amplification (36%), p16(INK4a) deletion (31%), and PTEN mutations (25%). Secondary GBMs progress from lower grade astrocytomas that are diagnosed in younger patients. These are associated with TP53 mutations and a G:C–>A:T mutation at CpG sites (60%). When these lower grade lesions progress to GBM, they acquire additional mutations including loss of heterozygosity 10q (70%). Primary GBM are associated with a much worse prognosis than secondary GBM. Despite recent therapeutic advances, GBM remains highly lethal when treated using standard measures [62, 66, 75]. The refractoriness of GBM probably relates, in part, to microscopic local extension which is beyond the areas treated by surgery and radiation therapy [49]. Also, there is a limited therapeutic index between brain and tumor which limits radiation therapy. Tumor cell heterogeneity [6], and the limitations of the blood brain barrier [76], conspire to impair the effectiveness of most systemically administered therapies. Surgery is still the mainstay of standard therapy, although a substantial number of patients are not candidates for surgery either because of the anatomic location of the tumor or because of comorbid medical conditions. Historically, following surgery alone, the median survival for GBM was only 35 weeks, with a 1-year survival of 33%, and a 2-year survival of 0% [41, 70]. The ability to perform a near-total resection has been enhanced by microsurgical techniques with navigational guidance, but GBM is rarely completely resected, which is why surgery alone seldom, if ever, is curative [10, 42, 84]. R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

External Beam Radiation therapy (EBRT) is a standard component of the initial treatment of GBM, preferably following surgical resection, but is also used as primary therapy in patients who are not surgical candidates [30, 46]. Improvements in the delivery of radiation therapy have decreased toxicity, but partial brain radiation to 6,000 cGy delivered over 5–6 weeks, which remains the standard of care for newly diagnosed GBM, and the use of hyperfractionation radiation therapy have had little effect on survival [82]. Stereotactic radiosurgery and gamma knife radiosurgery have enabled more specific delivery of even higher doses of radiation [51]. Although a randomized trial failed to show a survival advantage for the addition of a radiosurgery boost in the initial management of GBM [72], more advanced uses of this technique to areas around the edge of the radiographically defined tumor are being explored. Adding chemotherapy to surgery and radiation therapy does improve the survival of patients with newly diagnosed GBM. A meta-analysis of randomized trials of adjunctive chemotherapy determined that the 2-year survival for 884 patients treated with radiation therapy alone was only 16% compared to 23% for 1,538 patients who received radiation and chemotherapy [29]. Recently, concomitant and adjuvant chemoradiotherapy with oral temozolomide has become widely accepted as the standard treatment for newly diagnosed GBM based on a randomized trial that showed improved median survival from 12 months to 15 months, and the 2-year overall survival from 8% to 26% [74, 75] meta-analysis of 16 randomized trials comparing chemotherapy to no chemotherapy showed a survival advantage for the use of chemotherapy, but the impact of temozolomide was much greater than that of local or systemic carmustine therapy [71].

Biological Therapy of Glioblastoma The potential clinical role of immunotherapy in the management of glioblastomas is under investigation. The early forays with biological therapy, were not associated with improved outcomes [37, 81]. There has 723

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Biological therapy of glioblastoma

recently been a rekindling of enthusiasm for vaccine approaches, anti-angiogenesis agents, gene therapy strategies, newer monoclonal antibodies, and intracranial lymphocyte therapy, but these approaches are still under development.

Non-specific Immune Stimulators There was limited investigation of the early non-specific immune stimulants such as bacillus Calmette-Guerin (BCG), Corynbacterium parvum, or levamisole. In one small study five patients with malignant gliomas who had undergone BCG inoculation were injected intratumorally with PPD in an effort to induce an intratumoral delayed type hypersensitivity reaction [2]. There was a subjective increase in inflammation based on biopsies obtained before and after the PPD injections, but there was no evidence of an antitumor effect. Other studies combined these agents with other treatment. In one trial BCG, and in another levamisole, were combined with nitrosourea chemotherapy following surgery and/or radiotherapy, but neither agent appeared to enhance the anti-tumor effect [93]. No survival benefit was obtained in a trial in which 25 patients with malignant glioma were randomized to receive radiation therapy alone or with oral levamisole [94].

Interferons As summarized in Table 1, limited anti-tumor activity has been documented in clinical trials in malignant gliomas exploring the activity of lymphoblastoid interferon [56], interferon alpha [Chang et al. 1998, 54], or interferon beta [78, 90, 92].

Interferon-alpha Intratumor injections of leukocyte interferon were not associated with significant responses in 17 patients with malignant gliomas, 12 primary and five recurrent [40]. Most trials have utilized i.v. or s.c. routes to deliver IFN-α systemically. In a trial in which IFN-α was given for 8 weeks, three patients with glioblastoma exhibited a clinical response [54]. There appeared to be a possible relationship between IFN-α gene status and tumor response. No responses were noted in 18 patients with recurrent glioma who received thrice weekly s.c. IFN-α with oral tamoxifen (Chang et al. 1998). In 29 patients with recurrent anaplastic astrocytoma and glioblastoma, the combination of IFN-α with eflornithine, an irreversible inhibitor of ornithine decarboxylase was associated with no objective tumor responses [15]. As in other tumor types, IFN-α was combined with chemotherapy for patients with malignant gliomas. In one phase II trial the combination of BCNU and IFN-α produced objective response in 7/21 (33%) [13]. In another phase II trial in 35 patients with recurrent malignant glioma, the combination of IFN-α and BCNU chemotherapy produced an objective response rate of 29% [14]. This encouraging results led to a trial in which 383 malignant glioma patients were treated with radiation therapy and BCNU, then 275 who had experienced disease progression at the end of therapy, were randomized to receive more BCNU alone or in combination with IFN-α [16]. There was increased toxicity associated with the use of IFN-α but no improvement in outcome. Several trials have explored the use of IFN-α in combination with radiation therapy. A retrospective review of 175 patients with malignant gliomas suggested that the highest complete remission rates were in patients

Table 1. Single-agent activity of various biologicals in patients with malignant gliomas Modality class

Biological agent

Lead author

Patients

Response rate (%)

Retinoid Retinoid Retinoid Retinoid Cytokine Cytokine Cytokine Cytokine Cytokine Cytokine Cytokine Cytokine Monoclonal antibody

13-cis retinoic acid (CRA) 13-CRA and celecoxib All-trans retinoic acid All-trans retinoic acid Interferon-α + tamoxifen Interferon-α + eflornithine Interferon-α Lymphoblastoid interferon Interferon-β Interferon-β Interferon-β Interferon-β EMD 55,900 (Mab 425) anti-EGFR

[69] [43] [96] [97] [95] [15] [54] [56] [92] [78] [3] [92] [73]

82 25 34 30 18 29 30 14 14 13 21 65 16

4 0 3 10 0 0 10 0 0 0 19 28 0

EGFR = epidermal growth factor receptor.

Robert O. Dillman who received the combination of radiation therapy, nitrosourea, and IFN-α [87]. In 19 patients with primary glioblastoma, only two of whom were able to undergo a near total resection, the combination of radiation therapy and thrice weekly 3–5 MIU s.c. of IFN-α was associated with a median survival of 7.5 months [22]. In vitro experiments suggested that combining 13-cis retinoic acid with IFN-α was associated with even greater radiosensitizing effects against glioblastoma cell lines [45]. A subsequent trial of these agents was combined with radiation therapy in 40 patients, only four of whom underwent near total resection, and resulted in a median survival of 9.3 months with a 1-year survival of 42% [23]. Several patients experienced severe radiation necrosis, suggesting that enhanced radiosensitization had been achieved in vivo. Systemic IFN-α was given concurrently with intralesional interleukin-2 (IL-2) in five patients with recurrent malignant glioma [48]. IL-2 was given by this route because of the induction of cerebral edema by high doses of systemic IL-2. As part of this study four patients had received IL-2 alone and cerebral edema was observed in two patients who received 50,000 IU intralesionally. In the combination study, systemic IFN-α was given at dose of 3 MIU to 50 MIU i.v. weekly and IL-2 was limited to 10,000 IU intralesionally. The combination IL-2 + IFN-α was associated with weakness and fatigue, and two patients exhibited increased peritumoral edema on radiologic scans. Because of toxicity, this combination has not been pursued.

