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

v

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

vii

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

ix

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.

References 1. Abrams P, Oldham RK. Monoclonal antibody therapy of solid tumors. In: Foon KA, Morgan AC, Jr., eds. Monoclonal antibody therapy of human cancer. The Hague: Martinus Nijihoff, 1985; 103–120. 2. Abrams PG, Morgan AC, Schroff RW, et al. Monoclonal antibodies in cancer therapy: Drug-antibody conjugates. In: Reisfeld RA, Sell S, eds. Localization and biodistribution studies of a monoclonal antibody in patients with melanoma. New York: Alan R. Liss, 1985; 27:233–236. 3. Avner BP, Liao SK, Avner B, et al. Therapeutic murine monoclonal antibodies developed for individual cancer patients. J Biol Response Modif 1989; 8(1):25–36. 4. Baldwin, RW. Relevant animal models for immunotherapy. Cancer Immunol Immunother 1976; 1:97–206. 5. Balkwill FR, Burke F. The cytokine network. Immunol Today 1989; 10(9):29–304. 6. Balkwill FR, Lee A, Aldam G, et al. Human tumor xenografts treated with recombinant human tumor necrosis factor alone or in combination with interferons. Cancer Res 1986; 46:3990–3993. 7. Bernhard MI, Foon KA, Oeltmann TN, et al. Guinea pig line 10 hepatocarcinoma model: characterization of monoclonal antibody and in vivo effect of unconjugated antibody and antibody conjugated to diphtheria toxin A chain. Cancer Res 1983; 43:4420–4428. 8. Bollon AP, Barron EA, Berent SL, et al. Recombinant DNA techniques: Isolation, cloning and expression of genes. In: Bollon AP, ed. Recombinant DNA products: Insulin interferon and growth hormone. Boca Raton, Florida: CRC Press, 1984; 1–35. 9. Bonnem EM, Spregel RJ. Interferon-alpha: Current status and future promise. J Biol Response Modif 1984; 3:580. 10. Boss BD, Langman R, Trowbridge I, Dulbecco R, eds. Monoclonal antibodies and cancer. New York: Academic Press, 1983; 1–200. 11. Boyd MR. Status of the NCI preclinical antitumor drug discovery screen. Prin Pract Oncol Suppl 1989; 3(10):1–12. 12. Bunn PA, Foon KA, Ihde DC, et al. Recombinant leukocyte A interferon: An active agent in advanced cutaneous T-cell lymphomas. Ann Intern Med 1984; 101:484–487.

14 13. Carrasquillo JA, Bunn PA, Jr., Kennan AM, et al. Radioimmunodetection of cutaneous T-cell lymphoma with 111In-T101 monoclonal antibody. New Engl J Med 1986; 315:673–680. 14. Carrasquillo JA, Abrams PG, Schroff R, et al. Effect of antibody dose on the imaging and biodistribution of indium-111 9.2.27 antimelanoma monoclonal antibody. J Nucl Med 1988; January, 29(1):39–47. 15. Cheever MA, Greenberg PD, Fefer A, Gillis S. Augmentation of the antitumor therapeutic efficacy of long-term cultured T lymphocytes by in vivo administration of purified interleukin-2. J Exp Med 1982; 155:968–980. 16. Cheever MA, Greenberg PD, Fefer A. Potential for specific cancer therapy with immune T lymphocytes. J Biol Response Modif 1984; 3:113–127. 17. Cumberlin R, DeMoss E, Lassus M, Friedman M. Isolation perfusion for malignant melanoma of the extremity: A review. J Clin Oncol 1985; 3:1022–1031. 18. DeVita VT. Progress in cancer management: Keynote address. Cancer 1983; 51:2401–2409. 19. DeVita VT. The relationship between tumor mass and resistance to chemotherapy: Implication for surgical adjuvant treatment of cancer. Cancer 1983; 51:1209–1220. 20. DeVita VT, Hellman S, Rosenberg SA, eds. Important advances in oncology 1989. New York: J.B. Lippincott, 1995; 1–118. 21. DeVita VT, Jr., Hellman S, Rosenberg SA, eds. In: Biologic therapy of cancer, second edition. Philadelphia, Pennsylvania: J.B. Lippincott, 1995; 295–327. 22. DeVita VT, Jr., Hellman S, Rosenberg SA, eds. In: Biologic therapy of cancer, second edition. Philadelphia, Pennsylvania: J.B. Lippincott, 1995; 329–345. 23. DeVita VT, Jr., Hellman S, Rosenberg SA, eds. In: Biologic therapy of cancer, second edition. Philadelphia, Pennsylvania: J.B. Lippincott, 1995; 427–433. 24. Devos R, Cheroutre H, Taya Y, et al. Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells. Nucl Acids Res 1982; 10:2487–2501. 25. Dillman RO, Beauregard JC, Mendelsohn J, et al. Phase I trials of thymosin fraction 5 and thymosin alpha-1. J Biol Response Modif 1982; 1:35–41. 26. Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-ablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Onc 2005; 23:2346–2357. 27. Eberlein TJ, Rosenstein M, Spiess P, et al. Adoptive chemoimmunotherapy of a synergeneic murine lymphoma with long-term lymphoid cell lines expanded in T-cell growth factor. Cancer Immunol Immunother 1982; 13:5–13. 28. Eggermont AM, Schraffordt Koops H, Klausner JM, et al. Isolated limb perfusion with tumor necrosis factor and for limb salvage in 186 patients with locally advanced extremity sarcomas. The cumulative multicenter Euro experience. Ann Surg Dec, 1996; v.224(6). 29. Emery AEH. Recombinant DNA technology. Lancet 1981; 2:1406–1409. 30. Evans CH, Lymphotoxin: An immunologic hormone with anticarcinogenic and antitumor activity. Cancer Immunol Immunother 1982; 12:181–190. 31. Feinberg B, Kurzrock R, Talpaz M, et al. A phase I trial of intravenously-administered recombinant tumor necrosis factor-alpha in cancer patients. J Clin Oncol 1988; 6:1328–1334. 32. Fidler IJ, Gersten DM, Hart IR. The biology of cancer invasion and metastasis. Adv Cancer Res 1978; 28:149–250.

Cancer biotherapy: general principles 33. Fidler IJ, Berendt M, Oldham RK. The rationale for and design of screening assays for the assessment of biological response modifiers for cancer treatment. J Biol Response Modif 1982; 1:15–26. 34. Foon KA, Bernhard MI, Oldham RK. Monoclonal antibody therapy: Assessment by animal tumor models. J Biol Response Modif 1982; 1:277–304. 35. Foon KA, Schroff R, Bunn PA, et al. Effects of monoclonal antibody therapy in patients with chronic lymphocytic leukemia. Blood 1984; 64:1085–1093. 36. Foon KA, Sherwin SA, Abrams PG, et al. Treatment of advanced non-Hodgkin’s lymphoma with recombinant leukocyte A interferon. N Engl J Med 1984; 311:1148–1152. 37. Gamm H, Lindemann A, Mertelsmann R, Herrmann F. Phase I trial of recombinant human tumour necrosis factor α in patients with advanced malignancy. Eur J Cancer 1991; 27:856–863. 38. Giantonio BJ, Catalano PJ, Meropol NJ, et al. Bevacizumab in combination with oxaliplatin, fluorouracil and leucovorin (FLOFOX4) for previously treated metastatic colorectal cancer: Results from the Eastern Cooperative Oncology Group Study E3200. J Clin Oncol 2007; 25:1539–1544. 39. Goeddel DV, Yelverton E, Ullrich A, et al. Human leucocyte interferon produce by E. coli is biologically active. Nature 1980; 287:411–416. 40. Goldstein AL, Chirigos MA, eds. Lymphokines and thymic hormones; their potential utilization in cancer therapeutics. Progress in cancer research and therapy, vol. 20. New York: Raven Press, 1981. 41. Goldstein AL, Chirigos MA. In: Progress in cancer research and therapy, vol. 20. New York: Raven Press, 1982; 1–324. 42. Gray PSW, Leung DW, Pennica D, et al. Expression of human immune interferon cDNA in E. coli and monkey cells. Nature 1982; 295:503–508. 43. Grem JL, Hoth D, Hamilton MJ, et al. An overview of the current status and future direction of clinical trials of 5-fluorouracil and folinic acid. Cancer Treat Rep 1987; 71:1249–1264. 44. Grem JL, McAtee N, Murphy RF, et al. A pilot study of interferon alfa-2a in combination with fluorouracil plus high-dose leucovorin in metastatic gastrointestinal carcinoma. J Clin Oncol 1991; 9:1811–1820. 45. Hanna MG, 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. 46. Hanna MG, Jr., Brandhorst JS, Peters LC. Active specific immunotherapy of residual micrometastasis: An evaluation of sources, doses and ratios of BCG with tumor cells. Cancer Immunol Immunother 1979; 7:165–174. 47. Hanna MG, Jr., Key ME. Immunotherapy of metastases enhances subsequent chemotherapy. Science 1982; 217:367–370. 48. Haranaka K. Macrophage Symposium 1987, Tumor Necrosis Factor. Japan, January 5–8, 1987. 49. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350:2335–2342. 50. Hwang KM, Foon KA, Cheung PH, et al. Selective antitumor effect on L-10 hepatocarcinoma cella 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. 51. Hwang KM, Keenan AM, Frincke J, et al. Dynamic interaction of 111 indium-labeled monoclonal antibodies with surface of solid tumors visualized in vivo by external scintigraphy. J Natl Cancer Inst 1986; 76:849–855. 52. Kavvinaran FF, Hambleton J, Mass RD, et al. Combined analysis of efficacy: The addition of bevacizumab to fluorouracil/leucovorin

Robert K. Oldham

53.

54. 55.

56.

57. 58.

59.

60.

61.

62.

63. 64. 65.

66.

67. 68.

69. 70. 71. 72.

improves survival for patients with metastatic colorectal cancer. J Clin Oncol 2005; 23:3706–3712. Kahn JO, Kaplan LD, Volberding PA, et al. Intralesional recombinant tumor necrosis factor-α for AIDS-associated Kaposi’s sarcoma. A randomized, double-blind trial. J Acquir Immune Defic Syndr 1989; 2:217–223. Kallman RF, ed. Rodent tumor models in experimental cancer therapy. New York: Pergamon Press, 1987; 1–310. Key ME, Bernhard MI, Hoyer LC, et al. Guinea pig 10 hepatocarcinoma model for monoclonal antibody serotherapy: In vivo localization of a monoclonal antibody in normal and malignant tissues. J Immunol 1983; 139:1451–1457. Key ME, Brandhorst JS, Hanna MC, Jr. Synergistic effects of active specific immunotherapy and chemotherapy in guinea pigs with disseminated cancer. J Immunol 1983; 130:2987–2992. Kirkwood JM, Ernstoff MS. Interferon in the treatment of human cancer. J Clin Oncol 1984; 2:336–352. 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. Lienard D, Ewalenko P, Delmitti JJ, et al. High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J Clin Oncol 1992; 10:52–60. Mavligit GM, Zukiwski AA, Charnsargavej C. Regional biologic therapy: Hepatic arterial infusion of recombinant human tumor necrosis factor in patients with liver metastases. Cancer 1992; 69:557–561. McCormick DL, Adamowski CB, Fiks A, Moon RC. Lifetime dose-response relationships for mammary tumor induction by a single administration of N-methyl-N-nitrosourea. Cancer Res 1981; 41:1690–1694. Ogden JR, Leung K, Kundra SA, et al. Immunoconjugates of doxorubicin and murine antihuman breast carcinoma monoclonal antibodies prepared via an n-hydroxysuccinimide active ester intermediate of cis-aconityl-doxorubicin: Preparation and in vitro cytotoxicity. Mol Biother 1989; 1(3):170–174. Oldham, RK. Monoclonal antibodies in cancer therapy. J Clin Oncol 1983; 1:582–590. Oldham RK. Biologicals: New horizons in pharmaceutical development. J Biol Response Modif 1983; 2:199–206. Oldham RK. Biologicals and biological response modifiers: New strategies for clinical trials. In: Finter NB, Oldham RK, eds. Interferons IV. Amsterdam: Elsevier Science, 1985; 235–249. Oldham RK. Interferon: A model for future biological. In: Burke D, Cantell K, Gresser I, DeMaeyer E, Landy M, Revel M, Vilcek J, eds. Interferon VI. New York: Academic Press, 1985; 127–143. Oldham RK. In vivo effects of interleukin 2. J Biol Response Modif 1984; 3:455–532. Oldham RK. Biologicals and biological response modifiers: The fourth modality of cancer treatment. Cancer Treatment Rep 1984; 68:221–232. Oldham RK. Biologicals for cancer treatment: Interferons. Hospital Pract 1985; 20:72–91. Oldham RK. Biotherapy: The fourth modality of cancer treatment. J Cell Physiol Suppl 1986; 4:91–99. Oldham RK, Smalley RV. Immunotherapy: The old and the new. J Biol Response Modif 1983; 2:1–37. Oldham RK, Smalley RV. The role of interferon in the treatment of cancer. In: Zoon KC, Noguchi PC, Lui T-Y, eds. Interferon: Research, clinical application and regulatory consideration. Amsterdam: Elsevier Science, 1984; 191–205.

15 73. Oldham RK, Smalley RV. Biological and biological response modifiers. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer: Principles and practice of oncology. Philadelphia, Pennsylvania: J.B. Lippincott, 1985; 2223–2245. 74. 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. 75. 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. 76. Oldham RK. Perspectives on the use of immunotoxins in clinical medicine. In: Vogel C-W, ed. Immunoconjugates: Antibody conjugates in radioimaging and therapy of cancer. New York, Oxford University Press. 1987; 281–289. 77. Oldham RK. Therapeutic monoclonal antibodies: Effects of tumor cell heterogeneity. In: Therapeutic monoclonal antibodies: Effects of tumor cell heterogeneity. S. Karger Ag. Cancer Treatment Symposium (Germany), 1988. 78. 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. 79. Oldham RK, Lewis M, Orr DW, et al. Individually specified drug immunoconjugates in cancer treatment. In: Ceriani RL, ed. Breast cancer immunodiagnosis and immunotherapy. New York: Plenum Publishing 1990; 219–230. 80. Oldham RK. Gene therapy and cancer: Is it for everyone? Cancer Investigation 1992; 10(6):607–609. 81. Oldham RK. Cancer biotherapy: 1993 to the millennium and more! Cancer Biother 1993; 8(1):1–2. 82. Oldham RK. Cancer biotherapy: The first year. Cancer Biother 1994; 9(3):179–181. 83. 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. 84. Oldham RK, Dillman RO. JCO 2008; 26(11):1774–1777. 85. Lewko WM, Hall PB, Oldham RK. Growth of tumor-dervice activated T cells for the treatment of advanced cancer. Can Bio and Radio 2000; 15(4):357–366. 86. Oppenheim JJ, Stadler BM, Siraganian RP, et al. Lymphokines: Their role in lymphocyte responses properties of interleukin 1. Fed Proc 1982; 41:257–262. 87. 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. 88. Palladino MA, Shalaby MR, Kramer SM, et al. Characterization of the antitumor activities of human tumor necrosis factor-alpha, and the comparison with other cytokines: Induction of tumor specific immunity. J Immunol 1987; 138:4023–4032. 89. Paranasivam G, Pearson JW, Bohn W, et al. Immunotoxins of a human melanoma-associated antigen: Comparison of gelonin with ricin and other a-chain conjugates. Cancer Res 1987; 47:3169–3173. 90. Pfreundschuh MG, Steinmetz HT, Tuschen R, et al. Phase I study of intratumoral application of recombinant human tumor necrosis factor. Eur J Cancer Clin Oncol 1989; 25:379–388. 91. Regenass U, Muller M, Curschellas E, Matter A. Anti-tumor effects of tumor necrosis factor in combination with chemotherapeutic agents. Int J Cancer 1987; 39:266–273. 92. Rosenau W. Lymphotoxin: Properties, role and mode of action. Int J Immunopharm 1981; 3:1–8. 93. 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. 94. Rosenberg SA. Principles and practices of the biologic therapy of cancer. Philadelphia, Pennsylvania: J.B. Lippincott, 2001.

16 95. Schiller JH, Witt PL, Storer B, et al. Clinical and biologic effects of combination therapy with gamma-interferon and tumor necrosis factor. Cancer 1992; 69:562–571. 96. Sherwin SA, Knost JA, Fein S, et al. A multiple dose Phase I trial of recombinant leukocyte A interferon in cancer patients. J Am Med Assoc 1982; 248:2461–2466. 97. Smalley RV, Long CW, Sherwin SA, Oldham RK. Biological response modifiers: Current status and prospects as anticancer agents. In: Herberman RB, ed. Basic and clinical tumor immunology. The Hague: Martinus Hijhoff, 1983; 257–300. 98. Smalley RV, Oldham RK. Biological response modifiers: Preclinical evaluation and clinical activity. CRC Crit Rev Oncol/ Hematol 1984; 1:259–280. 99. Smalley RV, Talmadge JA, Oldham RK, Thurman GB. The thymosins: Preclinical and clinical studies with fraction V and alpha-1. Cancer Treatment Rev 1984; 11:69–84. 100. Smith JW, Urba WJ, Clark JW, et al. Phase I evaluation of recombinant tumor necrosis factor given in combination with recombinant interferon-gamma. J Immunother 1991; 10:355–362. 101. Spriggs DR, Sherman ML, Michie H, et al. Recombinant human tumor necrosis factor administered as a 24-hour intravenous infusion. A Phase I and Pharmacologic Study. J Natl Cancer Inst 1988; 80:1039–1044. 102. Stevenson HC, Ochs JJ, Halverson L, et al. Recombinant alpha interferon in retreatment of two patients with pulmonary lymphoma, dramatic responses with resolution of pulmonary complications. Am J Med 1984; 77:355–358.

Cancer biotherapy: general principles 103. Stevenson HC, eds. Adoptive cellular immunotherapy of cancer. New York: Marcel Dekker, 1989; 1–236. 104. Stringfellow DA, Smalley RV. Interferon inducers for clinical use. In: Finter N, Oldham RK, eds. In vivo application of interferons. Amsterdam: Elsevier Science, 1984. 105. Talmadge JE, Maluish AE, Collins M, et al. Immunomodulation and antitumor effects of MVE-2 in mice. J Biol Response Modif 1984; 3:634–652. 106. Taniguchi T, Mantei N, Schwarzstein M, et al. Human leukocyte and fibroblast interferons are structurally related. Nature 1980; 285:2848–2852. 107. Tanguichi T, Matsui H, Fujita T, et al. Structure and expression of cloned cDNA for human interleukin-2. Nature 1983; 302: 305–310. 108. Taguchi T. Phase I study of recombinant human tumor necrosis factor (rHu-TNF:PT-050). Cancer Detect Prev 1988; 12:561–572. 109. Terry MD, Rosenberg SA, eds. Immunotherapy of cancer. New York: Excerpta Medica, 1982; 1–398. 110. Wadler S, Lembersky B, Atkins M, et al. Phase II trial of fluorouracil and recombinant interferon alfa-2a in patients with colorectal carcinoma: An Eastern Cooperative Oncology Group study. J Clin Oncol 1991; 9:1806–1810. 111. Weissman C, Nagata S, Boll W, et al. Structure and expression of human alpha interferon genes. In: Miwa M, et al., eds. Primary and tertiary structure of nucleic acids and cancer research. Tokyo: Japan Science Society Press, 1982; 1–22. 112. 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.

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

17

18

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

1. Arteaga CL. The epidermal growth factor receptor: from mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol 2001; 19: 32s–40s. 2. Atlas I, Mendelsohn J, Baselga J, Fair WR, Masui H, Kumar R. Growth regulation of human renal carcinoma cells: role of transforming growth factor α. Cancer Res 1992; 52: 3335–9. 3. Auerbach W, Auerbach R. Angiogenesis inhibition: a review. Pharmacol Ther 1994; 63: 265–311. 4. Augustin HG, Kmeta J, Alves F, et al. Quantitating angiogenesis and assessing the causal relationship between angiogenesis and tumorigenesis: problems and progress. Ann Hematol 2002; 81(Suppl 2): 68–72. 5. Baker CH, Fidler IJ. Tumor microenvironment and angiogenesis. In: Davis DW, Herbst RS, Abbruzzese JL, eds. Antiangiogenic cancer therapy. Boca Raton, FL: CRC Press, 2008: 131–47. 6. Baker CH, Kedar D, McCarty MF, Tsan R, Weber KL, Bucana CD, Fidler IJ. Blockade of epidermal growth factor receptor signaling on tumor cells and tumor-associated endothelial cells for therapy of human carcinomas. Am J Pathol 2002; 161: 929–38. 7. Baker CH, Solorzano CC, Fidler IJ. Blockade of the vascular endothelial growth factor receptor and epidermal growth factor receptor signaling for therapy of metastatic human pancreatic cancer. Cancer Res 2002; 62: 1996–2003. 8. Bancroft CC, Chen Z, Yeh J, Sunwoo JB, Yeh NT, Jackson S, Jackson C, Van Waes C. Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines. Int J Cancer 2002; 99: 538–48. 9. Barrier A, Boelle PY, Roser F, et al. Stage II colon cancer prognosis prediction by tumor gene expression profiling. J Clin Oncol 2006; 24: 4685–91. 10. Beer DG, Kardia SL, Huang CC, et al. Gene expression profiles predict survival of patients with lung adenocarcinoma. Nat Med 2002; 8: 816–24. 11. Beer T, Raghavan D. Chemotherapy for hormone refractory prostate cancer: beauty is in the eye of the beholder. Prostate 2000; 45: 183–93. 12. Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodeling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 1998; 125: 1591–8. 13. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003; 3: 401–10.

Sun-Jin Kim et al. 14. Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 2003; 111: 1287–95. 15. Bergsten E, Uutela M, Li X, Pietras K, Ostman A, Heldikn CH, Alitalo K, Eriksson U. PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor. Nat Cell Biol 2001; 3: 512–6. 16. Black MM, Freeman C, Mork T, Harvei S, Cutler SJ. Prognostic significance of microscopic structure of gastric carcinomas and their regional lymph nodes. Cancer 1971; 27: 703–10. 17. Bogaerts J, Cardoso F, Buyse M, et al. Gene signature evaluation as a prognostic tool: challenges in the design of the MINDACT trial (Review). Nat Clin Pract Oncol 2006; 3: 540–51. 18. Bradley G, Juranka PE, Ling V. Mechanism of multidrug resistance. Biochim Biophys Acta 1988; 984: 87–128. 19. Bröcker EB, Reckenfeld C, Hamm H, Ruiter DJ, Sorg C. Macrophages in melanocytic naevi. Arch Dermatol Res 1992; 284: 127–31. 20. Bruns CJ, Harbison MT, David DW, et al. Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin Cancer Res 2000; 6: 1936–48. 21. Bruns CJ, Solorzano CC, Harbison MT, et al. Blockade of epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res 2000; 60: 2926–35. 22. Buchdunger E, Cioffi CL, Law N, et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000; 295: 139–45. 23. Busby JE, Kim SJ, Yazici S, et al. Therapy of multidrug resistant human prostate tumors in the prostate of nude mice by simultaneous targeting of the epidermal growth factor receptor and vascular endothelial growth factor receptor on tumor-associated endothelial cells. Prostate 2006; 66: 1788–98. 24. Busse D, Yakes FM, Lenferink AE, Arteaga CL. Tyrosine kinase inhibitors: rationale, mechanisms of action, and implications for drug resistance. Semin Oncol 2001; 28: 47–55. 25. Byers RM. Modified neck dissection: a study of 967 cases from 1970 to 1980. Am J Surg 1985; 150: 414–21. 26. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249–57. 27. Carr I. Lymphatic metastasis. Cancer Metastasis Rev 1983; 22: 307–19. 28. Chang JC, Wooten EC, Tsimelzon A, et al. Gene expression profiling for the prediction of therapeutic response to docetaxel in patients with breast cancer. Lancet 2003; 362: 362–9. 29. Chen CN, Lin JJ, Chen JJ, et al. Gene expression profile predicts patient survival of gastric cancer after surgical resection. J Clin Oncol 2005; 23: 7286–95. 30. Ciardiello F, Bianco R, Caputo R, et al. Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to anti-epidermal growth factor receptor therapy. Clin Cancer Res 2004; 10: 784–93. 31. Cifone MA, Fidler IJ. Increasing metastatic potential is associated with increasing genetic instability of clones isolated from murine neoplasms. Proc Natl Acad Sci USA 1982; 78: 6949–52. 32. Clark WH, Elder DE, Guerry D, Epstein ME, Green MH, VanHorn M. A study of tumor progression: the precursor lesions of superficial spreading and nodular melanoma. Hum Pathol 1984; 15: 1147–62. 33. Clark WH, Elder DE, VanHorn M. The biologic forms of malignant melanoma. Hum Pathol 1986; 17: 443–69.

35 34. Cole SP, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 1992; 258: 1650–4. 35. Colombel M, Symmans F, Gil S, et al. Detection of the apoptosissuppressing oncoprotein Bcl-2 in hormone-refractory human prostate cancers. Am J Pathol 1993; 143: 390–400. 36. Cox CE, Pendas S, Cox JM, et al. Guidelines for sentinel node biopsy and lymphatic mapping of patients with breast cancer. Ann Surg 1998; 227: 645–53. 37. Debnath J, Brugge JS. Modeling glandular epithelial cancers in three-dimensional cultures. Nat Rev Cancer 2005; 5(9): 675–88. 38. Ding A, Hwang S, Schwab R. Effect of aging on murine macrophages: diminished response to IFN-γ for enhanced oxidative metabolism. J Immunol 1994; 153: 2146–52. 39. Dinjens WN, van der Weiden MM, Schroeder FH, Bosman FT, Trapman J. Frequency and characterization of p53 mutations in primary and metastatic human prostate cancer. Int J Cancer 1994; 56: 630–3. 40. DiPietro L, Polverini PJ. Angiogenic macrophages produce the angiogenic factor thrombospondin 1. Am J Pathol 1993; 143: 678–84. 41. Donelli MG, Russo R, Garattini S. Selective chemotherapy in relation to the site of tumor transplantation. Int J Cancer 1967; 2: 421–4. 42. Dong Z, Kumar R, Yang X, Fidler IJ. Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell 1997; 88: 801–10. 43. Dong Z, Radinsky R, Fan D, et al. Organ-specific modulation of steady-state mdr-1 gene expression and drug resistance in murine colon cancer cells. J Natl Cancer Inst 1994; 86: 913–20. 44. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996; 2: 561–6. 45. Dvorak HF. Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing. N Engl J Med 1986; 315: 1650–9. 46. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995; 146: 1029–39. 47. Dvorak HF, Seneger DR, Dvorak AM. Fibrin as a component of the tumor stroma: origins and biological significance. Cancer Metastasis Rev 1983; 2: 41–75. 48. Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. Cancer Res 2000; 60: 1388–93. 49. Erber R, Thurnher A, Katsen AD, et al. Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms. FASEB J 2004; 18: 338–40. 50. Ewing J. Neoplastic diseases. 6th edt. Philadelphia, PA: WB Saunders, 1928. 51. Fabra A, Nakajima M, Bucana CD, Fidler IJ. Modulation of the invasive phenotype of human colon carcinoma cells by fibroblasts from orthotopic or ectopic organs of nude mice. Differentiation 1992; 52: 101–10. 52. Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat Med 1999; 5: 1359–64. 53. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocrinol Rev 1997; 18: 4–25. 54. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003; 9: 669–76.

36 55. Fidler IJ. Angiogenic heterogeneity: regulation of neoplastic angiogenesis by the organ microenvironment (Editorial). J Natl Cancer Inst 2001; 93: 1040–1. 56. Fidler IJ. Biology of cancer metastasis. In: Abeloff MD, Armitage JO, Niederhuber JE, Kastan MB, McKenna WG, eds. Clinical oncology, 3rd edt. Philadelphia, PA: Elsevier Sciencem 2004; 59–79. 57. Fidler IJ. Critical determinants of metastasis. Semin Cancer Biol 2002; 12: 89–96. 58. Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth G H A Clowes Memorial Award Lecture. Cancer Res 1990; 50: 6130–8. 59. Fidler IJ. Lymphocytes are not only immunocytes (Guest Editorial). Biomedicine 1980; 32: 1–3. 60. Fidler IJ. Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125I-5-iodo-2´-deoxyuridine. J Natl Cancer Inst 1970; 45: 773–82. 61. Fidler IJ. Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of cancer metastasis. Cancer Metastasis Rev 1991; 10: 229–43. 62. Fidler IJ. The organ microenvironment and cancer metastasis. Differentiation 2002; 70: 498–505. 63. Fidler IJ. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited (Timeline). Nat Rev Cancer 2003; 3: 453–8. 64. Fidler IJ. Rationale and methods for the use of nude mice to study the biology and therapy of human cancer metastasis. Cancer Metastasis Rev 1986; 5: 29–49. 65. Fidler IJ. “Seed and soil” revisited: contribution of the organ microenvironment to cancer metastasis. In: Brodt P, ed. Surgical oncology clinics of NA: cancer metastasis: biological and clinical aspects. Philadelphia, PA: WB Saunders, 2001: 257–69. 66. Fidler IJ. Selection of successive tumor lines for metastasis. Nature (New Biol) 1973; 242: 148–9. 67. Fidler IJ, Bucana C. Mechanism of tumor cell resistance to lysis by syngeneic lymphocytes. Cancer Res 1977; 37: 3945–56. 68. Fidler IJ, Gersten DM, Kripke ML. Influence of the immune status on the metastasis of three murine fibrosarcomas of different immunogenicities. Cancer Res 1979; 39: 3816–21. 69. Fidler IJ, Gruys E, Cifone MA, Barnes Z, Bucana C. Demonstration of multiple phenotypic diversity in a murine melanoma of recent origin. J Natl Cancer Inst 1981; 67: 947–56. 70. Fidler IJ, Kim SJ, Langley RR. The role of the organ microenvironment in the biology and therapy of cancer metastasis. J Cell Biochem 2007; 101: 927–36. 71. Fidler IJ, Kripke ML. Metastasis results from pre-existing variant cells within a malignant tumor. Science 1977; 197: 893–5. 72. Fidler IJ, Langley RR, Kerbel RS, Ellis LM. Biology of cancer: angiogenesis. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: principles and practice of oncology, 7th edt. Philadelphia, PA: Lippincott & Wilkins, 2005; 129–37. 73. Fidler IJ, Nicolson GL. Organ selectivity for implantation survival and growth of B16 melanoma variant tumor lines. J Natl Cancer Inst 1976; 57: 1199–202. 74. Fidler IJ, Poste G. The cellular heterogeneity of malignant neoplasms: implications for adjuvant chemotherapy. Semin Oncol 1985; 12: 207–21. 75. Fidler IJ, Talmadge JE. Evidence that intravenously derived murine pulmonary metastases can originate from the expansion of a single tumor cell. Cancer Res 1986; 46: 5167–71. 76. Fidler IJ, Wilmanns C, Staroselsky A, Radinsky R, Dong Z, Fan D. Modulation of tumor cell response to chemotherapy by the organ environment. Cancer Metastasis Rev 1994; 13: 209–22.

The pathogenesis of cancer metastasis: relevance to therapy 77. Fidler IJ, Yano S, Zhang R-D, Fujimaki T, Bucana CD. The seed and soil hypothesis: vascularization and brain metastases (Personal View). Lancet Oncol 2002; 3: 53–7. 78. Folkman J. How is blood vessel growth regulated in normal and neoplastic tissue? G H A Clowes Memorial Award Lecture. Cancer Res 1986; 46: 467–73. 79. Folkman J. The role of angiogenesis in tumor growth. Semin Cancer Biol 1992; 3: 65–71. 80. Folkman J, Klagsburn M. Angiogenic factors. Science 1987; 235: 444–7. 81. Foulds L. The experimental study of tumor progression: a review. Cancer Res 1954; 14: 327–39. 82. Fracasso PM, Westervelt P, Fears CL, et al. Phase I study of paclitaxel in combination with a multidrug resistance modulator, PSC 833 (Valspodar), in refractory malignancies. J Clin Oncol 2000; 18: 1124–34. 83. Freeman MR, Schneck FX, Gagnon ML, et al. Peripheral blood T-lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res 1995; 55: 4140–5. 84. Furuhashi M, Sjoblom T, Abramsson A, et al. Platelet-derived growth factor production by B16 melanoma cells leads to increased pericyte abundance in tumors and an associated increase in tumor growth rate. Cancer Res 2004; 64: 2725–33. 85. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998; 273: 13313–6. 86. Gerber HP, McMurtrey A, Kowalski J, et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3´-kinase/Akt signal transduction pathway: requirement for Flk-1/KDR activation. J Biol Chem 1998; 273: 30336–43. 87. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res 2003; 314: 15–23. 88. Gerritsen ME. Functional heterogeneity of vascular endothelial cells. Biochem Pharmacol 1987; 36: 2701–11. 89. Ghadimi BM, Grade M, Difilippantonio MJ, et al. Effectiveness of gene expression profiling for response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. J Clin Oncol 2005; 23: 1826–38. 90. Gisselsson D, Pettersson L, Hoglund M, et al. Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity. Proc Natl Acad Sci USA 2000; 97: 5357–62. 91. Gohji K, Fidler IJ, Tsan R, Radinsky R, von Eschenbach AC, Tsuruo T, Nakajima M. Human recombinant interferons-beta and -gamma decrease gelatinase production and invasion by human KG-2 renal carcinoma cells. Int J Cancer 1994; 58: 380–4. 92 Goidl E, ed. Aging and the immune response: cellular and humoral aspects. New York: Marcel Dekker, 1987. 93. Gorunova L, Hoglund M, Andren-Sandberg A, et al. Cytogenetic analysis of pancreatic carcinomas: intratumor heterogeneity and nonrandom pattern of chromosome aberrations. Genes Chromosomes Cancer 1998; 23: 81–99. 94. Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 1993; 62: 385–427. 95. Griffin CA, Hruban RH, Morsberger LA, et al. Consistent chromosome abnormalities in adenocarcinoma of the pancreas. Cancer Res 1995; 55: 2394–9. 96. Guo P, Hu B, Gu W, et al. Platelet-derived growth factor-β enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment. Am J Pathol 2003; 162: 1083–93.