Interferon-beta Results with interferon-beta (IFN-β) may be somewhat better than what has been reported with IFN-α. No responses were observed in 14 patients with relapsed high grade gliomas who were treated with lymphoblastoid interferon [56]. IFN-β (Betaseron) was given by i.v. infusion over 30 min thrice weekly to 29 pediatric patients with a variety of primary malignant brain tumors, including 12 high-grade astrocytomas and nine brain stem gliomas [3]. Objective responses were noted in 4/21 evaluable patients. No objective responses were recorded in ten patients in a pilot study in which IFN-β (Betaseron) was i.v. thrice weekly at a dose of 90 MIU to 14 adult patients with recurrent malignant glioma [90]. In a larger trial using higher i.v. thrice weekly dosing, 15/65 patients with recurrent or non-responsive malignant glioma had an objective response [92]. Histologically, 41 had glioblastoma and 24 had anaplastic astrocytoma. A thrice weekly dose of 180 MIU was felt to be the optimal dose based on toxicity

725 and clinical activity. No activity was noted in a small study of 13 patients treated with a different IFN-β preparation (Fiblaferon) [78]. No anti-tumor effects were observed in six patients with glioblastoma multiforme who were treated with local injection of IFN-β through an Ommaya reservoir [8]. In another trial, 55 of 109 patients with newly diagnosed supratentorial glioblastoma had stable disease following 60 Gy radiation therapy, and were then treated with adjuvant IFN-β [19]. Their median survival of 13.4 months was encouraging based on comparisons to historical controls in the Radiation Therapy Oncology Group glioma historical database. Japanese studies have combined IFN-β with other agents resulting in encouraging survival results [4, 80], but randomized trials are need to see whether IFN-β adds anything to current regimens that utilize temozolomide.

Interferon-gamma (IFN-γ) In a small randomized trial, 31 patients with high-grade gliomas were randomized to receive intralesional recombinant IFN-γ prior to and after radiation therapy, or standard RT after surgical debulking [31]. Intratumoral IFN-γ was given thrice weekly for 4 weeks until radiotherapy, with doses escalated from 5 to 50 micrograms, and after radiotherapy, was resumed at a dose of 50 micrograms twice a week for up to 9 weeks. Median survival was 54–55 weeks in both arms.

Retinoids Several laboratories have shown that retinoic acid has growth-inhibitory activity against glioma cells in vitro in addition to immune modulating and cell differentiation effects. A small number of trials have examined the activity of 13-cis-retinoic acid (13-CRA) as a single agent in the treatment of malignant gliomas. In a phase II trial conducted in 50 patients with progressive or recurrent disease after radiation and chemotherapy, there were three objective responses [91]. These same patients were included in a retrospective radiographic analysis of 85 patients with recurrent GBM who were treated with 13-CRA at MD Anderson Cancer Center, and an objective response rate of 4% was noted for 82 patients [69]. In a small pilot study, 13 patients with GBM and ten patients with anaplastic astrocytoma who had achieved a complete response after primary surgery, radiation therapy, and chemotherapy, were given 13-CRA at a dose of 60 mg/m2 daily for 3 weeks of each 4-week cycle with a dose escalation of up to 100 mg/m2 [85]. Treatment was well tolerated,

726 but even in this highly selected group of patients with a high representation of non-GBM histology, the median survival was still only 17 months from the time of entry into the study. Other trials have combined 13-CRA with other agents. 13-CRA plus the COX-2 inhibitor celecoxib yielded no responses in 25 patients with recurrent glioblastoma [43]. In a large phase II trial, 88 patients with recurrent or progressive malignant glioma were treated with a combination of temozolomide plus 13-CRA [38]. Histolologies included 40 glioblastoma multiforme, 28 astrocytoma, 14 oligodendroglioma, and six mixed glioma. There were ten responses among 84 evaluable patients (15%). 13-CRA was added to radiation therapy and temozolomide in the treatment of 61 adults with newly diagnosed supratentorial glioblastoma [17]. The median survival was 12.2 months and 1-year survival was estimated to be 57%. 13-CRA was added to radiation therapy and IFN-α in the treatment of 40 adults with newly diagnosed supratentorial glioblastoma, only four of whom underwent near total resection [23]. The median survival was 9.3 months, and 1-year survival was 42%. All-trans-retinoic acid (ATRA) has also been explored as a biotherapy for malignant gliomas. In a phase II trial, one of 34 evaluable patients with recurrent glioma had an objective response after two cycles of 120–150 mg/m2 given daily for 3 weeks of each 4-week cycle (Kaba 1997). The authors concluded that single agent ATRA has no significant activity against recurrent cerebral gliomas. In another trial, 30 patients with malignant gliomas, including 14 with glioblastoma multiforme and 14 with anaplastic astrocytoma, three (10%) had an objectivew response after treatment with all trans-retinoic acid (ATRA) [97].

Adoptive Cell Therapy The use of LAK and other cell therapies are being explored in the management of malignant brain tumors [77]. Systemic IL-2 often causes edema around brain tumors and sometimes hemorrhage. For this reason, patients with brain tumors have been excluded from most treatment protocols utilizing systemic IL-2. However, many investigators have explored the feasibility of direct instillation of activated cells, with or without IL-2, directly into brain tumors or areas in which brain tumors have been excised. The validity of alleged objective tumor regressions in many of these studies is in question, since they LAK were injected into a post-surgical tumor bed making interpretation of response quite difficult.

Biological therapy of glioblastoma

Intravenous adoptive cell therapy approaches Although the blood brain barrier is perceived as a limitation to systemic infusional approaches for adoptive cellular therapy, some investigators have attempted this as well. In terms of systemic adoptive cell therapy, one group has tried i.v. approaches with two different cell products. In the first study, following resection of recurrent anaplastic astrocytoma or GBM, 15 patients were immunized with BCG and their own irradiated tumor cells, then 2 weeks later peripheral blood mononuclear cells were harvested by leukopheresis, cultured in vitro with irradiated autologous tumor cells and IL-2 then infused i.v. without additional IL-2 [33]. Feasibility and safety were confirmed, but clinical benefit was not apparent. In the second study, these same investigators took nine patients with recurrent, but surgically resectable anaplastic astrocytoma or GBM, tapered them off steroids after total surgical resection and immunized them with autologous cancer cells from the resected disease that had been admixed with BCG [86]. They then collected peripheral blood mononuclear cells which were stimulated and expanded in vitro with anti-CD3 monoclonal antibody and IL-2, then 1010–1011 activated cells were infused into the patients. The study design makes it hard to interpret claims of objective tumor response in these patients, but two patients were considered disease-free more than 4 years later. T cells derived from lymph nodes have also been infused i.v. Following surgery, 12 patients with newly diagnosed gliomas (half lower grade, half GBM) were vaccinated i.d. with GM-CSF + short-term cultured autologous irradiated tumor cells, then lymphocytes from resected draining lymph nodes were stimulated with staphylococcal enterotoxin A for 48 h and then cultured in IL-2-containing medium 6–8 days, then 109–1010 cells were administered i.v. to the patients [55]. The cell products were mostly CD4+. Four patients were interpreted as having partial regression of residual tumor.

Localized Adoptive Cell Therapy Approaches Most efforts with adoptive cellular therapy for malignant gliomas have utilized localized approaches. In a phase I trial, nine patients received LAK and/or IL-2, and one patient received both together [36]. Escalating doses of LAK cells, 108–1010 or recombinant IL-2, 104–106 units were suspended in 5 ml of Hanks balanced salt solution and were directly injected into the tissue surrounding the surgical cavity remaining after tumor extirpation using multiple injections via 20 gauge needles. LAK cells were

Robert O. Dillman developed within 2–3 days of surgery. There was no comment regarding whether patients received steroids before and/or immediately after surgery and immunotherapy. None of the ten patients were felt to have exhibited toxicity beyond that normally seen post-craniotomy. In another trial, at the time of tumor resection, a catheter was implanted in the cavity and used to deliver 6 MIU/ day IL-2 by continuous infusion for 5 days, with or without LAK [11]. In five patients, LAK cells were infused into the cavity on days 1, 3, and 5 after surgery, and eight patients received IL-2 alone. Fever, confusion, and cerebral edema were observed in all patients and there was no evidence of clinical benefit. Several trials utilized instillation of LAK with IL-2. In one trial, 23 recurrent glioma patients were treated with 107–108 LAK cells with 50–400 units of a recombinant IL-2 via Ommaya reservoir [88]. LAK were generated 4–6 days prior to instillation. Many patients had substantial neurological deficits at the time of treatment.. Side effects noted were chills and fever. Substantial improvement occurred in at least three patients. In another small study, 13 recurrent glioma patients were treated with LAK generated 3–5 days prior to craniotomy and suspended in 5–10 ml saline with 106 units of Cetus IL-2 [47]. Daily injections of 106 units of IL-2 were given for 3 additional days. A second cycle of IL-2/LAK was infused via Ommaya reservoir 1–2 weeks later. All patients had symptoms of aseptic meningitis, including increased intracranial pressure, headache, fever, and malaise. The authors commented on the difficulty of attributing some of these effects to IL-2 as opposed to post-craniotomy. They felt that the approach was sufficiently safe to try on newly diagnosed patients, although all of these patients were on dexamethasone, which is known to inhibit IL-2 and LAK activity. Barba et al. treated ten recurrent glioma patients with LAK cells and IL-2 given via a catheter stereotactically placed in the tumor bed and connected to an Ommaya reservoir [5]. Three weeks after surgery, therapy was begun with 1010 LAK cells in 10 cc of saline. IL-2 was infused for 5 days via the same catheter at 10,000–60,000 U/kg/dose, three times per day. Neurologic side effects occurred in all patients because of intracerebral edema. Glucocortocoid therapy was limited during the immunotherapy, but patients were then treated with high-dose dexamethasone after completing IL-2/LAK. One of nine evaluable patients was considered to have had a beneficial response. Lillehei et al. treated 11 patients with recurrent high-grade gliomas [98]. Peripheral blood lymphocytes were stimulated in one of two different culture conditions and placed into the tumor cavity with IL-2 in a plasma clot. The survival

727 following therapy ranged from 3.5 months to >3 years, with a median survival of 4.5 months. Three of nine patients with recurrent GBM, who were treated with LAK and IL-2 administered directly into the tumor cavity via an Ommaya reservoir, were interpreted as having an objective response to treatment, although long-term survival was not achieved [9]. One of ten patients with recurrent GBM, who were treated similarly with local instillations of LAK and IL-2, were felt to have had a partial response [67]. Hayes et al. gave LAK cells to 19 out of 44 possible patients with recurrent glioma who were considered candidates for resection [32]. Fifteen tumors were classified as gliomas (grade 4) and four as anaplastic astrocytomas (grade 3). Patients underwent leukapheresis within 1–6 weeks of resection after discontinuation or reduction of corticosteroids. LAK cells were produced by incubating the mononuclear cells in 6 million international units/ml of IL-2 for 4 days. A total of 0.5 – 1 × 106 LAK were infused via Ommaya reservoir on day 1, with a maximum-tolerated IL-2 dose of 1.2 MIU/ml IL-2. The same dose of IL-2 was infused without cells on days 3, 5, 8, 10 and 12, which constituted one treatment cycle. The 12-day cycle of therapy was repeated about 2 weeks later. Patients who had not progressed after two treatment cycles could be re-treated at 12-week intervals. Since all of these patients had maximum tumor debulking at the time of surgery, tumor response could not have been a meaningful endpoint of this trial, although the authors reported one complete and two partial responses. However, median survival from the date of operation was 53 weeks for the 15 glioblastoma patients, with a 1-year survival of 53% compared to a median survival of only 26 weeks, versus a 1-year survival rate of 5% for 18 contemporary controls who underwent debulking and then received chemotherapy. The largest phase II trial to date enrolled 40 patients with pathologically confirmed GBM at the time of surgery, who subsequently had placement of autologous LAK cells into the tumor cavity [25]. LAK cells were generated by incubating peripheral blood mononuclear cells with interleukin-2 (IL-2) for 3–5 days in vitro. Because of the inherent difficulties in interpreting a tumor response in such patients who have undergone surgical resection, survival was compared to contemporary matched controls. Median survival post-LAK was 9.0 months; 1-year survival was 34%. Gender, age, location of tumor, and number of cells implanted were unrelated to outcome. Inclusion of IL-2 at the time of cell instillation was associated with improved survival (p = .097). Median survival from date of original diagnosis for 31 patients who had GBM at initial diagnosis

728 was 17.5 months versus 13.6 months for a control group of 41 contemporary GBM patients (p = .012). The median survival rates were higher than reported in most published series of patients who had undergone re-operation for recurrent GBM. This approach is currently being combined with surgery, radiation therapy and temozolomide and preliminary results are encouraging [26]. In a slightly different approach, peripheral blood lymphocytes were stimulated with both phytohemagglutin (PHA) and IL-2 to produce what were called mitogen activated killer (MAK) cells. In a phase I/II trial involving 55 patients with various glioma histologies, MAK were generated over 7–10 days prior to craniotomy, then suspended in 15 ml of autologous EDTA-plasma (total volume 15–20 ml) for placement in the tumor bed following surgery [34]. Therapy was generally well tolerated, other than fever and nausea, and most patients were discharged within 5–7 days after craniotomy. There was no effort made to judge tumor response, but survival for the patient who underwent treatment was considered encouraging. MAK were also used to treat 19 patients with high-grade gliomas [39] At the time of treatment both PHA and IL-2 were placed in the tumor site as well in 16 patients. Three patients were felt to have a significant decrease in radiologic enhancement of the tumor area. Because of the paucity of lymphocytes found in malignant gliomas, few investigators have explored the use of tumor infiltrating lymphocytes (TIL) as adoptive cell therapy for GBM. Following surgical resection and placement of an Ommaya reservoir, six patients were treated with low doses of TIL every 2 weeks concurrent with thrice weekly low doses of IL-2 for 1 month [57]. Three patients were interpreted as having a partial response to the treatment.

Antibody Therapy The epidermal growth factor receptor (EGFR) is expressed on malignant glioma cells; and therefore has been a target for antibody-based therapy of glioblastoma. EMD55900 (Mab 425) is a murine IgG2A Mab directed against EGFR. Mab 425 was given i.v. to 16 patients previously treated with surgery, radiotherapy and chemotherapy for high grade supratentorial malignant gliomas (11 GBM) [73]. There were no tumor responses including the last six patients who received 200 mg thrice weekly for 4 weeks. Single doses of 20, 40, 100, 200, or 400 mg of the murine MAb 425 were given i.v. before surgery to 30 patients with malignant brain tumors [28]. Treatment was well-tolerated and in

Biological therapy of glioblastoma vivo binding was confirmed. Eight patients with primary or recurrent EGF-R-positive glioblastomas were treated intratumorally twice weekly through an implantable catheter with total doses of 4 mg and 120 mg of Mab 425 [83]. The treatment induced an intense inflammatory reaction and a substantial necrosis. In an early pilot study, 25 patients with primary malignant glioma were given i.v. or intra-arterial I-131-labeled anti EGFR (425) 4–6 weeks following initial surgery and radiation therapy [12]. The median survival was 16 months for the 15 patients with GBM. During 1987–1997, this group treated 180 patients following surgery and radiation therapy, with and without chemotherapy, with an average total dose of 140 mCi given typically as three weekly injections [27]. The median survival for patients with GBM was 13 months. The humanized anti-EGFR Mab h-R3 was given to 29 patients with newly diagnosed malignant gliomas including 16 glioblastoma, 12 anaplastic astrocytoma and one anaplastic oligodendroglioma [58]. Following debulking surgery, patients received six 200 mg infusions of h-R3 weekly during radiotherapy. Interestingly, no patients developed acneiform rash, and there were no allergic reactions. Median survival was 17 months for the glioblastoma patients. A trial of the anti-EGFR mab cetuximab with temozolomide and radiation therapy is being conducted in patients with previously untreated glioblastoma [21]. For many years investigators at Duke University have been testing monoclonal antibodies (Mab) that react tenascin, which is a large, disulfide-bonded glycoprotein of the extracellular matrix that is highly expressed in the stroma of gliomas, but not normal brain tissue. Radiommunotherapy trials have been carried out with murine Mab 81C6, but a chimeric version has also been tested. In a phase I trial conducted in 34 previously irradiated patients with recurrent or metastatic brain tumors, the maximum tolerated dose for 131I-labeled 81C6 administered through an Ommaya reservoir into the surgical resection cavity was 120 mCi based on doselimiting delayed neurologic toxicities [7]. Patients with recurrent GBM had an encouraging survival of about 13 months. The same MTD was determined in a phase I trial involving 42 newly diagnosed patients who had not received prior radiation therapy or chemotherapy [18]. Median survival for patients with GBM was 16 months. A small number of patients had to undergo surgical excision of symptomatic radionecrosis. In a subsequent trial, newly diagnosed patients were treated with direct injections of 131-I-labeled anti-tenascin murine 81C6 into the surgical cavity followed by conventional external-beam radiotherapy and chemotherapy [1].