Sun-Jin Kim et al. 97. Gutman M, Singh RK, Xie K, Bucana CD, Fidler IJ. Regulation of IL-8 expression in human melanoma cells by the organ environment. Cancer Res 1995; 55: 2470–5. 98. Gutman, M, Singh RK, Yoon S, Xie K, Bucana CD, Fidler IJ. Leukocyte-induced angiogenesis and subcutaneous growth of B16 melanoma. Cancer Biother 1994; 9: 163–70. 99. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 57–70. 100. Harris AL. Hypoxia – a key regulatory factor in tumour growth. Nat Rev Cancer 2002; 2: 38–47. 101. Hart IR, Fidler IJ. Role of organ selectivity in the determination of metastatic patterns of B16 melanoma. Cancer Res 1980; 40: 2281–7. 102. Hedley D, Pintilie M, Woo J, et al. Carbonic anhydrase IX expression, hypoxia, and prognosis in patients with uterine cervical carcinomas. Clin Cancer Res 2003; 9: 5666–74. 103. Heldin CH, Ostman A, Ronnstrand L. Signal transduction via platelet-derived growth factor receptors. Biochim Biophys Acta 1998; 1378: F79–F113. 104. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999; 79: 1283–316. 105. Herbst RS, Yano S, Kuniyasu H, et al. Differential expression of E-cadherin and type IV collagenase genes predicts outcome in patients with stage I non-small cell lung carcinoma. Clin Cancer Res 2000; 6: 790–7. 106. Herlyn M. Human melanoma: development and progression. Cancer Metastasis Rev 1990; 9: 101–29. 107. Herlyn M, Clark WH, Rodeck U, Mancianti ML, Jambrosic J, Koprowski H. Biology of tumor progression in human melanocytes. Lab Investigation 1987; 56: 461–74. 108. Heuchel R, Berg A, Tallqauist M, et al. Platelet-derived growth factor β receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3′ kinase signaling. Proc Natl Acad Sci USA 1999; 86: 11410–5. 109. Hewitt RE, Powe DG, Carter GI, Turner DR. Desmoplasia and its relevance to colorectal tumour invasion. Int J Cancer 1993; 53: 62–9. 110. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor-growth and angiogenesis. J Clin Oncol 2005; 23: 1011–27. 111. Hofmockel G, Riess S, Bassukas ID, Dammrich J. Epidermal growth factor family and renal cell carcinoma: expression and prognostic impact. Eur Urol 1997; 4: 478–84. 112. Huang S, Van Arsdall M, Tedjarati S, et al. Contributions of stromal metalloproteinase-9 to angiogenesis and growth of human ovarian carcinoma in mice. J Natl Cancer Inst 2002; 94: 1134–42. 113. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350: 2335–42. 114. Hwang RF, Yokoi K, Bucana CD, et al. Inhibition of plateletderived growth factor receptor phosphorylation by STI571 (Gleevec) reduces growth and metastasis of human pancreatic carcinoma in an orthotopic nude mouse model. Clin Cancer Res 2003; 9: 6534–44. 115. Iizuka N, Oka M, Yamada-Okabe H, et al. Oligonucleotide microarray for prediction of early intrahepatic recurrence of hepatocellular carcinoma after curative resection. Lancet 2003; 361: 923–9. 116. Ikeda N, Adachi M, Taki T, et al. Prognostic significance of angiogenesis in human pancreatic cancer. Br J Cancer 1999; 79: 1553–63. 117. Ito M, Yoshida K, Kyo E, et al. Expression of several growth factors and their receptor genes in human colon carcinomas. Virchows Arch B Cell Pathol Incl Mol Pathol 1990; 59: 173–8. 118. Jain R. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005; 307: 58–62. 119. Joseph E, Brobeil A, Glass F, et al. Results of complete lymph node dissection in 83 melanoma patients with positive sentinel nodes. Ann Surg Oncol 1998; 5: 119–25.

37 120. Kadowaki T, Shiozaki H, Inoue M, et al. E-cadherin and α-catenin expression in human esophageal cancer. Cancer Res 1994; 54: 291–6. 121. Kerbel RS. Antiangiogenic therapy: a universal chemosensitization strategy for cancer? Science 2006; 312: 1171–5. 122. Kerbel RS. Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anticancer therapeutic agents. BioEssays 1991; 13: 31–6. 123. Kerbel RS, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2002; 2: 727–39. 124. Killion JJ, Radinsky R, Fidler IJ. Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer Metastasis Rev 1999; 17: 279–84. 125. Kim SJ, Uehara H, Karashima T, Shepherd DL, Killion JJ, Fidler IJ. Blockade of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice. Clin Cancer Res 2003; 9: 1200–10. 126. Kim SJ, Uehara H, Yazici S, et al. Targeting platelet-derived growth factor receptor on endothelial cells of multidrug resistant prostate cancer. J Natl Cancer Inst 2006; 98: 783–93. 127. Kim SJ, Uehara H, Yazici S, et al. Modulation of bone microenvironment with zoledronate enhances the therapeutic effects of STI571 and paclitaxel against experimental bone metastasis of human prostate cancer. Cancer Res 2005; 65: 3707–15. 128. Kim SJ, Uehara H, Yazici S, et al. Simultaneous blockade of platelet-derived growth factor-receptor and epidermal growth factorreceptor signaling and systemic administration of Paclitaxel as therapy for human prostate cancer metastasis in bone of nude mice. Cancer Res 2004; 64: 4201–8. 129. Kitadai Y, Ellis LM, Takahashi Y, et al. Multiparametric in situ mRNA hybridization analysis to detect metastasis-related genes in surgical specimens of human colon carcinoma. Clin Cancer Res 1995; 1: 1095–102. 130. Kitadai Y, Ellis LM, Tucker SL, et al. Multiparametric in situ mRNA hybridization analysis to predict disease recurrence in patients with colon carcinoma. Am J Pathol 1996; 149: 1541–51. 131. Kitadai Y, Sasaki T, Kuwai T, et al. Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential. Int J Cancer 2006; 119: 2567–74. 132. Kitadai Y, Sasaki T, Kuwai T, et al. Targeting the expression of platelet-derived growth factor receptor by reactive stroma inhibits growth and metastasis of human colon carcinoma. Am J Pathol 2006; 169: 2054–65. 133. Kreisle RA, Stebler BA, Ershler WB. Effect of host age on tumorassociated angiogenesis in mice. J Natl Cancer Inst 1990; 82: 44–7. 134. Kripke ML. Speculation on the role of ultraviolet radiation in the development of malignant melanoma. J Natl Cancer Inst 1979; 63: 541–5. 135. Kripke ML. Immunologic mechanisms in UV radiation carcinogenesis. Adv Cancer Res 1981; 34: 69–105. 136. Kripke ML, Fidler IJ. Enhanced experimental metastasis of UV-induced fibrosarcomas in UV-irradiated syngeneic mice. Cancer Res 1980; 40: 625–9. 137. Kumar R, Dong Z, Fidler IJ. Differential regulation of metalloelastase activity in murine peritoneal macrophages by GM-CSF and M-CSF. J Immunol 1996; 157: 5104–11. 138. Kuniyasu H, Ellis LM, Evans DB, et al. Relative expression of E-cadherin and type IV collagenase genes predicts disease outcome in patients with resectable pancreatic carcinoma. Clin Cancer Res 1999; 5: 25–33. 139. Kuniyasu H, Troncoso P, Johnston D, et al. Relative expression of type IV collagenase, E-cadherin, and vascular endothelial growth factor/vascular permeability factor in prostatectomy specimens distinguishes organ-confined from pathologically advanced prostate cancers. Clin Cancer Res 2000; 6: 2295–308. 140. Kuwai T, Kitadai Y, Tanaka S, et al. Expression of hypoxiainducible factor-1alpha is associated with tumor vascularization in human colorectal carcinoma. Int J Cancer 2003; 105: 176–81.

38 141. Kuwai T, Nakamura T, Kim SJ, et al. Intratumoral heterogeneity for expression of tyrosine kinase growth factor receptors in human colon cancer surgical specimens and orthotopic tumors. Am J Pathol 2008; 172: 358–66. 142. Langley RR, Fan D, Tsan RZ, et al. Activation of the plateletderived growth factor receptor enhances survival of murine bone endothelial cells. Cancer Res (Adv in Brief) 2004; 64: 3727–30. 143. Langley RR, Ramirez KM, Tsan RZ, Van Arsdall M, Nilsson MB, Fidler IJ. Tissue-specific microvascular endothelial cell lines from H-2kb-tsA58 mice for studies of angiogenesis and metastasis. Cancer Res 2003; 63: 2971–6. 144. LaRochelle WJ, Jeffers M, McDonald WF, et al. PDGF-D, a new protease-activated growth factor. Nat Cell Biol 2001; 3: 517–21. 145. Lazard D, Sastre X, Frid MG, Glukhova MA, Thiery JP, Koteliansky VE. Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc Natl Acad Sci USA 1993; 90: 999–1003. 146. Lee WH, Morton RA, Epstein JI, et al. Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci USA 1994; 91: 11733–7. 147. Lehnert M. Clinical multidrug resistance: a multifactorial problem. Eur J Cancer 1996; 32A: 912–20. 148. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989; 246: 1306–9. 149. Li X, Ponten A, Aase K, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat Cell Biol 2000; 2: 302–9. 150. Lieubeau B, Garrigue L, Barbieux I, Meflah K, Gregoire M. The role of transforming growth factor beta 1 in the fibroblastic reaction associated with rat colorectal tumor development. Cancer Res 1994; 54: 6526–32. 151. Lindmark G, Sundberg C, Glimelius B, Pahlman L, Rubin K, Gerdin B. Stromal expression of platelet-derived growth factor beta-receptor and platelet-derived growth factor B-chain in colorectal cancer. Lab Invest 1993; 69: 682–9. 152. Lingen MW, Polverini PJ, Bouck N. Inhibition of squamous cell carcinoma angiogenesis by direct interaction of retinoic acid with endothelial cells. Lab Invest 1996; 74: 476–83. 153. Lingen MW, Polverini PJ, Bouck N. Retinoic acid induces cells cultured from oral squamous cell carcinomas to become antiangiogenic. Am J Pathol 1996; 149: 247–58. 154. Liotta LA. Tumor invasion and metastases – Role of the extracellular matrix: Rhoads memorial award lecture. Cancer Res 1986; 46: 1–7. 155. Liotta LA, Kohn EC. The microenvironment of the tumor-host invasion field. Nature 2001; 375–9. 156. Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 1991; 64: 327–36. 157. Luria SE, Delbruck M. Mutations of bacteria from virus sensitivity to virus resistant. Genetics 1943; 28: 491–511. 158. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350: 2129–39. 159. Man S, Bocci G, Francia G, Green S, et al. Anti-tumor and antiangiogenic effects in mice of low-dose (metronomic) cyclophosphamide administered continuously through the drinking water. Cancer Res 2002; 62: 2731–5. 160. Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L. The origin and function of tumor-associated macrophages. Immunol Today 1992; 13: 265–70. 161. Mareel M, Vleminck K, Vermeulen S, et al. Homotypic cell-cell adhesion molecules and tumor invasion. In: Graumann W, Drukker J, eds. Progress in histo- and cytochemistry: Histochemistry of receptors, vol 26. Sutttgart: Fischer Verlag, 1992; 95–106. 162. Martin-Orozco E, Ferragut JA, Garcia-Penarrubia P, FerrerMontiel A. Acquisition of multidrug resistance by L1210 leukemia cells decreases their tumorigenicity and enhances their

The pathogenesis of cancer metastasis: relevance to therapy

163. 164. 165. 166. 167. 168.

169. 170.

171.

172.

173.

174. 175. 176. 177. 178.

179. 180. 181.

182. 183. 184.

susceptibility to the host immune response. Cancer Immunol Immunother 2005; 54: 328–36. Mattern J, Bak M, Volm M. Intrinsic and acquired multidrug resistance in human lung carcinomas grown in nude mice. Anticancer Res 1990; 10: 177–80. Menininger IJ, Zetter BR. Mast cells and angiogenesis. Semin Cancer Biol 1992; 3: 73–9. Micke P, Ostman A. Tumour-stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy? Lung Cancer 2004; 45(Suppl 2): S163–75. Mickisch GH, Noordzij MA, vd Gaast A, et al. Dexverapamil to modulate vinblastine resistance in metastatic renal cell carcinoma. J Cancer Res Clin Oncol 1995; 121(Suppl 3): R11–6. Mickisch GH, Pai LH, Gottesman MM, Pastan I. Monoclonal antibody MRK16 reverses the multidrug resistance of multidrugresistant transgenic mice. Cancer Res 1992; 52: 4427–32. Mickisch GH, Rahman A, Pastan I, Gottesman MM. Increased effectiveness of liposome-encapsulated doxorubicin in multidrug-resistant-transgenic mice compared with free doxorubicin. J Natl Cancer Inst 1992; 84: 804–5. Miguez M, Davel L, deLustig ES. Lymphocyte-induced angiogenesis: correlation with the metastatic incidence of two murine mammary adenocarcinomas. Inv Metastasis 1986; 6: 313–20. Moch H, Sauter G, Buchholz N, et al. Epidermal growth factor receptor expression is associated with rapid tumor cell proliferation and renal cell carcinoma. Hum Pathol 1997; 11: 1255–9. Morikawa K, Walker SM, Jessup JM, Fidler IJ. In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinoma implanted into nude mice. Cancer Res 1988; 48: 1943–8. Morikawa K, Walker SM, Nakajima M, Pathak S, Jessup JM, Fidler IJ. The influence of organ environment on the growth, selection, and metastasis of human colon cancer cells in nude mice. Cancer Res 1988; 48: 6863–71. Moroni M, Veronese S, Benvenuti S, et al. Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to anti-EGFR treatment in colorectal cancer: a cohort study. Lancet Oncol 2005; 6: 279–86. Morrow CS, Cowan KH. Glutathione S-transferase and drug resistance. Cancer Cells 1990; 2: 15–22. Morton DL, Wen D-R, Wong JH, Cochran AJ. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 1992; 127: 392–9. Motzer RJ, Lyn P, Fischer P, et al. Phase I/II trial of dexverapamil plus vinblastine for patients with advanced renal cell carcinoma. J Clin Oncol 1995; 13: 1958–65. Nakajima M, Irimura T, Nicolson GL. Heparanases and tumor metastasis. J Cell Biochem 1988; 36: 157–64. Nakajima M, Morikawa K, Fabra A, Bucana CD, Fidler IJ. Influence of organ environment on extracellular matrix degradative activity and metastasis of human colon carcinoma cells. J Natl Cancer Inst 1990; 82: 1890–8. Nakamura T, Fidler IJ, Coombes KR. Gene expression profile of metastatic human pancreatic cancer cells depends on the organ microenvironment. Cancer Res 2007; 67: 139–48. Nakamura T, Kuwai T, Kitadai Y, et al. Zonal heterogeneity for gene expression in human pancreatic carcinoma. Cancer Res 2007; 67: 7597–604. Nanus DM, Schmitz-Dräger BJ, Motzer RJ, et al. Expression of basic fibroblast growth factor in primary human renal tumors: correlation with poor survival. J Natl Cancer Inst 1994; 85: 1598–9. Nels V, Denzer K, Drenchahn D. Pericyte involvement in capillary sprouting during angiogenesis in situ. Cell Tissue Res 1992; 270: 469–74. Nesbit M, Herlyn M. Adhesion receptors in human melanoma progression. Inv Metastasis 1994–1995; 14: 131–8. Nguyen M, Watanabe H, Budson AE, Richie JP, Hayes DF, Folkman J. Elevated levels of an angiogenic peptide, basic fibroblast growth factor, in the urine of patients with a wide spectrum of cancers. J Natl Cancer Inst 1994; 86: 356–61.

Sun-Jin Kim et al. 185. Nicolson GL. Metastatic tumor cell attachment and invasion assay utilizing vascular endothelial cell monolayer. J Histochem Cytochem 1982; 30: 214–20. 186. Nicolson GL. Tumor cell instability, diversification, and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res 1987; 47: 1473–87. 187. Nicolson GL, Dulski KM. Organ specificity of metastatic tumor colonization is related to organ-selective growth properties of malignant cells. Int J Cancer 1986; 38: 289–94. 188. Nigro JM, Baker SJ, Preisinger AC, et al. Mutations in the p53 gene occur in diverse human tumour types. Nature 1989; 342: 705–8. 189. Nor JE, Polverni PJ. Role of endothelial cell survival and death signals in angiogenesis. Angiogenesis 1999; 3: 101–16. 190. Nowell PC. The clonal evolution of tumor cell populations. Science 1976; 194: 23–8. 191. Ohmori H, Fujii K, Sasahira T, et al. Determinants for prediction of malignant potential by metalloproteinase: E-cadherin ratio in prostate core needle biopsy. Pathobiology 2006; 73: 98–104. 192. Ostman A. PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma. Cytokine Growth Factor Rev 2004; 15: 275–86. 193. Paget S. The distribution of secondary growths in cancer of the breast. Lancet 1889; 1: 571–3. 194. Pasqualini R, Arap W, McDonald DM. Probing the structural and molecular diversity of tumor vasculature. Trends Mol Med 2002; 8: 563–71. 195. Patel PH, Chaganti RS, Motzer RJ. Targeted therapy for metastatic renal cell carcinoma. Br J Cancer 2006; 94: 614–9. 196. Pauli BU, Schwartz DE, Thonar EJM, Kuttner KE. Tumor invasion and host extracellular matrix. Cancer Metastasis Rev 1983; 2: 129–53. 197. Petrylak DP. Chemotherapy for advanced hormone refractory prostate cancer. Urology 1999; 54: 30–5. 198. Petrylak DP, MacArthur RB, O’Connor J, et al. Phase I trial of Docetaxel with estramustine in androgen-independent prostate cancer. J Clin Oncol 1999; 17: 958–67. 199. Pietras K. Increasing tumor uptake of anticancer drugs with imatinib. Semin Oncol 2004; 31: 18–23. 200. Pietras K, Hanahan D. A multi-targeted, metronomic, and maximum-tolerated dose “chemo-switch” regimen is antiangiogenic, producing objective responses and survival benefit in a mouse model of cancer. J Clin Oncol 2005; 23: 939–52. 201. Pietras K, Ostman A, Sjoquist M, et al. Inhibition of plateletderived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors. Cancer Res 2001; 61: 2929–34. 202. Pietras K, Rubin K, Sjoblom T, et al. Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy. Cancer Res 2002; 62: 5476–84. 203. Pietras K, Stumm M, Hubert M, et al. STI571 enhances the therapeutic index of epothilone B by a tumor selective increase of drug uptake. Clin Cancer Res 2003; 9: 3779–87. 204. Polverini PJ. How the extracellular matrix and macrophages contribute to angiogenesis-dependent diseases. Eur J Cancer 1996; 32A: 2430–7. 205. Polverini PJ, Cotran PS, Gimbrone MA Jr. Activated macrophages induce vascular proliferation. Nature 1977; 269: 804–6. 206. Polverini JJ, Leibovich SJ. Induction of neovascularization in vivo and endothelial cell proliferation in vitro by tumor-associated macrophages. Lab Invest 1984; 51: 635–42. 207. Pomeroy SL, Tamayo P, Gaasenbeek M, et al. Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature 2002; 415: 436–42. 208. Poste G. Pathogenesis of metastatic disease: implications for current therapy and for the development of new therapeutic strategies. Cancer Treat Rep 1986; 70: 183–99. 209. Poste G, Fidler IJ. The pathogenesis of cancer metastasis. Nature 1979; 283: 139–46. 210. Poste G, Doll J, Fidler IJ. Interactions among clonal subpopulations affect stability of the metastatic phenotype in polyclonal

39

211.

212.

213. 214.

215. 216. 217.

218. 219. 220. 221. 222. 223. 224. 225.

226. 227.

228.

229.

230. 231.

232.

populations of B16 melanoma cells. Proc Natl Acad Sci USA 1981; 78: 6226–30. Ramadori G, Veit T, Schwogler S, et al. Expression of the gene of the alpha-smooth muscle-actin isoform in rat liver and in rat fatstoring (ITO) cells. Virchows Arch B Cell Pathol Incl Mol Pathol 1990; 59: 349–57. Ripple M, Mulcahy RT, Wilding G. Characteristics of the glutathione/glutathione-S-transferase detoxification system in melphalan resistant human prostate cancer cells. J Urol 1993; 150: 209–14. Risau W, Drexler H, Mironov V, Smits A, Siegbahn A, Funa K, Heldin CH. Platelet-derived growth factor is angiogenic in vivo. Growth Factors 1992; 7: 261–6. Rittmann-Grauer LS, Yong MA, Sanders V, Mackensen DG. Reversal of Vinca alkaloid resistance by anti-P-glycoprotein monoclonal antibody HYB-241 in a human tumor xenograft. Cancer Res 1992; 52: 1810–6. Rønnov-Jessen L, Petersen OW, Bissell MJ. Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction. Physiol Rev 1996; 76: 69–125. Rosenwald A, Wright G, Chan WC, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse largeB-cell lymphoma. N Engl J Med. 2002; 346: 1937–47. Ruiter DJ, Bhan AK, Harrist TJ, Sober AJ, Mihm MC Jr. Major histocompatibility antigens and mononuclear inflammatory infiltrate in benign nevomelanocytic proli and malignant melanoma. J Immunol 1982; 129: 2808–15. Ruoslahti E. Fibronectin and its α5β1 integrin receptor in malignancy. Inv Metastasis 1994–1995; 14: 87–94. Schackert G, Fidler IJ. Site-specific metastasis of mouse melanomas and a fibrosarcoma in the brain or the meninges of syngeneic animals. Cancer Res 1988; 48: 3478–84. Schackert G, Price JE, Zhang RD, Bucana CD, Itoh K, Fidler IJ. Regional growth of different human melanoma as meta-stases in the brain of nude mice. Am J Pathol 1990; 136: 95–102. Sharifi N, Gulley JL, Dahut WL. Androgen deprivation therapy for prostate cancer. J Am Med Assoc 2005; 294: 238–44. Shimizu S, Yamada N, Sawada T, et al. In vivo and in vitro interactions between human colon carcinoma cells and hepatic stellate cells. Jpn J Cancer Res 2000; 91: 1285–95. Sidky YA, Auerbach R. Lymphocyte-induced angiogenesis in tumor-bearing mice. Science 1976; 192: 1237–8. Sieweke MH, Bissell MJ. The tumor-promoting effect of wounding: a possible role for TGF-beta-induced stromal alterations. Crit Rev Oncol 1994; 5: 297–311. Sigalotti L, Fratta E, Coral S, et al. Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2′deoxycytidine. Cancer Res 2004; 64: 9167–71. Singh RK, Bucana CD, Gutman M, Fan D, Wilson MR, Fidler IJ. Organ-site dependent expression of bFGF in human renal cell carcinoma cells. Am J Pathol 1994; 145: 365–74. Singh RK, Gutman M, Bucana CD, Sanchez R, Llansa N, Fidler IJ. Interferons alpha and beta downregulate the expression of basic fibroblast growth factor in human carcinomas. Proc Natl Acad Sci USA 1995; 92: 4562–6. Singh RK, Gutman M, Radinsky R, Bucana CD, Fidler IJ. Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice. Cancer Res 1994; 54: 3242–7. Slaton JW, Perrotte P, Inoue K, Dinney CPN, Fidler IJ. Interferonalpha-mediated downregulation of angiogenesis-related genes and therapy of bladder cancer are dependent on optimization of dose and schedule. Clin Cancer Res 1995; 2726–34. Sloane BF, Honn KV. Cysteine proteinase and metastasis. Cancer Metastasis Rev 1984; 3: 249–65. Smith DC, Esper P, Strawderman M, Redman B, Poenta KJ. Phase II trial of oral estramustine, oral etoposide, and intravenous paclitaxel in hormone-refractory prostate cancer. J Clin Oncol 1999; 17: 1664–71. Solorzano CC, Baker CH, Tsan R, et al. Optimization for the blockade of epidermal growth factor receptor signaling for

40

233.

234. 235. 236. 237.

238.

239. 240. 241. 242.

243. 244. 245. 246. 247.

248. 249. 250. 251.

252.

253.

The pathogenesis of cancer metastasis: relevance to therapy therapy of human pancreatic carcinoma. Clin Cancer Res 2001; 7: 2563–72. Stetler-Stevenson WG. Invasion and metastasis. In: DeVita WT Jr, Hellman S, Rosenberg SA, eds. Cancer: principles and practice of oncology, 7th edt. Philadelphia, PA: Lippincott, Williams & Wilkins, 2005; 113–126. Strohmeyer D, Rossing C, Bauerfeind A, et al. Vascular endothelial growth factor and its correlation with angiogenesis and p53 expression in prostate cancer. Prostate 2000; 45: 216–24. Sugarbaker EV. Cancer metastasis: a product of tumor-host interactions. Curr Probl Cancer 1979; 3: 1–59. Sugarbaker EV, Cohen AM, Ketcham AS. Do metastases metastasize? Ann Surg 1971; 174: 161–70. Sullivan GF, Amenta PS, Villanueva JD, Alvarez CJ, Yang JM, Hait WN. The expression of drug resistance gene products during the progression of human prostate cancer. Clin Cancer Res 1998; 4: 1393–403. Sundberg C, Ljungstrom M, Lindmark G, Gerdin B, Rubin K. Microvascular pericytes express platelet-derived growth factorbeta receptors in human healing wounds and colorectal adenocarcinoma. Am J Pathol 1993; 143: 1377–88. Sunderkötter C, Goebeler M, Schulze-Osthoff K, Bhardwaj R, Sorg C. Macrophage-derived angiogenesis factors. Pharmacol Ther 1991; 51: 195–216. Sunderkötter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol 1994; 55: 410–22. Takahashi Y, Bucana CD, Liu W, et al. Platelet derived endothelial cell growth factor in human colon cancer angiogenesis: role of infiltrating cells. J Natl Cancer Inst 1996; 88: 1091–2. Takahashi Y, Kitadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 1995; 55: 3964–8. Talmadge JE, Fidler IJ. Enhanced metastatic potential of tumor cells harvested from spontaneous metastases of heterogeneous murine tumors. J Natl Cancer Inst 1982; 69: 975–80. Talmadge JE, Wolman SR, Fidler IJ. Evidence for the clonal origin of spontaneous metastasis. Science 1982; 217: 361–3. Tarin D, Price JE, Kettlewell MGW, Souter RG, Vass ACR, Crossley B. Mechanisms of human tumor metastasis studied in patients with peritoneovenous shunts. Cancer Res 1984; 44: 3584–92. Terranova VP, Hujanen ES, Martin GR. Basement membrane and the invasive activity of metastatic tumor cells. J Natl Cancer Inst 1986; 77: 311–6. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebocontrolled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 2005; 366: 1527–37. Theyer G, Schirmbock M, Thalhammer T, et al. Role of the MDR-1-encoded multiple drug resistance phenotype in prostate cancer cell lines. J Urol 1993; 150: 1544–7. Tsuruo T. Mechanisms of multidrug resistance and implications for therapy. Jpn J Cancer Res 1988; 79: 285–96. Tuxhorn JA, Ayala GE, Rowley DR. Reactive stroma in prostate cancer progression. J Urol 2001; 166: 2472–83. Uehara H, Kim SJ, Karashima T, et al. Effects of blocking platelet-derived growth factor-receptor signaling in a mouse model of prostate cancer bone metastasis. J Natl Cancer Inst 2003; 95: 458–70. Uemura A, Ogawa M, Hirashima M, et al. Recombinant angiopoietin-1 restores higher-order architecture of growing blood vessels in mice in the absence of mural cells. J Clin Invest 2002; 110: 1619–28. Uhlman DL, Nguyen P, Manicel JC, et al. Epidermal growth factor receptor and transforming growth factor α expression in papillary and non papillary renal cell carcinoma: correlation with metastatic behavior and prognosis. Clin Cancer Res 1995; 1: 913–20.

254. van Brussel JP, Busstra MB, Lang MS, Catsburg T, Schroder FH, Mickisch GH. A phase II study of temozolomide in hormonerefractory prostate cancer. Cancer Chemother Pharmacol 2000; 45: 509–12. 255. van Brussel JP, Mickisch GHJ. Multidrug resistance in prostate cancer. Onkologie 2003; 26: 175–81. 256. van Brussel JP, van Steenbrugge GJ, Romijn JC, Schroder FH, Mickisch GH. Chemosensitivity of prostate cancer cell lines and expression of multidrug resistance-related proteins. Eur J Cancer 1999; 35: 664–71. 257. van Brussel JP, van Steenbrugge GJ, Van Krimpen C, et al. Expression of multidrug resistance related proteins and proliferative activity is increased in advanced clinical prostate cancer. J Urol 2001; 165: 130–5. 258. van de Vijver MJ, He YD, van’t Veer LJ, et al. A gene expression signature as a predictor of survival in breast cancer. N Engl J Med 2002; 347: 1999–2009. 259. van ‘t Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002; 415: 530–6. 260. Vaupel P, Mayer A, Hockel M. Tumor hypoxia and malignant progression. Meth Enzymol 2004; 381: 335–54. 261. Vermeulen SJ, Bruyneel EA, Bracke ME, et al. Transition from the noninvasive to the invasive phenotype and loss of α-catenin in human colon cancer cells. Cancer Res 1995; 55: 4722–8. 262. Volm M, Koomagi R, Mattern J. Prognostic value of vascular endothelial growth factor and its receptor Flt-1 in squamous cell lung cancer. Int J Cancer 1997; 74: 64–8. 263. Wang Y, Jatkoe T, Zhang Y, et al. Gene expression profiles and molecular markers to predict recurrence of Dukes’ B colon cancer. J Clin Oncol 2004; 22: 1564–71. 264. Weiss L. Metastasis of cancer: a conceptual history from antiquity to the 1990s. Cancer Metastasis Rev 2000; 19: 193–400. 265. Weiss L. Principles of metastasis. Orlando, FL: Academic Press, 1985. 266. Wilhelm SM, Carter C, Tang L, et al. BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/ MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004; 64: 7099–109. 267. Wilmanns C, Fan D, O’Brian CA, Radinsky R, Bucana CD, Tsan R, Fidler IJ. Modulation of doxorubicin sensitivity and level of P-glycoprotein expression in human colon carcinoma cells by ectopic and orthotopic environments in nude mice. Int J Oncol 1993; 3: 413–22. 268. Xu L, Xie K, Mukaida N, Matsushima K, Fidler IJ. Hypoxiainduced elevation in interleukin-8 expression by human ovarian carcinoma cells. Cancer Res 1999; 59: 5822–9. 269. Yokoi K, Kim SJ, Thaker P, et al. Induction of apoptosis in tumorassociated endothelial cells and therapy of orthotopic human pancreatic carcinoma in nude mice. Neoplasia 2005; 7: 696–704. 270. Yokoi K, Sasaki T, Bucana CD, et al. Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model. Cancer Res 2005; 65: 10371–80. 271. Yoshida K, Tosaka A, Takeuchi S, Kobayashi N. Epidermal growth factor receptor content in human renal cell carcinomas. Cancer 1994; 73: 1913–8. 272. Yuan F, Chen Y, Dellian M, Safabakhsh N, Ferrara N, Jain RK. Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody. Proc Natl Acad Sci USA 1996; 93: 14765–70. 273. Zhai BJ, Wu F, Shao ZY, Hu K, Wang ZB. Establishment of human multidrug-resistant hepatocellular carcinoma cell line (HepG2/Adm) and biological characteristics evaluation. Ai Zheng 2004; 23: 391–5.

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

41

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.

References 1. Avner B, Swindell L, Sharp E, et al. Evaluation and clinical relevance of patient immune responses to intravenous therapy with murine monoclonal antibodies conjugated to Adriamycin. Mol Biother 1991; 3(1):14–21. 2. Bernhard MI, Foon KA, Oeltman TN, et al. Guinea pig line 10 hepatocarcinoma model: characterization of monoclonal antibody and in vivo effect of unconjugated antibody and antibody conjugated to diphtheria toxin A chain. Cancer Res 1983; 43:4420–4428. 3. Bernhard MI, Hwang KM, Foon KA, et al. Localization of 111Inand 125I-labeled monoclonal antibody in guinea pigs bearing line 10 hepatocarcinoma tumors. Cancer Res 1983; 43:4429–4433. 4. Bunn PA Jr., Foon KA, Ihde DC, et al. Recombinant leukocyte alpha interferon: an active agent in advanced cutaneous T-cell lymphomas. Ann Intern Med 1982; 101:484–487. 5. Cantell K. In: Burke D, Cantell K, DeMaeyer E, Landy M, Revel M, Vilcek J, eds. Interferon 1 New York: Academic Press, 1981. 6. Carter SK. The clinical trial evaluation strategy for interferons and other biological response modifiers – not a simple task [editorial]. J Biol Response Modif 1982; 1:101–105. 7. Cheever MA, Greenberg PD, Fefer A. Potential for specific cancer therapy with immune T lymphocytes. J Biol Response Modif 1984; 3:113–127. 8. Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002; 346:235–242. 9. Curran WJ, Reasonableness and randomization in clinical trials: fundamental law and governmental regulation [editorial]. N Engl J Med 1979; 300:1273–1274. 10. DeVita VT Jr, Hellman S, Rosenberg SA, eds. Biologic therapy of cancer, second edition. Philadelphia, PA: J.B. Lippincott, 1995; 553–607. 11. DeVita VT Jr, Hellman S, Rosenberg SA, eds. Biologic therapy of cancer, second edition. Philadelphia, PA: J.B. Lippincott, 1995; 553–565. 12. DeVita VT Jr, Hellman S, Rosenberg SA, eds. Biologic therapy of cancer, second edition. Philadelphia, PA: J.B. Lippincott, 1995; 879–890. 13. Dillman RO, Oldham RK, Barth NM, et al. Continuous interleukin-2 and tumor infiltrating lymphocytes as treatment of advanced melanoma. Cancer 1991; 68(1):1–8.

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

66

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,

Kapil Mehta et al.

67

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

Kapil Mehta et al.

69

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

70

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

72

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.