Robert O. Dillman Biopsies from 15 patients all showed tumor and/or radionecrosis. Subsequently, a phase II trial was carried out in 33 newly diagnosed patients (27 GBM) using 120 mCi of 131-I-labeled murine 81C6 injected directly into the surgical resection cavity prior to receiving conventional external-beam radiotherapy, followed by a year of alkylator-based chemotherapy [59]. Median survival for the GBM patients was 18 months. In a phase II trial in 43 patients with recurrent gliomas (33 GBM), 100 mCi of 131-I-labeled murine 81C6 was injected directly into the surgical resection cavity. The median overall survival for patients with recurrent GBM was 15 months [60]. In animal models, a 131-I-labeled IgG2/mouse chimeric antitenascin 81C6 (ch81C6) construct exhibited higher tumor accumulation and enhanced stability compared to the murine antibody, but in 43 patients the maximum tolerated dose was only 80 mCi because of dose-limiting hematologic toxicity associated with a longer serum half life [61]. Another group has conducted trials with murine antitenascin Mabs called BC-2 and BC-4, which were radiolabeled with I-131. In one trial, an early analysis of 17 of 23 patients with recurrent malignant gliomas who had received intracavitary doses ranging from 15 mCi to 57 mCi, showed a median survival of about 16 months [63]. In an expanded group of 50 patients (24 with primary and 26 with recurrent disease) the median survival was 18 months for 26 patients with recurrent disease and 20 months in 24 patients with newly diagnosed lesions [64]. BC-4 was labeled with Y-90 and administered to another 20 patients with recurrent high grade gliomas (18 GBM) using doses ranging from 5–30 mCi [65]. The maximum tolerated dose to the brain was 25 mCi delivering 3,200 cGy/mCi. In patients who have experienced recurrence of GBM, encouraging results for the combination of irinotecan and the anti-vascular endothelial growth factor monoclonal antibody bevacizumab have been reported. In a phase II trial, adult patients with recurrent anaplastic astrocytoma or glioblastoma (grade III or IV glioma) received bevacizumab at 10 mg/kg and irinotecan i.v. every 2 weeks of a 6-week cycle [79]. The dose of irinotecan was determined based on whether patients were taking antiepileptic drugs that induced hepatic enzymes that accelerate metabolism of irinotecan. Patients taking enzyme-inducing antiepileptic drugs received irinotecan at 340 mg/m2, while other patients received irinotecan at 125 mg/m2. Although the significance of radiographic changes in treated gliomas is questionable, based on study design, the authors alleged an overall response rate of 63% for all patients. In other trials of bevacizumab, patients with brain metastases were

729 excluded because of concerns that there might be an increased risk of intracerebral hemorrhage. However, in patients with malignant gliomas, there were no instances of central nervous system hemorrhage, but four patients (12%) experienced thromboemboic complications.

Vaccine Therapy A variety of vaccine approaches are being explored in patients with glioblastomas. These include NewcastleDisease-Virus (NDV) infected autologous tumor cells derived from short term cultures [68], irradiated autologous tumor cells from continuously proliferating cell cultures [24], autologous dendritic cells pulsed with lysates of autologous tumor [89], autologous dendritic cells pulsed with of acid-eluted peptides from autologous tumor [44], autologous tumor vaccines prepared from formalin-fixed and/or paraffin-embedded tumor tissue obtained at the time of surgery [35], autologous glioma cells and interleukin (IL)-4 gene transfected fibroblasts [53] and intratumor injection of Herpses Simplex retroviral vector-producing cells, followed by intravenous ganciclovir [20]. These were mostly small pilot studies to demonstrate safety, involving only 10–15 patients in most instances.

Summary Currently there are no biological products that have a regulatory approval for marketing as treatment for malignant gliomas, although several appear to have some activity. Whether any of these products become widely available and paid for by payment providers will depend on the choices made by large biotechnology companies. Because of current treatment paradigms, a successful biological will almost certainly have to be integrated into existing treatment algorithms as part of treatment of newly diagnosed glioblastoma. Unintentional patient selection issues and difficulties in measuring a tumor response, especially as these relate to local instillation of cell therapies and antibodies, makes it difficult to determine the clinical significance of apparent improvements in survival. Randomized trials will be needed to determine if any of these therapies are adding benefit.

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732 75. Stupp R, Hegi ME, Gilbert MR, Chakravarti A. Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 2007;25:4127–36. 76. Tardridge WM, Oldendorf WH, Cancilla P, Frank HJL. Bloodbrain barrier: interface between internal medicine and the brain. Ann Intern Med 1986;105:82–95. 77. Terzis AJ, Niclou SP, Rajcevic U, et al. Cell therapies for glioblastoma. Expert Opin Biol Ther 2006;6:739–49. 78. Von Wild KR, Knocke TH. The effects of local and systemic interferon beta (Fiblaferon) on supratentorial malignant cerebral glioma – a phase II study. Neurosurg Rev 1991;14:203–13. 79. Vredenburgh JJ, Desjardins A, Herndon JE 2nd, et al. Phase II trial of bevacizumab and irinotecan in recurrent malignant glioma. Clin Cancer Res 2007;13:1253–9. 80. Watanabe T, Katayama Y, Yoshino A, et al. Human interferon beta, nimustine hydrochloride, and radiation therapy in the treatment of newly diagnosed malignant astrocytomas. J Neurooncol 2005;72: 57–62. 81. Weller M, Fontana A. The failure of current immunotherapy for malignant glioma. Tumor derived TGF-beta, T-cell apoptosis, and the immune privilege of the brain. Brain Res Rev 1995;21: 128–51. 82. Werner-Wasik M, Scott CB, Nelson DF, et al. Final report of a phase I/II trial of hyperfractionated and accelerated hyperfractionated radiation therapy with carmustine for adults with supratentorial malignant gliomas. Radiation Therapy Oncology Group Study 83-02. Cancer 1996;77:1535–43. 83. Wersall P, Ohlsson I, Biberfeld P et al. Intratumoral infusion of the monoclonal antibody, mAb 425, against the epidermal-growthfactor receptor in patients with advanced malignant glioma. Cancer Immunol Immunother 1997;44:157–64. 84. Wirtz CR, Albert FK, Schwaderer M, et al. The benefit of neuronavigation for neurosurgery analyzed by its impact on glioblastoma surgery. Neurol Res 2000;22:354–60. 85. Wismeth C, Hau P, Fabel K, et al. Maintenance therapy with 13-cis retinoid acid in high-grade glioma at complete response after firstline multimodal therapy – a phase-II study. J Neurooncol 2004;68:79–86. 86. Wood GW, Holladay FP, Turner T, et al. A pilot study of autologous cancer cell vaccination and cellular immunotherapy using

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22 Speculations for 2009 and beyond ROBERT K. OLDHAM

It is now apparent that the genomic capacity of mammalian cells to produce biological substances that have medicinal value is enormous. Now that a substantial number of lymphokines/cytokines, growth and maturation factors, cellular therapies, and antibody-based approaches have been explored, there is clear evidence of clinical activity with respect to colony-stimulating factors (CSF), blocking factors for epidermal growth receptors (EGF) and vascular endothelial growth factor (VEGF), interferon (IFN), interleukin 2 (IL-2), activated cells, vaccines, monoclonal antibodies, and their immunoconjugates. If one analyzes the current level of clinical activity for biotherapy in the historical context of chemotherapy, it is obvious that much more rapid progress is being made in biotherapy, leading to development of improved, less toxic and more selective forms of treatment. From the earliest days of chemotherapy, massive searches were done among tens of thousands of chemical compounds, searching for the one right drug that might have antitumor activity without excessive toxicity for the patient. The successes were few. From analyzing more than a million chemical structures, less than 80 anticancer drugs have come to the clinic, and no more than 30 of these can be considered even moderately active. This is not to say that the drug development paradigm has not had some success. However, the search has been arduous, complex, expensive, and almost wholly based on the process of random screening of large numbers of compounds to find the rare active chemical. By contrast, the first genetically engineered biological to be approved as an anticancer agent, alpha-interferon, has been an unqualified success in the clinic. It has been the drug of choice for the treatment of hairy-cell leukemia and chronic myelogenous leukemia (until even better targeted therapies were developed) and is moderately active in other forms of leukemia and lymphoma. Substantial activity has been reported in renal cancer, melanoma, and selected other tumors. IL-2 is now approved for renal cancer and melanoma. Monoclonal antibodies and their immunoconjugates have proven broadly effective, and the first inhibitors of EGF receptor and VEGF have been approved. Thus, biotherapy and small molecule targeted therapy can