References 1. Aebersold R, Rist B, Gygi SP. Quantitative proteome analysis: methods and applications. Ann N Y Acad Sci 2000;919:33–47. 2. Aggarwal BB, Gutterman JU, eds. Human Cytokines: Handbook for Basic and Clinical Research, Boston, MA, Blackwell, 1992. 3. Aggarwal BB, Pocsik E. Cytokines: From clone to clinic. Arch Biochem Biophys 1992;292:335–359. 4. Aggarwal BB, Puri R. Common and uncommon features of cytokines and cytokine receptors: an overview. In: Human Cytokines: Their Role in Disease and Therapy (Aggarwal BB, Puri R, eds), Cambridge, MA, Blackwell, 1994:pp3–24. 5. Aggarwal BB.Tumour necrosis factors receptor associated signalling molecules and their role in activation of apoptosis, JNK and NF-kappaB. Ann Rheum Dis. 2000;59 Suppl 1:i6–16. 6. Aggarwal BB, Puri R. Common and uncommon features of cytokines and cytokine receptors: an overview. In: Human Cytokines: Their Role in Disease and Therapy (Aggarwal BB, Puri R, eds), Cambridge, MA, Blackwell, 1994:pp3–24. 7. Ahuja SK, Murphy PM. Molecular piracy of mammalian interleukin-8 receptor type B by herpes virus saimiri. J Biol Chem 1993;268:20691–20694. 8. Akira S, Taga T, Kishimoto T.Interleukin-6 in biology and medicine. Adv Immunol, 1993;54: 1–78. 9. Alizadeh AA, Ross DT, Perou CM, van De Rijn M. Towards a novel classification of human malignancies based on gene expression patterns. J Pathol 2001;195:41–52. 10. Alizadeh A, Eisen M, Davis RE. The lymphochip: a specialized cDNA microarray for the genomic-scale analysis of gene expression in normal and malignant lymphocytes. Cold Spring Harb Symp Quant Biol 1999;64:71–78. 11. Alton K, Stabinsky Y, Richards R, et al. Production, characterization and biological effects of recombinant DNA derived human IFN-α and IFN-α analogues. In: The Biology of Interferon System (De Maeyer E, Schellekens H, eds), Elsevier, North Holland, Amsterdam 1983;pp119–127. 12. Ambrus JL, Pippin J, Joseph A, et al. Identification of a cDNA for a human high molecular weight B-cell growth factor. Proc Natl Acad Sci USA 1993;90:6330–6334. 13. Andus T, Gross V, Holstege A, et al. High concentrations of soluble tumor necrosis factor receptors in ascites. Hepatology 1992;16: 749–755. 14. Apelgren LD, Zimmerman DL, Briggs SL, Bumol TF. Antitumor activity of the monoclonal antibody-vinca alkaloid immunoconjugate LY203725 in a nude mouse model of human ovarian cancer. Cancer Res 1990;50:3540–3544. 15. Arai K, Lee F, Miyajima A, et al. Cytokines: Coordinators of immune and inflammatory responses. Annu Rev Biochem 1990; 59:783–836. 16. Arai N, Nomura D, Villaret D, et al. Complete nucleotide sequence of the chromosomal gene for human interleukin 4 and its expression. J Immunol 1989;142: 274–282. 17. Arend WP. Interleukin-1 receptor antagonist. Adv Immunol. 1993;54:167–227. 18. Bajorin DF, Cheung NKV, Houghton AN. Macrophage colony stimulating factor: biological effects and potential applications for cancer therapy. Semin Hematol 1991;28:42–48. 19. Baltathakis I, Alcantara O, Boldt DH. Expression of different NF-kappaB pathway genes in dendritic cells (DCs) or macrophages assessed by gene expression profiling. J Cell Biochem 2001;83:281–290.

76 20. Batra JK, Kasprzyk PG, Bird RE, Pastan I, King RC. Recombinant anti-erbB2 immunotoxins containing Pseudomonas exotoxin. Proc Natl Acad Sci USA 1992;89:5867–5871. 21. Baynes RD, Reddy GK, Shih YJ, Skikne BS, Cook JD. Serum form of the erythropoietin receptor identified by a sequence-specific peptide. Blood 1993;82:2088–2095. 22. Baselga J. Clinical trials of Herceptin (R) (trastuzumab). Eur J Cancer 2001; 37:S18–S24. 23. Bell R. Duration of therapy in metastatic breast cancer: management using Herceptin. Anti-Cancer Drugs 2001;12:561–568. 24. Blumberg H, Conklin D, Xu WF et al. Interleukin −20: discovery, receptor identification, and role in epidermal function. Cell. 2001;104:9–19. 25. Brinkmann U, Pai LH, FitzGerald DH, Willingham MC, Pastan I. B3(Fv)-PE38KDEL, a single chain immunotoxin that causes complete regression of human carcinoma in mice. Proc Natl Acad Sci USA 1991;88:8616–8620. 26. Brinkmann U, Reiter Y, Jung SH, Lee B, Pastan I. A recombinant immunotoxin containing a disulfide-stabalized Fv fragment. Proc Natl Acad Sci USA 1993;90:7538–7542. 27. Brown F. From Jenner to genes-the new vaccines. Lancet 1990;335:587–590. 28. Brown PO, Botstein D. Exploring the new world of the genome with DNA microarrays. Nat Genet 1999;21:33–37. 29. Broxmeyer HE, Benninger L, Cooper S, Huge N, Benjamin RS, Vadhan-Raj S. Effects of in vivo treatment with PIXY321 on proliferation kinetics of bone marrow and blood myeloid progenitor cells in patients with sarcoma. Exp Hematol 1995;23:335–340. 30. Campbell HD, Tucker WQJ, Hort Y, et al. Molecular cloning, nucleotide sequence, and expression of gene encoding human eosinophil differentiation factor (IL-5). Proc Natl Acad Sci USA 1987;84:6629–6633. 31. Cantell K, Hirvonen S, Mogensen KT, Pyhala L. Human leukocyte interferon: production, purification, stability, and animal experiments. Monograph. 1977;3:35–139. 32. Carter P, Presta L, Gorman CM, et al. Humanization of antip185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA 1992;89:4285–4289. 33. Chaffee S, Mary A, Stiehm ER, Girault D, Fischer A, Hershfield MS. IgG antibody response to ethylene glycol-modified adenosine deaminase in patients with ADA deficiency. J Clin Invest 1992;89:1643–1651. 34. Chari RVJ, Martell BA, Gross JL, et al. Immunoconjugates containing novel maytansinoids: promising anticancer drugs. Cancer Res 1992;52:127–131. 35. Chaudhary VK, FitzGerald DJ, Adhaya S, Pastan I. Activity of a recombinant fusion protein between transforming growth factoralpha and Pseudomonas toxin. Proc Natl Acad Sci USA 1987; 84:4538–4542. 36. Chaudhary VK, Queen C, Junghans RP, Waldman TA, FitzGerald DJ, Pastan I. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 1989;339:394–396. 37. Chong CF, Scuderi P, Grimes WJ, et al. Tumor tragets stimulate IL-2 activated killer cells to produce IFN-gamma and TNF. J Immunol 1989;142:2133–2139. 38. Clackson T, Hoogenbbom HR, Griffiths AD, Winter G. Making antibody fragments using phage display libraries. Nature 1991;352: 624–628. 39. Clark BD, Collins KL, Gandy MS, Webb AC, Auron PE. Genomic sequence for human interleukin-1 beta gene. Nucleic Acid Res 1986;14:7897–7914. 40. Claverie JM. Gene number. What if there are only 30,000 human genes? Science 2001; 291: 1255–1257. 41. Coller HA, Grandori C, Tamayo P, Colbert T, Lander ES, Eisenman RN, Golub TR. Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc Natl Acad Sci USA 2000;97: 3260–3265.

Recombinant proteins and genomics in cancer therapy 42. Colombo MP, Forni G. Cytokine gene transfer in tumor inhibition and tumor therapy: where are we now? Immunol Today 1994;15:48–51. 43. Colombo MP, Modesti A, Parmiani G, Forni G. Local cytokine availability elicits tumor rejection and systemic immunity through granulocyte-T lymphocyte cross talk. Cancer Res 1992;52:4853–4857. 44. Conry RM, LoBugilio A, Wright M, et al. Charaterization of a messenger RNA polynucleotide vaccine vector. Cancer Res 1995;55:1397–1400. 45. Conry RM, LoBuglio AF, Kantor J, et al. Immune response to a carcinoembryonic antigen polynucleotide vaccine. Cancer Res 1994;54:1164–1168. 46. Cosman D, Lyman SD, Idzerda RL, Beckmann MP, Park LS, Goodwin RG, March CJ. A new cytokine receptor superfamily. Trends Biochem Sci. 1990;7:265–270. 47. Coussens L, Yang-Feng TL, Liau YC, et al. Tyrosine kinase recptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 1985;230:1132–1139. 48. Crawford J, Ozer H, Stoller R. Reduction by granulocyte colony stimulating factor of fever and neutropenia induced by chemotherapy in patients with small cell lung cancer. N Engl J Med 1991;325:164–170. 49. Cruikshank WW, Kornfeld H, Center DM. Interleukin-16. J Leukoc Biol. 2000;67:757–766. 50. Curtis BM, Williams DE, Broxmeyer HE, et al. Enhanced hematopoietic activity of a human granulocyte/macrophage colony stimulating factor-interleukin-3 fusion protein. Proc Natl Acad Sci USA 1991;88:5809–5813. 51. Cwirla SE, Peters EA, Barrett RW, Dower WJ. Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci USA 1990;87:6378–6382. 52. Darnay BG, Aggarwal BB.Signal transduction by tumour necrosis factor and tumour necrosis factor related ligands and their receptors. Ann Rheum Dis. 1999;58 Suppl 1:I2-I13. 53. Das M, Burge CB, Park E, Colinas J, Pelletier J. Assessment of the total number of human transcription units. Genomics 2001;77:71–78. 54. Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 1989;10:68–91. 55. Debinski W, Puri RK, Kreitman RJ, Pastan I. A wide range of human cancers express interleukin-4 receptors that can be targeted with chimeric toxin composed of IL-4 and Pseudomonas exotoxin. J Biol Chem 1993;268:14065–14070. 56. Derynck R. The physiology of transforming growth factor-β. Adv Cancer Res 1992; 58:27–52. 57. Derynck R, Jarrett JA, Chen EY, et al. Human transforming growth factor-beta cDNA sequence and expression in tumor cell lines. Nature 1985;316:701–705. 58. Dinarello CA. The interleukin-1 family: 10 years of discovery. FASEB J 1994;8:1314–1325. 59. Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood. 1991 Apr 15;77(8):1627–52. 60. DiStefano PS, Johnson EM. Identification of a truncated form of nerve growth factor receptor. Proc Natl Acad Sci USA 1988;85:270–274. 61. Dorai H, McCartney JE, Hudziak RM, Tai M-S, Laminet AA, Houston LL, Huston JS, Oppermann H. Mammalian cell expression of a single chain Fv (sFv) antibody protein and their C-terminal fusions with interleukin-2 and other effector domains. Biotechnology 1995,12:890–897. 62. Downing JR, Rousesel MF, Sherr CJ. Ligand and protein kinase C downmodulate the colony stimulating factor 1 receptor by independent mechanism. Mol Cell Biol 1989;9:2890–2896. 63. Dranoff G, Mulligan C. Gene transfer as cancer therapy. Adv Immunol 1995;58:417–454. 64. Du XX, Williams DA.Interleukin-11: a multifunctional growth factor derived from the hematopoietic microenvironment. Blood 1994;83:2023–2030.

Kapil Mehta et al. 65. Edman JC, Halliwell RA, Valenzuela P, Goodman HM, Rutter WJ. Synthesis of hepatitis B surface and core antigens in E. coli. Nature 1981;291: 503–506. 66. Edwards GM, DeFeo-Jones D, Tai JY, et al. Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion protein. Mol Cell Biol 1989;9:2860–2867. 67. Eschbach JW, Egrie JC, Downing MR, Browne JK, Adamson JW. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. N Engl J Med 1987;316:73–78. 68. Estrov Z, Kurzrock R, Wetzler M, et al. Suppression of chronic myelogenous leukemia colony growth by IL-1 receptor antagonist and soluble IL-1 receptors: a novel application for inhibitors of IL-1 activity. Blood 1991;78:1476–1484. 69. Fantes KH, Allen GJ. Specific activity of pure human interferons and a non-biological method for estimating the purity of highly purified interferon preparations. Interferon Res 1981;1:465–472. 70. Fehniger TA, Caliguiri MA. Interleukin-15; biology and relevance to human diseases. Blood 2001;97:14–32. 71. Feng GS, Gray PW, Shepard HM, Taylor MW. Antiproliferative activity of a hybrid protein between interferon- and tumor necrosis factor-ß. Science 1988;241:1501–1503. 72. Fenton RG, Taub DD, Kwak LW, Smith MR, Longo DL. Cytotoxic T cell response and in vivo protection against tumor cells harboring activated ras prot-oncogenes. J Natl Cancer Inst 1993;85:1294–1302. 73. Fernandez-Botran R. Soluble cytokine receptors: their role in immunoregulation. FASEB J 1191;5:256–2574. 74. Fernadez-Borton R, Vitetta ES. A soluble, high affinity interleukin-4 binding protein is present in the biological fluids of mice. Proc Natl Acad Sci USA 1990;87:4202–4206. 75. FitzGerald DJ, Bjorn MJ, Ferris R, et al. Antitumor activity of an immunotoxin in nude mouse model of human ovarian cancer. Cancer Res 47:1407–1410. 76. FitzGerald DJ, Pastan I. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 1989;339:394–397. 77. Fletcher FA, Williams DE. Recent progress in the discovery and invention of novel hematopoietic cytokines. Crit Rev Oncol Hematol 1992;13:1–15. 78. Fujiwara K, Yoshino T, Miyake K, Ohara N, Akagi T. Expression of lymphocyte adhesion molecule (CD44) in malignant lymphomas. Relevance to primary site, histological subtype and clinical stage. Acta Med Okayama 1993;47:215–222. 79. Fukunaga R, Seto Y, Mizushima S, Nagata S. Three different mRNAs encoding human granulocyte colony-stimulating factor receptor. Proc Natl Acad Sci USA 1990;87:8702–8706. 80. Fulton RJ, Uhr JW, Vitetta ES. In vivo therapy of the BCL1 tumor: effect of immunotoxin valency and deglycosylation of the ricin A chain. Cancer Res 1988;48;2626–2631. 81. Furr BJA, Hutchinson FG. A biodegradable delivery system for peptides: preclinical experience with the gonadotropin-releasing hormone agonist Zoladex. J Controlled Release 1992;21:117–128. 82. Furukawa Y, Ohata M, Miura Y, Saito M. Interleukin-1 production by monocytic leukemia cells and its role in coagulation abnormalities. Leuk Res 1991;15:1133–1137. 83. Furutani Y, Notake M, Fukui T, et al. Complete nucleotide sequence of the gene for human interleukin-1 alpha. Nucleic Acid Res 1986;143:3167–3179. 84. Gabrilove J, Jakubowski A, Grous J, et al. Initial results of a study of rhG-CSF in cancer patients. Exp Hematol 1987;15:461–465. 85. Gadina M, Hilton D, Johnston JA et al. Signaling by type I and II cytokine receptors: ten years after. Curr Opin Immunol 2001;13: 363–373. 86. Gallagher G, Dickensheets H, Eskdale J, Izotova LS, Mirochnitchenko OV, Peat JD, Vazquez N, Pestka S, Donnelly RP, Kotenko SV. Cloning, expression and initial characterization of interleukin-19 (IL-19), a novel homologue of human interleukin-10 (IL-10). Genes Immun. 2000;1:442–450. 87. Ganser A, Lindemann A, Seipelt G, et al. Clinical effects of recombinant interleukin-3. Am J Clin Oncol 1991;14:51–63.

77 88. Gatanaga T, Hwang C, Kohr W, et al. Purification and characterization of an inhibitor (soluble TNF receptor) for TNF and lymphotoxin obtained from the serum infiltrates of human cancer patients. Proc Natl Acad Sci USA 1990;87:8781–8784. 89. Ghetie MA, Vitetta ES. Recent developments in immunotoxin therapy. Curr Opin Immunol 1994;6:707–714. 90. Giri JG, Anderson DM, Kumaki S, Park LS, Grabstein KH, Cosman D. IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2. J Leukoc Biol. 1995;57:763–766. 91. Godon C, Lagniel G, Lee J, Buhler JM, Kieffer S, Perrot M, Boucherie H, Toledano MB, Labarre J. The H2O2 stimulon in Saccharomyces cerevisiae. J Biol Chem 1998;273:22480–22489. 92. Golub TR, Slonim DK, Tamayo P et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999;286: 531–537. 93. Golumbek PT, Lazenby AJ, Levitsky HI, et al. Treatment of established renal cell cancer by tumor cells engineered to secrete interleukin-4. Science 1991;254:713–716. 94. Goodnough LT, Rudnick S, Price TH, et al. Increased preoperative collection of autologous blood with recombinant human erythropoietin therapy. N Engl J Med 1989;321:1163–1168. 95. Goodwin R, Friend D, Ziegler SF, et al. Cloning of the murine and human interleukin receptors: demonstration of a soluble form and homology to a new receptor superfamily. Cell 1990;60:941–951. 96. Goodwin RG, Lupton S, Schmierer A, et al. Human interleukin-7: molecular cloning and growth factor activity on human and murine B-lineage cells. Proc Natl Acad Sci USA 1989; 86:302–306. 97. Gorman CM. Mammalian cell expression. Curr Opin Biotech 1990;1:36–43. 98. Gospodarowicz D. Fibroblast growth factor. Crit Rev Oncog 1989;1:1–26. 99. Gough NM, Gearing DP, King JA, et al. Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia inhibitory factor. Proc Natl Acad Sci USA 1988;85:2623–2627. 100. Grabstein KH, Eisenman J, Shanebeck K, et al. Cloning of a novel T cell growth factor that interacts with the ß chain of the interleukin-2 receptor. Science 1994;264:965–968. 101. Gray A, Dull TJ, Ullrich A. Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000-molecular weight protein precursor. Nature 1983;303:722–725. 102. Gray PW, Aggarwal BB, Benton C, et al. Cloning and expression of cDNA for human lymphotoxin, a lymphokine with tumor necrosis factor activity. Nature 1984;312:721–724. 103. Gray PW, Goeddel DV. Structure of the human immune interferon cDNA. Nature 1982;298:859–863. 104. Griffin JD, Rambaldi A, Vallenga E, Young DC, Ostapovicz D, Cannistra SA. Secretion of interleukin-1 by acute myeloblastic leukemia cells in vitro induces endothelial cells to secrete colony stimulating factors. Blood 1987;70:1218–1221. 105. Griffin TW, Richardson C, Houston LL, LePage D, Bogden A, Raso V. Antitumor activity of intraperitoneal immunotoxins in a nude mouse model of human malignant mesothelioma. Cancer Res 1987;47:4266–4270. 106. Grossbard ML, Press OW, Appelbaum FR, Bernstein ID, Nadler LM. Monoclonal antibody-based therapies of leukemia and lymphoma. Blood 1992;80:863–878. 107. Gubler U, Chua AO, Schoenhaut DS, et al. Coexpression of two distinct genes is required to generate secreted, bioactive cytotoxic lymphocyte maturation factor. Proc Natl Acad Sci USA 1991;88:4143–4147. 108. Gunther N, Betzel C, Weber W. The secreted form of the epidermal growth factor receptor. J Biol Chem 1990;265:22082–22085. 109. Gurney AL, Marsters SA, Huang RM. Identification of a new member of the tumor necrosis factor family and its receptor, a human ortholog of GITR. Curr Biol. 1999;9:215–218.

78 110. Gutterman JU, Blumenshein GR, Alexanian R, et al. Leukocyte interferon-induced tumor regression in human metastatic breast cancer, multiple myeloma, and malignant lymphoma. Ann Intern Med 1980;93:399–406. 111. Gutterman JU, Fine S, Quesada J, et al. Recombinant leukocyte A interferon: pharmacokinetics, single-dose tolerance, and biological effects in cancer patients. Ann Intern Med 1982;96:549–556. 112. Gutterman JU. Cytokine therapeutics: lessons from interferon alpha. Proc Natl Acad Sci USA 1994;91:1198–1205. 113. Hall WA, Merill MJ, Walbridge S, Youle RJ. Epidermal growth factor receptors on ependymomas and other brain tumors. J Neurosurg 1990;72:641–646. 114. Han ZC, Caen JP. Cytokines acting on committed hematopoietic progenitors. Clin Hematol 1994;7:65–89. 115. Heaney ML, Golde DW. Soluble hormone receptors. Blood 1993; 82:1945–1948. 116. Heldin C-H. Structural and functional studies on platelet derived growth factor. EMBO J 1992;11:4251–4259. 117. Heller J. Polymers for controlled parenteral delivery of peptides and proteins. Adv Drug Deliv Rev 1993;10:163–204. 118. Heller RA, Song K, Fan N, et al. The p70 tumor necrosis factor receptor mediates cytotoxicity. Cell 1992;70:47–56. 119. Hellstrom I, Hellstrom KE. Anti-idiotypic antibodies as tumor vaccines. In: New Generation Vaccines (Woodrow GC, Levine MM, eds), New York, Marcel Dekker, 1990;pp863–870. 120. Hersch EM, Metch BS, Muggia FM, et al. Phase II studies of recombinant tumor necrosis factor alpha in patients with malignant disease: a summary of the Southwest Oncology Group experience. J Immunother 1991;10:426–431. 121. Hill ADK, Redmond HP, McCarthy J, Croke DT, Grace PA, Bouchier-Hayes D. Antineoplastic effects of granulocyte macrophage colony stimulating factor. Br J Surg 1992;79:459 (Abstr). 122. Hill ADK, Redmond HP, Croke DT, Grace PA, Bouchier-Hayes D. Cytokines in tumor therapy. Br J Surg 1992;79:990–997. 123. Hill CS, Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 1995;80:199–211. 124. Hinman LM, Hamann PR, Wallace R, Menedez TA, Durr FE, Upeslacis J. Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res 1993;53:3336–3342. 125. Hirano T, Yasukawa K, Harada H, et al. Complementary DNA for a novel human interleukin (BSF-2) that induces B-lymphocytes to produce immunoglobulin. Nature 1986;324:73–76. 126. Hitzeman RA, Chen CY, Hagie FE, Lugovoy JM, Singh A. Yeast: an alternative organism for foreign protein production. In: Recombinant DNA Products (Bollon, AP, ed), Boca Raton, FL, CRC Press, 1983; pp 47–65. 127. Hock H, Dorsch M, Kunzendorf U, et al. Vaccination with tumor cells genetically engineered to produce different cytokines: effectivity not superior to a classical adjuvant. Cancer Res 1993;53:714–716. 128. Horuk R. The interleukin-8-receptor family: from chemokines to malaria. Immunol Today. 1994;15:169–174. 129. Hoshino T, Kawase Y, Okamoto M et al. IL-18 transgenic mice: in vivo evidence of a broad role for IL-18 modulating immune function. J Immunol 2001;166:1014–7018. 130. Ideker T, Thorsson V, Ranish J A. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 2001;292:929–934. 131. Ihle JN. Signaling by the cytokine receptor superfamily in normal and transformed hematopoietic cells. Adv Cancer Res. 1996;68:23–65. 132. International Human Genome Sequence Consortium. Initial sequencing and analysis of the human genome. Nature 2001; 409: 860. 133. Itoh N, Yonehara S, Ishii A, et al. The polypeptide encoded by the cDNA for the human cell surface antigen Fas can mediate apoptosis. Cell 1991;66:233–243. 134. Jacobs AJ, Fer M, Su FM, et al. A phase I trial of a Rhenium 186-labeled monclonal antibody administered intraperitoneal in

Recombinant proteins and genomics in cancer therapy

135. 136. 137.

138. 139. 140. 141. 142. 143. 144.

145. 146. 147.

148. 149.

150. 151. 152. 153.

154.

155.

ovarian carcinoma: toxicity and clinical response. Obstet Gynecol 1993;82:586–593. Jacobs K, Shoemaker C, Rudersdorf R, et al. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 1985;313:806–810. Jinno Y, Chaudhary VK, Kondo T, Adhya S, FitzGerald D, Pastan I. Mutational analysis of domain I of Pseudomonas exotoxin. J Biol Chem 1988;263:13203–13207. Jurcic JC, Scheinberg DA, Houghton AN. Monoclonal antibody therapy of cancer. In: Cancer Chemotherapy and Biological Response Modifiers Annual 14 (Pinedo HM, Longo DL, Chabner BA, eds), Amsterdam Elsevier 1993;pp129–149. Jurcic JG, Scheinberg DA. Recent developments in the radiotherapy of cancer. Curr Opin Immunol 1994;6:715–721. Kalinkowich A, Engelmann H, Harpaz N, et al. Elevated serum levels of soluble tumor necrosis factor in patients with HIV infection. Clin Exp Immunol 1992;89:351–355. Kaplan EH, Leslie WT. Cutaneous T-cell lymphomas. Curr Opin Oncol 1993;5:812. Kaptain S, Tan LK, Chen BY. Her-2/neu and breast cancer. Diag Mol Pathol 2001;10:139–152. Karp JE, Broder S. New directions in molecular medicine. Cancer Res 1994;54:653–665. Katoh M, Yazaki Y, Sugimura T, Terada M. C-erbB3 gene encodes secreted as well as transmembrane receptor tyrosine kinase. Biochem Biophys Res Commun 1993;192:1189–1197. Katre NV. The conjugation of proteins with polyethylene glycol and other polymers: altering properties of proteins to enhance their therapeutic potential. Adv Drug Deliv Rev 1993;10:91–114. Kawasaki ES, Ladner MB, Wang AM, et al. Molecular cloning of a complementary DNA encoding human macrophage-specific colony stimulating factor (CSF-1). Science 1985;230:291–296. Klein B, Wijdenes J, Xang XG, et al. Murine anti-interleukin-6 monoclonal antibody therapy for a patient with plasma cell leukemia. Blood 1991;78:1198–1204. Koff WC, Paige C, Gutterman J, Fidler IJ. Efficient activation of human blood monocytes to a tumoricidal state by liposomes containing human recombinant gamma interferon. Cancer Immunol Immunther 1985;19:85–89. Koppel GA. Recent advances with monoclonal antibody drug targeting for the treatment of human cancer. Bioconjug Chem 1990;1:13–23. Kotenko SV, Izotova LS, Mirochnitchenko OV, Esterova E, Dickensheets H, Donnelly RP, Pestka S. Identification, cloning, and characterization of a novel soluble receptor that binds IL-22 and neutralizes its activity. J Immunol. 2001;166:7096–7103. Kozak RW, Lorberboum GH, Jone L, et al. IL-2-PE40 prevents the development of tumors in mice injected with IL-2 receptor expressing EL4 transfectant tumor cells. J Immunol 1990;145:2766–2771. Krammer PH. CD95’s deadly mission in the immune system. Nature 2000;407:789–795. Kreeger KY. Cancer immunotherapies: an old idea sparks new studies, industry interest. The Scientist 1995;9:1. Kreitman RJ, Bailon P, Cahudhary VK, FitzGerald DJ, Pastan I. Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of interleukin-2 receptor-expressing human carcinoma. Blood 1994;83:426–434. Kreitman RJ, Chang CN, Hudson DV, Queen C, Bailon P, Pastan I. Anti-Tac(Fab)-PE40, a recombinant double chain immunotoxin which kills interleukin-2 receptor bearing cells and induces complete remission in an in vivo tumor model. Int J Cancer 1994;57:856–864. Kreitman RJ, Chaudhary VK, Waldman T, Willingham MC, FitzGerald DJ, Pastan I. The recombinant immunotoxin anti-Tac (Fv)-Pseudomonas exotoxin 40 is cytotoxic towards peripheral blood malignant cells from patients with adult T cell leukemia. Proc Natl Acad Sci USA 1990;87:8291–8299.

Kapil Mehta et al. 156. Kreitman RJ, Hansen HJ, Jones AL, FitzGerald D, Goldenberg DM, Pastan I. Pseudomonas exotoxin-based immunotoxin containing the antibody LL2 or LL2 Fab’ induce regression of subcutaneous human B-cell lymphoma in mice. Cancer Res 1993; 53:819–825. 157. Lachman LB, Dinarello CA, Llansa LD, Fidler IJ. Natural and recombinant human interleukin-1 is cytotoxic for human melanoma cells. J Immunol 1986;136: 3098–3102. 158. Lalli E, Sassone-Corsi P. Signal transduction and gene regulation: the nuclear response to cAMP. J Biol Chem 1994;269:17359–17362. 159. Layton MJ, Cross BA, Metcalf D, Ward LD, Simpson RJ, Nicola NA. A major binding-protein for human leukemia inhibitory factor in normal mouse serum: identification as a soluble form of the cellular receptor. Proc Natl Acad Sci USA 1992;89:8616–8620. 160. Lee VHL. Peptide and protein drug delivery: opportunities and challenges. Pharm Int 1986;208–212. 161. Leitzel K, Teramoto Y, Sampson E, et al. Elevated soluble c-erb-2 antigen levels in the serum and effusions of a proportion of a breast cancer patients. J Clin Oncol 1992;10:1436–1443. 162. Leonard WJ, Shores EW, Love PE. Role of the common cytokine receptor gamma chain in cytokine signaling and lymphoid development. Immunol Rev. 1995 Dec;148:97–114. 163. Lewis R. End of century marks dawn of clinical trial era for cancer vaccines. The Scientist 1995;9:15. 164. Lewis GD, Figari L, Fendly B, Carter P, Gorman C, Shepard M. Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol Immunother 1993;37:255–263. 165. Lewis R. Herceptin earns recognition in breast cancer arsenal. Scientist 2001;15:10–11. 166. Li H, Chen J, Huang A, Stinson J, Heldens S, Foster J, Dowd P, Gurney AL, Wood WI. Cloning and characterization of IL-17B and IL-17C, two new members of the IL-17 cytokine family. Proc Natl Acad Sci U S A 2000;97:773–778. 167. Lienard D, Ewalenko P, Delmotte JJ, et al. High dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J Clin Oncol 1992;10:52–60. 168. Lim VS, DeGowin RL Zavala D, et al. Recombinant human erythropoietin treatment in pre-dialysis patients. Ann Intern Med 1989;110:108–118. 169. Lipshutz RJ, Fodor SP, Gingeras TR, Lockhart DJ. High density synthetic oligonucleotide arrays. Nat Genet 1999;21:20–24. 170. Lippman SM, Parkinson DR, Itri LM, et al. 13-Cis retinoic acid and interferon-α-2a: effective combination therapy for advanced squamous cell carcinoma of the skin. J Natl Cancer Inst 1992;84:235–241. 171. Lodato RF, Feig B, Akimaru K, Soma GI, Klostergaard J. Hemodynamic evaluation of recombinant human tumor necrosis factor (TNF), TNF-SAM2 and liposomal TNFSAM2 in an anesthetized dog model. J Immunother 1995;17:19–29. 172. Lorberboum GH, FitzGerald DJ, Chaudhary VK, Adhya S, Pastan I. Cytotoxic activity of an interleukin 2-Pseudomonas exotoxin chimeric protein produced in Escherichia coli. Proc Natl Acad Sci USA 1988;85:1922–1926. 173. Lord MJ, Roberts LM, Robertus JD. Ricin: structure, mode of action, and some current applications. FASEB J 1994;8:201–208. 174. Lotze MT, Custer MC, Bolton ES, Wiebke EA, Kwakami Y, Rosenberg SA. Mechanisms of immunological antitumor activity: lessons from the laboratory and clinical application. Hum Immunol 1990;28:198–207. 175. Lotze MT, Matory YL, Rayner AA. Clinical effects and toxicity of interleukin-2 in patients with cancer. Cancer 1986;58:2764–2772. 176. Lowman HB, Bass S, Simpson S, Wells JA. Selecting high affinity binding proteins by monovalent phage display. Biochemistry 1991;30:10832–10838. 177. Luckow VA. Recombinant DNA Technology and Applications (Prokop A, Bajpai RK, Ho CS, eds), New York, McGraw Hill, 1991;pp97–152.

79 178. Luckow VA, Summers MD. Trends in the development of baculovirus expression vectors. Biotechniques 1988;6:47–55. 179. Malik N, Kallestad JC, Gunderson NL, et al. Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M. Mol Cell Biol 1989;9:2847–2853. 180. Markussen J, Diers I, Engesgaard A, et al. Soluble, prolonged acting insulin derivatives. II Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B27 and B30. Protein Eng 1987;1:215–223. 181. Marton MJ, DeRisi JL, Bennett HA et al. Drug target validation and identification of secondary drug target effects using DNA microarrays. Nat Med 1998;4:1293–1301. 182. McLaughlin P, Cabanillas, F, Hagemeister FB, et al. CHOP-Bleo plus interferon for stage IV low-grade lymphoma. Ann Oncol 1993;4:205–211. 183. Meredith RF, Khaxaeli MB, Lui T, et al. Dose fractionation of radiolabeled antibodies in patients with metastatic colon cancer. J Nucl Med 1992;33:1648–1653. 184. Meredith RF, Khazaeli MB, Plott WE, et al. Phase I trial of iodine-131 chimeric B72.3 (human IgG4) in metastatic colorectal cancer. J Nucl Med 1992;33:23–29. 185. Miller L. Current status of G-CSF in support of chemotherapy and radiotherapy. Oncology 1993;7:67–88. 186. Milstein C. Monoclonal antibodies (review). Sci Am 1980;243:66–74. 187. Minty A, Chalon P, Derocq JM, et al. Interleukin 13 is a new human lymphokine regulating inflammatory and immune responses. Nature 1993;362:248–250. 188. Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683–765. 189. Morgan AC, Comezoglu FT, Manger R, Jarvis B, Abrams PJ, Sivam G. Immunoconjugates of a protein-synthesis-inhibiting drugs. In: Therapeutic Monoclonal Antibodies (Borrebaeck C, Larrick J, eds), New York, Stockton Press, 1990:pp143–158. 190. Mori I, Hossain MJ, Takeda K, Okamura H, Imai Y, Koshaka S, Kimura Y. Impaired microglial activation in the brain of IL-18 gene-disrupted mice after neurovirulent influenza A virus infection. Virol 2001;287:163–170. 191. Mosley B, Beckman MP, March CJ, et al. The murine interleukin-4 receptor: a molecular cloning and characterization of secreted and membrane-bound forms. Cell 1989;59:335–348. 192. Motoyshi K, Takaku F. Human monocytic colony stimulating factor, phase I/II clinical studies. In: Hematopoietic Growth Factors in Clinical Applications. (Metelsmann R, Hermann F, eds), New York: Marcel Dekker, 1990; pp 161–175. 193. Mukaida N, Shiroo M, Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8. J Immunol 1989;143:1366–1371. 194. Mule JJ, Smith CA, Rosenberg SA. Interleukin 4 (B cell stimulatory factor 1) can mediate the induction of lymphokine activated killer activity directed against fresh tumor cells. J Exp Med 1987;166:792–798. 195. Mullberg J, Schooltink H, Stoyan T, et al. The soluble interleukin-6 receptor is generated by shedding. Europ J Immunol 1993;23:473–480. 196. Nagao M, Masuda S, Abe S, Ueda M, Sasaki R. Production and ligand-binding characteristics of the soluble form of murine erythropoietin receptor. Biochem Biophys Res Commun 1992;188:888–897. 197. Nagata S, Tsuchiya M, Asano S, et al. Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor. Nature 1986;319:415–418. 198. Nakajima T, Yamamoto S, Cheng M, et al. Soluble interleukin-6 receptor is released from receptor-bearing cell lines in vitro. Japan J Cancer Res 1992;83:373–378. 199. Nakamura T, Nishizawa T, Hagiya M, et al. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989;342:440–443.