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, © Springer Science + Business Media B.V. 2009

claim a high success rate in terms of new substances that have come through the complete testing program with approval for clinical use. The process of drug development was historically based on the idea that sufficient random testing would identify those active substances that could selectively kill cancer cells. Ancillary was the hope that specific processes, unique to cancer cells, would be identified that might allow chemicals to be selectively active on those cells. A secondary hypothesis that rational drug development could be based on structure–function relationships, receptor information, or analog development has proven equally difficult. These concepts have been difficult to validate, and the level of selectivity for chemotherapy is very low. In fact, for every chemotherapeutic agent, there are normal tissues that are probably more affected by the drug than the cancer in which the drug is active. While there is still hope for the discovery of some unique chemotherapeutic drug that is selectively active against cancer, this goal seems much less likely to be achieved than it was a decade ago. In contrast, biotherapy and targeted therapy work through physiologic molecules for which the body has receptors and known mechanisms of action. These substances are often used in pharmacological amounts, but they are message molecules known to our cellular systems. Emerging evidence in laboratory animal model systems raises the hope that cancer cells are not completely without the ability to be controlled by appropriate growth, differentiation, and antiproliferative influences. Most of the data for interferon assumed an antiproliferative activity on cancer cells, in that higher doses have generally been more effective, and tumors resistant to lower doses sometimes respond to an increased dose. In addition, the doses active in many cancers are well above the level where interferon is most active as an immunomodulator. As the process of developmental therapeutics for biotherapy is expanded, there is a great need for all those involved to remain open-minded with respect to new paradigms for drug development. Simply to presume that biologicals must fit into the mold used for chemotherapy drug development has been a major error, since the process of developmental therapeutics for biologics is

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734 very different from that for drugs. Even in the preclinical screening programs now in use for biologicals, the process is more one of rational selection than of random screening – the need is to exploit the activities of known molecules rather than to search among the thousands for the singularly active substance. As the information base in cancer biology grows, it will become increasingly important to exploit what we know of the power of biological molecules rather than to search among unknown chemicals for active substances.

Beyond Interferon Alpha-interferon is an approved agent for hairy-cell leukemia, chronic myelogenous leukemia, Kaposi’s sarcoma, renal cancer and melanoma; it is also active against a variety of other cancers. Beta- and gammainterferon are both now approved for clinical use. Beyond these three interferons, the process of genetic engineering is bringing forth a huge number of interferon molecules for testing. Given any small protein of 140– 160 amino acids, the possibility now exists for making an infinite number of molecules simply for altering portions of the amino acid chain. This process is underway for the interferons, and will demonstrate whether more active interferons can be developed to enhance the immunomodularity, antiviral, or antiproliferative activity of the natural molecules.

Lymphokines/cytokines Interleukin 2 came into the clinic in the mid-1980s by way of a process that used the growth factor to activate lymphocytes that could subsequently be used therapeutically as effector cells in patients. The cancer-killing capacity of these activated cells was apparent from their in-vitro activity, and the major questions related to their in-vivo use, distribution, and anticancer activity. Interleukin 2 is being used in amounts far in excess of its normal physiologic role, and toxicities are the natural result. This particular approach was very expensive; it required hospitalization and truly enormous doses of both IL-2 and the activated cells. The future may bring a better strategy for using this approach in patients, whereby repeated treatment may become less toxic and more active than the current intensive regimens. Outpatient regimens that are much less toxic are now used more frequently. Interleukin 2 is part of a broad cascade of biological molecules that have activities in cellular activation and

Speculations for 2009 and beyond the control of cellular proliferation. These molecules include IL-1-32 and beyond. The future may allow the use of these components in combination or sequence to strengthen what are natural but ineffective bodily responses to the problem of cancer growth and metastasis. Although induction of such a cascade by one particular substance may not be feasible, the artificial programming and the medicinal use of these lymphokines are now both feasible and practical. As the process of administration of biotherapy becomes better defined, so will the programmability of therapeutic regimens using these substances. One can envision pre-programmed infusions of combinations of lymphokines designed for maximal exploitation of their biological activities.

Antigen-specific Lymphokines Over and above the use of lymphokines in a more general sense, as just described, there is evidence that certain lymphokines act in antigen-specific ways. As the proteins constituting tumor-associated antigens become more thoroughly characterized, as a result of monoclonal antibody technology and genetic engineering, one can envision the use of specific antigens with specific lymphokines, thus allowing oncologists to mount antigenspecific, and perhaps cancer-specific, immunologic responses in individual patients. It is likely that these antigen-specific lymphokines will have to be used in ways that are selected specifically for individual patients. If, as will be reasoned below, the array of tumor-associated antigens present on the tumor cells of each individual is slightly different from the array present on cancers of a like type in other individuals, the use of antigen-specific lymphokines with specific antigenic preparations will of necessity be unique to each individual. Parallels for the clinic are already being explored in animal models for transplantation and other specific immune responses. The application of this technology to human therapeutics will occur in the not-too-distant future.

Growth and Differentiation Factors Cancer might be likened to a juvenile delinquent; a derivative of one’s own self but without the controls necessary for appropriate socialization. If cancer cells were to have only a limited spread and growth and if subsequent therapeutic approaches could simply limit further growth, there would be no need to eradicate the

Robert K. Oldham cancer cells completely. Such an approach has an obvious parallel in the use of physiologic mediators for endocrinopathies. For example, diabetes did not disappear by virtue of the discovery of insulin. The genetic anomaly continues to exist, and diabetes has become a more frequent clinical problem in our society. However, insulin has been used to control the pathologic manifestations of this disorder. Is it possible there are growth and differentiation factors, coded for by the mammalian genome, that could be used similarly for treating cancer? There is early evidence of such mediators in vitro and in animal models. The control of growth factors, as well as agonists and antibodies to those factors, provide substance to the argument that molecular manipulation of growth and differentiation is a major area for developmental therapeutics for cancer. If one takes the growth and differentiation of cells in vitro as a model, the use of epidermal growth factor, nerve growth factor, insulin, and other hormones has a profound effect on both the rate of replication and the degree of deviance of replicating cells from their ‘normal’ counterparts. Similarly, cancer growth, proliferation, and spread may be controllable by manipulation of growth factors made by normal tissues and/or by the cancer cells themselves. Similar approaches are now in the clinic as targeted therapy using both monoclonal antibodies and small molecules interacting with EGF, VEGF and their receptors. Rudimentary attempts using certain chemicals that cause in-vitro differentiation, have had interesting effects on cell lines and experimental modes; however, the use of retinoids, butyrates, and other factors that might regulate growth and differentiation have had minimal impact in the clinic. With almost daily discovery of substances that support growth of cancer cells in vitro, one might imagine that the antithesis of such factors might also be developed, exploited, and utilized for clinical therapeutics. In biology, as in physics, there seems to be a reaction for every action. For many of the molecules that support growth, experimental data also indicates that other molecules exist to offset that increased growth. As these molecules are isolated and characterized, and subsequently produced by genetic engineering, they will broaden our potential arsenal for cancer biotherapy.

Monoclonal Antibodies The current evidence to support the therapeutic use of monoclonal antibodies and their immunoconjugates has been presented in earlier chapters. While it is early in

735 the clinical application of this biotherapeutic approach, it is already apparent that monoclonal antibodies offer the probability of selective cancer treatment that has never before been available [34]. The search for the perfect ‘magic bullet’ is probably futile. With the exception of anti-idiotypic antibodies, which can be quite specific for the B cell clone that has deviated and become a malignant lymphoma or leukemia, it seems unlikely that such singular and specific antigens are represented on all cancers. Rather, the evidence seems to support a bewildering array of cancerassociated antigens present in different quantities on cancers from different individuals. Data are accumulating concerning the use of typing panels of monoclonal antibodies. They indicate that one can assess the antigenic array on each individual’s tumor cells and decide which antibodies should compose a cocktail to cover the available specificities of that cancer. This testing mechanism reveals cancer to be individualistic. It is different for every patient and perhaps even among the clones of cancer present within an individual patient. This should not be surprising, since the genetic apparatus that governs each of us is individualistic. With the exception of identical twins, no two humans are exactly alike, and one would be amazed if cancers were exactly alike. Unless a specific genetic lesion is identified as the cause of each histological type of cancer, each malignancy in each patient may be an individual problem in biology. It is unlikely that a singular change leads to all forms of lung cancer, breast cancer, or colon cancer. It is possible that genetic changes will result in specific markers for each of those cancers, which can be exploited in treatment. However, it is perfectly obvious that the cells in which those changes occur are genetically distinct from one person to another. Many genetic alterations, caused by the same chemical or physical carcinogen, result in the outgrowth of cells that markedly differ from each other. If such is the case, one might postulate that cancer in each patient is the individualistic expression of a response to that multistep, complex carcinogenic impulse, thereby embodying the genetic characteristics of both the host and the carcinogenic influence. If such is the case, then the actual problem in developmental therapeutics will be individualistic and each patient will have to be analyzed as an individual problem in cancer biology. This concept of individuality of cancers has been difficult for many cancer biologists and clinicians to accept. This concept was first present in the 1st Edition of this text book in 1987 and is still being debated. Recently, it has been “rediscovered” as “Personalized Medicine”. We have learned and use the histological classification systems

736 embedded in the minds of pathologists and transmitted through textbooks of medicine. These concepts classify cancers categorically according to tissue of origin and histological features. In spite of the clinical and laboratory observation that phenotypic analysis and even ‘genotype’ in cancer biology confer great diversity within cancers of the same histological type, we continue to develop therapeutics as if all breast cancer, all lung cancer, and all colon cancer are replicates. This is the simplest to teach, the simplest to learn, and the simplest to practice. However, it is fundamentally and biologically incorrect. Heretofore, there has never been a technology that allows cancer biologists to understand cancer on an individualistic basis. Monoclonal antibody technology, proteonomics, and genomics represent what may be the solutions to the problem. If cancer is indeed diverse and individualistic, might not the immune system, itself individualistic and diverse, be able to solve the problem? Each person’s immune system has the ability to produce several hundred thousand to several million different antibody molecules. Taken together, the diversity of the immune system from the broader perspective of a population of individuals is gigantic. By the use of in-vitro techniques and the cellular diversity inherent in the immune system, it is probable that the response capabilities in antibody technology exceed the diversity implicit to cancer. It may well be postulated that each cancer in each patient presents with an individual array of antigens. If such is the case, it might be possible to generate antibodies and/or type tumors with a multiphasic approach, leading to the generation of cocktails of antibody to respond to the diversity inherent in cancer biology. Clearly, T cells could be generated and manipulated in a similar manner for specific cellular therapy.

Cancer Treatment: The Future It has been categorically stated by various authorities that cancer is a problem that should be largely solved or under control within 10–20 years. The National Cancer Institute set a national priority of reducing the cancer death rate by at least 50% by the year 2000. These projections were certainly optimistic, and they were not achieved. On the horizon for surgery, radiotherapy, and chemotherapy, there seems little to support the goal of reducing the cancer death rate by 50% by any future date. Although it is doubtful that the cancer death rate can easily be reduced by this magnitude, it is likely that the approaches available through biotherapy will play a major role in developmental therapeutics in the next decade. If the field is allowed to progress by using

Speculations for 2009 and beyond the best minds of science rather than proceeding in an overly structured and rigidified way, new paradigms for developmental therapeutics may allow for greater strides to be made using biotherapy. End-point reductions in cancer death rates are difficult to achieve, or even to detect, because of time factors. To know that the death rate from breast cancer has been reduced by 50%, one would have to have at least a 5-year follow up of the patients at risk since the recurrence rate, although highest in the first 2 years, continues to be significant for several years after diagnosis. For lung cancer, the problem is somewhat more straightforward, in that end point of an effective change in therapeutics can be discerned within 3–5 years. Breast, colon, and lung cancers being the most common malignancies, the reduction of death rate by 50% in these three tumors by the year 2000 would have required that the actual change would have to have occurred between 1990 and 1995. That simply did not occur. Although biotherapy is likely to play an increasingly important role within the next decade, it is doubtful that changes of such magnitude can be produced in the near term. Rather, it seems more likely that the first 2 decades of the 2000s will be those in which the early phases of such changes may occur, and the actual end point for significant death-rate reduction will then fall well after the year 2020. It is 2009. Interferon, interleukin 1–32, growth and maturation factors, tumor necrosis factor, colony stimulating factors, activated cells, monoclonal antibodies, immunoconjugates, vaccines, and gene therapy are clinical realities – but they are all in an early phase of developmental therapeutics. If developed as drugs have been tested in the past, the average time to take these from the laboratory to an approved therapeutic is 10 years, at a cost of over $800 million each. If we are to move forward in developmental therapeutics for biologicals, we cannot take 10 years to develop each approach individually. Certainly, it is impossible to imagine a series of events that will allow an end-point reduction of 50% by the year 2020 or even 2050 if each biopharmaceutical takes 10 years to be brought from concept to widespread clinical use.

Biotherapy is not Chemotherapy and it not just Immunotherapy The fourth modality of cancer treatment is constituted more broadly to include all of the factors described in this book [3]. To take maximum advantage of the opportunities available through biotherapy, major structural changes are necessary in our system of translation of

Robert K. Oldham developmental therapeutics from concept to the laboratory and then to the clinic [2]. We cannot afford to develop biologicals in the protracted, expensive unidimensional manner of drug development. We have a huge number of new biological substances, and the current system of access and opportunity for patients, the system of funding of research, our method of government regulation, and our reimbursement system for developmental therapeutics must undergo major change [4–11, 15, 18, 19, 21–27, 29–33]. We are now faced with the reality of many more opportunities for effective cancer therapy than mechanisms by which these opportunities can be brought to clinical reality. For more than 2 decades, cancer research and treatment have operated on the ‘kill and cure’ hypothesis. Developmental therapeutic programs have functioned under a format where a new drug is brought to the clinic test in phase I for toxicity and phase II for activity with the presumption that short-term effects on cancer (response rates) will ultimately lead, if positive, to survival benefit. While this paradigm has been useful in developing cytotoxic drugs, there is much to suggest that we should now broaden our concept of developmental therapeutics, as cancer biotherapy comes to the fore, to include the idea of cancer control. Much as one treats diabetes with insulin, it may soon be possible, through the use of growth and maturation factors and other forms of cancer biotherapy, to arrest the growth and spread of cancer and thus make these diseases more amenable to long-term control even in the absence of tumor eradication. The implications in terms of cost and reimbursement for clinical services are obvious. We cannot afford to pay for developmental therapeutics as we have in the past. How is society going to pay for the very long-term clinical trials, perhaps constituted over a 3–5 year period, to test the various hypotheses for cancer control? Witness the difficulty in establishing clinical trials for cancer prevention and the high cost intrinsic to these programs as examples of the difficulty in envisioning clinical research for long-term cancer control projects. Obviously, this is going to require a major restructuring of developmental therapeutics if society is to take advantage of these opportunities [8–10, 12, 13, 34]. For more than 3 decades, the major factors slowing developmental therapeutics have been the regulations promulgated by Congress and the Food and Drug Administration (FDA) for the development of new drugs [14]. These regulations have resulted in a system of drug development costing more than $800 million per new drug taken through to commercial availability. In the last 10 years, it has become apparent that the

737 funding for clinical research is becoming less available. Much of the funding was derived from insurance reimbursement for clinical trials, and with the current pressure on insurers to reduce costs, at the behest of employers and patients who pay for their insurance premiums, it is becoming increasingly difficult to fund clinical research through third-party reimbursement. Thus, while the regulation and the intrinsic cost were the major impediments to rapid drug development prior to 2000, reimbursement for clinical trials and even reimbursement for the ‘off-label’ use of new forms of therapy will become the major limiting factor in the future. We are now faced with the probability that cancer cures are being developed that will not be reimbursed by government or private insurance programs [13, 16, 17, 20]. With our current reimbursement system, autologous bone marrow transplantation for certain forms of cancer has reached a level of cure of 20% of patients with selected neoplasms at a cost of $100,000–150,000 per procedure. Thus, the upper limit of the price for a human life is becoming more clear. New technology tends to be expensive early and becomes less expensive later. However, we now face the probability that certain new technologies will be available and highly effective while still very expensive [20]. Society must decide how much life is worth and what percentage of the gross national product should be allocated to healthcare. This is an area that needs public debate and should not be simply left to ‘social planners and thought leaders’. Therefore, it is apparent that cancer biotherapy brings cancer research and developmental therapeutics to a crossroads. In this millennium, we will not be limited by our ability to conceptualize and develop highly effective treatment. We will be limited by broader social considerations of who shall pay for such approaches and how much each life is worth [20, 28]. Thus, it is apparently that the many opportunities biotherapy brings to scientists and clinicians will have to be moved at a much faster rate from laboratory to clinic if our patients are to derive benefit from these new approaches. Perhaps the use of the laboratory in conjunction with the clinical practice of medicine, as suggested by the Nobel Laureate, Dr. Peter Medawar, is the true ‘breakthrough’ in the developmental therapeutics of cancer [1]: “The cure of cancer is never going to be found. It is far more likely that each tumor in each patient is going to present a unique research problem for which laboratory workers and clinicians between them will have to work out a unique solution.”