80 200. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine Growth Factor Rev. 2001;12(1)57–72. 201. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol. 2001;19:423–474. 202. Narazaki M, Yasukawa K, Saiton T, et al. Soluble form of IL-6 signal transducing receptor component gp130 in human serum processing a potential to inhibit signals through membrane anchored gp130. Blood 1993;82:1120–1126. 203. Nedwin GE, Jarrett-Nedwin J, Smith D, et al. Structure and chromosomal localization of the human lymphotoxin gene. J Cell Biochem 1985;29:171–182. 204. Nedwin GE, Naylor SL, Sakaguchi AY, et al. Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acid Res 1985;13:6361–6371. 205. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. The IL-4 receptor: signaling mechanisms and biologic functions. Annu Rev Immunol 1999;17:701–738. 206. Nemunaitis J, Rabinowe SN, Singer JW, et al. Recombinant granulocyte-macrophage colony stimulating factor after autologous bone marrow transplantation for lymphoid cancer. N Engl J Med 1991;324:1773–1778. 207. Niskanen E. Hematopoietic growth factors in clinical hematology. Ann Med 1991;23:615–624. 208. Novick D, Cohen B, Rubinstein M. Soluble interferon-alpha receptor molecules are present in body fluids. FEBS Lett 1993;314:445–448. 209. Novick D, Engelmann H Rubinstein M, Wallach D. Soluble cytokine receptors are present in normal huamn urine. J Exp Med 1989;170:1409–1414. 210. Obreg K, Norheim I, Lind E. Treatment of malignant carcinoid tumors with human leukocyte interferon: long term results. Cancer Treat Rev 1986;70:1297–1304. 211. Oehm A, Behrmann I, Falk W, et al. Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily; sequence identity with the Fas antigen. J Biol Chem 1992;267:10932–10937. 212. Old LJ, ed. Tumor necrosis factor and related cytotoxins. CIBA Foundation Symp. 1987;131:131–137. 213. Oppmann B, Lesley R, Blom B, Timans JC et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity. 2000;13:715–725. 214. Ostade XV, Vandenabeele P, Everaerdt B, et al. Human TNF mutants with selective activity on the p55 receptor. Nature 1993;361:266–269. 215. Ostrand-Rosenberg S. Tumor immunotherapy: the tumor cell as an antigen-presenting cell. Curr Opin Immunol 1994;6:722–727. 216. Pardoll D. Cancer Vaccines. Immunol Today 1993;14:310–316. 217. Parrish-Novak J, Dillon SR, Nelson A et al. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 2000;408:57–63. 218. Park JW, Kirpotin DB, Hong K et al. Tumor targeting using anti her2 immunoliposomes. J Cont Release 2001;74:95–113. 219. Park CC, Morel JCM, Amin MA, Connors MA, Harlow LA, Koch AE. Evidence of IL-18 as a novel angiogenic mediator. J Immunol 2001;167:1644–1653. 220. Pastan I, FitzGerald DJ. Recombinant toxins for cancer treatment. Science 1991;254:1173–1177. 221. Pastan I, Chaudhary V, FitzGerald DJ. Recombinant toxins as novel therapeutic agents. Annu Rev Biochem 1992;61:331–354. 222. Paul SR, Bennet F, Calvetti JA, et al. Molecular cloning of a cDNA encoding interleukin 11, a stromal cell-derived lymphpoietic and hematopoietic cytokine. Proc Natl Acad Sci USA 1990;87: 7512–7516. 223. Pennica D, Nedwin GE, Hayflick JS, et al. Human tumor necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 1984;312:724–729.

Recombinant proteins and genomics in cancer therapy 224. Peoples GE, Goedegebuure PS, Smith R, Linehen DC, Yoshino I, Eberlein TJ. Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same HER1/neu-derived peptide. Proc Natl Acad Sci USA 1995;92:432–436. 225. Pestka S. The human interferons-from protein purification and sequence to cloning and expression in bacteria: before, between and beyond. Arch Biochem Biophys 1983;221:1–37. 226. Pestka S. The purification and manufacture of human interferons. Sci Am 1983;249:36–43. 227. Pestka S, Langer JA, Zoom KC, Samuel CE. Interferons and their actions. Annu Rev Biochem 1987; 56:727–777. 228. Petch L, Harris J, Raymond VW, Blasband A, Lee DC, Earp HS. A truncated secreted form of the epidermal growth factor receptor is encoded by an alternatively spliced transcript in normal rat tissue. Mol Cell Biol 1990;10:2973–2982. 229. Peters K, Vejlsgaard GL. Basal cell and squamous cell carcinoma and Kaposi’s carcinoma. Curr Opin Oncol 1992;4:380–385. 230. Petersen BH, DeHerdt SV, Schneck DW, Bumol TF. The human immune response eto KS1/4-desacetylvinblastin (LY256787) and KS1/4-desacetylvinblastin hydrazide (LY203728) in single and multiple dose clinical studies. Cancer Res 1991;51:2286–2290. 231. Pietersz GA, McKenzie IFC. Antibody conjugates for the treatment of cancer. Immunol Rev 1992;129:57–80. 232. Plautz GE, Yang ZY, Wu BY, Gao X, Huang L, Nabel GJ. Immunotherapy of malignancy by in vivo gene transfer into tumors. Proc Natl Acad Sci USA 1993;10:4645–4649. 233. Pluckthun A. Antibodies from Escherichia coli. Nature 1990; 347:497–498. 234. Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. A model for p53-induced apoptosis. Nature 1997;389:300–305. 235. Porgador A, Tzehoval E, Katz A, et al. Interleukin 6 gene transfection into Lewis lung carcinoma tumor cells suppresses the malignant phenotype and confers immunotherapeutic competence against parental metastatic cell. Cancer Res 1992;52:3679–3686. 236. Press OW, Eary JF, Appelbaum FR, et al. Radiolabeled antibody therapy of B cell lymphoma with autologous bone marrow support. New Engl J Med 1993;329:1219–1224. 237. Quesada JR, Hersh EM, Manning J, et al. Treatment of hairy cell leukemia with recombinant -interferon. Blood 1986;68:493–497. 238. Ramakrishnam S, Bjorn MJ, Houston LL. Recombinat ricin A chain conjugated to monoclonal antibodies: improved tumor cell inhibition in the presence of lysosomotropic compounds. Cancer Res 1989;49:613–617. 239. Renauld JC, Druez C, Kermouni A, et al. Expression cloning of the murine and human interleukin 9 receptor cDNAs. Proc Natl Acad Sci USA 1992;89:5690–5694. 240. Renauld JC, Houssiau F, Louahed J, Vink A, Van Snick J, Uyttenhove C. Interleukin-9. Adv Immunol. 1993;54:79–97. 241. Rinderknecht E, O’Connor BH, Rodriguez H. Natural human interferon gamma. J Biol Chem 1984;259:6790–6797. 242. Rosenberg SA. Immunotherapy and gene therapy of cancer. Cancer Res 1991;51:5074s–5079s. 243. Rosenberg SA, Lotze MT, Muul LM, et al. Observations on the systemic administration of autologous lymphokine activated killer cells and recombinant interleukin-2 of high dose IL-2 alone. N Engl J Med 1985;313:1485–1490. 244. Rosenberg SA, Lotze MT, Yang JC, et al. Combination therapy with interleukin-2 and alpha-interferon for treatment of patients with advanced cancer. J Clin Oncol 1989;7:1863–1874. 245. Rosenberg SA, Spiess P, Lafreniere RA. A new approach to the adoptive immunotherapy of cancer with tumor infiltrating lymphocytes. Science 1986;233:1318–1321. 246. Rosenblum RG, Unger BW, Gutterman JU, Hersh EM, David GS, Frincke JM. Modification of human leukocyte interferon pharmacology with a monoclonal antibody. Cancer Res 1985;45:2421–2424. 247. Ross C, Tingsgaard P, Jorgensen H, Vejlsgaard GL. Interferon treatment of cutaneous T-cell lymphoma. Eur J Haematol 1993;51:63–72. 248. Samakrishnan S, Bjorn MJ, Houston LL. Recombinant ricin A chain conjugated to monoclonal antibodies: improved tumor cell

Kapil Mehta et al.

249. 250. 251.

252. 253.

254. 255. 256. 257. 258. 259. 260. 261.

262.

263. 264.

265.

266.

267. 268. 269. 270. 271.

inhibition in the presence of lysosomotropic compounds. Cancer Res 1989;49:613–617. Sambrook J, Fritsch EF, Manatis T. Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989; pp 5.30–5.90. Sanderson CJ. The biological role of interleukin-5. Int J Cell Cloning 1990;8 (suppl 1): 147–153. Sasaki K, Chiba S, Mano H, Yazaki Y, Hirai H. Identification of a soluble GM-CSF binding-protein in the supernatant of a human choriocarcinoma cell line. Biochem Biophys Res Commun 1992;183:252–257. Scambia G, Panici PB, Battaglia F, et al. Receptors for epidermal growth factor and steroid hormones in primary lyrangeal tumors. Cancer 1991;67:1347–1351. Sceinberg DA, Straus DJ, Yeh SD, et al. A phase I toxicity, pharmacology, and dosimetery trial of monoclonal antibody OKB7 in patients with non-Hodgkin’s lymphoma: effect of tumor burden and antigen expression. J Clin Oncol 1990;8:792–803. Scherf U, Ross DT, Waltham M. A gene expression database for the molecular pharmacology of cancer. Nat Genet 2000;24:236–244. Shaffer AL, Rosenwald A, Hurt EM et al. Signatures of the immune response. Immunity 2001;15:375–385. Sherlock G, Hernandez-Boussard T, Kasarskis A et al. The Stanford Microarray Database. Nucleic Acids Res 2001;29:152–155. Schiro R, Longoni D, Rossi V, et al. Suppression of juvenile chronic myelogenous leukemia colony growth by IL-1 receptor antagonist. Blood 1994;83:460–465. Seigall CB, Xu YH, Cahudary VK, Adhya S, Fitzgerald D, Pastan I. Cytotoxic activities of a fusion protein compromised of TGFα and Pseudomonas exotoxin. FASEB J 1989;3:2647–2652. Sen GC, Lengyel P. The interferon system. J Biol Chem 1992;267:5017–5020. Sharma S, Mehta S, Morgan J, Maizel A. Molecular cloning and expression of a human B-cell growth factor gene in E. coli. Science 1985;235:1489–1492. Shoyab M, Plowman GD, McDonald VL, Bradley JG, Todaro GJ. Structure and function of human amphiregulin: a member of the epidermal growth factor family. Science 1989; 243: 1074–1076. Sica GL, Zhu G, Tamada K, Liu D, Ni J, Chen L. RELT, a new member of the tumor necrosis factor receptor superfamily, is selectively expressed in hematopoietic tissues and activates transcription factor NF-kappaB. Blood. 2001;97:2702–2707. Siegall CB, FitzGerald DJ, Pastan I. Cytotoxicity of IL6-PE40 and derivatives on tumor cells expressing a range of interleukin 6 receptor levels. J Biol Chem 1990;265:16318–16323. Siegall CB, FitzGerald DJ, Pastan I. Selective killing of IL6 receptor bearing myeloma cells using recombinant IL6Pseudomonas toxin. Curr Top Microbiol Immunol 1990;166: 63–69. Siegall CB, Kreitman RJ, FitzGerald DJ, Pastan I. Antitumor effects of interleukin-6 Pseudoman exotoxin chimeric molecules against the human hepatocellular carcinoma. Cancer Res 1991;51:2831–2836. Sievers EL, Appelbaum FR, Spielberger RT et al. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: A phase I study of an anti-CD33 Calicheamicin immunoconjugate. Blood 1999;93:3678–3684. Sims JE, Acres RB, Grubin CE, et al. Cloning of the interleukin-1 receptor from human T-cells. Proc Natl Acad Sci USA 1989;86:8946–8950. Sims JE, March CJ, Cosman D, et al. cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily. Science 1988;241:585–589. Singh A, Ni J, Aggarwal BB.Death domain receptors and their role in cell demise. J Interferon Cytokine Res. 1998;18:439–450. Skolnick AA. First immunotoxin therapy for many common solid tumors enters phase I clinical trial. JAMA 1993;270:2280. Slamon D et al. Use of chemotherapy plus a monoclonal antibody against Her-2 for metastatic breast cancer that overexpresses Her-2. New Eng J Med 2001;344:783–792.

81 272. Smalley RV, Andersen JW, Hawkins MJ, et al. Interferon alpha combined with cytotoxic chemotherapy for patients with nonHodgkin’s lymphoma. N Engl J Med 1992;327:1336–1341. 273. Smith CM Bioinformatics, genomics, and proteomics. Scientist 2000;14:26–28. 274. Smith CA, Davis T, Anderson D, et al. A receptor for human tumor necrosis factor defines an unusual family of cellular and viral proteins. Science 1990;248:1019–1102. 275. Solal-Celigny P, Lepage E, Brousse N, et al. Recombinant interferon alfa-2b combined with a regimen containing doxorubicin in patients with advanced follicular lymphoma. N Engl J Med 1993;329:1608–1614. 276. Souza LM, Boone TC, Gabrilove J, et al. Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 1986;232:61–65. 277. Spencer VA, Davie JR. Signal transduction pathways and chromatin structure in cancer cells. J Cell Biochem (Suppl) 2000; 27–35. 278. Spitler LE. Cancer vaccines: the interferon analogy (editorial). Cancer Biother 1995;10:1–3. 279. Spriggs MK, Hruby DE, Maliszewski CR, et al. Vaccinia and cowpox viruses encode a novel secreted interleukin-1 binding protein. Cell 1992; 71:145–152. 280. Stebbing N, Weck PK. Preclinical assessment of biological properties of recombinant DNA derived interferons. In: Recombinant DNA Products: insulin, interferon, and growth hormone (Bollon AP, ed), Boca Raton, FL, CRC Press, 1984;pp75–114. 281. Stills HF, Bullock BC, Clarkson TB. Increased atherosclerosis and glomerulonephritis in cynomolgus monkeys given injections of BSA over an extended period of time. J Pathol 1983;113:222–234. 282. Strander H. Interferon treatment of human neoplasia. Adv Cancer Res 1986;46:1–265. 283. Strander H, Cantell K, Jakobsson PA, Nilsonn U, Soderberg G. Exogenous interferon therapy of osteogenic sarcoma. Oerhop Scand 1975;45:958–959. 284. Strom TB, Anderson PL, Rubin-Kelley VE, Williams DP, Kijokawa T, Murphy JR. Immunotoxins and cytotokine toxin fusion proteins. Semin Immunol 1990;2:467–479. 285. Su AI, Welsh JB, Sapinoso LM et al. Molecular classification of human carcinomas by use of gene expression signatures. Cancer Res 2001;61:7388–7393. 286. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993;75:1169–1178. 287. Suzuki H, Yasukawa K, Saito T, et al. Anti-human IL-6 receptor antibody inhibits human myeloma growth in vivo. Eur J Immunol 1992;22:1189–1193. 288. Takatsu K. Interleukin 5 and B cell differentiation. Cytokine Growth Factor Rev. 1998 Mar;9(1):25–35. 289. Takaki S, Tominaga A, Hitoshi Y, et al. Molecular cloning and expression of the murine interleukin-5 receptor. EMBO J 1990;9:4367–4374. 290. Talpaz M, Kantarjian HM, McCredie K, Trujillo JM, Keating MJ, Gutterman JU. Hematological remission and cytogenetic improvement induced by recombinant human interferon alpha in chronic myelogenous leukemia. N Engl J Med 1986;314:1065–1069. 291. Talpaz M, O’Brien S, Rose E et al. Phase 1 study of polyethylene glycol formulation of interferon alpha-2B (Schering 54031) in Philadelphia chromosome positive chronic myelogenous leukemia. Blood 2001;98:1708–1713. 292. Talapatra S, Thompson CB. Growth factor signaling in cell survival: implications for cancer treatment. J Pharmacol Exp Ther 2001;298:873–878. 293. Tanaguchi T, Ohno S, Fujii-Kuriyama Y, Murmatsu M. The nucleotide sequence of human fibroblast interferon cDNA. Gene 1980;10:11–15. 294. Tanaka H, Satake IR, Ishikawa M, Matsuki S, Asano K. Pharmacokinetics of recombinant human granulocyte colony stimulating factor conjugated to polyethylene glycol in rats. Cancer Res 1991;51:3710–3714.

82 295. Tao MH, Levy R. Idiotype/granulocyte-macrophage colony stimulating factor fusion protein as a vaccine for a B-cell lymphoma. Nature 1993;362:755–758. 296. Tartaglia LA, Goeddel DV. Two TNF receptors. Immunol Today 1992;13:151–153. 297. TeVelde AA, Huijbens RJF, DeVries JE, Figdor CG. Interleukin 4 inhibits secretion of IL-1, tumor necrosis factor and IL-6 by human monocytes. Blood 1990;76:1392–1398. 298. The International Chronic Granulomatous Disease Comparative Study Group. N Engl J Med 1991;324:509–516. 299. Tiesman J, Hart CE. Identification of a soluble receptor for platelet-derived growth factor in cell-conditioned medium and human plasma. J Biol Chem 1993;268:9621–9628. 300. Tong J, Gordon MS, Srour EF, et al. In vivo administration of recombinant human stem cell factor expands the number of hematopoietic stem cells. Blood 1993;82:784–791. 301. Trail PA, Willner D, Lasch SJ, et al. Cure of xenografted human carcinomas by BR96-doxorubicin conjugates. Science 1993;261: 212–214. 302. Trauth BC, Klas C, Peters AMJ, et al. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 1989;245:301–305. 303. Trillet-Lenoir V, Green J, Manegold C, et al. Recombinant granulocyte colony stimulating factor reduces the infectious complications of cytotoxic chemotherapy. Eur J Cancer 1993;29A:319–324. 304. Trinchieri G. Interleukin-12 and its role in generation of Th1 cells. Immunol Today 1993;14:335–338. 305. Truitt GA, Brunda MJ, Levitt D, Anderson TD, Sherman MI. The therapeutic activity in cancer of interleukin-2 in combination with other cytokines. Current Surv 1989; 8:875–889. 306. Uckun FM, Evans WE, Forsyth CJ, et al. Biotherapy of B-cell precursor leukemia by targeting Geninstein to CD19-associated tyrosine kinases. Science 1995;267:886–891. 307. Uckun FM, Frankel A. The current status of immunotoxins: an overview of experimental and clinical studies as presented at the 3rd International Symposium on Immunotoxicins. Leukemia 1993;7:341–348. 308. Upton C, Mossman K, McFadden G. Encoding of a homolog of the IFN-gamma receptor by myxoma virus. Science 1992; 258:1369–1372. 309. Ushio S, Namba M, Okura T, et al. Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 1996;156:4274–4279. 310. Vadhan-Raj S, Kudelka AP, Garrison L, et al. Effect of interleukin-1 on carboplatin-induced thrombocytopenia in patients with recurrent ovarian cancer. J Clin Oncol 1994;12:707–714. 311. Vadhan-Raj S, Papadopoulous NE, Burgess MA, et al. Effects of PIXY321, a GM-CSF/IL-3 fusion protein, on chemotherapyinduced multilineage myelosuppression in patients with sarcoma. J Clin Oncol 1994;12:71–724. 312. Vadhan-Raj S. PIXY321 (GM-CSF/IL-3 fusion protein) biology and early clinical development. Stem Cells 1994;12:253–261. 313. Valezuela P, Medina A, Rutter WJ, Ammerer G, Hall BD. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 1982;298:347–350. 314. Vallera DA. Immunotoxicins: will their clinical promise be fulfilled? Blood 1994;83:309–314. 315. Van Der Bruggen P, Traversari C, Chomez P, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991; 254:1643–1647. 316. Velculescu VE, Madden SL, Zhang L et al. Analysis of human transcriptomes. Nat Genet 1999;23:387–388. 317. Venter JC et al. The sequence of the human genome.Science Feb 16, 2001: 291. 318. Vieira P, deWaal Malefyt R, Dang W, et al. Isolation and expression of human cytokine synthesis inhibitory factor (CSIF/IL-10) cDNA clones: homology to Epstein Barr virus open reading frame BCRF1. Proc Natl Acad Sci USA 1991;88:1172–1176.

Recombinant proteins and genomics in cancer therapy 319. Vitetta ES, Thrope PE, Uhr JW. Immunotoxicins: magic bullets or misguided missiles. Immunol Today 1993;14:252–259. 320. Vitetta ES, Uhr JW. Monoclonal antibodies as agonists: an expanded role for their use in cancer therapy. Cancer Res 1994;54:5301–5309. 321. Waldmann TA, Goldman CK, Bongiovanni KF, et al. Therapy of patients with human T-cell lymphotropic virus I-induced adult T cell leukemia with anti-Tac, a monoclonal antibody to the receptor for interleukin-2. Blood 1988;72:1805–1816. 322. Waldmann T, Tagaya Y, Bamford R. Interleukin-2, interleukin-15, and their receptors. Int Rev Immunol 1998;16:205–226. 323. Wang H, Marsters SA, Baker T et al. TACI-ligand interactions are required for T cell activation and collagen-induced arthritis in mice. Nat Immunol. 2001;2:632–637. 324. Watson, J (ed). Recombinant DNA in medicine and industry. 1992;453–471. 325. Weber J, Yang JC, Topalian SL, et al. Phase I trial of subcutaneous interleukin-6 in patients with advance malignancies. J Clin Oncol 1993;11:499–506. 326. Welsh JB, Sapinoso LM, Su AI et al. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res 2001;61:5974–5978. 327. Welsh JB, Zarrinkar PP, Sapinoso S et al. Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer. Proc Natl Acad Sci U S A 2001;98:1176–1181. 328. Wetzel R, Heyneker HL, Goeddel DV, et al. Production of biologically active N-desacetylthymosin in E. coli through expression of a chemically synthesized gene. Biochemistry 1980;19: 6096–6104. 329. Whetton AD. The biology and clinical potential of growth factors that regulate myeloid cell production. Trends Pharmacol Sci 1990;11:285–289. 330. Williams DE, Dunn JT, Park LS, Frieden EA, Seiler FR, Farese AM, MacVittie TJ. GM-CSF/IL-3 fusion protein promotes neutrophil and platelet recovery in sublethally irradiated rhesus monkeys. Biotechnol Ther 1993;4:17–29. 331. Williams DE, Eisenman J, Baird A, et al. Identification of a ligand for the c-kit protooncogene. Cell 1990; 63:167–174. 332. Williams GT, Smith CA, Spooncer E, Dexter TM, Taylor DR. Hematopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 1990;343:76–79. 333. Winter G, Milstein C. Man-made antibodies. Nature 1991;349: 293–299. 334. Wong GG, Witek J, Temple PA, et al. Human GM-CSF: molecular cloning and the complementary DNA and purification of the natural and recombinant proteins. Science 1985;228:810–815. 335. Xie MH, Aggarwal S, Ho WH, Foster J, Zhang Z, Stinson J, Wood WI, Goddard AD, Gurney AL. Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2–4 and IL-22R. J Biol Chem. 2000;275: 31335–31339. 336. Xu F, Lupu R, Rodriguez GC, et al. Antibody induced growth inhibition is mediated through immunologically and functionally distinct epitopes on the extracellular domain of the erbB-2R gene product p185. Int J Cancer 1993;53:401–408. 337. Xu W, Presnell SR, Parrish-Novak J, Kindsvogel W et al. A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist. Proc Natl Acad Sci U S A. 2001;98:9511–16. 338. Yan M, Marsters SA, Grewal IS, Wang H, Ashkenazi A, Dixit VM. Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity. Nat Immunol. 2000;1:37–41. 339. Yan M, Wang H, Chan B, Roose-Girma M, Erickson S, Baker T, Tumas D, Grewal IS, Dixit VM. Activation and accumulation of B cells in TACI-deficient mice.Nat Immunol. 2001;2:638–643. 340. Yang YC, Ciarletta AB, Temple PA, et al. Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to human IL-3. Cell 1986;47:3–10.

Kapil Mehta et al. 341. Yanuck M, Carbone DP, Pendleton CD, et al. A mutant p53 tumor suppressor protein is a target for peptide-induced CD8+ cytotoxic T cells. Cancer Res 1993;53:3257–3261. 342. Yin M, Gopal V, Banavali S, Gartside P, Preisler H. Effects of IL-1 receptor antagonist on acute myeloid leukemia. Leukemia 1992;6:898–901. 343. Young DC, Wagner K, Griffin JD. Constitutive expression of the granulocyte macrophage colony stimulating factor gene in acute myeloblastic leukemia. J Clin Invest 1987;79:100–106. 344. Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, Vogelstein B. Identification and classification of p53-regulated genes. Proc Natl Acad Sci U S A 1999;96:14517–14522. 345. Zhu N, Leggitt D, Liu Y, Debs R. Systemic gene expression after intravenous DNA delivery into adult mice. Science 1993; 261:209–211. 346. Zurawski G, de Vries JE.Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today. 1994;15:19–26. 347. Lebedeva IV, Emdad L, Su ZZ, Gupta P, Sauane M, Sarkar D, Staudt MR, Liu SJ, Taher MM, Xiao R, Barral P, Lee SG, Wang D, Vozhilla N, Park ES, Chatman L, Boukerche H, Ramesh R, Inoue S, Chada S, Li R, De Pass AL, Mahasreshti PJ, Dmitriev IP, Curiel DT, Yacoub A, Grant S, Dent P, Senzer N, Nemunaitis JJ, Fisher PB. mda-7/IL-24, novel anticancer cytokine: focus on bystander antitumor, radiosensitization and antiangiogenic properties and overview of the phase I clinical experience. Int J Oncol. 2007 31(5):985–1007. 348. Weiss JM, Subleski JJ, Wigginton JM, Wiltrout RH. Immunotherapy of cancer by IL-12-based cytokine combinations. Expert Opin Biol Ther. 2007;7(11):1705–21.

83 349. Imamichi T, Yang J, Huang DW, Brann TW, Fullmer BA, Adelsberger JW, Lempicki RA, Baseler MW, Lane HC. IL-27, a novel anti-HIV cytokine, activates multiple interferon-inducible genes in macrophages. AIDS. 2008;22(1):39–45. 350. Liu LH, Shan BE, Shao LL, Wang SJ. Inhibitory effect of IL-27 gene on tumor formation activity of Eca109 cells in nude mice and its mechanisms. Ai Zheng. 2008;27(1):12–17. 351. Kim SH, Han SY, Azam T, Yoon DY, Dinarello CA. Interleukin-32: a cytokine and inducer of TNFalpha. Immunity 2005;22(1): 131–142. 352. Marcondes AM, Mhyre AJ, Stirewalt DL, Kim SH, Dinarello CA, Deeg HJ. Dysregulation of IL-32 in myelodysplastic syndrome and chronic myelomonocytic leukemia modulates apoptosis and impairs NK function. Proc Natl Acad Sci U S A. 2008;105(8):2865–2870. 353. Smyth MJ, Teng MW, Sharkey J, Westwood JA, Haynes NM, Yagita H, Takeda K, Sivakumar PV, Kershaw MH. Interleukin 21 enhances antibody-mediated tumor rejection. Cancer Res. 2008 5;68(8):3019–3025. 354. Luo J, Xia Q, Zhang R, Lv C, Zhang W, Wang Y, Cui Q, Liu L, Cai R, Qian C. Treatment of the established tumors induced high level of local expression of IL-24 in tumor cells and resulted in tumor regression. Clin Cancer Res. 2008 15;14(8):2450–2457. 355. Sarkar D, Dent P, Curiel DT, Fisher PB. Acquired and innate resistance to the cancer-specific apoptosis-inducing cytokine, mda-7/IL-24: not insurmountable therapeutic problems. Cancer Biol Ther. 2008;7(1):109–112. 356. Chen C, Sun L, Yu Y, Xue Y and Yang P. Amino acid-coded tagging approaches in quantitative proteomics. Expert RevProteomics 2007; 4(1):25–37.

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

85

86

Current concepts in immunology

Bone Marrow Pluripotent Stem Cell

GEMM–CFC

L-CFU

GM–CSF IL–3 CFU–GM GM–CSF IL–3 IL–5

IL–3 IL–4

GM–CSF IL–3 IL–4 EP

Bursa of Fabricius or equivalent

Thymus

Eosinophil

IL–3 GM–CSF G–CSF

IL–1 IL–2 IL–4 IL–6

IL–3 GM–CSF M–CSF

Basophil

IL–2 IFN–γ

B IL–1 IL–2 IL–4 IL–6

LGL Mφ

BFU–E

GM–CSF IL–3 IL–4

RBC

Pc Th/i PMN

Megakaryocyte

Platelets

Ts

Tc

Ab

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

Robert K. Oldham

87

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

Plt

Monocyte

IL-1 PDGF

LOCAL EFFECTS

Vascular Permeability Leukocyte Adherence Anticoagulant IL-1/ Chemotaxis PGI Synthesis Prolif. Endothelial Cells

Proliferation Proteolytic Enzymes Collagen Synthesis PGE Production

PMN

TNF-a

IMMUNE RESPONSE

IL – 1

IL – 6

TNF – a

T Lymphocyte Activation Cytokine Production Antibody Sythesis

TGF – β T Lymphocyte Proliferation

SYSTEMIC EFFECTS

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

88

Current concepts in immunology

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

Mφ IL–1 IL–6

Ag IL–1 IL–6

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

II IL–4; IFN–γ GM–CSF

TGF–β

Ag

Tind

IL–2; IL–4 IFN–γ

Tact

IL–2; IL–3; IL–4 IL–5; IL–6; IFN–γ

Bact

IL–2; IL–4 IFN–γ

Proliferation Differentiatioin

Proliferation Differentiatioin

Proliferation Differentiation LGL

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

Ts

Ts

Tc

Tc

Tm

Pc

Pc

Th Ab

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.

89

APC MHC Class I or II

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

94

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.

References 1. Allison JP, Lanier LL. Structure, function and serology of the T-cell antigen receptor complex. Annu Rev Immunol 1987; 5:503–540. 2. Asherson GL, Colizzi V, Zembala M. An overview of T-suppressor cell circuits. Annu Rev Immunol 1986; 4:37–68. 3. Baehner RL, Boxer LA, Davis J. The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leukocytes. Blood 1976; 48:309–313. 4. Barlozzari T, Reynolds CW, Herberman RB. In vivo role of natural killer cells: involvement of large granular lymphocytes in the clearance of tumor cells in anti-asialo GM1-treated rats. J Immunol 1983; 131:1024–1027. 5. Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Jr. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci USA 1987; 84:9238–9242. 6. Bich Thuy LT, Dukovich M, Peffer NJ, et al. Direct activation of human resting T cells by IL 2: The role of an Il 2 receptor distinct from the Tac protein. J Immunol 1987; 139:1550–1556. 7. Bierer BE, Mentzer SJ, Greenstein JL, Burakoff SJ. The role of functional cell surface antigens in T cell activation. Year Immunol 1986; 2:39–59. 8. Buckley CE III. Delayed hypersensitivity skin testing. In: Rose NR, Friedman H, Fahey JL, eds. Manual of clinical laboratory immunology. Washington, DC: American Society for Microbiology, 1986; 259–273. 9. Carpenter CB. Lymphocyte-mediated cytotoxicity. In: Rose NR, Friedman H, Fahey JL, eds. Manual of clinical laboratory immunology. Washington, DC: American Society of Microbiology, 304–307. 10. Ceuppens JL, Baroja ML, Lorre K, et al. Human T cell activation with phytohemagglutinin. The function of IL-6 as an accessory signal. J Immunol 1988; 141:3868–3874. 11. Chiu CP, Moulds C, Coffman RL, et al. Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells. Proc Natl Acad Sci USA 1988; 85:7099–7103. 12. Clevers H, Alarcon B, Wileman T, Terhorst C. The T cell receptor/ CD3 complex: a dynamic protein ensemble. Annu Rev Immunol 1988; 6:629–662. 13. Crabtree GR. Contingent genetic regulatory events in T lymphocyte activation. Science 1989; 243:355–361. 14. Crump WL 3d, Own Schaub LB, Grimm EA. Synergy of human recombinant interleukin 1 with interleukin 2 in the generation of lymphokine-activated killer cells. Cancer Res 1989; 49:149–153. 15. Dean JH, Connor R, Herberman RB, et al. The relative proliferation index as a more sensitive parameter for evaluating lymphoproliferative responses of cancer patients to mitogens and alloantigens. Int J Cancer 1977; 20:359–370. 16. Dillman RO, Oldham RK, Tauer KW, et al. Continuous interleukin-2 and lymphokine activated killer cells for advanced cancer: an NBSG trial. J Clin Onc 1991; 9:1233–1240.