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References 1. Medawar P, ed. The Life Sciences. New York: Harper & Row, 1977. 2. Oldham, RK. Biologicals: new horizons in pharmaceutical development. J Biol Response Modif 1983; 2:199–206. 3. Oldham RK. Biologicals and biological response modifiers: the fourth modality of cancer treatment. Cancer Treat Rep 1984;68: 221–232. 4. Oldham RK. The cure for cancer. J Biol Response Modif 1985;4:111–116. 5. Oldham RK. Whose rights come first? J Biol Response Modif 1985;4:211–212. 6. Oldham R`K. The government-academic ‘industrial’ complex. J Biol Response Modif 1986;5:109–111. 7. Oldham RK. Profits: who benefits? J Biol Response Modif 1986;5:203–205. 8. Oldham RK. Patient-funded cancer research. N Engl J Med 1987;316:46–47. 9. Oldham RK. Drug development: who foots the bill? Biotechnology 1987;5:648. 10. Oldham RK. False hope vs. opportunity. Cope 1987; April:66. 11. Oldham RK. Whose life is it anyway? Wall Street J 1987;April 24. 12. Oldham RK. Letter to the Editor: patient-funded cancer research. N Engl J Med 1987;316:172. 13. Oldham RK. Who pays for new drugs? Nature 1988;332:795. 14. Oldham RK. Regularity hierarchies (editorial). Mol Biother 1988;1:3–6. 15. Oldham RK. Fundamentally flawed? Mol Biother 1988;1:58–60. 16. Oldham RK. Why deny health care? The Freeman 1989;March: 94–95. 17. Oldham RK. Clinical research in cancer: a time for consensus. Pharm Exec 1989;July:23–34.

Speculations for 2009 and beyond 18. Oldham RK, Avent RA. Clinical research: who pays the bill? Oncol Issues 1989;4:13–14. 19. Oldham RK. Clinical research in cancer: a time for consensus. Mol Biother 1989;1:242–243. 20. Oldham RK. Cancer cures. By the people, for the people, at what cost? (editorial) Mol Biother 1990;2:2–3. 21. Oldham RK. Cancer and diabetes: are there similarities? Mol Biother 1990;2:130–131. 22. Oldham RK. Whose rights come first? Mol Biother 1991;3: 58–59. 23. Oldham RK. Cancer research: a pubic trust. BioPharm 1991;4: 8–9. 24. Oldham RK. Is informed consent a function of who pays? Mol Biother 1991:3:2–5. 25. Oldham RK The rights and wrongs of national heath care. Pharm Exec 1991;11:92–93. 26. Oldham RK. Cancer research for whom? BioTechnology 1991; 9:772. 27. Oldham RK. Peer review. Mol Biother 1992;4:2–3. 28. Oldham RK. BioEthics: Opportunities, Risks and Ethics: The Privatization of Cancer Research. Franklin, TN: Media American, 1992. 29. Oldham RK. Fundamentally flawed. Cancer Biother 1993;8: 111–114. 30. Oldham RK. What’s the score? Cancer Biother 1993;8:187–188. 31. Oldham RK. Cancer research: does it deliver for the patient? Cancer Biother 1994;9:99–102. 32. Oldham RK. The war on cancer: new battle plan needed. Cancer Biother 1994;9:289–290. 33. Oldham RK, Dillman RO. Gold standard or wrong standard (editorial). Cancer Biother Radiopharm 2004;19(3):271–272. 34. Oldham RK, Dillman RO. Monoclonal antibodies in cancer therapy: twenty-five years of progress. JCO 26(11):1774–1777.

Index

1,25-(OH)2D3 544 4-1BB ligand 199 activation-induced cell death (AICD) 169, 204, 276 active specific immunotherapy 5 acute leukemia 584 acute lymphocytic leukemia (ALL) 366 acute myelocytic leukemia (ALM) 366 acute myeloid leukemia 548 adeno-associated viruses (AAV) 589–591 adenoviral vectors 591 adjuvant chemotherapy 128 adoptive cellular therapy 652, 684 AIDS/HIV, 118, 576 alemtuzumab 340 alkylating agents 112 all-trans retinoic acid (ATRA) 527, 528 allergic reactions 321 allogeneic bone-marrow transplantation (ALBMT) 497, 500 allogeneic stem cells 652 allogeneic tumor cells 512 alpha interferon 465 alpha particles 647, 660, 681, 699, 715, 724 amifostine 685 anaphylactoid reactions 320 angiogenesis 18, 28, 29, 179, 191, 203, 216, 599 animal models 305 anthracyclines 596 anthrax 408 anti-angiogenesis 292 antibiotics 112 antibodies 12, 67, 68, 71, 303, 305–312, 315, 338, 363, 377, 640, 661, 672, 686, 696, 701, 702, 711, 735 see also monoclonal antibodies anti-idiotype 363 current trends 640, 711, 728, 735 heterogeneity 451 radiolabelled 375 antibody-dependent cell-mediated cytotoxicity (ADCC) 312 anti-CD20 antibodies 469, 497 antigen-specific lymphokines 734 antigenic modulation 322 antigens 306, 363, 364, 372, 373, 380, 381, 509, 669 anti-idiotype antibodies 363 anti-idiotype vaccines 314 antimetabolites 111, 596 antiproliferative action 290 antisera 303, 305 antitumor cocktails 453 antiviral activity 290 apoptosis 200, 204, 207, 213, 216, 371

autologous bone-marrow transplantation (ABMT) autologous tumor cells 507, 511, 585 azocytidine 548

498

B cells 279, 333 cytokines 200, 208, 211, 212 interleukins 194 lymphocytes 85, 88 Bacillus Calmete Guerin (BCG) 669, 679, 713 bacterial toxins 409 bestatin 126, 128 beta particles 464 bifunctional antibodies 307 biological heterogeneity 25, 23, 33 biological response modifiers (BRM) 1, 41, 101, 364, 484 biological 2, 5, 41, 117, 121, 123, 125, 338, 342, 349, 613, 616, 660, 671, 681, 700, 715, 724 bispecific antibodies 307 blood transfusions 109 bone cells 207 bone marrow 497, 575, 576, 583 breast cancer 344, 347, 348, 473, 669 camptothecins 596 cancer vaccines see vaccines carbohydrates 148, 150 CD20, 325, 366 CD27 ligand 200 CD30 ligand 200 CD40 ligand 201, 369 CD52, 340 CEA gene expression 472 cell cycle gene therapy 594 cell functions 95 chemoattractants 203 chemokines 203 chemosensitization 603 chemotactic cytokines 179 chemotherapy 29, 113, 126 differentiation 547 ex-vivo purging 498 immunoconjugates 455 immunosuppression 111, 127, 128, 130 interferions 285 monoclonal antibodies 332, 333 renal cancer 645 chemotherapy-induced immunosuppression 111 chemotherapy-induced neutropenia 574, 583 chimeric monoclonal antibodies 325 chimeric proteins 71 chronic lymphocytic leukemia (CLL) 122, 329, 333, 702

739

740 chronic myeloid leukemia (VML) 284 cimetidine 128 clinical remission 114 clinical trials 41, 326, 340, 344, 351, 357, 361, 457, 623 antisera 305 immunoconjugates 455, 457 immunotoxins 426, 427 monoclonal antibodies 326, 340, 344, 351, 357, 361 cloning 53 colon cancer 122, 659 colony-stimulating factors (CSF) 62, 551, 569, 581 colorectal cancer 351, 357, 359 combination chemotherapy 113 combined-modality studies 126 complement-mediated cytotoxicity (CMC) 312 conjugates 67 copper-64, 465 Corynebacterium parvum 679, 714 cytokines 8, 57, 155, 549, 734 see also individual cytkines current trends 647, 651, 734 cytolytic functions 106 cytosine arabinoside 548 cytotoxicity 95, 312 daclizumab 367 delayed allergic reactions 321 delayed-type hypersensitivity (DTH) 93, 105 delivery see drug delivery systems dendritic cells 165, 204, 513 differentiation 174, 292, 527, 734 dimethyl sulfoxide (DMSO) 527 dose response 324 drug delivery systems 74 drug development 42, 625 effector cells 102, 595 embryonic development 206 empirical clinical testing 46 endothelia 213 enzyme-prodrug systems 596, 605 eosinophils 174 epidermal growth factor receptor 314, 356, 372, 373 escalating-dose trials 46 ex-vivo purging 498 expression of genes 25, 27, 53 Fas ligand 204 febrile neutropenia 332, 347, 571 FISP, 196, 276 Flt-3 ligand 171, 177, 178, 205, 206 free antigen 323 fungal toxins 415 gamma interferon 684, 715, 725 gastrointestinal cancer 472 gene expression 25, 27 gene therapy 589, 594, 604, 639