Robert K. Oldham 17. Dinarello CA. Interleukin-1 and its biologically related cytokines. Adv Immunol 1989; 44:153–206. 18. Dinarello CA, Mier JW. Lymphokines. N Engl J Med 1987; 317:940–945. 19. Dorf ME, Benacerraf B. Suppressor cells and immunoregulation. Annu Rev Immunol 1984; 2:127–157. 20. Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-ablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoms. J Clin Onc 2005; 23:2346–2357. 21. Dukovich M, Wano Y, Le thi BT, et al. A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors. Nature 1987; 327:518–522. 22. Durum SK, Schmidt JA, Oppenheim JJ. Interleukin 1: an immunological perspective. Annu Rev Immunol 1985; 3:263–287. 23. Dustin ML, Springer TA. Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule- (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J Cell Biol 1988; 107:321–331. 24. Dvorak HF, Galli SJ, Dvorak AM. Cellular and vascular manifestations of cell-mediated immunity. Hum Pathol 1986; 17: 122–137. 25. Fauci AS, Pratt KR. Activation of human B lymphocytes. I. Direct plaque-forming cell assay for the measurement of polyclonal activation and antigenic stimulation of human B lymphocytes. J Exp Med 1976; 144:674–684. 26. Gallin JI, Quie PG. Leukocyte chemotaxis: methods, physiology and clinical implications. New York: Raven Press, 1978. 27. Ganser A, Ottmann OG, Erdmann H, et al. The effect of recombinant human granulocyte-macrophage colony-stimulating factor on neutropenia and related morbidity in chronic severe neutropenia. Ann Intern Med 1989; 111:887–892. 28. Gearing AJ, Johnstone AP, Thorpe R. Production and assay of the interleukins. J Immunol Methods 1985; 83:1–27. 29. Geller RL, Gromo G, Inverardi L, et al. Stepwise activation of T cells. Role of the calcium ionophore A23187. J Immunol 1987; 139:3930–3934. 30. Gillis S. Interleukin 2: biology and biochemistry. J Clin Immunol 1983; 3:1–13. 31. Goldfarb RH, Serrate SA. Natural killer cells. In: Yoshida T, ed. Investigation of cell-mediated immunity. New York: ChurchillLivingston, 1985; 65–80. 32. Grey Hm, Sette A, Buus S. How T cells see antigen. Sci Am 1989; 261:56–64. 33. Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA. Lymphokineactivated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med 1982; 155:1823–1841. 34. Grimm EA, Owen Schaub LB, Loudon WG, Yagita M. Lymphokine-activated killer cells. Induction and function. Ann NY Acad Sci 1988; 532:380–386. 35. Gromo G, Geller RL, Inverardi L, Bach FH. Signal requirements in the step-wise functional maturation of cytotoxic T lymphocytes. Nature 1987; 327:424–426. 36. Gromo G, Inverardi L, Geller RL, et al. The stepwise activation of cytotoxic T lymphocytes. Immunol Today 1987; 8:259–261. 37. Groopman JE, Molina JM, Scadden DT. Hematopoietic growth factors. Biology and clinical applications. N Engl J Med 1989; 321:1449–1459. 38. Han X, Itoh K, Balch CM, Pellis NR. Recombinant interleukin 2 (RIL-4) inhibits interleukin 2-induced activation of peripheral blood lymphocytes. Lymphokine Res 1988; 7:227–235.

97 39. Hanna N, Fidler IJ. Role of natural killer cells in the destruction of circulating tumor emboli. J Natl Cancer Inst 1980; 65:801–809. 40. Harada N, Matsumoto M, Koyama N, et al. T cell replacing factor/ interleukin 5 induces not only B-cell growth and differentiation, but also increased expression of interleukin 2 receptor on activated B-cells. Immunol Lett 1987; 15:205–215. 41. Haynes BF. Human T lymphocyte antigens as defined by monoclonal antibodies. Immunol Rev 1981; 57:127–161. 42. Henkart PA. Mechanism of lymphocyte-mediated cytotoxicity. Annu Rev Immunol 1985; 3:31–58. 43. Herberman RB, Thurman GB. Approaches to the immunological monitoring of cancer patients treated with natural or recombinant interferons. J Biol Response Modif 1983; 2:548–562. 44. Hirano T, Taga T, Yamasaki K, et al. A multifunctional cytokine (IL-6/BSF-2) and its receptor. Int Arch Allergy Appl Immunol 1989; 88:29–33. 45. Hong R. Immunodeficiency. In: Rose NR, Friedman H, Fahey JL, eds. Manual of clinical laboratory immunology. Washington, DC: American Society for Microbiology, 1986; 702–722. 46. IUIS-WHO Nomenclature Subcommittee. Announcement. J Immunol 1985; 134:659–660. 47. Kawase I, Urdal DL, Brooks CG, Henney CS. Selective depletion of NK cell activity in vivo and its effect on the growth of NK-sensitive and NK-resistant tumor cell variants. Int J Cancer 1982; 29:567–574. 48. Kehrl JH, Muraguchi A, Butler JL, et al. Human B cell activation, proliferation and differentiation. Immunol Rev 1984; 78:75–96. 49. Kishimoto T. Factors affecting B-cell growth and differentiation. Annu Rev Immunol 1985; 3:133–157. 50. Kleinerman ES, Schroit AJ, Fogler WE, Fidler IJ. Tumoricidal activity of human monocytes activated in vitro by free and liposome-encapsulated human lymphokines. J Clin Invest 1983; 72:304–315. 51. Koj A. The role of interleukin-6 as the hepatocyte stimulating factor in the network of inflammatory cytokines. Ann NY Acad Sci 1989; 557:1–8. 52. Kovacs EJ, Beckner SK, Longo DL, et al. Cytokine gene expression during the generation of human lymphokine-activated killer cells: Early induction of interleukin 1β by interleukin 2. Cancer Res 1989; 49:940–944. 53. Kumar A, Moreau JL, Baran D, Theze J. Evidence for negative regulation of T cell growth by low affinity interleukin 2 receptors. J Immunol 1987; 138:1485–1493. 54. Kupfer A, Singer SJ. Cell biology of cytotoxic and helper T cell functions: immunofluorescence microscopic studies of single cells and cell couples. Annu Rev Immunol 1989; 7:309–337. 55. Lafreniere R, Rosenberg SA. Adoptive immunotherapy of murine hepatic metastases with lymphokine activated killer (LAK) cells and recombinant interleukin 2 (RIL 2) can mediate the regression of both immunogenic and nonimmunogenic sarcomas and an adenocarcinoma. J Immunol 1985; 135:4273–4280. 56. Lafreniere R, Rosenberg SA. Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant interleukin 2. Cancer Res 1985; 45: 3735–3741. 57. Lasser A. The mononuclear phagocytic system: a review. Hum pathol 1983; 14:108–126. 58. Lipton JN, Nathan DJ. Interactions between lymphocytes and macrophages in hematopoiesis. In: Golde DW, Takaku F, eds. Hematopoietic stem cells. New York: Marcel Dekker, 1980; 145–202. 59. Lotzova E, Herberman RB. Immunobiology of natural killer cells. Boca Raton, FL: CRC Press, 1986.

98 60. Liu W, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4(+) Treg cells. J Exp Med 2006; 203:1701–1711. 61. Maluish AE, Strong DM. Lymphocyte proliferation. In: Rose NR, Friedman H, Fahey JL, eds. Manual of clinical laboratory immunology. Washington, DC: American Society of Microbiology, 1986; 274–281. 62. Mancini G, Carbonara AO, Heremans JF. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1965; 2:235–254. 63. Meriney DK. Methodology of immunologic assays relating to humoral components. In: Grieco MH, Meriney DK, eds. Immunodiagnosis for clinicians. Chicago, IL: Year Book Medical Publishers, 1983; 19–39. 64. Meuer SC, Acuto O, Hercent T, Schlossman SF, Reinherz EL. The human T-cell receptor. Annu Rev Immunol 1984; 2:23–50. 65. Michel RH, Pancake SJ, Noseworthy J, Karnovsky ML. Measurement of rates of phagocytosis: the use of cellular monolayers. J Cell Biol 1969; 40:216–224. 66. Millard PJ, Henkart MP, Reynolds CW, Henkart PA. Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors. J Immunol 1984; 132:3197–3204. 67. Miyajima A, Miyatake S, Schreurs J, et al. Coordinate regulation of immune and inflammatory responses by T cell-derived lymphokines. FASEB J 1988; 2:2462–2473. 68. Moller G. Concanavalin-A-activated lymphocytes suppress immune responses in vitro but are helper cells in vivo. Scand J Immunol 1985; 21:31–34. 69. Mosmann TR, Coffman RL. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989; 7:145–173. 70. Mule JJ, Shu S, Rosenberg SA. The antitumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo. J Immunol 1985; 135:646–652. 71. Muller Eberhard HJ. The molecular basis of target cell killing by human lymphocytes and of killer cell self-protection. Immunol rev 1988; 103:87–98. 72. Munthe-Kaas AC, Kaplan G. Endocytosis by macrophages. In: Carr I, Daems WT, eds. The reticuloendothelial system: a comprehensive treatise. New York: Plenum, 1980; 19–55. 73. Nagler A, Lanier LL, Phillips JH. The effects of IL-4 on human natural killer cells. A potent regulator of IL-2 activation and proliferation. J Immunol 1988; 141:2349–2351. 74. Nathan CF. Secretory products of macrophages. J Clin Invest 1987; 79:319–326. 75. Neckers LM, Cossman J. Transferrin receptor induction in mitogen-stimulated human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2. Proc Natl Acad Sci USA 1983; 80:3494–3498. 76. Ochoa AC, Gromo G, Alter BJ, et al. Long-term growth of lymphokine-activated killer (LAK) cells: role of anti-CD3, beta-IL 1, interferon-gamma and -beta. J Immunol 1987; 138:2728–2733. 77. Ochoa AC, Hasz DE, Rezonzew R, et al. Lymphokine-activated killer activity in long-term cultures with anti-CD3 plus interleukin 2: Identification and isolation of effector subsets. Cancer Res 1989; 49:963–968. 78. OGarra A, Umland S, DeFrance T, Christiansen J. ‘B-cell factors’ are pleiotropic. Immunol Today 1988; 9:45–54. 79. OGarra A, Warren DJ, Sanderson CJ, et al. Interleukin-4 (B cell growth factor-II/eosinophil differentiation factor) is a mitogen and differentiation factor for preactivated murine B lymphocytes. Curr Top Microbiol Immunol 1986; 132:133–141.

Current concepts in immunology 80. Oldham RK. Natural killer cells: history and significance. J Biol Response Modif 1982; 1:217–231. 81. Oldham RK. NK cells: artifact to reality, an odyssey in biology. Can Metas Rev 1983; 2:232–336. 82. Oldham RK, Gail MH, Baker MA, et al. Immunological studies in a double blind randomized trial comparing intrapleural BCG against placebo in patients with resected stage I non-small cell lung cancer. Cancer Immunol Immunother 1982; 13:164–173. 83. Oldham RK, Weese JL, Herberman RB, et al. Immunological monitoring and immunotherapy in carcinoma of the lung. Int J Cancer 1976; 18:739–749. 84. Ortaldo JR, Herberman RB. Heterogeneity of natural killer cells. Annu Rev Immunol 1984; 2:359–394. 85. Ortaldo JR, Mason A, Overton R. Lymphokine-activated killer cells. Analysis of progenitors and effectors. J Exp med 1986; 164:1193–1205. 86. Owen-Schaub L, Yagita M, Tsudo M, et al. Evidence for distinct IL-2 receptors in induction versus maintenance of LAK function. Ann NY Acad Sci 1988; 532:480–481. 87. Paetkau V, Bleackley RC, Riendeau D, et al. Toward the molecular biology of IL-2. Contemp Top Mol Immunol 1985; 10:35–61. 88. Palacios R, Henson G, Steinmetz M, McKearn JP. Interleukin-3 supports growth of mouse pre-B-cell clones in vitro. Nature 1984; 309:126–131. 89. Pasternack MS, Verret CR, Liu MA, Eisen HN. Serine esterase in cytolytic T lymphocytes. Nature 1986; 322:740–743. 90. Perlmutter DH. IFNβ2/IL-6 is one of several cytokines that modulate acute phase gene expression in human hepatocytes and human macrophages. Ann NY Acad Sci 1989; 557:332–342. 91. Poulter LW, Seymour GJ, Duke O, et al. Immunohistological analysis of delayed-type hypersensitivity in man. Cell Immunol 1982; 74:358–369. 92. Raulet DH. The structure, function, and molecular genetics of the gamma/delta T cell receptor. Annu Rev Immunol 1989; 7: 175–207. 93. Reed JC, Alpers JD, Nowell PC, Hoover RG. Sequential expression of proto-oncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc Natl Acad Sci USA 1986; 83:3982–3986. 94. Reed JC, Prystowsky MB, Kern JA, et al. Regulation of protooncogene expression during lymphocyte activation and proliferation. In: Gupta S, Paul WE, Fauci AS, eds. Advances in experimental medicine and biology. New York: Plenum, 1986; 249–262. 95. Reinherz EL, Schlossman SF. Current concepts in immunology: regulation of the immune response – inducer and suppressor T-lymphocyte subsets in human beings. N Engl J Med 1980; 303:370–373. 96. Reinherz EL, Schlossman SF. The characterization and function of human immunoregulatory T lymphocyte subsets. Immunol Today 1981; 2:69–73. 97. Robb RJ, Greene WC. Internalization of interleukin 2 is mediated by the beta chain of the high-affinity interleukin 2 receptor. J Exp Med 1987; 165:1201–1206. 98. Robb RJ, Rusk CM, Yodoi J, Greene WC. Interleukin 2 binding molecule distinct from the Tac protein: analysis of its role in formation of high-affinity receptors. Proc Natl Acad Sci USA 1987; 84:2002–2006. 99. Rocklin RE, Meyers OL, David JR. An in vitro assay for cellular hypersensitivity in man. J Immunol 1970; 104:95–102. 100. Rosenberg SA, Eberlein TJ, Grimm EA, et al. Development of long-term cell lines and lymphoid clones reactive against murine and human tumors: a new approach to the adoptive immunotherapy of cancer. Surgery 1982; 92:328–336.

Robert K. Oldham 101. 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. 102. Rosenberg SA, Mule JJ. Immunotherapy of cancer with lymphokine-activated killer cells and recombinant interleukin-2. Surgery 1985; 98:437–444. 103. Rosenberg SA, Mule JJ, Spiess PJ, et al. Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2. J Exp Med 1985; 161:1169–1188. 104. Rosenblum MG, Donato NJ. Tumor necrosis factor α: A multifaceted peptide hormone. CRC Crit Rev Immunol 1989; 9:21–44. 105. Schatten S, Granstein RD, Drebin JA, Greene MI. Suppressor T cells and the immune response to tumors. CRC Crit Rev Immunol 1984; 4:335–379. 106. Schwartz RH. T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Annu Rev Immunol 1985; 3:237–261. 107. Seddiki N, et al. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006; 203:1693–1700 108. Shipp MA, Reinherz EL. Differential expression of nuclear proto-oncogenes in T cells triggered with mitogenic and nonmitogenic T3 and T11 activation signals. J Immunol 1987; 139:2143–2148. 109. Sieff CA. Hematopoietic growth factors. J Clin Invest 1987; 79:1549–1557. 110. Smith CW, Rothlein R, Hughes BJ, et al. Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration. J Clin Invest 1988; 82:1746–1756. 111. Smith KA. Dissection of the molecular events occurring during T cell cycle progression. In: Gupta S, Paul WE, and Fauci AS, eds. Advances in experimental medicine and biology. New York: Plenum, 1986; 125–128. 112. Smith KA. The interleukin 2 receptor. Adv Immunol 1988; 42:165–179. 113. Smith KA. Interleukin-2: inception, impact, and implications. Science 1988; 240:1169–1176. 114. Spencer RP, Pearson HH. Radionuclide studies of the spleen. Cleveland: CRC Press, 1975. 115. Springer TA, Dustin ML, Kishimoto TK, Marlin SD. The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system. Annu Rev Immunol 1987; 5:223–252. 116. Taniguchi T. Regulation of cytokine gene expression. Annu Rev Immunol 1988; 6:439–464. 117. Te Velde AA, yard BA, Klomp JP, et al. Modulation of phenotypic and functional properties of human peripheral blood

99

118.

119.

120.

121. 122.

123. 124.

125. 126. 127.

128. 129.

130.

131. 132.

133. 134.

monocytes by interleukin-2 (IL-4). Agents Actions 1989; 26:199–200. Teshigawara K, Wang HM, Kato K, Smith KA. Interleukin 2 high-affinity receptor expression requires two distinct binding proteins. J Exp Med 1987; 165:223–238. Tosato G, Pike SE. Interferon-beta 2/interleukin 6 is a co-stimulant for human T lymphocytes. J Immunol 1988; 141: 1556–1562. Tracey DE. Macrophage mediated injury. In: Rose NR, Siegel BV, eds. The reticuloendothelial system: a comprehensive treatise. New York: Plenum, 1983; 77–101. Trinchieri G, Perussa B. Human natural killer cells: biologic and pathologic aspects. Lab Invest 1984; 50:489–513. Tsudo M, Kozak RW, Goldman CK, Waldmann TA. Demonstration of a non-Tac peptide that binds interleukin 2: a potential participant in a multichain interleukin 2 receptor complex. Proc Natl Acad Sci USA 1986; 83:9694–9698. Unanue ER. Antigen-presenting function of the macrophage. Annu Rev Immunol 1984; 2:395–428. Wahl SM, McCartney-Francis N, Mergenhagen SE. Inflammatory and immunomodulatory roles of TGF-β. Immunol Today 1989; 10:258–261. Waldmann TA, Broder S. Suppressor cells in the regulation of the immune response. Prog Clin Immunol 1977; 3:155–199. Weiss A, Imboden JB. Cell surface molecules and early events involved in human T lymphocyte activation. Adv Immunol 1987; 41:1–38. 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. Whitlock C, Denis K, Robertson D, Witte O. In vitro analysis of murine-B-cell development. Annu Rev Immunol 1985; 3:213–235. Woodruff JJ, Clarke LM, Chin YH. Specific cell-adhesion mechanisms determining migration pathways of recirculating lymphocytes. Annu Rev Immunol 1987; 5:201–222. Yang SC, Owen Schaub L, Grimm EA, Roth JA. Induction of lymphokine-activated killer cytotoxicity with interleukin-2 and tumor necrosis factor-alpha against primary lung cancer targets. Cancer Immunol Immunother 1989; 29:193–198. Young JD, Liu C. Multiple mechanisms of lymphocyte mediated killing. Immunol Today 1988; 9:140–144. Zlotnik A, Fischer M, Roehm N, Zipori D. Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages. J Immunol 1987; 138:4275–4279. Zola H. The surface antigens of human B lymphocytes. Immunol Today 1987; 8:308–310. Zucali JR, Broxmeyer HE, Gross MA, Dinarello CA. Recombinant human tumor necrosis factors alpha and beta stimulate fibroblasts to produce hemopoietic growth factors in vitro. J Immunol 1988; 140:840–844.

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

101

102

Therapeutic approaches to cancer-associated immune suppression

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

104

Therapeutic approaches to cancer-associated immune suppression

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

106

Therapeutic approaches to cancer-associated immune suppression

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.

107

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].

108

Therapeutic approaches to cancer-associated immune suppression

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

110

Therapeutic approaches to cancer-associated immune suppression

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

112

Therapeutic approaches to cancer-associated immune suppression

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

114

Therapeutic approaches to cancer-associated immune suppression

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.

116

Therapeutic approaches to cancer-associated immune suppression

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

118

Therapeutic approaches to cancer-associated immune suppression

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

120

Therapeutic approaches to cancer-associated immune suppression

[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.

122

Therapeutic approaches to cancer-associated immune suppression

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].

123

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

124

Therapeutic approaches to cancer-associated immune suppression

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.

125

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

126

Therapeutic approaches to cancer-associated immune suppression

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].

Robert K. Oldham

127

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].

128

Therapeutic approaches to cancer-associated immune suppression

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].

129

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

130

Therapeutic approaches to cancer-associated immune suppression

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.

References 1. Addison I, Baggage J, Gandossini M, Souhami R. Assessment of host defense against infection during chemotherapy of Hodgkin’s disease. Cancer Chemother Pharmacol 1978; 1:129–133. 2. Adler A, Stein JA, Ben-Efraim S. Immunocompetence, immunosuppression, and human breast cancer, II. Further evidence of initial immune impairment by integrated assessment effect of nodal involvement (N) and of primary tumor size (T). Cancer 1980; 45:2061–2073. 3. Adler A, Stein JA, Ben-Efraim S. Immunocompetence, immunosuppression, and human breast cancer. III. Prognostic significance of initial level of immunocompetence in early and advanced disease. Cancer 1980; 45:2074–2083. 4. Ahmed K, Turk JL. Effect of anticancer agents neothramycin, aclacinomycin, FK-565 and FK-156 on the release of interleukin-2 and interleukin-1 in vitro. Cancer Immunol Immunother 1989; 28:87–92. 5. Alberola V, Gonzalez-Molina A, Trenor A, et al. Mechanism of suppression of the depressed lymphocyte response in lung cancer patients. Allergol Immunopathol (Madr) 1985; 13:213–219. 6. Albin RJ, Gordon MJ, Soane WA. Levels of immunoglobulins in the serum of patients with carcinoma of the prostate. Neoplasma 1972; 19:57–60. 7. Alexopoulos LG, Wiltshaw E. Immunological monitoring during chemotherapy for advanced Hodgkin’s disease. Cancer 1978; 42:2631–2640. 8. Alsabti EA. In vivo and in vitro assays of incompetence in bronchogenic carcinoma. Oncology 1979; 36:171–175. 9. Amery WK, Butterworth BS. The dosage regimen of levamisole in cancer: Is it related to efficacy and safety? Int J Immunopharmacol 1983; 5:1–9. 10. Amery WK, Cosemans J, Gooszen HC, et al. Four-year results from double-blind study of adjuvant levamisole treatment in resectable lung cancer. In: Terry WD, Rosenberg SA, eds. Immunotherapy of human cancer. New York: Elsevier/North-Holland, 1982: 123–133. 11. Amlot PL, Anger A. Binding of phytohaemagglutinin to serum substances and inhibition of lymphocyte transformation in Hodgkin’s disease. Clin Exp Immunol 1976; 26:520–527. 12. Anderson TC, Jones SE, Sochnlen BJ, et al. Immunocompetence and malignant lymphomas: Immunologic status before therapy. Cancer 1981; 48:2702–2709. 13. Anderson TM, Ibayashi Y, Tokuda Y, et al. Natural killer activity of lymphocytes infiltrating human lung cancers following preoperative systemic recombinant interleukin-2. Arch Surg 1987; 122:12; 1446–1450.

Robert K. Oldham 14. Anderson TM, Ibayashi Y, Holmes EC, Golub SH. Modification of natural killer activity of lymphocytes infiltrating human lung cancers. Cancer Immunol Immunother 1987; 25:65–68, 15. Anthony HM, Templeman GH, Madsen KE, Mason MK. The prognostic significance of DHS skin tests in patients with carcinoma of bronchus. Cancer 1974: 34(6) 1901–1906. 16. Anthony HM. Yorkshire trial of adjuvant therapy with levamisole in surgically treated lung cancer. In: Terry WD, Rosenberg SA, eds. Immunotherapy of human cancer. New York: Elsevier/NorthHolland, 1982:135–440. 17. Aparicio-Pages NM, Verspaget HW, Pena SA, Lamers CB. Impaired local natural killer-cell activity in human colorectal carcinomas. Cancer Immunol Immunother 1989; 28:4; 301–304. 18. Aparicio-Pages NM, Verspaget HW, Pena SA, Lamers CBHW. Impaired local natural killer cell activity in human colorectal carcinomas. Cancer Immunol Immunother 1989; 28:301–304. 19. Aranha GV, McKhann CF, Simmons RL, Grage TB. Recall skin test antigens and the prognosis of stage I melanoma. J Surg Oncol 1976; 11:13–16. 20. Arinaga S, Akiyoshi T, Tsuji H. Augmentation of the generation cell mediated cytotoxicity after a single dose of adriamycin in cancer patients. Cancer Res 1986; 46:4213–4216. 21. Arinaga S, Akiyoshi T, Tsuji H. Augmentation of the cell-mediated cytotoxic response induced in mixed cell culture by adriamycin. Jpn J Cancer Res 1985; 76:414–419. 22. Armstrong D, Chmel M. Infectious complications of Hodgkin’s disease. In: Lacher MJ, ed. Hodgkin’s disease. New York: Wiley, 1976:267–290. 23.Arnaud JP, Buyse M, Nordlinger B, et al. Adjuvant therapy of poor prognosis colon cancer with levamisole: Results of an EORTC double-blind randomized clinical trial. Br J Surg 1989; 76:284–289. 24. Ascher MS, Schneider WJ, Valentine FT, Lawrence HS. In vitro properties of leukocyte dialysates containing transfer factor. Proc Natl Acad Sci USA 1974; 74:1178–1182. 25. Ashorn RG, Vandenbard AA, Acott KM, Krohn KJ. Dialysable leukocyte extracts (transfer factor) augment non-specifically keyhole limpet haemocyanin and horseshoe crab haemocyanin skin reactivity in immunized human recipients. Scand J Immunol 1976; 23:161–167. 26. Balaram P, Remani P, Padmanabhan TK, Vasudevan DM. Role of levamisole immunotherapy as an adjuvant to radiotherapy in oral cancer: Immune responses. Neoplasma 1988; 35:6; 617–625. 27. Balaram P, Padmanabhan TK, Vasudevan DM. Role of levamisole immunotherapy as an adjuvant to radiotherapy in oral cancer. II. Lymphocyte subpopulations. Neoplasma 1988; 35:2; 235–242. 28. Balch CM, Doughtery PA, Cloud GA, Tilden AB. Prostaglandin E2-mediated suppression of cellular immunity in colon cancer patients. Surgery 1984; 95:71–77. 29. Balch CM, Tilden AB, Dougherty PA, Cloud GA. Depressed levels of granular lymphocytes with natural killer (NK) cell function in 247 cancer patients. Ann Surg 1983; 198:192–199. 30. Balch CM, Dougherty PA, Tilden AB. Excessive prostaglandin E2 production by suppressor monocytes in head and neck cancer patients. Ann Surg 1982; 196:645–650. 31. Balm FA, Drexhage HA, von Blomberg M, et al. Mononuclear phagocytic function in head and neck cancer. Cancer 1984; 54:1010–1015. 32. Bancewicz J, Calman K, MacPherson SG, et al. Adjuvant chemotherapy and immunotherapy for colorectal cancer; preliminary communication. J R Soc Med 1980; 73:197–199. 33. Baral E, Blomgren H, Petrini B, Wasserman J. Blood lymphocytes in breast cancer patients following radiotherapy and surgery. Int J Radiat Oncol Biol Phys 1977; 2:289–295.

131 34. Baral E, Blomgren H, Petrini B, et al. Prognostic relevance of immunologic variables in breast carcinoma. Acta Radiol Ther Phys Biol 1977; 16:417–426. 35. Baseler MW, Maxim PE, Veltri RW. Circulating IgA immune complexes in head and neck cancer, nasopharyngeal carcinoma, lung cancer, and colon cancer. Cancer 1987; 59:1727–1731. 36. Baskies AM, Chretien PB, Weiss JF, et al. Serum glycoproteins in cancer patients: First report of correlations with in vitro and in vivo parameters and cellular immunity. Cancer 1980; 45:3050–3060. 37. Bast RC, Reinherz EL, Maven C, et al. Contrasting effects of cyclophosphamide and prednisolone on the phenotype of human peripheral blood leukocytes. Clin Immunol Immunopathol 1983; 28:101–114. 38. Baumann MA, Milson TJ, Patrick CW, et al. Correlation of circulating natural killer cell count with prognosis in large cell lymphoma. Cancer 1986; 57:2309–2312. 39. Bean MA, Schelhammer PS, Her HW, et al. Immunocompetence of patients with transitional cell carcinoma as measured by DNCB skin tests and in vitro lymphocyte function. Natl Cancer Inst Monogr 1978; 49:111–114. 40. Bedikian AY, Patt YZ, Murphy WK, et al. Prospective evaluation of thymosin fraction V immunotherapy in patients with non-small cell lung cancer receiving vindesine, doxorubicin, and cisplatin (VAP) chemotherapy. Am J Clin Oncol 1984; 7:399–404. 41. Benkovic B, Burek B, Jaksic B, Vitale B. Modulation of chronic lymphocytic leukemia (CLL) lymphocyte phenotypes by in vitro incubation with alpha-1 thymosin. Blood Cells 1987; 12:2; 441–455. 42. Bennett A, Berstock A, Harris M, et al. Prostaglandins and their relationship to malignant and benign breast tumors. In: Sammuelsson B, Ramuell PW, Paoletti R, eds. Advances in prostaglandin and thromboxane research vol 6. New York: Raven Press, 1980:595–599. 43. Berd D, Macguire HC Jr, Mastrangelo MJ. Induction of cell-mediated immunity to autologous melanoma cells and regression of metastases after treatment with a melanoma cell vaccine preceded by cyclophosphamide. Cancer Res 1986; 46:2572–2577. 44. Berd D, Mastrangelo MJ, Enstron PF, et al. Augmentation of the human immune response by cyclophosphamide. Cancer Res 1982; 42:4862–4866. 45. Berd D, Mastrangelo MJ. Effect of low dose cyclophosphamide on the immune system of cancer patients: Reduction of T-suppressor function without depletion of the CD8+ subset. Cancer Res 1987; 47:3317–3321. 46. Berd D, Maguire HC, Mastrangelo MJ. Impairment of concanavalin A-inducible suppressor activity following administration to patients with advanced cancer. Cancer Res 1984; 44:1275–1280. 47. Berenyl E, Kavai M, Szabolcsi M, Szegedi G. Levamisole in the treatment of Hodgkin’s disease. Acta Med Acad Sci Hung 1979; 36:177–185. 48. Bergmann L, Mitrou PS, Demmer-Dieckmann M, et al. Impaired T- and B-cell functions in patients with Hodgkin’s disease. Cancer Immunol Immunother 1987; 25:59–64. 49. Berkarda B, Bouffard-Eyaboglu H, Derman U. The effect of coumarin derivatives on the immunological system of man. Agents Actions 1983; 13:50–52. 50. Berlinger NT. Deficient immunity in head and neck cancer due to excessive monocyte production of prostaglandins. Laryngoscope 1984; 94:11, Pt.1; 1407–1410. 51. Bernengo MG, Fra P, Lisa F, Meregilli M, Zina G. Thymostimulin therapy in melanoma patients: Correlation of immunologic effects with clinical course. Clin Immunol Immunopathol 1983; 28:311–324.

132

Therapeutic approaches to cancer-associated immune suppression

52. Bernengo MG, Lisa F, Meregalli M, Doveil GC. Changes in Tand B-lymphocyte subpopulations before, during and after chemotherapy for malignant melanoma. Int J Tissue React 1984; 6:505–511. 53. Bicker U. Immunomodulation by 2-cyanaziridine derivatives. In: Serrou B, Rosenfeld C, Daniels JC, Saunder JP, eds. Current concepts in human immunology and cancer immunomodulation. Amsterdam: Elsevier Biomedical, 1982:521–534. 54. Bier J, Nicklisch U, Platz H. The doubtful relevance of nonspecific immune reactivity in patients with squamous-cell carcinoma of the head and neck region. Cancer 1983; 52:1165–1172. 55. Bjorkholm M, Wedelin C, Holm G, et al. Lymphocytotoxic serum factors and lymphocyte functions in untreated Hodgkin’s disease. Cancer 1982; 50:2044–2048. 56. Bjorkholm M, Holm G, Mellstedt H. Persisting lymphocyte deficiencies during remission in Hodgkin’s disease. Clin Exp Immunol 1977; 28:389–393. 57. Bjorkholm M, Holm G, Mellstedt H. Immunologic profile of patients with cured Hodgkin’s disease. Scand J Hematol 1977; 18:361–368. 58. Bjorkholm M, Holm G, Mellsted H, Johansson B. Immunodeficiency and prognosis in Hodgkin’s disease. Acta Med Scand 1975; 198:275–279. 59. Block JB, Haynes HA, Thompson WL, Neiman PE. Delayed hypersensitivity in chronic lymphocytic leukemia. J Natl Cancer Inst 1969; 42:973–980. 60. Blomgren H, Edsmyr F, Esposti PL, et al. Influence of bestatin, a new immunomodifier, on human lymphoid cells. In: Serrou B, Rosenfeld C, Daniels JC, Saunders JP, eds. Current concepts in human immunology and cancer immunomodulation. Amsterdam: Elsevier Biomedical, 1982:593–610. 61. Blomgren H, Edsmyr F, Esposti PL, Naslund I. Immunological and haematological monitoring in bladder cancer patients receiving adjuvant bestatin treatment following radiation therapy. A prospective randomized trial. Biomed Pharmacother 1984; 38:143–149. 62. Blomgren SE, Wolberg WH, Kisken WA. Effect of fluoropyrimidines on delayed cutaneous hypersensitivity. Cancer Res 1965; 25:977–979. 63. Blomgren H, Wasserman J, Rotstein S, et al. Possible role of prostaglandin-producing monocytes in the depression of mitogenic response of blood lymphocytes following radiation therapy. Radiother Oncol 1984; 1:255–261. 64. Blomgren H, Baral E, Edsmyr F, et al. Natural killer activity in peripheral blood lymphocyte populations following local radiation therapy. Acta Radiol Oncol 1980; 2:139–143. 65. Blomgren H, Wasserman J, Littbrand B. Blood lymphocytes after radiation therapy of carcinoma of prostate and urinary bladder. Acta Radiol Ther Phys Biol 1974; 13:357–367. 66. Blomgren H, Naslund I, Esposti PL, et al. Adjuvant bestatin immunotherapy in patients with transitional cell carcinoma of the bladder. Clinical results of a randomized trial. Cancer Immunol Immunother 1987; 25:1; 46. 67. Blomgren H, Strender LE, Petrini B, Wasserman J. Changes of the spontaneous cytotoxicity of the blood lymphocyte population following local radiation therapy for breast cancer. Eur J Cancer Clin Oncol 1982; 18:637–643. 68. Blomgren H, Wasserman J, Baral E, Petrini B. Evidence for the appearance of non-specific suppressor cells in the blood after local irradiation therapy. Int J Radiol Oncol Biol Phys 1978; 4:249–253. 69. Blomgren H, Strender LE, Edsmyr F. Bestatin treatment and the peripheral lymphocyte population in cancer patients. Recent Results Cancer Res 1980; 75:133–138.

70. Blomgren H, Edsmyr F, von Stedingk L-V, Wasserman J. Bestatin treatment enhances the recovery of radiation-induced impairments of the immunological reactivity of the blood lymphocyte population in bladder and cancer patients. Biomed Pharmacother 1986; 40:50–54. 71. Bobrove AM, Fuks Z, Strober S. Quantitation of T- and B-lymphocytes and cellular immune function in Hodgkin’s disease. Cancer 1975; 36:169–179. 72. Bockman RS. Prostaglandins in cancer: A review. Cancer Invest 1983; 1:485–493. 73. Boetcher DA, Leonard EJ. Abnormal monocyte chemotactic response in cancer patients. J Natl Cancer Inst 1974; 52:1091–1098. 74. Bollag W, Matta A. From vitamin A to retinoids in experimental and clinical oncology: Achievements, failures, and outlook. Ann NY Acad Sci 1981; 359:9–23. 75. Bolton PM, Teasdale C, Mander AM, et al. Immune competence in breast cancer: Relationships of pretreatment immunologic tests to diagnosis and tumor stage. Cancer Immunol Immunother 1976; 1:251–258. 76. Bolton PM, Mander AM, Davidson JM, et al. Cellular immunity in cancer: Comparison of delayed hypersensitivity skin tests in three common cancers. Br J Med 1975; 3(5974):18–20. 77. Bone G, Appleton DR, Venebles CW. The prognostic value of the cutaneous delayed hypersensitivity response to 2,4-dinitrochlorobenzene in gastrointestinal cancer. Br J Cancer 1974; 29:403–406. 78. Borden EC, Davis TE, Crowley JJ, et al. Interim analysis of trial of levamisole and 5-fluorouracil in metastatic colorectal carcinoma. In: Terry WD, Rosenberg SA, eds. Immunotherapy of human cancer. New York: Elsevier/North-Holland, 1982:231–235. 79. Bosworth JL, Ghossein NA, Brooks TL. Delayed hypersensitivity in patients treated by curative radiotherapy. Its relation to tumor response and short-term survival. Cancer 1975; 36:353–358. 80. Braun DP, Harris JE, Rugenstein M. Relationships of arachidonic acid metabolism to indomethacin-sensitive immunoregulatory function and lymphocyte PGE sensitivity in peripheral blood mononuclear cells of disseminated solid-tumor cancer patients. J Immunopharmacol 1984; 6:227–236. 81. Braun DP, Harris JE. Effects of combination chemotherapy on immunoregulatory cells in peripheral blood of solid-tumor cancer patients: Correlation with rebound overshoot immune function recovery. Clin Immunol Immunopathol 1981; 20:193–214. 82. Braun DP, Harris JE. Modifications of the effects of cytotoxic chemotherapy on the immune responses of cancer patients with a non-steroidal, anti-inflammatory drug, piroxicam. (Abstr.) Proc Am Soc Clin Oncol 1985; 4:223. 83. Brenner BG, Friedman G, Margolese RG. The relationship of clinical status and therapeutic modality to natural killer cell activity in human breast cancer. Cancer 1985; 56:1543–1548. 84. Brooks WH, Caldwell HD, Mortara RH. Immune responses in patients with gliomas. Surg Neurol 1974; 2:419–423. 85. Brosman S, Elhilali M, Vescura C, Fahey J. Immune response in bladder cancer patients. J Urol 1979; 121:162–169. 86. Brosman S, Hausman M, Shacks SJ. Studies on the immune status of patients with renal adenocarcinoma. J Urol 1975; 114:375–380. 87. Browder JP, Cretien PB. Immune reactivity in head and neck squamous carcinoma and relevance to the design of immunotherapy trials. Sem Oncol 1977; 4:4; 431–439. 88. Brown AE, Badger AM, Poste G. The effects of cimetidine on immune cell function and host response to tumors. In: Serrou B, Rosenfeld C, Daniels JC, and Sanders JP, eds. Current concepts of human immunology and cancer immunomodulation. Amsterdam: Elsevier Biomedical, 1982:513–519.

Robert K. Oldham 89. Bruckner HW, Mokyr MB, Mitchell MS. Effect of imidazole-4carboxamide-5-(3,3-dimethyl-1-triazeno) on immunity in patients with malignant melanoma. Cancer Res 1974; 34:181–183. 90. Bukowski RM, Deodhar S, Hewlett JS, Greenstreet R. Randomized controlled trial of transfer factor in stage II malignant melanoma. Cancer 1983; 51:269–272. 91. Buroker TR, Moertel CG, Fleming TR, et al. A controlled evaluation of recent approaches to biochemical modulation or enhancement of 5-fluorouracil therapy in colorectal carcinoma. J Clin Oncol 1985; 3:1624–1631. 92. Burtin C, Noirot C, Scheinmann P, et al. Clinical improvement in advanced cancer disease after treatment combining histamine and H2-antihistaminics (rantidine or cimetidine). Eur J Cancer Clin Oncol 1988; 24:2; 161–167. 93. Busutti L, Blotta A, Mastrorilli M, et al. Transfer factor adjuvant therapy in non-small-cell lung carcinoma (NSCLC) after surgery and radiotherapy. J Exp Pathol 1987; 3:4; 565–568. 94. Butterworth C, Oon CJ, Westbury G, Hobbs JR. T-lymphocyte response in patients with malignant melanoma. Eur J Cancer 1977; 10:639–646. 95. Cairo MS, Mallett C, Vande Ven C, et al. Impaired in vitro polymorphonuclear function secondary to the chemotherapeutic effects of vincristine, adriamycin, cyclophosphamide, and actinomycin D. J Clin Oncol 1986; 5:798–804. 96. Cameron DJ, O’Brien P, Majeski JA. Effects of in vivo treatment with ibuprofen on macrophage function in breast and colon cancer patients. J Clin Lab Immunol 1986; 20:1; 23–28. 97. Cannon GB, Dean JH, Herberman RB, et al. Association of depressed postoperative lymphoproliferative responses to alloantigens with poor prognosis in patients with stage I lung cancer. Int J Cancer 1980; 25:9–17. 98. Carmignani G, Belgrano E, Puppo P, Cornaglia P. T-lymphocyteinhibiting factors in renal cancer: Evaluation of differences between arterial and renal venous blood levels of E-rosetteforming cells. J Urol 1978; 120:673–675. 99. Carpenter JT Jr, Smalley RV, Raney M, et al. Ineffectiveness of levamisole in prolonging remission or survival of women treated with cyclophosphamide, doxorubicin, and 5-fluorouracil for good-risk metastatic breast carcinoma: A Southeastern Cancer Study Group Trial. Cancer Treat Rep 1986; 70:9; 1073–1079. 100. Case DC, Hansen JA, Corrales E, et al. Depressed in vitro lymphocyte responses to PHA in patients with Hodgkin’s disease in continuous long remissions. Blood 1977; 49:771–778. 101. Catalona WJ, Chretien PB. Abnormalities of quantitative dinitrochlorobenzine sensitization in cancer patients: Correlation with tumor stage and histology. Cancer 1973; 31:353–356. 102. Catalona WJ, Sample WF, Chretien PB. Lymphocyte reactivity in cancer patients: Correlation with tumor histology and clinical stage. Cancer 1973; 31:65–71. 103. Catalona WJ, Tarpley JL, Potvin C, Chretien PB. Correlations among cutaneous reactivity to DNCB, PHA-induced lymphocyte blastogenesis and peripheral blood E-rosettes. Clin Exp Immunol 1975; 19:327–332. 104. Catalona WJ, Ratliff TL, McCool R. Concanavalin-A-activated suppressor cell activity in peripheral blood lymphocytes of urologic cancer patients. J Natl Cancer Inst 1980; 65:553–557. 105. Caulfield MJ, Luce KJ, Proffitt MR, Cevy J. Induction of idiotypespecific suppressor T-cells with antigen/antibody complexes. J Ex Med 1983; 157:1713–1725. 106. Cervical Cancer Immunotherapy Study Group. Immunotherapy using the streptococcal preparation OK-432 for the treatment of uterine cervical cancer. Cancer 1987; 60:2394–2402.

133 107. Chahinian AP, Goldberg J, Holland JF, et al. Chemotherapy vs. Chemoimmunotherapy with levamisole or Corynebacterium parvum in advanced lung cancer. Cancer Treat Rep 1982; 66:1291–1297. 108. Cheema AR, Hersh EM. Patient survival after chemotherapy and its relationship to in vitro lymphocyte blastogenesis. Cancer 1971; 28:851–855. 109. Chihara G, Hamuro J, Maeda YY, et al. Antitumor and metastasisinhibitory activities of lentinan as an immunomodulator: An overview. Cancer Detect Prev Suppl 1987; 1:423–443. 110. Chirhara G. The antitumor polysaccharide lentinan: An overview. In: Aoki T, Urushizaki I, and Tsubara E, eds. Manipulation of host defense mechanisms. Excerpta Medica 27(9) 1981:1–16. 111. Chirigos MA, Mastrangelo MJ. Immunorestoration by chemicals. In: Milhich E, ed. Immunological approaches to cancer therapeutics. New York: Wiley, 1982:191–239. 112. Chisesi T, Capnist G, Rancan L, et al. The effect of thymic substances on T-circulating cells of patients treated for Hodgkin’s disease. J Biol Regul Homeost Agents 1988; 2:4,193–198. 113. Chlebowski RT, Nystrom S, Reynolds R, et al. Long-term survival following levamisole or placebo adjuvant treatment of colorectal cancer: A Western Cancer Study Group trial. Oncology 1988; 45:3:141–143. 114. Clemens LE, Siiteri PK, Stities DP. Mechanism of immunosuppression of progesterone of natural lymphocyte activation during pregnancy. J Immunol 1979; 122:5; 1978–1985. 115. Cochran AJ, Mackie RM, Grant RM, et al. An examination of the immunology of cancer patients. Int J Cancer 1976; 18:298–309. 116. Cohen MH, Chretien PB, Johnston-Early A, et al. Thymosin fraction V prolongs survival of intensively treated small-cell lung cancer patients. In: Terry WD, Rosenberg SA, eds. Immunotherapy of human cancer. New York: Elsevier/North-Holland, 1982:141–145. 117. Cohen JHD, Danel L, Cordier G, et al. Sex steroid receptors in peripheral T-cell: Absence of androgen receptors to OKT8positive cells. J Immunol 1983; 131:2767–2771. 118. Cohen MH, Chretien PB, Indo DC, et al. Thymosin fraction V and intensive combination chemotherapy prolonging the survival of patients with small-cell lung cancer. JAMA 1979; 241:1813–1815. 119. Colozza M, Tonato M, Belsanti V, et al. 5-Fluorouracil and isoprinosine in the treatment of advanced colorectal cancer: A limited phase I, II evaluation. Cancer 1988; 62:1049–1052. 120. Cone L, Uhr JW. Immunological deficiency disorders associated with chronic lymphocyte leukemia and multiple myeloma. J Clin Invest 1964; 43:2241–2249. 121. Consolini R, Cei B, Cini P, et al. Circulating thymic hormone activity in young cancer patients. Clin Exp Immunol 1986; 66:1; 173–180. 122. Costanzi JJ, Fletcher WS, Balcerzak SP, et al. Combination chemotherapy plus levamisole in the treatment of disseminated malignant melanoma. A Southwest Oncology Group Study. Cancer 1984; 53:833–836. 123. Cozzolino F, Torcia M, Carossino AM, et al. Characterization of cells from invaded lymph nodes in patients with solid tumors. J Exp Med 1987; 166:303–318. 124. Cullen BF, Van Belle G. Lymphocyte transformation and changes in leukocyte count. Effect of anesthesia and operation. Anesthesiology 1975; 43:563–569. 125. Dagan S, Tzehoval E, Fridkin M, Feldman M. Tuftsin and tuftsin conjugates potentiate immunogenic processes: Effects and possible mechanisms. J Biol Response Mod 1987; 6:6; 625–636. 126. Danel L, Sonweine G, Monier JC, Saez S. Specific estrogen binding sites in human lymphoid cells and thymic cells. J Steroid Biochem 1983; 18:5; 559–563.

134

Therapeutic approaches to cancer-associated immune suppression

127. Danish Breast Cancer Cooperative Group. Increased breast cancer recurrence rate after adjuvant therapy with levamisole. Lancet 1980; 2:824–827. 128. Das SN, Khanna NN, Khanna S. A multiparametric observation of immune competence in breast cancer and its correlation with tumor load and prognosis. Ann Acad Med Singapore 1985; 14:374–381. 129. Davis S, Mietlowski W, Rohweder JJ, et al. Levamisole as an adjuvant to chemotherapy in extensive bronchogenic carcinoma. A Veterans Administration Lung Cancer Group study. Cancer 1982; 50:646–651. 130. Dawson DE, Everts EC, Vetto RM, Burger DR. Assessment of immunocompetent cells in patients with head and neck squamouscell carcinoma. Ann Otol Rhinol Laryngol 1985; 94:342–345. 131. DeBoer KP, Braun DP, Harris JE. Natural cytotoxicity and antibody-dependent cytotoxicity in solid-tumor cancer patients: Regulation by adherent cells. Clin Immunol Immunopathol 1982; 23:133–144. 132. Del Giacco GS, Cengiarotti L, Mantovani G, et al. Advanced lung cancer treated with combination chemotherapy with or without thymostimulin. In: Byrom NA, Hobbs JR, eds. Thymic factor therapy Serono Symposium Publications. New York: Raven Press, 1982:321–328. 133. Dellon AL, Potvin C, Chretien PB. Thymus-dependent lymphocyte levels during radiation therapy for bronchogenic and esophageal carcinoma: Correlations with clinical course in responders and nonresponders. Am J Roentgenol Radiat Ther Nucl Med 1975; 123:500–511. 134. Dillman RO, Beauregard JC, Zavanelli MI, et al. In vivo immune restoration in advanced cancer patients after administration of thymosin fraction V or thymosin alpha-1. J Biol Response Mod 1983; 2:139–147. 135. Dimitrov NV, Arnold D, Munson J, et al. Phase II study of thymosin fraction 5 in the treatment of metastatic renal cell carcinoma. Cancer Treat Rep 1985; 69:137–138. 136. Dizon QS, Southam CM. Abnormal cellular response in skin abrasion in cancer patients. Cancer 1963; 16:1288–1292. 137. Domellof L. Athlin L, Berghem L. Effects of long-term combination chemotherapy on the reticuloendothelial system. Cancer 1984; 53:2073–2078. 138. Donoran AJ. Reticuloendothelial function in patients with cancer: Initial observations. Am J Surg 1967; 111:230–238. 139. Droller MJ, Schneider MU, Perlmann P. A possible role of prostaglandins in the inhibition of natural and antibody-dependent cell-mediated cytotoxicity against tumor cells. Cell Immunol 1978; 39:165–177. 140. Droller MJ, Perlmann P, Schneider MU. Enhancement of natural and antibody-dependent lymphocyte cytotoxicity by drugs which inhibit prostaglandin production by tumor target cells. Cell Immunol 1978; 39:154–164. 141. Ducos J, Mingueres J, Colombio P, et al. Lymphocyte response to PHA in patients with lung cancer. Lancet 1970; 1:111–112. 142. Dupont B, Ballon M, Hansen JA, et al. Effects of transfer factor therapy on mixed lymphocyte culture reactivity. Proc Natl Acad Sci USA 1974; 71:867–871. 143. Edelman GM, Cunningham BA, Gall WE, et al. The covalent structure of and entire gamma G immunoglobulin molecule. Proc Natl Acad Sci USA 1969; 63:78–85. 144. Ehrke MJ, Mihich E, Berd D, Mastrangelo MJ. Effects of anticancer drugs on the immune system in humans. Sem Oncol 1989; 16:3; 230–253. 145. Eilber FR, Nizze HA, Morton DL. Sequential evaluation of general immune competence in cancer patients: Correlation with clinical course. Cancer 1975; 35:660–665.

146. Eilber FR, Morton DL. Impaired immunologic reactivity and recurrence following cancer surgery. Cancer 1970; 25:362–367. 147. Eilber FR, Morton DL, Ketcham AS. Immunologic abnormalities in head and neck cancer. Am J Surg 1974; 128:534–538. 148. Eisenthal A, Monselise J, Zinger R, Adler A. The effect of cimetidine on PBL from healthy donors and melanoma patients: Augmentation of T-cell responses to TCGF mitogens and alloantigens and of TCGF production. Cancer Immunol Immunother 1986; 21:2; 141–147. 149. Ershler WB, Hacker MP, Burroughs BJ, et al. Cimetidine and the immune response. In vivo augmentation of nonspecific and specific immune response. Clin Immunol Immunopathol 1983; 26:10–17. 150. Estervez ME, Ballart IJ, Diez RA, et al. Normalization of monocyte candidacidal deficiency by cyclooxygenase inhibitors in Hodgkin’s disease. Cancer 1985; 55:2774–2778. 151. Eura M, Maehara T, Tsutomo I, Ishikawa T. Suppressor cells in the effector phase of autologous cytotoxic reactions in cancer patients. Cancer Immunol Immunother 1988; 27:147–153. 152. Ezdinli EZ, Simonson KL, Smith RA. Comparison of the effects of single vs. multiple agent chemotherapy on lymphocytes assayed by the rosette techniques. Cancer 1978; 42:2234–2243. 153. Faguet GB, Davis HC. Survival in Hodgkin’s disease: The role of immunocompetence and other major risk factors. Blood 1982; 59:938–945. 154. Fisher RI, Bostick-Bruton F. Depressed T-cell proliferative responses in Hodgkin’s disease: Role of monocyte-mediated suppression via prostaglandins and hydrogen peroxide. J Immunol 1982; 129:1770–1774. 155. Fisherman EW. Does the allergic diathesis influence malignancy? J Allergy 1960; 31:74–78. 156. Flodgren P, Sjogren HO. Influence in vitro on NK and K-cell activities by cimetidine and indomethacin with and without simultaneous exposure to interferon. Cancer Immunol Immunother 1985; 19:28–34. 157. Florentin I, Kraus L, Mathé G, Hadden JW. In vivo study in mice of the immunopharmacological properties of NPT-15392. In: Serrou B, Rosenfeld C, Daniels JB, Saunders JP, eds. Current concepts of human immunology and cancer. Amsterdam: Elsevier Biomedical, 1982:463–469. 158. Forbes JT, Greco FA, Oldham RK. Human natural cell-mediated cytotoxicity: II. Levels in neoplastic disease. Cancer Immunol Immunother 1982; 11:147–153. 159. Ford RJ, Tsao J, Kouttab NM, et al. Association of an interleukin abnormality with the T-cell defect in Hodgkin’s disease. Blood 1984; 64:386–392. 160. Fracchia A, Pacetti M, Barberis M, et al. Determination of T-lymphocyte subpopulations in patients with lung cancer. A comparison between lung lavage and peripheral blood by monoclonal antibodies and flow cytometry. Respiration 1987; 51:3; 161–169. 161. Frydecka I. Natural killer-cell activity during the course of disease in patients with Hodgkin’s disease. Cancer 1985; 56:2799–2803. 162. Fujisama T, Yamaguchi Y, Kimura H, et al. Adjuvant immunotherapy of primary resected lung cancer with transfer factor. Cancer 1984; 54:663–669. 163. Fujisawa T, Yamaguchi Y, Kimura H. Transfer factor in restoration and cell-mediated immunity in lung cancer patients. Jpn J Surg 1983; 13:304–311. 164. Fuks Z, Strober S, Kaplan HS. Interaction between serum factors and T-lymphocytes in Hodgkin’s disease. N Eng Med 1976; 295:1273–1278. 165. Fuks Z, Strober W, Bobrove AM, et al. Long-term effects of radiation on T- and B-lymphocytes in peripheral blood of patients with Hodgkin’s disease. J Clin Invest 1976; 58:803–814.

Robert K. Oldham 166. Garay GE, Pavlovsky S, Sasiain MC, et al. Immunocompetence and prognosis in children with acute lymphoblastic leukemia: Combination of two different maintenance therapies. Med Pediatr Oncol 1976; 2:403–415. 167. Gatti RA, Good RA. Occurrence of malignancy in immunodeficiency disease: A literature review. Cancer 1971; 28:89–98. 168. Geffner JR, Serebrinsky G, Isturiz MA. Normal human serum restores the expression of Fc8 receptors in immune complex: Blocked human mononuclear cells. Immunology 1986; 59:239–243. 169. Giulivi A, Cilano L, Roncoroni L, et al. Effects of cimetidine on in vitro transformation of peripheral monocytes to macrophages in healthy volunteers and cancer patients. Int J Immunopharmacol 1986; 8:5; 517–523. 170. Goldenberg GJ, Brandes LJ, Lau WH, et al. Cooperative trial of immunotherapy for nasopharyngeal carcinoma with transfer factor from donors with Epstein-Barr virus antibody activities. Cancer Treat Rep 1985; 69:761–767. 171. Goldrosen MH, Han T, Jung D, et al. Impaired lymphocyte blastogenic response in patients with colon adenocarcinoma: Effects of disease and age. J Surg Oncol 1977; 9:229–234. 172. Goldsmith HS, Levin AG, and Southam CM. A study of cellular responses in cancer patients by quantitative and qualitative Rebuck tests. Surg Forum 1965; 16:102–104. 173. Goldstein AL, Low TLK, Thurman GB, et al. Thymosins and other hormone-like factors of the thymus gland. In: Mihich E, ed. Immunological cancer therapeutics. New York: Wiley, 1982: 137–190. 174. Golub SH, Rangel DM, Morton D. In vitro assessment of immunocompetence in patients with malignant melanoma. Int J Cancer 1977; 20:873–880. 175. Goodwin JS, Messner RP, Bankhurst AD, et al. Prostaglandinproducing suppressor cells in Hodgkin’s disease. N Engl J Med 1977; 297:963–968. 176. Gottlieb AA, Farmer JL, Matzura CT, et al. Modulation of human T-cell production of migration inhibitory lymphocytes by cytokines derived from human leukocyte dialysates. J Immunol 1984; 132:1; 256–260. 177. Green AA, Borella L. Immunologic rebound after cessation of long-term chemotherapy in acute leukemia. II. In vitro response to phytohemagglutinin and antigens by peripheral blood and bone marrow lymphocytes. Blood 1973; 42:99–110. 178. Greenwald RJ, et al. The B7 family revisited. Annu Rev Immunol 2005; 23:515–548. 179. Groveman DS, Borden EC. In vitro and in vivo effects of levamisole on monocyte chemotaxis in normal donors and patients with colorectal carcinoma. J Biol Response Mod 1983; 2: 167–174. 180. Grzelak I, Olszewski WL, Engeset A. Decreased suppressor cell activity after surgery. J Clin Lab Immunol 1985; 16:201–205. 181. Grzelak I, Olszewski WL, Engeset A. Suppressor cell activity in peripheral blood in cancer patients after surgery. Clin Exp Immunol 1983; 51:149–156. 182. Guiliano AE, Rangel D, Golub SH, et al. Serum-mediated immunosuppression in lung cancer. Cancer 1979; 43:917–924. 183. Gupta S, Cunningham-Rundles S. Deficient concanavalin-Ainduced suppressor cell activity in women with untreated breast cancer. J Clin Lab Immunol 1982; 9:159–161. 184. Gupta S, Good RA. Subpopulations of human T-lymphocytes: II. Effect of thymopoietin, corticosteroids, and irradiation. Cell Immunol 1977; 34:10–18. 185. Haas GS, Halperin E, Doseretz D, et al. Differential recovery of circulating T-cell subsets after nodal irradiation for Hodgkin’s disease. J Immunol 1984; 132:1026–1030.

135 186. Hadden JW. Chemically-defined immunotherapeutic drugs under development for the treatment of cancer. In: Serrou B, Rosenfeld C, Daniels JC, and Saunders JP, eds. Current concepts in human immunology and cancer immunomodulation. Amsterdam: Elsevier Biomedical, 1982:439–448. 187. Hadden JW. Recent advances in the preclinical and clinical immunopharmacology of interleukin-2: emphasis on IL-2 as an immunorestorative agent. Cancer Detect Prev 1988; 12:537–552. 188. Hadjipetrou-Kourounakis L, Manikou H, Tsougranis A. Restoration of immunosuppression in lung cancer by normal sera. J Clin Lab Immunol 1985; 16:149–153. 189. Haim N, Meidav A, Samuelly B, et al. Prostaglandin-related and adherent cell suppressor system in apparently cured Hodgkin’s disease patients. J Biol Response Mod 1984; 3:219–225. 190. Hakim AA. Peripheral blood lymphocytes from patients with cancer lack interleukin-2 receptors. Cancer 1988; 61:4; 689–701. 191. Halili M, Bosworth J, Romney S, et al. The long-term effect of radiotherapy on the immune status of patients cured of a gynecologic malignancy. Cancer 1976; 37:2875–2878. 192. Hall HRS, Goldstein AL. The endocrine thymus. In: Becker KC, ed. Principles and practice of endocrinology and metabolism. New York: Lippincott, 1990. 193. Han T, Takita H. Indomethacin-mediated enhancement of lymphocyte response to mitogens in healthy subjects and lung cancer patients. Cancer 1980; 46:2416–2420. 194. Han T, Nemoto T, Ledesma EJ, Bruno S. Enhancement of T-lymphocyte response to mitogens by indomethacin in breast and colorectal cancer patients. Int J Immunopharmacol 1983; 5:11–15. 195. Herr HW. Adherent suppressor cells in the blood of patients with bladder cancer. J Urol 1981; 126:457–460. 196. Hakim AA. Lipid-like agent from human neoplastic cells suppresses cell-mediated immunity. Cancer Immunol Immunother 1980; 8:1330. 197. Han T, Takita H. Immunologic impairment in bronchogenic carcinoma: A study of lymphocyte response to phytohemagglutinin. Cancer 1972; 30:616–620. 198. Hancock BW, Bruce L, Sokol RJ, Clark A. Transfer factor in Hodgkin’s disease: A randomized clinical and immunological study. Eur J Cancer Clin Oncol 1988; 24:5; 929–933. 199. Harris J, Copeland D. Impaired imunoresponsiveness in tumor patients. Ann NY Acad Sci 1974; 230:56–85. 200. Harris JE, DeBoer KP, Vahey AC, Braun DP. The measurement of leukocyte subsets in the peripheral blood of cancer patients using monoclonal antibody reagents. Med Pediatr Oncol 1982; 10:185–194. 201. Harris JE, Stewart THM. Recovery of mixed lymphocyte reactivity (MLR) following cancer chemotherapy in man. In: Schwartz MR, ed. Proceedings of the Sixth Leukocytes Culture Conference. New York: Academic Press, 1979:555–580. 202. Haskell CM. Immunologic aspects of cancer chemotherapy. Ann Rev Pharmacol Toxicol 1977; 17:179–195. 203. Hausman MS, Brossman SA, Snyderman R, et al. Defective monocyte function in patients with genitourinary carcinoma. J Natl Cancer Inst 1975; 55:1047–1054. 204. Hayashi Y, Nishida T, Yoshida H, et al. Peripheral T-gamma lymphocyte population in head and neck cancer. Cancer Immunol Immunother 1984; 17:160–164. 205. Heal JM, Chuang C, Blumberg N. Perioperative blood transfusions and prostate cancer recurrence and survival. Am J Surg 1988; 156:374–379. 206. Hersey P, Bindon C, Czerniecki M, et al. Inhibition of interleukin-2 production by factors released from tumor cells. J Immunol 131:2837–2842.

136

Therapeutic approaches to cancer-associated immune suppression

207. Hersh EM, Gutterman JU, Mavligit GM, Reed RC. Immunologic aspects of chemotherapy. In: Cancer Chemotherapy-Fundamental Concepts and Recent Advances. Chicago, Illinois: Yearbook Medical, 1975: 279–294. 208. Hersh EM. Vinca alkaloids. In: Sartorelli AC, Johns DG, eds. Antineoplastic and immunosuppressive agents, part I. New York: Springer Verlag, 1974:601–602. 209. Hersh EM, Gutterman JU, Mavligit GM. Immunotherapy of leukemia. Med Clin North Am 1976; 60:1019–1042. 210. Hersh EM, Gutterman JU, Mavligit GM, et al. Serial studies of immunocompetence of patients undergoing chemotherapy for acute leukemia. J Clin Invest 1974; 54:401–408. 211. Hersh EM, Whitecan JP Jr, McCredie JB, et al. Chemotherapy, immunocompetence, immunosuppression and prognosis in acute leukemia. N Engl J med 1971; 285:1211–1216. 212. Hersh EM, Irwin WS. Blastogenic responses of lymphocytes from patients with untreated and treated lymphomas. Lymphology 1969; 2:150–160. 213. Hersh EM. Immunosuppressive agents. In: Sartorelli AC, Johns DG, eds. Antineoplastic and immunosuppressive agents, part I. New York: Springer Verlag, 1974:555–576. 214. Hersh EM, Oppenheim J. Inhibition of in vitro lymphocyte transformation during chemotherapy in man. Cancer Res 1979; 27:98–105. 215. Herskovic A, Bauer M, Seydel G, et al. Postoperative thoracic irradiation with or without levamisole in non-small-cell lung cancer: Results of a radiation therapy oncology group study. Int J Radiation Oncology Biol Phys 1988; 14:1; 37–42. 216. Hilgard P, Pohl J, Stekar J, Voegeli R. Oxazaphosphorines as biological response modifiers: Experimental and clinical perspective. Cancer Treat Rev 1985; 12:155–162. 217. Hillinger SM, Herzig GP. Impaired cell-mediated immunity in Hodgkin’s disease mediated by suppressor lymphocytes and monocytes. J Clin Invest 1978; 61:1620–1627. 218. Hiraoka N, et al. Prevalence of FOXP3 regulatory T cells increases during progression of pancreatic ductal adenocarcinoma and its premalignant lesion. Clin Cancer Res 2006; 12:5423–5434 219. Hirsh B, Johnson JT, Rabin BS, Thearle PB. Immunostimulation of patients with head and neck cancer. In vitro and preliminary clinical experience. Arch Otolaryngol 1983; 109:298–301. 220. Hirshaut Y, Pinsky CM, Frydecka I, et al. Effect of short-term levamisole therapy on delayed hypersensitivity. Cancer 1980; 45:362–366. 221. Hoagland JG, Scoggin S, Giavazzi R, et al. Tumor-derived suppressor factors (TDSFs) in normal and neoplastic colon and rectum. J Surg Res 1986; 40:5; 467–474. 222. Hodgson WJB, Lowenfels AB. Blood transfusion and recurrence rates in colonic malignancy. Lancet 1982; 2:1047. 223. Hojo H, Hashimoto Y. Cytotoxic cells induced in tumor-bearing rats by a streptococcal preparation (OK-432). Gann 1981; 72:62–69. 224. Holm G, Mellstedt H, Bjorkholm M, et al. Lymphocyte abnormalities in untreated patients with Hodgkin’s disease. Cancer 1976; 37:751–762. 225. Holmes EC. Immunology of tumor-infiltrating lymphocytes. Ann Surg 1985; 201:158–163. 226. Holmes E, Sibbitt WL, Bankhurst AD. Serum factors which suppress natural cytotoxicity in cancer patients. Int Arch Allergy Appl Immunol 1986; 80:39–43. 227. Holmes EC, Golub SH. Immunologic defects in lung cancer patients. J Thoracic Cardiovasc Surg 1976; 71:161–168. 228. Hoon DSB, Bowker RJ, Cochran AJ. Suppressor-cell activity in melanoma-draining lymph nodes. Cancer Res 1987; 47:1529–1533. 229. Hoon DSB, Korn EL, Cochran AJ. Variations in functional immunocompetence of individual tumor-draining lymph nodes in humans. Cancer Res 1987; 47:1740–1744.

230. Hoon DS, Irie RF, Cochran AJ. Gangliosides from human melanoma immunomodulate responses of T-cells to interleukin-2. Cell Immunol 1988; 111:2; 410–419. 231. Horiguchi M, Saito A, Sonoda H. A gastric cancer showing marked improvement (stage IV) with lentinan immunotherapy and 5-FU, MMC-chemotherapy. Gan To Kagaku Ryoho 1988; 15:6; 1973–1977. 232. Horrino N. Impaired PWM-induced polyclonal B-cell activation in patients with malignancies treated with various intermittent combination chemotherapies including doxorubicin. Cancer 1982; 50:659–667. 233. Hortobagyi GN, Hersh EM, Papadopoulos EJ, et al. Initial clinical studies with copovithane. J Biol Response Mod 1986; 5:319–329. 234. Hsu CC, Logerfo P. Correlation between serum alpha globulin and plasma inhibitory effect of PHA-stimulated lymphocytes in colon cancer patients. Proc Soc Exp Biol Med 1972; 139:575–578. 235. Huang AT, Mold NG, Fisher Sr, et al. A prospective study of squamous head and neck carcinoma. Cancer 1987; 59:1721–1726. 236. Hubbard GW, Wanebo H, Fukuda M, Pace R. Defective suppressorcell activity in cancer patients: A defect in immune regulation. Cancer 1981; 47:2177–2184. 237. Ninneman JL. Melanoma-associated immunosuppression through B-cell activation of suppressor T-cells. J Immunol 1978; 120:1573–1579. 238. Hughes NR. Serum concentrations of gamma G, gamma A and gamma M immunoglobulins in patients with carcinoma, melanoma, and sarcoma. J Natl Cancer Inst 1971; 46:1015–1028. 239. Humphrey LJ, Humphrey MA, Singla O, Volenec FJ. Immunologic responsiveness of patients with cancer: Relationship to tumor type, stage and prognosis. Ann Surg 1981; 193:574–578. 240. Hyman GA. Increased incidence of neoplasia in association with chronic lymphocytic leukemia. Scand J Haematol 1969; 6:99–104. 241. Iaffaioli RV, Frasci G, Tortora G, et al. Effect of thymic extract ‘thymostimulin’ on the incidence of infections and myelotoxicity during adjuvant chemotherapy for breast cancer. Thymus 1988; 12:69–75. 242. Ichimura O, Suzuki S, Saito M, et al. Augmentation of interleukin-1 and interleukin-2 production by OK-432. Int J Immunopharmacol 1985; 7:263–270. 243. Inagaki T, Morise K, Matsunaga H. Effects of endoscopic intratumoral injection of lentinan in patients with gastric cancer. Gan To Kagaku Ryoho 1988; 15:2; 319–324. 244. Intergropu 5-FU/levamisole study positive; untreated arms stopped, clinical alert sent. Clin Trials – Clin Cancer Lett 1989; October. 245. Israel L, Mugica J, Chahinian PH. Prognosis of early bronchogenic carcinoma: Survival cures of 451 patients after resection of lung cancer in relation to the results of preoperative tuberculin skin test. Biomed Exp 1973; 19:68–72. 246. Itoh K, Pellis NR, Balch C. Monocyte-dependent serum-borne suppressor of induction of lymphokine-activated killer cells in lymphocytes from melanoma patients. Cancer Immunol Immunother 1989; 29:57–62. 247. Jacobson DR, Zolla-Pazner S. Immunosuppression and infection in multiple myeloma. Sem Oncol 1986; 13:3; 282–290. 248. Jansen HM, The TH, deGast GC, et al. The primary immune response of patients with different stages of squamous-cell bronchial carcinoma. Thorax 1978; 33:755–760. 249. Jenkins VK, Ray P, Ellis HN, et al. Lymphocyte response in patients with head and neck cancer: Effect of clinical stage and radiotherapy. Arch Otolaryngol 1976; 102:596–600. 250. Jerrells TR, Dean JH, Richardson GL, et al. Role of suppressor cells in depression of in vitro lymphoproliferative responses of

Robert K. Oldham

251.