Index gene-directed enzyme-prodrug therapy (GDEPT) 595 genetic immunopotentiation 595 genetically modified tumor cells 654 genitourinary cancer 645 genomics 53, 65 glioma 474 graft-versus-host disease (GVHD) 497 graft-versus-tumor (GVT) effects 500 granulocyte-colony stimulating factor (G-CSF) 569 granulocyte-macrophage colony-stimulating factor (GM-CSF) 581 growth factors 10, 527, 549, 555, 734 hair-cell leukemia (HCL) 279, 329, 693 head cancer 123, 358, 359, 713 helper T cells see T-helper cells hematopoiesis 165, 174, 183, 191, 544 interleukins 165, 174, 183, 191 malignancies 107, 109, 114 HER-2, 68, 343, 373, 430, 453, 646, 573 herpes simplkex virus (HSV) 591 histamine receptor antagonists 116 historical perspective 1 HIV/AIDS, 118, 576 Hodgkin’s disease 120, 330 hormones 113, 680 host-tumor interactions 68 human anti-immunoglobulin response (HAMA/HACA/ HAHA) 321 human monoclonal antibodies 309 humanized monoclonal antibodies 310 humoral immunity 94 hybridoma technology 307 idiotype network 313 immune system 1, 85, 429, 505, 633, 645 colony-stimulating factors 551 complexes 321 cytokines 180 immunoselection 323 immunotoxins 69, 407, 426, 427, 698, 701 interferons 291 potentiation 595 stimulation 183, 669 suppression 101 immunity 85, 94, 183, 194, 195, 515 immunoconjugates 451, 452, 455 immunoglobulin 95, 321 immunosuppression 101 chemotherapy-induced 111 radiation therapy-induced 110 in vitro assays 94, 96 in vivo assays 93 in vivo binding 315 inflammation cytokines 201, 208, 213, 216 interleukins 165, 174, 183, 191 infusion reactions 318

Index

741

interferons 9, 45, 57, 197, 277, 649, 660, 715 alpha 647, 660, 681 current trends 671 gamma 161, 191, 684, 715, 725 interleukin-1 (IL-1) 60, 156, 165, 552 interleukin-2 (IL-2) 61, 156, 169, 637, 638, 649, 661, 671, 684, 695, 701, 715 interleukin-3 (IL-3) 61, 171 interleukin-4 (IL-4) 61, 172, 156, 685 interleukin-5 (IL-5) 157, 174 interleukin-6 (IL-6) 157, 174, 552 interleukin-7 (IL-7) 157, 177 interleukin-8 (IL-8) 157, 179 interleukin-9 (IL-9) 157, 180 interleukin-10 (IL-10) 157, 180 interleukin-11 (IL-11) 158, 182 interleukin-12 (IL-12) 158,183 interleukin-13 (IL-13) 158, 185 interleukin-14 (IL-14) 158, 186 interleukin-15 (IL-15) 158, 186 interleukin-16 (IL-16) 158, 187 interleukin-17A (IL-17A) 158, 189 interleukin-17B (IL-17B) 159, 190 interleukin-17C (IL-17C) 159, 191 interleukin-17E (IL-17E) 159, 191 interleukin-17F (IL-17F) 159, 191 interleukin-18 (IL-18) 159, 191 interleukin-19 (IL-19) 159, 193 interleukin-20 (IL-20) 159, 194 interleukin-21 (IL-21) 159, 194 interleukin-22 (IL-22) 159, 195 interleukin-23 (IL-23) 159, 195 interleukin-24 (IL-24) 159, 196 interleukin-24 (IL-25) 160, 196 interleukin-26(IL-26) 160, 197 interleukin-27 (IL-27) 160, 197 interleukin-28A (IL-28A) 160, 197 interleukin-28B (IL-28B) 160, 197 interleukin-29 (IL-29) 160, 197 interleukin-31(IL-31) 160, 198 interleukin-32 (IL-32) 160, 198 interleukin-33 (IL-33) 160, 198 interleukin-35 (IL-35) 160, 199 interleukins 8, 60, 62, 119, 715 iodine-131, 464, 483 isolating genes 53 Isoprinosine

124

Kaposi’s sarcoma 286 kidney see renal cancer lentinan 128 leukemia 284, 329, 333, 470, 471, 487, 532, 540, 548, 551, 584, 693, 702 leukemia inhibitory factor (LIF) 206, 553 levamisole 115, 123, 126, 129, 659, 670, 680, 713 ligand conjugation 416

LIGHT, 207, 161 lipopolysaccharide (LPS) 163 lung cancer 122, 123, 679, 684 lutetium-177, 464, 465 lym-1 antibody 466, 467 lymph nodes 209 lymphocytes 88, 91, 498, 652 lymphoid organogenesis 207, 208 lymphokine-activated (LAK) cells 93, 505, 652 lymphokines 8, 95, 734 lymphoma 180, 281, 326 lymphoproliferative responses (LPR) 94, 105 lymphotoxins 207, 208, 554 macrophages 92, 177 malignant melanoma 121, 123, 633 mast cells 174, 180, 212 maturation factors 5, 10 melanoma 121, 123, 380, 381, 385, 633 metastasis 17, 20–22, 24, 32 migration 203 ML-1 191, 555, 556 molecular chemotherapy 589, 590, 595, 599 monoclonal antibodies 12, 71, 303, 305, 307, 309–311, 315, 338, 378, 661, 686, 696, 701, 702, 735 monocytes 92 multidrug resistance 32 multiple myeloma 280, 329, 711 mutation compensation 593 myelodysplastic syndrome (MDS) 548 natural killer (NK) cells 92, 169, 194, 208 neck cancer 123, 358 neutropenia 574, 576, 583 neutrophils 189 nitric oxide synthetase-inhibiting cytokine 210 non-hematological tumors 472 non-Hodgkin’s lymphoma (NHL) 281 non-small cell lung cancer (NSCLC) 684 nonspecific immunomodulators 1, 5 nonsteriodal anti-inflammatory and antipyretic drugs (NSAIDs) 117 non-viral vectors 590, 592 novel neurotophin 209 NPT 15392, 117 OKT-432, 126, 129, 680, 714 oncogenes 594 oncostatin M, 209 osteoblasts 213 osteoclasts 212 osteopontin 210 ovarian cancer 355 OX40 ligand 211 pancreatic cancer 355 pathogenesis 17 patient selection 48

742 PEG interferon 649 peptide cytotoxins 407 peptides 476, 477, 479 perioperative immunosuppression 109 phagocytes 95 plant toxins 407 podophyllotoxins 113 polar-planar compounds 528, 546 preclinical models 4, 426 pro-apoptotic gene therapy 589 proallergic cytokines 185 prostaglandin antagonists 124 prostate cancer 32, 355, 380, 472 proteomics 65 radiation therapy-induced immunosuppression 110 radioimmunotherapy (RIT) 71, 463, 466, 469 radiolabelled antibodies 463 radiosensitization 603 RANKL, 212 recombinant DNA, 7, 53, 571 recruitment 203 regulatory approaches 313 remission 114 renal cancer 288, 353, 380, 645 replicative vector oncolysis 601 reticuloendothelial system 94 retinoids 119, 125, 672, 682, 701, 716, 725 retroviral vectors 590 rhenium-186/188, 464 rituximab 325–333, 337, 38, 616 sarcomas 356 screening 4 serum sickness 321 severe chronic neutropenia 576 sickle-cell anemia 577 skin development 194 small cell lung cancer (SCLC) 684 solid tumors 105, 108, 114, 121 soluble cytokine receptors 64 stem cell factor (SCF) 162, 212 stem cells bone-marrow transplantation 497, 500, 575 colony-stimulating factors 569 cytokines 204 radioimmunotherapy 469 renal cancer 645

Index stomach cancer 310 surgical adjuvant studies

122

T cells 638 cytokines 200, 206, 209, 211 interleukins 169, 174, 177, 180, 186, 194, 195 lymphocytes 88, 147 lymphoma 699 reconstituting factor 118, 125 T-cytotoxic factor 91 T-helper cells 90, 172, 209 T-suppressor cells 90 technetium-99m 478 thrombocytosis 182 thrombopoiesis 174 thymic factors 7, 117, 125, 128, 129 thymic hormones 680, 714 toll-like receptors 163 TRAIL, 216 TRANCE, 212 transductal targeting 592 transfer factor 119, 125, 129 transforming growth factor β 555 transcriptional targeting 592 trastuzumab 343, 344–346, 348, 349, 672 tumor cell burden 105 tumor infiltrating lymphocytes (TIL) 505 tumor lysis syndrome 319 tumor necrosis factors (TNF) 63, 165, 207, 213, 371, 554 type I toxins 415, type II toxins 415 type III toxins 415 unconjugated monoclonal antibodies 303 urologic cancer 122 vaccines. 71, 147, 374, 385, 510, 513, 634, 653, 664, 675, 687, 698, 701, 718 current trends 634, 653, 664, 675 vertebrate toxins 414 vinca alkaloids 113 viral vectors 589 vitamin A analogs 532 vitamin D analogs 540, 543, 544 wound repair

165, 209, 468

Y2B8 antibody 468 yttrium90, 464–477, 481–485, 487, 488