252.

253.

254. 255.

256.

257.

258.

259.

260.

261.

262.

263. 264. 265. 266.

267.

268. 269.

270.

271.

lung cancer and breast cancer patients. J Natl Cancer Inst 1978; 61:1001–1009. Jerrells TR, Dean JH, Herberman RB. Relationship between T-lymphocyte levels and lymphoproliferative responses to mitogens and alloantigens in lung and breast cancer patients. Int J Cancer 1978; 21:282–290. Job G, Pfeundschuh M, Bauer M, et al. The influence of radiation therapy on T-lymphocyte subpopulations defined by monoclonal antibodies. Int J Radiat Oncol Biol Phys 1984; 10:2077–2081. Joensun H, Nordman E, Toivanen A. Long-term effect of levamisole on the immune functions in melanoma patients. Strahlenther Onkol 1986; 162:12; 753–756. Joensun H, Toivanen A, Nordman E. Effect of tamoxifen on immune function. Cancer Treat Rep 1986; 7:3; 381–382. Johnson JD, Summersgill JT, Raff MJ. The in vitro effects of methotrexate on the phagocytosis and intracellular killing of Staphylococcus aureus by human neutrophils. Cancer 1986; 57:2343–2345. Johnston-Early A, Cohen MH, Fossieck BE, et al. Delayed hypersensitivity skin testing as a prognostic indicator in patients with small-cell lung cancer. Cancer 1983; 52:1395–1400. Jondal M, Merrill J, Ullberg M. Monocyte-induced human natural killer cell suppression followed by increased cytotoxic activity during short-term in vitro culture in autologous serum. Scand J Immunol 1981; 14:555–563. Jones SE, Griffith K, Dombrowski P, Gaines JA. Immunodeficiency in patients with non-Hodgkin’s lymphomas. Blood 1977; 49:335–344. Jose DG, Seshadri R. Circulating immune complexes in neuroblastoma: Direct assay and role in blocking specific cellular immunity. Int J Cancer 1974; 13:824–838. Jubert AV, Lee ET, Hersh EM, McBridge CM. Effects of surgery, anesthesia and intraoperative blood loss on immunocompetence. J Surg Res 1973; 15:399–403. Jung TT, Berlinger NT, Juhn SK. Prostaglandins in squamous-cell carcinoma of the head and neck: A preliminary study. Laryngoscope 1985; 95:307–312. Kadish AS, Doyle AT, Steinhaur EH, Ghossein NA. Natural cytotoxicity and interferon production in human cancer: Deficient natural killer activity and normal interferon production in patients with advanced disease. J Immunol 1981; 127:1817–1822. Kaneko T. Effects of lentinan on cytotoxic functions of human lymphocytes. Immunopharmacol Immunotoxicol 1988; 10:2; 157–163. Kaplan HS. Hodgkin’s disease. Cambridge, Massachusetts: Harvard University Press, 1980. Kaplan HS. Hodgkin’s disease. Cambridge, Massachusetts: Harvard University Press, 1980. Karavodin LM, Guilliano AE, Golub SH. T-lymphocyte subsets in patients with malignant melanoma. Cancer Immunol Immunother 1981; 11:251–254. Kaszubowski PA, Husky G, Tung KS, Williams RC Jr. T-lymphocyte subpopulations in peripheral blood and tissues of cancer patients. Cancer Res 1980; 40:4684–4657. Katz AE. Immunobiologic staging of patients with carcinoma of the head and neck. Laryngoscope 1983; 93:445–463. Kawaguchi T, Suematsu M, Koizumi HM, et al. Activation of macrophage function by intraperitoneal administration of the streptococcal anti-tumor agent OK-432. Immunopharmacology 1983; 6:1077–1089. Kay RG, Mason BH, Stephans EJ, et al. Levamisole in primary breast cancer: A controlled study in conjunction with L-phenylalanine mustard. Cancer 1983; 51:1992–1997. Keller SM, Groshen S, Martini N, Kaiser LR. Blood transfusion and lung cancer recurrence. Cancer 1988; 62:606–610.

137 272. Kenady DE, Chretien PB, Potvin C, et al. Effect of thymosin in vitro on T-cell levels during radiation therapy. Cancer 1972; 39:642–652. 273. Kerman RH, Stefani SS. Phytohemagglutinin stimulation of lymphocytes in lung cancer patients. Oncology 1977; 34:10–12. 274. Khoo SK, Whitaker SV, Jones IS, Thomas DA. Levamisole as adjuvant to chemotherapy of ovarian cancer. Results of a randomized trial and 4-year follow-up. Cancer 1984; 54:986–990. 275. Kikuchi Y, Kizawa I, Oomori K, et al. Effects of cimetidine on interleukin-2 production by peripheral blood lymphocytes in advanced ovarian carcinoma. Eur J Cancer Clin Oncol 1988; 24:7; 1185–1190. 276. Kirsh MM, Orringer MB, McAuliffe S, et al. Transfer factor in the treatment of carcinoma of the lung. Ann Thorac Surg 1984; 38:140–145. 277. Klefstrom P, Holsti P, Grohn P, et al. Levamisole in the treatment of stage II breast cancer. Five-year follow-up of a randomized double-blind study. Cancer 1985; 55:2753–2757. 278. Klefstrom P. Combination of levamisole immunotherapy and polychemotherapy in advanced breast cancer. Cancer Treat Rep 1980; 64:65–72. 279. Klefstrom P, Nuortio L, Taskinen E. Post-operative radiation therapy and adjuvant chemoimmunotherapy in breast cancer. Aspects of timing and immune competence. Acta Radiol [Oncol] 1986; 25:3; 161–166. 280. Klefstrom P, Grohn P, Heinonen E, et al. Adjuvant postoperative radiotherapy, chemotherapy, and immunotherapy in stage III breast cancer. II. Five-year results and influence of levamisole. Cancer 1987; 60:5; 936–942. 281. Klefstrom P, Holsti P, Grohn P, Heinonem E. Combination of levamisole immunotherapy with conventional treatments in breast cancer. In: Terry WD, ed. Proceedings of Second International Conference on Immunotherapy of Cancer, Bethesda, Maryland, 1982; 19:4128–4130. 282. Kleinerman ES, Zwelling LA, Schwartz R, et al. Effect of L-phenylalanine mustard, adriamycin and actinomycin D, and 4′-(9-acridinylamino) methanesulfon-m-anisidide on naturally occurring human spontaneous monocyte-mediated cytotoxicity. Cancer Res 1982; 42:1692–1695. 283. Koba F, Akiyoshi T, Tsuji H. Natural killer-cell activity in the perigastric lymph nodes from patients with gastric carcinoma or benign lesions. J Clin Lab Immunol 1987; 23:4; 191–195. 284. Kova J, Ninger E, Zemanov D, Lauerov P. A prospective study of lymphocyte responses to phytohemagglutinin in melanoma (lack of prognostic value of correlation with minimal tumor burden). Neoplasma 1980; 27:575–582. 285. Kraus S, Comas F, Perez C, et al. Treatment of inoperable nonsmall-cell carcinoma of the lung with radiation therapy, with or without levamisole. Am J Clin Oncol 1984; 7:405–412. 286. Kreienberg R, Boerner D, Melchert F, and Lemmell EM. Reduction of the immunosuppressive action of chemotherapeutics in patients with mammary carcinoma by Azimexon. J Immunopharmacol 1983; 5:49–64. 287. Kripke ML. Immunoregulation of carcinogenesis: Past, present, and future. J Natl Cancer Inst 1988; 80:722–727. 288. Kumano N, Suzuki S, Oizumi K, et al. Imbalance of T-cell subsets in cancer patients and its modification with bestatin, a small molecular immunomodifier. Tohoku J Exp Med 1985; 147:125–133. 289. Kurosu Y, Tanaka N, Furusho Y, Morita K. Cimetidine-mediated augmentation of lymphocyte responses to phytohemagglutinin in gastric cancer patients. Jpn J Clin Oncol 1989; 19:1; 56–61. 290. Lauria F, Raspadori D, Tura S. Effect of a thymic factor on T-lymphocytes in B-cell chronic lymphocytic leukemia: In vitro and in vivo studies. Blood 1984; 64:667–671.

138

Therapeutic approaches to cancer-associated immune suppression

291. Lauria F, Foa R, Gobbi M, et al. Increased proportion of suppressor/cytotoxic (OKT8+) cells in patients with Hodgkin’s disease in long-lasting remission. Cancer 1983; 52:1385–1388. 292. Lauria JA, Moertel CG, Fleming TR, et al. Surgical adjuvant therapy of large-bowel carcinoma: An evaluation of levamisole and the combination of levamisole and fluorouracil. J Clin Oncol 1989; 7:1447–1456. 293. Laurie J, Moertel C, Fleming T, et al. Surgical adjuvant therapy of poor prognosis colorectal cancer with levamisole alone or combined levamisole and 5-fluorouracil (5-FU) (Abstract). Proc ASCO 1986; 5:81. 294. Lawrence HS. The transfer in humans of delayed skin sensitivity to streptococcal M substance and to tuberculin with disrupted leukocytes. J Clin Invest 1955; 34:219–230. 295. Lee ET, Ishmael R, Bottomley RH, Murray JL. An analysis of skin tests and their relationship to recurrence and survival in stage III and stage IV melanoma patients. Cancer 1982; 49:2336–2341. 296. Lee YT. Delayed cutaneous hypersensitivity, lymphocyte count, and blood tests, in patients with breast carcinoma. J Surg Oncol 1984; 27:135–140. 297. Lee AKY, Rowley M, MacKay IR. Antibody-producing capacity in human cancer. Br J Cancer 24:454–463. 298. Lee YT, Sparks FC, Eilber FR, Morton DL. Delayed cutaneous hypersensitivity and peripheral lymphocyte counts in patients with advanced cancer. Cancer 1975; 35:748–755. 299. Leibler GA, Concannon JP, Magorern GJ, et al. Immunoprofile studies for patients with bronchogenic carcinoma. J Thorac Cardiovasc Surg 1977; 74:506–518. 300. Levin RH, Landy M, Frei E. The effect of 6-mercaptopurine on immune response in man. N Engl J Med 1964; 271:16–22. 301. Levo Y, Rotter V, Ramot B. Restoration of cellular immune response by levamisole in patients with Hodgkin’s disease. Biomedicine 1975; 23:198–200. 302. Lewinski UH, Mavligit GM, Hersh EM. Cellular immune modulation after a single high dose of levamisole. Cancer 1980; 46:2185–2194. 303. Liberati AM, Bragia M, Edwards BS, et al. Immunorestorative properties of thymostimulin (TS) in patients with Hodgkin’s disease in clinical remission. Cancer Immunol Immunother 1985; 19:136–141. 304. Liberata AM, Ballatori E, Fizzotti M, et al. A randomized trial to evaluate the immunorestorative properties of thymostimulin in patients with Hodgkin’s disease in complete remission. Cancer Immunol Immunother 1988; 26:87–93. 305. Liberata AM, Voekel JG, Borden EC, et al. Influence of non-specific immunologic factors on prognosis in advanced bronchogenic carcinoma. Cancer Immunol Immunother 1982; 13:140–144. 306. Lichtenstein A, Zighelboim J, Dorey R, et al. Comparison of immune derangements in patients with different malignancies. Cancer 1980; 45:2090–2095. 307. Liu W, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CO4+ Treg cells. J Exp Med 2006; 203:1701–1711. 308. Lotan R. Effects of vitamin A and its analogs (retinoids) on normal and neoplastic cells. Biochim Biophys Acta 1980; 605:33–91. 309. Loutfi A, Shakr A, Jerry M, et al. Double-blind randomized prospective trial of levamisole/placebo in stage I cutaneous malignant melanoma. Clin Invest Med 1987; 10:4; 325–328. 310. Ludwig CU, Hartmann D, Landmann R, et al. Unaltered immunocompetence in patients with nondisseminated breast cancer at the time of diagnosis. Cancer 1985; 55:1673–1678. 311. Lundy J, Raaf JH, Deakins S, Wanebo HJ. The acute and chronic effects of alcohol on the human immune system. Surg Gynecol Obstet 1975; 141:212–218.

312. Luzi G, Troopea F, Seminara R, et al. Clinical and immunological evaluation in non-resectable lung cancer patients treated with thymostimulin. In: Byrom NA, Hobbs JR, eds. Thymic factor therapy. New York: Raven Press, 1984:309–320. 313. Maca RM, Panje WR. Indomethacin-sensitive suppressor-cell activity in head and neck cancer patients pre- and post-irradiation therapy. Cancer 1982; 50:483–489. 314. Macchiarini P, Danesi R, Del Tacca M, Angeletti CA. Effects of thymostimulin on chemotherapy-induced toxicity and long-term survival in small-cell lung cancer patients. Anticancer Res 1989; 9:1; 193–196. 315. Mackay JR, Goodyear MD, Riglar C, et al. Effect on immunologic and other indices of adjuvant cytotoxic chemotherapy including melphalan in breast cancer. Cancer 1984; 53:2619–2627. 316. Maguire LC, Roszman TL, Lackey S. Failure of cimetidine as an immunomodulator in cancer patients and normal subjects. South Med J 1985; 78:1078–1080. 317. Mahaley MS Jr, Brooks WH, Roszman TL, et al. Immunobiology of primary intracranial tumors. I. Studies of the cellular immunity of brain tumor patients. J Neurosurg 1977; 46:467–476. 318. Maisel RH, Ogura JH. Dinitrochlorobenzene skin sensitization and peripheral lymphocyte count: Predictors of survival in head and neck cancer. Ann Otol Rhinol Laryngol 1976; 85:517–522. 319. Mandel MA, Dvorak K, Decasse JJ. Salivary immunoglobulins in patients with oropharyngeal and bronchopulmonary cancer. Cancer 1973; 31:1408–1413. 320. Mandeville R, Lamoureux G-T, Poisson SL, Poisson R. Biological markers and breast cancer: A multiparameter study. II. Depressed immune competence. Cancer 1982; 50:1280–1288. 321. Mantovani G, Floris C, Maccio A, et al. Role of interleukin-2 (IL-2) in cancer-related immune deficiency: In vitro response to IL-2, production of IL-2, and IL-2 receptor expression in patients with advanced cancer. Cancer Detect Prev 1988; 12:149–159. 322. Marshall ME, Mendelsohn L, Butler K, et al. Treatment of metastatic renal cell carcinoma with coumarin (1,2-benzopyrone) and cimetidine: A pilot study. J Clin Oncol 1987; 5:6; 862–866. 323. Marshall ME, Riley LK, Rhoades J, et al. Effects of coumarin (1,2-benzopyrone) and cimetidine on peripheral blood lymphocytes, natural killer cells, and monocytes in patients with advanced malignancies. J Biol Response Mod 1989; 8:1; 62–69. 324. Martelli MF, Velardi A, Rambotti P, et al. The in vivo effect of a thymic factor (thymostimulin) in immunologic parameters of patients with untreated Hodgkin’s disease. Cancer 1982; 50:490–497. 325. Mastrangelo MJ, Berd D, Maguire H Jr. Cell-mediated immunity: The immunoaugmenting effects of cancer chemotherapeutic agents. Sem Oncol 1986; 13:2; 186–194. 326. Mathé G, Amiel JL, Schwarzenberg L, et al. Active immunotherapy for acute lymphoblastic leukemia. Lancet 1969; 1:697–699. 327. Mathé G. Do tuftsin and bestatin constitute a biopharmacological immunoregulatory system? Cancer Detect Prev Suppl 1987; 1:445–455. 328. Mathé G, Umezawa H, Misset JL, et al. Immunomodulating properties of bestatin in cancer patients. A phase II trial. Biomed Pharmacother 1986; 40:10; 379–382. 329. Mavligit GM. Immunologic effects of cimetidine: Potential uses. Pharmacotherapy 1987; 7:120S–124S. 330. Mavligit GM, Raphael LS, Calvo DB III, Wong WL. Indomethacin-induced monocyte-dependent restoration of local graft-versus-host reaction among cells from cancer patients. J Natl Cancer Inst 1980; 65:317–320. 331. McGeorge MB, Russel EC, Mohanakumar T. Immunologic evaluation of long-term effects of childhood ALL chemotherapy: Analysis of in vitro NK- and K-cell activities of peripheral blood lymphocytes. Am J Hematol 1982; 12:19–27.

Robert K. Oldham 332. McGinnes K, Florence J, Penny R. The effect of radiotherapy on the natural killer (NK) cell activity of cancer patients. J Clin Immunol 1987; 7:3; 210–217. 333. Menconi E, Barzi A, Greco M, et al. Immunological profile of breast cancer patients in early of advanced disease. Experimentia 1979; 35:820–822. 334. Merrin C, Han T. Immune responses in bladder cancer. J Urol 1974; 111:170–172. 335. Messerschmidt GL, Henry DH, Snyder HW Jr, et al. Protein A immunoadsorption in the treatment of malignant disease. J Clin Oncol 1988; 6:2; 203–212. 336. Metzher Z, Hoffeld JT, Oppenheim JJ. Evidence for the involvement of monocyte-derived toxic oxygen metabolites in the lymphocyte dysfunction of Hodgkin’s disease. Clin Exp Immunol 1981; 46:313–320. 337. Micksche M, Cerni C, Kokron K, et al. Stimulation of the immune response in lung cancer patients by vitamin A. Oncology 1977; 34:234–239. 338. Micksche M, Colot M, Kucera H. Radioimmunotherapy in advanced cervical cancer. In: Dubois JB, Serrou B, Rosenfeld C, eds. Immunopharmacologic effects of radiation therapy. New York: Raven Press, 1981:499–518. 339. Mihich E. Biological response modifiers in cancer therapeutics: Potentialities and limitations. Cancer Detect Prev 1988; 12:531–535. 340. Mihich E, Ehreke MJ, Ishizuka M. Immunomodulation by antibiotics. In: Mihich E, Sakurai Y, eds. Biological responses in cancer, vol 3: immunomodulation by anti-cancer drugs. New York: Plenum Press, 1985:71–73. 341. Miller HC, Vito C. Action of a thymic cytokine TsIF in reversing the autoimmune disease state of the MRL/1pr mouse. Mol Biother 1989; 1:4; 213–217. 342. Miller LL, Spitler LE, Allen RE, Minot DR. A randomized, double-blind, placebo-controlled trial of transfer factor as adjuvant therapy for malignant melanoma. Cancer 1988; 61:1543–1549. 343. Miller FR, Kataoka T. Interactions of antimetabolites with tumors and the immune system. In: Mihich E, Sakurai Y, eds. Biological responses in cancer, vol 3: immunomodulation by anti-cancer drugs. New York: Plenum Press, 1985:33–69. 344. Mitchell MS, Kempf RA, Harel W, et al. Effectiveness and tolerability of low-dose cyclophosphamide and low-dose intravenous interleukin-2 disseminated melanoma. J Clin Oncol 1988; 6:3; 409–424. 345. Mitchell MS, Defonti RC. Immunosuppression by 5-fluorouracil. Cancer 1970; 26:884–889. 346. Mitchell MS, Wade ME, Deconti RC, et al. Immunosuppressive effects of cytosine arabinoside and methotrexate in man. Ann Int Med 1969; 70:535–547. 347. Miwa H, Orita K. Immunotherapy of gastrointestinal cancer patients with levamisole. Acta Med Okayama 1979; 33:29–42. 348. Miwa H, Orita K. Cancer immunotherapy with levamisole. Acta Med Okayama 1978; 32:239–245. 349. Miwa H, Orita K. Reactivating effect of levamisole on cell-mediated immunity in gastrointestinal cancer patients. Acta Med Okayama 1977; 31:325–329. 350. Modini C, Ziparo V, Cicconetti F, et al. Role of delayed cutaneous hypersensitivity reaction in classifying patients with bronchial carcinoma. Tumori 1985; 71:277–281. 351. Moertel CG, Fleming TR, MacDonald JS, et al. Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma. N Engl J Med 1990; 322:6; 352–358.

139 352. Moertel CG, Ritts RE, O’Connell MJ, Silvers A. Nonspecific immune determinants in the patient with unresectable gastrointestinal carcinoma. Cancer 1979; 43:1483–1492. 353. Monson JR, Ramsden C, Guillou PJ. Decreased interleukin-2 production in patients with gastrointestinal cancer. Br J Surg 1986; 73:6; 483–486. 354. Monte JE, Bukowski RM, James RE, et al. A critical review of immunotherapy of disseminated renal adenocarcinoma. J Surg Oncol 1982; 21:5–8. 355. Morales A, Eidinger D. Immune reactivity in renal cancer. J Urol 1976; 115:510–513. 356. Morin A, Ballet J, Touraine J, Hadder JW. Current status of isoprinosine. In: Serrou B, Rosenfeld C, Daniels JC, Saunders JP, eds. Current concepts in human immunology and cancer immunomodulation. Amsterdam: Elsevier Biomedical, 1982:479–489. 357. Morton DL, Eilber FR, Malmgren RA, Wood WC. Immunological factors which influence response to immunotherapy in malignant melanoma. Surgery 1970; 68:158–164. 358. Mukherji B, Guha A, Loomis R, Ergin MT. Cell-mediated amplification and down regulation of cytotoxic immune response against autologous human cancer. J Immunol 1987; 138:1987–1991. 359. Mukhopadhyaya R, Advani SH, Gangal SG. Functional evaluation of T-lymphocytes from peripheral blood and spleens in Hodgkin’s disease. Br J Cancer 1987; 56:800–802. 360. Munster AM. Post-traumatic immunosuppression is due to activation of suppressor T-cells. Lancet 1976; 1:1329. 361. Munzarova M, Mechl Z, Kovarik J, Kolcova V. Skin tests with autologous cholesteryl hemisuccinate-treated tumor cells, DNCB and PPD and their relationship to prognosis in malignant melanoma patients. Neoplasma 1988; 35:2; 229–234. 362. Murphy RM, Colton CK, Yarmush ML. Staphylococcal protein A adsorption in neoplastic disease: Analysis of physiochemical aspects. Mol Biother 1989; 1:4; 186–207. 363. Murray JL, Kollmorgen GM. Inhibition of lymphocyte response by prostaglandin-producing suppressor cells in patients with melanoma. J Clin Immunol 1983; 3:268–276. 364. Murray JL, Reuben JM, Smith TL, et al. A pilot clinical trial of the toxicity and immunorestorative effects of T-cell reconstituting factor (SR 270258) in immunosuppressed cancer patients. J Biol Response Mod 1987; 6:1; 56–68. 365. Najjar VA. Biochemistry and physiology of tuftsin – Thr-LysPro-Arg. In: Sbarra S, Strauss R, eds. The reticuloendothelial system. New York: Plenum Press, 1980:45–71. 366. Nathanson SD, Zamfirescu PL, Portaro JK, et al. Acute effects of orally administered levamisole on random monocyte motility and chemotaxis in man. J Natl Cancer Inst 1978; 61:301–306. 367. Niimoto M, Hattori T, Ito I, et al. Levamisole as postoperative adjuvant immunochemotherapy for gastric cancer. A randomized controlled study of the MMC+ Tegafur regimen with or without levamisole. Report I. Cancer Immunol Immunother 1984; 18:13–18. 368. Noda T, Asano M, Yoshie O, et al. Interferon-γ induction of human peripheral blood mononuclear cells by OK-432, a killed preparation of Streptococcus pyogenes. Microbiol Immunol 1985; 30:81–88. 369. Noma T, Yoshimura N, Yata J. Depressed lymphocyte functions of cancer patients and their correlation by bestatin (product of Streptomyces olivereticuli). In: Serrou S, Rosenfeld C, Daniels JC, Saunders JP, eds. Current concepts in human immunology and cancer immunomodulation. Amsterdam: Elsevier Biomedical, 1982:611–616. 370. Nordman E, Saarimaa H, Tolvanen A. The influence of 5-fluorouracil on cellular and humoral immunity in cancer patients. Cancer 1978; 41:64–69.

140

Therapeutic approaches to cancer-associated immune suppression

371. Nordman E, Tolvanen A. Effects of irradiation of the immune function in patients with mammary, pulmonary or head and neck carcinoma. Acta Radiol Oncol Radiat Phys Biol 1978; 17:3–9. 372. Norris RW, Byrom NA, Nagvekar NM, et al. Thymostimulin plus surgery in the treatment of primary truncal malignant melanoma: Preliminary results of a U.K. multicentre clinical trial. In: Byrom NA, Hobbs JR, eds. Thymic factor therapy. New York: Raven Press, 1984:341–348. 373. Oka S. A review of clinical studies of bestatin. Recent results. Cancer Res 1980; 75:126–132. 374. Olivari AJ, Glait HM, Guardo A, et al. Levamisole in squamouscell carcinoma of the head and neck. Cancer Treat Rep 1979; 63:983–990. 375. Onsrud M. Whole pelvic irradiation in stage I endometrial carcinoma: Changes in numbers and reactivities of some blood lymphocyte subpopulations. Gynecol Oncol 1982; 13:283–292. 376. Orita K, Miwa H, Fukuda H, et al. Preoperative cell-mediated immune status of gastric cancer patients. Cancer 1976; 38:2343–2348. 377. Osband M, Shen Y, Schlesinger M, et al. Successful tumor immunotherapy with cimetidine in mice. Lancet 1981; 1:636–638. 378. Oshimi K, Kano S, Takaku F, Okumura K. Augmentation of mouse natural killer cell activity by a streptococcal preparation OK-432. J Natl Cancer Inst 1980; 65:1265–1269. 379. Ota K, Kurita S, Yamada K, et al. Immunotherapy with bestatin for acute non-lymphocytic leukemia in adults. Cancer Immunol Immunother 1986; 23:1; 5–10. 380. Ozer H. Effects of alkylating agents on immunoregulatory mechanisms. In: Mihich E, Sakurai Y, eds. Biological responses in cancer, vol 3: immunomodulation by anti-cancer drugs. New York: Plenum Press, 1985:95–129. 381. Paavonen T, Anderson LC, Adlercrentz H. Estradiol enhances human B-cell maturation via inhibition of suppressor T-cells in pokeweed mitogen-stimulated cultures. J Exp Med 1981; 154:1935–1945. 382. 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; 34:5; 627–632. 383. Paik YK, Kimura L, Yanagihora E, et al. Inhibition of the phytohemagglutinin stimulation of leukocytes by C-reactive protein. J Reticuloendothel Soc 1972; 11:420. 384. Papatestas AE, Kark AE. Peripheral lymphocyte counts in breast carcinoma: An index of immune competence. Cancer 1978; 34:2014–2017. 385. Parkinson DR, Cano PO, Jerry LM, et al. Complications of cancer immunotherapy with levamisole. Lancet 1977; i:1129–1132. 386. Passwell J, Lavanon M, Davidsohn J, Ramot B. Monocyte PG E2 secretion in Hodgkin’s disease and its relation to decreased cellular immunity. Clin Exp Immunol 1983; 51:61–68. 387. Paterson AHG, Natting M, Takata L, et al. Chemoimmunotherapy with levamisole in metastatic breast carcinoma: a controlled clinical trial. Cancer Clin Trials 1980; 3:5–10. 388. Patt YZ, Hersh EM, Reuben J, et al. A phase-I study of intravenous azimexon therapy in human cancer. J Biol Response Mod 1986; 5:4; 313–318. 389. Pauwels R, Van der Straeter M. Delayed hypersensitivity in patients with bronchial carcinoma. Scand J Resp 1975; 56:160–164. 390. Pavlovsky S, Muriel FS, Garay G, et al. Chemoimmunotherapy with levamisole in acute lymphoblastic leukemia. Cancer 1981; 48:1500–1507. 391. Pavlovsky S, Garay G, Sackman MF, Svarch E. Levamisole therapy during maintenance of remission in patients with acute lymphoblastic leukemia. In: Terry WD, ed. Proceedings of Second

392.

393. 394. 395.

396.

397.

398.

399.

400.

401.

402.

403.

404.

405.

406. 407. 408.

409.

410.

411.

412.

International Conference on Immunotherapy of Cancer. Amsterdam: Elsevier/North-Holland, 1980:47–54. Payne JE, Meyer JH, Macpherson JG, et al. The value of lymphocyte transformation in carcinoma of the colon and rectum. Surg Gynecol Obstet 1980; 150:687–693. Penn I. Tumors of the immunocompromised patient. Annu Rev Med 1988; 39:63–73. Penn I. Occurrence of cancer in immune deficiency. Cancer 1974; 34:858–866. Perez CA, Bauer M, Emami BN, et al. Thoracic irradiation with or without levamisole (NSC #177023) in unresectable non-small-cell carcinoma of the lung: A phase III randomized trial of the RTOG. Int J Radiation Oncology Biol Phys 1988; 15:6; 1337–1346. Petrini B, Wasserman J, Blomgren H, Rotstein S. T-helper/ suppressor ratios in chemotherapy and radiotherapy. Clin Exp Immunol 1983; 53:255–256. Petrini B, Wasserman J, Glas U, Blomgren H. T-lymphocyte subpopulations in blood following radiation therapy for breast cancer. Eur J Cancer Clin Oncol 1982; 18:921–924. Petrini B, Wasserman J, Biberfeld G, et al. The effect of in vitro irradiation on PHA-mediated cytotoxicity and lymphocytes with receptors for the Fc pat of Ig. J Clin Lab Immunol 1979; 2:333–336. Pierri I, Cordone G, Rogna S, et al. T-lymphocyte phenotype and functions in patients with head and neck cancer. Laryngoscope 1985; 95:577–581. Pines A. BCG plus levamisole following irradiation of advanced squamous bronchial carcinoma. Int J Radiation Oncology Biol Phys 1980; 6:1041–1042. Pinsky CM, El-Domeiri A, Caron AA, et al. Delayed hypersensitivity reactions in patients with cancer. In: Mathé G, Weiner R, eds. Recent results in cancer research, 1970; 35: 37–41. Pinsky CM, Wanebo H, Mike V, Oettgen H. Delayed cutaneous hypersensitivity reactions and prognosis in patients with cancer. Ann NY Acad Sci 1976; 276:407–410. Pompidou A, Soubrane C, Cour V, et al. Immunological effects of isoprinosine as a pulse immunotherapy in melanoma and ARC patients. Cancer Detect Prev Suppl 1987; 1:457–462. Posner MR, Reinherz E, Lane H, et al. Circulating lymphocyte populations in Hodgkin’s disease after mantle and para-aortic irradiation. Blood 1983; 61:705–708. Powles TJ, Dady DJ, Williams J, et al. Use of inhibitor of prostaglandin synthesis and patients with breast cancer. In: Sammuelsson B, Ramwel PW, Paoletti R, eds. Advances in prostaglandin and thromboxane research, vol 6. New York: Raven Press, 1980 511–516. Prehn RT, Prehn LM. The flip side of tumor immunity. Arch Surg 1989; 124:102–106. Prehn RT, Prehn LM. The autoimmune nature of cancer. Cancer Res February 15, 1987; 47: 927–932. Pritchard DJ, Ritts RE Jr, Taylor WF, Miller GC. A prospective study of immune responsiveness in humans. I. Assessment of initial pretreatment status with stage of disease. Cancer 1978; 41:2165–2173. Pross HF, Baines MG. Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells: I. The effect of malignant disease. Int J Cancer 1976; 18:593–604. Pui C-H, Ip SH, Kung P, et al. High serum interleukin-2 receptor levels are related to advanced disease and a poor outcome in childhood non-Hodgkin’s lymphoma. Blood 1987; 70:3; 624–628. Pulay TA, Csomor S. Effect of levamisole treatment on immunological parameters and the early course of cervical cancer. Neoplasia 1980; 29:81–86. Quirt I, Shelley W, Bodurtha A, et al. Adjuvant levamisole improves survival and disease-free survival in patients with poor

Robert K. Oldham

413.

414.

415.

416.

417.

418.

419.

420.

421.

422.

423.

424.

425.

426.

427.

428.

429. 430.

431.

prognostics with malignant melanoma. (Abstr.) Proc ASCO 1986; 5:130. Raben M, Walch N, Galili U, Schlesinger M. The effect of radiation therapy on lymphocyte subpopulations in cancer patients. Cancer 1976; 37:1417–1421. Ramot B, Biniaminov M, Shoham C, Rosenthal E. The effect of levamisole on E-rosette-forming cells in vivo and in vitro in Hodgkin’s disease patients. N Engl Med 1976; 194:809–811. Ramot B, Rosenthal E, Biniaminov M, Ben-Bassat I. Effect of levamisole, thymic humoral factor, and indomethacin on E-rosette formation of lymphocytes in Hodgkin’s disease. Isr J Med Sci 1981; 17:232–235. Rangel DM, Golub SH, Morton DL. Demonstration of lymphocyte blastogenesis-inhibiting factors in sera of melanoma patients. Surgery 1977; 82:224–232. Rapson NT, Cornbleet MA, Chessells JM, Hardisity RM. Immunosuppression and serious infections in children with acute lymphoblastic leukemia: A comparison of the three chemotherapy regimens. Br J Hematol 1980; 45:41–52. Ravikumar T, Steele G, Rodrick M, et al. Effects of tumor growth on interleukins and circulating immune complexes. Cancer 1984; 53:1373–1378. Reed DM. On the pathological changes in Hodgkin’s disease with special references to its relation to tuberculosis. Johns Hopkins Hosp Rep 1902; 10:133–198. Rees JC, Rossio JL, Wilson HG, et al. Cellular immunity in neoplasia. Antigen and mitogen responses in patients with bronchogenic carcinoma. Cancer 1975; 36:2010–2015. Remacle-Bonnet MM, Rommier GJ, Luc C, et al. Nonspecific suppressive and cytostatic activities mediated by human colonic carcinoma tissue or cultured cell extract. J Immunol 1978; 121:44–52. Renoux G, Renoux M. DTC. A summary of current status. In: Serrou B, Rosenfeld C, Daniels JC, Saunder JP, eds. Current concepts in human immunology and cancer immunomodulation. Amsterdam: Elsevier Biomedical 1982:575–584. Renoux G, Renoux M. Effect immunostimulant d’un imidothiazole dans l’immunization des souris contre l’infecction par Brucella abortus. C R Acad Sci (Paris) 1971; 272:349–350. Ress JC, Rossio HL, Wilson HE, et al. Cellular immunity in neoplasia. Antigen and mitogen response in patients with bronchogenic carcinoma. Cancer 1975; 36:2010–2015. Retsas S, Thomas C, Hobbs JR. The effect of vindesine therapy on the in vitro immune response of patients with advanced malignant melanoma. Clin Oncol 1981; 7:33–37. Richtsmeier WJ, Eisele D. In vivo anergy reversal with cimetidine in patients with cancer. Arch Otolaryngol Head Neck Surg 1986; 112:10; 1074–1077. Rijswijk REN, Sybesma JPH, Kater L. A prospective study of the changes in the immune status before, during and after multiple agent chemotherapy for Hodgkin’s disease. Cancer 1983; 51:637–644. Rijswijk REN, Sybesma JPH, Kater L. A prospective study of the changes in immune status following radiotherapy for Hodgkin’s disease. Cancer 1984; 53:62–69. Roberts MM. Lymphocyte transformation in breast cancer. Br J Surg 1970; 57:381. Robinson BW. Asbestos and cancer: Human natural killer cell activity is suppressed by asbestos fibers but can be restored by recombinant interleukin-2. Am Rev Respir Dis 1978; 139:4; 897–901. Rojas AF, Feirstan JN, Glait HM, Olivari AJ. Levamisole action in breast cancer. In: Terry WD, Windhorst D, eds. Immunotherapy

141

432.

433. 434.

435.

436.

437.

438.

439.

440.

441.

442.

443.

444.

445.

446.

447.

448.

449.

of cancer: Present status on trials in man. New York: Raven Press, 1978:635–646. Romagnani S, Maggi E, Del Prete G, et al. Short- and long-term effects of radiation on T-cell subsets in peripheral blood of patients with Hodgkin’s disease. Cancer 1980; 46:2590–2595. Romagnani S, Ferrini PLR, Ricci M. The immune derangement in Hodgkin’s disease. Semin Oncol 1985; 22:41–55. Rosenberg SA, Seipp CA, White DE, Wesley R. Perioperative blood transfusions are associated with increased rates of recurrence and decreased survival in patients with high-grade soft-tissue sarcomas of the extremities. J Clin Oncol 1985; 3:5; 698–709. Rossen RD, Morgan AC. Blockage of the humoral immune response: Immune complexes in cancer. Cancer Immunol 1980; 210–280. Roth JA, Osborne BA, Ames RS. Immunoregulatory factors derived from human tumors. II. Partial purification and further immunobiochemical characterization of a human sarcoma-derived immunosuppressive factor expressing HLA-DR and immunoglobulin-related determinants. J Immunol 1983; 130:303–308. Roth JA, Golub SH, Grimm EA, et al. Effects of operation on immune response in cancer patients: Sequential evaluation of in vitro lymphocyte function. Surgery 1976; 79:47–51. Rotstein S, Blomgren H, von Stedingk LV, et al. Long-term effects on the immune system following local irradiation for breast cancer. Pokeweed mitogen-induced immunoglobulin secretion by blood lymphocytes and serum immunoglobulin levels. Eur J Surg Oncol 1985; 11:137–141. Rotstein S, Blomgren H, Nilsson B, et al. Prognostic value of various immunological tests in breast cancer patients treated with adjuvant postoperative chemotherapy. Chemotherapy 1984; 3:227–231. Rotstein S, Blomgren H, Petrini, et al. Blood lymphocyte counts with subset analysis in operable breast cancer. Cancer 1985; 56:1413–1419. Ryan RE Jr, Neel HB, Ritts RE. Correlation of pre-operative immunologic test results with recurrence in patients with head and neck cancer. Otolaryngol Head Neck Surg 1980; 88:58–63. Saijo N, Shimizu E, Shibuya M, et al. Effect of chemotherapy on natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity in carcinoma of the lung. Br J Cancer 1982; 46: 180–189. Saito M, Ebina T, Koi M, et al. Induction of interferon-gamma in mouse spleen cells by OK-432, a preparation of Streptococcus pyogenes. Cell Immunol 1982; 68:187–192. Sakaguchi S. Naturally arising CO4(+) regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004; 22:531–562. Salmon SE, Hunt A, Bonnet JD, et al. Alternating combination chemotherapy and levamisole improves survival in multiple myeloma: A Southwest Oncology Group study. J Clin Oncol 1983; 1:453–461. Sample WF, Gertner HR, Chretien PB. Inhibition of phytohemagglutinin-induced in vitro lymphocyte transformation by serum from patients with carcinoma. J Natl Cancer Inst 1971; 46:1291–1297. Santos GW, Owens AH, Sensenbrenner LL. Effects of selected cytotoxic agents on antibody production in man: A preliminary report. Ann NY Acad Sci 1964; 114:404–423. Santos LB, Yamada FT, Scheinberg MA. Monocyte and lymphocyte interaction in patients with advanced cancer. Evidence for deficient IL-1 production. Cancer 1985; 56:1553–1558. Sarin PS, San DK, Thornton AH, et al. Neutralization of HTLVIII/LAV replication by antiserum to thymosin-alpha-one. Science 1986; 232:1135–1137.

142

Therapeutic approaches to cancer-associated immune suppression

450. Sato E, et al. Intraepithelial CO8(+) tumor-infiltrating lymphocytes and a high CO8(+)/regulatory T cell ratio are associated with a favorable prognosis in ovarian cancer. Proc Natl Acad Sci USA 2005; 102:18538–18543. 451. Scambia G, Panici PB, Mancuso S, et al. Effects of antiestrogen and progestin on immune functions in breast cancer patients. Cancer 1988; 61:2214–2218. 452. Schantz SP, Savage HE, Racz T, et al. Immunologic determinants of head and neck cancer response to induction chemotherapy. J Clin Oncol 1989; 7:7; 857–864. 453. Schlechter GP, Wahl LM, Oppenheim JJ. Suppressor monocytes in human disease: A review. Adv Exp Med Biol 1979; 121B: 283–298. 454. Schechter GP, Soehlen F. Monocyte-mediated inhibition of lymphocyte blastogenesis in Hodgkin’s disease. Blood 1978; 52:261–271. 455. Scher HI, Shank B, Chapman R, et al. Randomized trial of combined modality therapy with and without thymosin fraction V in the treatment of small cell lung cancer. Cancer Res 1988; 48:1663–1670. 456. Schindler L, Leroux M, Zimmerman GF, et al. Reversal of defective lymphoproliferation in postoperative patients with colon cancer. J Cancer Res Clin Oncol 1987; 113:2; 166–170. 457. Schlelhammer PF, Bracken RB, Bean M, et al. Immune evaluator with skin testing. A study of testicular, prostatic, and bladder neoplasms. Cancer 1976; 38:149–156. 458. Schlumberger HD. BAY I 7433: a synthetic polymer with antitumor activity. In: Hersh EM, Cirigos MA, Mastrangelo, MJ, eds. Augmenting agents in cancer therapy. New York: Raven Press, 1981:373–390. 459. Schulof RS, Lloyf MJ, Cleary PA, et al. A randomized trial to evaluate the immunorestorative properties of thymosin-alpha-one in patients with lung cancer. J Biol Response Mod 1985; 4:147–158. 460. Schulof RS, Bockman RS, Garofalo JA, et al. Multivariate analysis of T-cell functional defects and circulating serum factors in Hodgkin’s disease. Cancer 1981; 48:964–973. 461. Schulof RS, Michitsch RW, Livingston PO, Gupta S. Monocytemediated suppression on lymphocyte cytotoxic activity for cultured autologous melanoma cells. Cell Immunol 1981; 57:529–532. 462. Schulof RS, Chorba TC, Cleary PA, et al. T-cell abnormalities after mediastinal irradiation for lung cancer. The in vitro influence of synthetic thymosin alpha-1. Cancer 1985; 55:974–983. 463. Schulof RS. Thymic peptide hormones: Basic properties and clinical applications in cancer. CRC Critical Rev Oncol Hematol 1985; 3:309–376. 464. Schulof RS, Lee BJ, Lacher MJ, et al. Concanavalin-A-induced suppressor cell activity in Hodgkin’s disease. Clin Immunol Immunopathol 1980; 16:454–462. 465. Schulof RS, Lloyd MJ, Ueno WM, et al. Phase II trial of thymosin fraction 5 in advanced renal cancer. J Biol Response Mod 1984; 3:151–159. 466. Schulof RS, Lacher MJ, Gupta S. Abnormal phytohemagglutinin-induced T-cell proliferative responses in Hodgkin’s disease. Blood 1981; 57:607–613. 467. Seddiki N, et al. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006; 203:1693–1700. 468. Seder RH, Katz AE, Keggins JJ, et al. Plasma exchange in six patients with advanced cancers of the head and neck. J Clin Apheresis 1985; 2:238–249. 469. Seder RH, Katz AE, Keggins JJ, et al. Plasma exchange in six patients with advanced cancers of the head and neck. J Clin Apheresis 1985; 2:238–249.

470. Seder RH, Vaughn CW, Oh S-K, et al. Tumor regression and temporary restoration of immune response after plasmapheresis in a patient with recurrent oral cancer. Cancer 1987; 60:318–325. 471. Sedman PC, Ramsden CW, Brennan TG, et al. Effects of lowdose perioperative interferon on the surgically induced suppression of antitumor immune response. Br J Surg 1988; 75:976–981. 472. Sen L, Borella L. Expression of cell surface markers on T- and B-lymphocytes after long-term chemotherapy of acute leukemia. Cell Immunol 1973; 9:84–95. 473. Serdengecti S, Buyukunal E, Molinas N, et al. Overall survival results of non-small cell lung cancer patients: Chemotherapy alone versus chemotherapy with combined immunomodulation. Chemioterapia 1988; 7:2; 122–126. 474. Sertoli MR, Guarneri D, Rubagotti A, et al. Adjuvant immunochemotherapy in colorectal cancer. Dukes C. Oncology 1987; 44:2; 78–81. 475. Shafir M, Bekesi JG, Papatestas A, et al. Preoperative and postoperative immunological evaluation of patients with colorectal cancer. Cancer 1980; 46:700–705. 476. Shank B, Scher H, Hilaris BS, et al. Increased survival with highdose multified radiotherapy and intensive chemotherapy in limited small cell carcinoma of the lung. Cancer 1985; 56:2771–2778. 477. Shiraki S, Mori H, Kadomoto N, et al. Adjuvant immunotherapy of carcinoma colli with levamisole. Prevention of immunological depression following surgical therapy and radiotherapy. Int J Immunopharmacol 1982; 4:73–80. 478. Shoham J, Theodore E, Brenner HJ, et al. Enhancement of the immune system of chemotherapy-treated cancer patients by simultaneous treatment with thymic extract, TP-1. Cancer Immunol Immunother 1980; 9:173–180. 479. Shukla HS, Hughes LE, Whitehead RH, Newcomb RG. Long-term follow-up of general immune competence in breast cancer. II. Sequential pre- and post-treatment levels: A 10-year study. Cancer Immunol Immunother 1986; 21:6–11. 480. Sibbit WL, Bankhurst AD, Williams RC. Studies of cell subpopulations mediating mitogen hyporesponsiveness in patients with Hodgkin’s disease. J Clin Invest 1978; 61:55–63. 481. Silverman NA, Alexander JC, Potvin C, Chretien PB. In vitro lymphocyte reactivity and T-cell levels in patients with melanoma: Correlations with clinical and pathological stage. Surgery 1976; 79:332–339. 482. Sinclair T, Ezdinli EZ, Boonlayangoor P, et al. Rosette and blastogenesis inhibition by plasma from Hodgkin’s disease and other malignancies. Positive correlation in stage I and II Hodgkin’s disease. Cancer 1983; 51:238–244. 483. Slade MS, Simmons RL, Yunis E, Greenberg LJ. Immunodepression after major surgery in normal patients. Surgery 1975; 78:363–372. 484. Slater JM, Ngo E, Lau BHS. Effect of therapeutic irradiation on the immune response. Am J Roentgenol Radium Ther Nucl Med 1976; 126:313–320. 485. Smalley RV, Talmadge J, Oldham RK, Thurman GB. The thymosins: Preclinical and clinical studies with fraction V and alpha-1. Cancer Treat Rev 1984; 11:69–84. 486. Smith RB, Dekernion J, Baron B, et al. Levamisole in the treatment of non-invasive and invasive bladder cancer: A preliminary report. J Urol 1978; 119:347–349. 487. Snyderman R, Siegler HF, Meadew R. Abnormalities of monocyte chemotaxis in patients with melanoma: Effects of immunotherapy and tumor removal. J Natl Cancer Inst 1977; 58:37–41. 488. Sokal JE, Aungst CW. Response to BCG vaccination and survival in advanced Hodgkin’s disease. Cancer 1969; 24:128–134.

Robert K. Oldham 489. Soto A, Hall T, Reed S. Trial of antiviral action of isoprinosine against rhinovirus infection of volunteers. Antimicrob Agent Chemother 1973; 3:332–334. 490. Soulillou JP, Douillard JU, Vie H, et al. Defects in lectin-induced interleukin-2 (IL-2) production by peripheral blood lymphocytes of patients with Hodgkin’s disease. Eur J Cancer Clin Oncol 1985; 21:935. 491. Spitler LE, Sagebiel R. A randomized trial of levamisole versus placebo as adjuvant therapy in malignant melanoma. N Engl J Med 1980; 303:1143–1147. 492. Spreafico F, Vecchi A. The imunomodulatory activity of certain cancer chemotherapeutic agents. In: Mihich E, Sakurai Y, eds. Biological responses in cancer, vol. 3: immunomodulation by anti-cancer drugs. New York: Plenum Press, 1985:131–149. 493. Steele G, Wang BS, Mannick JA. Immune RNA and transfer factor. In: Mihich E, ed. Immunological approaches to cancer therapeutics. New York: Wiley, 1982:257–276. 494. Stefani S, Kerman R, Abbate J. Serial studies of immunocompetence in head and neck cancer patients undergoing radiation therapy. Am J Roentgenol Radium Ther Nucl Med 1976; 126:880–886. 495. Stefano S, Kerman R, Abbate J. Serial studies of immunocompetence in head and neck cancer patients undergoing radiation therapy. Am J Roentgenol 1976; 126:880–886. 496. Stein JA, Adler A, Efraim SB, Maor M. Immunocompetence, immunosuppression and human breast cancer. Cancer 1976; 38:1171–1187. 497. Stephens EJW, Wood HF, Mason B. Levamisole as adjuvant to cyclic chemotherapy in end-stage mammary carcinoma. Recent Results Cancer Res 1978; 68:139–145. 498. Stratton JA, Byfield PE, Byfield JE, et al. A comparison of the acute effects of radiation therapy, including or excluding the thymus, or the lymphocyte subpopulations of cancer patients. J Clin Invest 1975; 56:88–97. 499. Stratton JA, Fast PE, Weintraub I. Recovery of lymphocyte function after radiation therapy for cancer in relationship to prognosis. J Clin Lab Immunol 1982; 7:147–153. 500. Strender LE, Blomgren H, Wasserman J, et al. Influence of adjuvant radiation therapy in breast cancer on PWM-induced Ig secretion by blood lymphocytes in vitro. Anticancer Res 1983; 3:41–45. 501. Strender LE, Blomgren H, Petrini B, et al. Immunologic monitoring in breast cancer patients receiving postoperative adjuvant chemotherapy. Cancer 1981; 48:1996–2002. 502. Sugden PJ, Lilleyman JS. Impairment of lymphocyte transformation by plasma from patients with advanced Hodgkin’s disease. Cancer 1980; 45:899–905. 503. Symoens J, Veys E, Mielants W, Pinals R. Adverse reactions to levamisole. Cancer Treat Rep 1978; 62:1721–1730. 504. Taguchi T. Clinical efficacy of lentinan on patients with stomach cancer: Endpoint results of a four-year follow-up survey. Cancer Detect Prev Suppl 1987; 1:333–349. 505. Takusugi M, Ramseyer A, Takasugi J. Decline of natural nonselective cell-mediated cytotoxicity in patients with tumor progression. Cancer Res 1977; 37:413–418. 506. Talmadge JE, Lenz BF, Pennington R, et al. Immunomodulatory and therapeutic properties of bestatin in mice. Cancer Res 1986; 46:9; 4505–4510. 507. Talmadge JE, Lenz B, Schneider M, et al. Immunomodulatory and therapeutic properties of FK-565 in mice. Cancer Immunol Immunother 1989; 28:93–100. 508. Talpaz M, Medina JE, Patt YZ, et al. The immune restorative effect of cimetidine administration in vivo on the local graft-vs-host

143

509.

510.

511

512.

513.

514.

515.

516. 517.

518. 519. 520.

521.

522.

523.

524. 525.

526.

527. 528.

529.

reaction of cancer patients. Clin Immunol Immunopathol 1982; 24:155–160. Tamura K, Shibata Y, Matsuda Y, Ishida N. Isolation and characterization of an immunosuppressive acidic protein from ascitic fluids of cancer patients. Cancer Res 1981; 41:3244–3252. Taramelli D, Mazzocchi A, Clemente C, et al. Lack of suppressive activity of human primary melanoma cells on the activation of autologous lymphocytes. Cancer Immunol Immunother 1988; 26:1; 61–66. Tarpley JL, Twomey PL, Catalona WJ, Cretien PB. Suppression of cellular immunity by anesthesia and operation. J Surg Res 1977; 22:195–201. Tartter PI, Burrows L, Kirshner P. Perioperative blood transfusion adversely affects prognosis after resection in stage I (subset No) non-oat cell lung cancer. J Thorac Cardiovasc Surg 1984; 88:659–662. Tartter PI, Martinelli G. Lymphocyte subsets, natural killer cytotoxicity, and perioperative blood transfusion for elective colorectal cancer surgery. Cancer Detect Prev (Suppl) 1987; 1:571–576. Tartter PI, Burrows L, Papatestas AE, et al. Perioperative blood transfusion has prognostic significance for breast cancer. Surgery 1985; 97:225–230. Taylor SA, Gordon MY, Currie GA, Shepherd VB. Irradiation and monocyte function in patients with breast cancer. Int J Rad Oncol 1979; 5:2063–2067. Theofilopoulos A, Dixon FJ. The biology and detection of immune complexes. Adv Immunol 1979; 28:89–220. Thienpont D, Vanparjis OFJ, Racymakers AHM, et al. Tetramasole (R8299): A new potent broad spectrum anthelmintic. Nature 1966; 209:1084–1086. Thornes RD, Lynch G, Sheehan MV. Cimetidine and coumarin therapy of melanoma. Lancet 1982; 2:328. Thornes RD, Lynch G. Combination of cimetidine with other drugs for the treatment of cancer. N Engl J Med 1983; 308:591. Tilden AB, Balch CM. Immune modulatory effects of indomethacin in melanoma patients are not related to prostaglandin E2-mediated suppression. Surgery 1982; 92:528–532. Toge T, Hamamoto S, Itagaki E, et al. Concanavalin-A-induced and spontaneous suppressor cell activities in peripheral blood lymphocytes and spleen cells from gastric cancer patients. Cancer 1983; 52:1624–1631. Toivanen A, Granberg I, Nordman E. Lymphocyte subpopulations in patients with breast cancer after postoperative radiotherapy. Cancer 1984; 54:2919–2923. Tom BH, Macek CM, Subramanian C, et al. In vitro expression of suppressogenic and enhancing activities in human colon cancer cells. J Biol Response Mod 1984; 3:435–444. Tonnesen H, Knigge U, Bulow S, et al. Effect of cimetidine on survival after gastric cancer. Lancet 1988; 29:2(8618); 990–992. Treurniet-Donker AD, Meischke-de Jongh ML, van Putten WLJ. Levamisole as adjuvant immunotherapy in breast cancer. Cancer 1987; 59:1590–1593. Tsang KY, Fudenberg HH, Pan JF, et al. An in vitro study on the effects of isoprinosine on immune responses in cancer patients. Int J Immunopharmacol 1983; 5:481–490. Tsuyuguchi I, Shiratsuchi H, Fukuoka M. T-lymphocyte subsets in primary lung cancer. Jpn J Clin Oncol 1987; 17:1; 13–17. Uchida A, Kolb R, Micksche M. Generation of suppressor cells for natural killer activity in cancer patients after surgery. J Natl Cancer Inst 1982; 68:735–741. Uchida A, Mickskche M. Generation of suppressor cells f or natural killer (NK) activity in cancer patients after surgery. Cancer Immunol Immunother 1981; 10:203–210.

144

Therapeutic approaches to cancer-associated immune suppression

530. Uchida A, Hoshino T. Clinical studies on well-mediated immunity in patients with malignant disease. I. Effects of immunotherapy with OK-432 on lymphocyte subpopulation and phytomitogen responsiveness in vitro. Cancer 1980; 45:476–483. 531. Umeda T, Yokoyama H, Kobayashi K, et al. Subsets of T-lymphocytes of patients with malignancies of the urogenital tract. Cell Mol Biol 1979; 25:95–99. 532. Uracz W, Mytar B, Zembala M, et al. Activated monocytes in gastric cancer patients. II. Suppressor and cytostatic activity in vitro. J Cancer Res Clin Oncol 1982; 104:307–313. 533. Van Rijswijk REN, DeMeijer A, Bob Sybesma JPH, Kater L. Five-year survival in Hodgkin’s disease. The prospective value of immune status at diagnosis. Cancer 1986; 57:1489–1496. 534. Van-Stolen K, Wiernick PH, Schiffer CA, Schimpff SC. Evaluation of levamisole as an adjuvant to chemotherapy for treatment of ANLL. Cancer 1983; 51:1576–1580. 535. Vanhaelen CPJ, Fisher RI. Increased sensitivity of T-cells to regulation by normal suppressor cells persists in long-term survivors with Hodgkin’s disease. Am J Med 1982; 72:385–390. 536. Vaudaux P, Kiefer B, Forni M, et al. Adriamycin impairs phagocytic function and induces morphologic alterations in human neutrophils. Cancer 1984; 59:400–410. 537. Venkataraman M, Rao DS, Levin RD, Westerman MP. Suppression of B-lymphocyte function by T-lymphocytes in patients with advanced lung cancer. J Natl Cancer Inst 1985; 74:37–41. 538. Verhaegen H, DeCree J, DeCock W, et al. Levamisole therapy in patients with colorectal cancer. In: Terry WD, Rosenberg SA, eds. Immunotherapy of human cancer. New York: Elsevier/ North-Holland, 1982:225–229. 539. Vinzenz K, Matejka M, Watzek G, et al. Modulation of NK activity in regional lymph nodes by preoperative immunotherapy with OK-432 in patients with cancer of the oral cavity. Cancer Detect Prev Suppl 1987; 1:463–475. 540. Von Roenn J, Harris JE, Braun DP. Suppressor cell function in solid-tumor cancer patients. J Clin Oncol 1987; 5:1; 150–159. 541. Voogt PJ, van de Velde CJH, Brand A, et al. Perioperative blood transfusion and cancer prognosis: Different effects of blood transfusion on prognosis of colon and breast cancer patients. Cancer 1987; 59:836–843. 542. Vose BM, Vanky F, Argrov S, Klein E. Natural cytotoxicity in man: Activity of lymph node and tumor-infiltrating lymphocytes and regional lymphocytes. Eur J Immunol 1977; 7:753–757. 543. Wagner DK, Kiwanuka J, Edwards BK, et al. Soluble interleukin-2 receptor levels in patients with undifferentiated and lymphoblastic lymphomas: Correlation with survival. J Clin Oncol 1987; 5:8; 1262–1274. 544. Wagner V, Janku O, Wagnerora M, et al. The production of complete and incomplete antibodies in patients with neoplastic disease. Neoplasma 1972; 19:75–87. 545. Wakasugi H, Kasahara T, Minato N, et al. In vivo potentiation of human natural killer cell activity by a streptococcal preparation: Interferon and interleukin-2 participation in the stimulation with OK-432. J Natl Cancer Inst 1982; 69:807–812. 546. Walter RJ, Danielson JR. Defective monocyte chemotaxis in patients with epidermoid tumors of the head and neck. Arch Otolaryngol 1985; 11:530–540. 547. Wanebo HJ, Rao B, Miyazawa N, et al. Immune reactivity in primary carcinoma of the lung and its relation to prognosis. J Thorac Cardiovasc Surg 1976; 72:339–347. 548. Wanebo HJ, Pace R, Hargett S, et al. Production of and response to interleukin-2 in peripheral blood lymphocytes of cancer patients. Cancer 1980; 57:656–662.

549. Wanebo HJ. Observations on the effects of adjuvant radiation on immune tests of patients with colorectal cancer and head and neck cancer. In: Dubois JB, Serrou B, Rosenfeld C, eds. Immunopharmacologic effects of radiation therapy. New York: Raven Press, 1980:241–252. 550. Wanego HJ, Riley T, Katz D, et al. Indomethacin-sensitive suppressor-cell activity in head and neck cancer patients. Cancer 1988; 61:462–474. 551. Wara WM, Phillips TL, Wara DW, et al. Immunosuppression following radiation therapy for carcinoma of the nasopharynx. Am J Roentgenol Radium Ther Nucl Med 1975; 123:482–485. 552. Wara WM, Wara DW, Philips TL, Amman AJ. Elevated IgA in cancer of the nasopharynx. Cancer 1975; 35:1313–1315. 553. Wara WM, Neely MH, Amman AJ, Wara DW. Biological modification of immunologic parameters in head and neck cancer patients with thymosin fraction V. In: Goldstein AL, Chirigos MA, eds. Lymphokines and thymic hormones: Their potential utilization in cancer therapeutics. New York: Raven Press, 1981:257–262. 554. Warren RP, McCall CA, Urban RW. Augmentation of natural killer cell activity by ImuVert: A biological response modifier derived from Serratia marcescens. Mol Biother 1989; 1:3; 145–151. 555. Wasserman J, Wallgren A, Blomgren H, et al. Prognostic relevance of post-irradiation lymphocyte reactivity in breast cancer patients. Cancer 1986; 58:348–351. 556. Wasserman J, Blomgren H, Petrini B, et al. Effect of radiation therapy and in vitro x-ray exposure on lymphocyte subpopulations and their functions. Am J Clin Oncol 1982; 5:195–208. 557. Watanabe Y, Takashi I. Clinical value of immunotherapy for lung cancer by the streptococcal preparation OK-432. Cancer 1984; 53:248–253. 558. Webster DJ, Whitehead RH, Richardson G, Hughes LE. Levamisole: A double-blind immunological study. Anticancer Res 1982; 2:29–32. 559. Weiden PL, Bean MA, Schultz P. Perioperative blood transfusion does not increase the risk of colorectal cancer recurrence. Cancer 1987; 60:870–874. 560. Wells SA Jr, Burdick JF, Christiansen C, et al. Demonstration of tumor-associated delayed cutaneous hypersensitivity reactions in lung cancer patients and in patients with carcinoma of the cervix. Natl Cancer Inst Monogr 1973; 37:197–203. 561. Wells SA Jr, Burdick JF, Christiansen C, et al. Demonstration of tumor-associated delayed cutaneous hypersensitivity reactions in lung cancer patients and in patients with carcinoma of t he cervix. Natl Cancer Inst Monogr 1973; 37:197–203. 562. Wells SA Jr, Burdick JF, Joseph WL, et al. Delayed cutaneous hypersensitivity reactions to tumor cell antigens and to nonspecific antigens. J Thorac Cardiovasc Surg 1973; 66:557–562. 563. Weppelmann B, Monkemeir D. The influence of prostaglandin antagonists on radiation therapy of carcinoma of the cervix. Gynecol Oncol 1984; 17:196–199. 564. Weese J, Oldham RK, Tormey DC, et al. Immunologic monitoring in carcinoma of the breast. Surg Gynecol Obstet 1977; 145: 209–218. 565. Wesselius LJ, Wheaton DL, Manahan-Wahl LJ, et al. Lymphocyte subsets in lung cancer. Chest 1987; 91:5; 725–729. 566. White JE, Chen T, Reed R, et al. Limited squamous-cell carcinoma of the lung: A Southwest Oncology Group randomized study of radiation with or without doxorubicin and with or without levamisole immunotherapy. Cancer Treat Rep 1982; 66:113–120. 567. Whitehead RH, Thatcher J, Teasdale C, et al. T- and B-lymphocytes in breast cancer: Stage relationship and abrogation of T-lymphocyte depression by enzyme treatment in vitro. Lancet 1976; 1:330–333.

Robert K. Oldham 568. Wilkins SA Jr, Olkowskii ZL. Immunocompetence of cancer patients treated with levamisole. Cancer 1977; 39:487–493. 569. Windle R, Bell PR, Shaw D. Five-year results of a randomized trial of adjuvant 5-fluorouracil and levamisole in colorectal cancer. Br J Surg 1987; 74:7; 569–572. 570. Windle R, Wood RFM, Bell PRF. The effect of levamisole on post-operative immunosuppression. Br J Surg 1979; 66:507–509. 571. Wleklik MS, Luczak M, Najjajr VA. Tuftsin-induced tumor necrosis activity. Mol Cell Biochem 1987; 75:2; 169–174. 572. Wolf GT, Lovett EJ III, Peterson KA, et al. Lymphokine production and lymphocyte subpopulations in patients with head and neck squamous carcinoma. Arch Otolaryngol 1984; 110:731–735. 573. Wrzesinki C, et al. Hematopoietic stem cells promote the expansion and function of adoptively transferred antitumor CD8(+) T cells. J Clin Invest 2007; 117:492–501. 574. Wybran J, Govaerts P. Levamisole and human lymphocyte surface markers. Clin Exp Immunol 1977; 27:319–321. 575. Wybran J, Serrou B, Belpomme D, et al. Immunomodulating of T-cell and NK function by NPT-15392 in cancer patients. A cooperative study by the tumor immunology group. In: Serrou B,

145

576.

577. 578. 579.

580.

581.

582.

Rosenfeld C, Daniels JC, Saunders JP, eds. Current concepts in human immunology and cancer immunomodulation. Amsterdam: Elsevier Biomedical, 1982:471–478. Yokoyama H, Umeda T, Kobayashi K, et al. Restoration of E-rosette formation by bestatin in patients with bladder cancer. C Cancer Res Clin Oncol 1980; 98:195–201. Young RC, Corder MP, Haynes HA, DeVita VT. Delayed hypersensitivity in Hodgkin’s disease. Am J Med 1972; 52:63–72. Zacharski LR, Linman JW. Lymphocytopenia: Its causes and significance. Mayo Clin Proc 1971; 46:168–173. Zamkoff KW, Reeves WG, Paolozzi FP, et al. Impaired interleukin regulation of the phytohemagglutinin response in Hodgkin’s disease. Clin Immunol Immunopathol 1985; 35:111–124. Zamkoff KW, Reeves WQ, Paolozzi FP, et al. Impaired interleukin regulation of the phytohemagglutinin response in Hodgkin’s disease. Clin Immunol Immunopathol 1985; 35:111. Zanker KS, Blumel G, Lange J, et al. Coumarin in malignant melanoma patients: An experimental and clinical study. Drug Exp Clin Res 1984; 10:767–774. Zou W. Regulatory T. Cells, tumour immunity and immunotherapy. Nat Rev Immunology 2006; 6:295–307.

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

147

148

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.

References 1. Brodsky FM, Guafgliardi LE. The cell biology of antigen processing and presentation and presentation. Annu Rev Immunol (1991) 9:707–44. 2. Osanto S. Vaccine trials for the clinician: prospects for tumor antigens. Oncologist (1997) 2:284–99. 3. Parham P. Antigen processing. Transporters of delight. Nature (Lond.) (1900) 348:674–5. 4. Monaco J. A molecular model of MHC class-I-restricted antigen processing. Immunol Today (1992) 13:173–9. 5. Townsend SE, Allison JP. Tumor rejection after direct constimulation of CD8+ T cells by B7-transfected melanoma cells. Science (Washington DC) (1993) 259:368–70. 6. Falk K, Rotzchke O. Consensus motifs and peptide ligands of MHC class I molecules. Semin Immunol (1993) 5:81–7. 7. Falk K, Rotzchke O, Stevanovic J. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature (Lond.) (1991) 351:290–4. 8. Kast WM, Bronklhorst AM, DeWaal LP, Melief CJ. Cooperation between cytotoxic and helper T lymphocytes in protection against lethal Sendai virus infection. J Exp Med (1986) 164:723–38. 9. Keene J, Forman J. Helper activity is required for the in vitro generation of cytotoxic T lymphocytes. J Exp Medi (1982) 150:1134–42. 10. Raulet D, Bevan M. Helper T cells for cytotoxic T lymphocytes need not be region restricted. J Exp Med (1982) 155:1766–84. 11. Allison JP, Hurwitz AA, Leach Dr. Manipulation of co-stimulatory signals to enhance antitumor T-cell responses. Curr Opin Immunol (1995) 7:682–6. 12. Tai T, Paulson JC, Cahan CD et al. Ganglioside CM2 as a human tumor antigen (OFA-I-1). Proc Natl Acad Sci USA (1983) 80:5392–6. 13. Fitch FW, Laneki DW, Gajewski TF. T-cell mediated immune regulation: help and suppression. In: Paul W, Nita E, eds. Fundamental Immunology. New York: Raven Press (1993) 733–61. 14. Zinkernagel R, Doherty P. MHC-restricted cytotoxic cell: studies on the biological role of polymorphic major transplantation antigens determining T cell restriction-specificity, function and responsiveness. Adv Immunol (1979) 27:51–106. 15. Grant EP, Rock KL. MHC class I-restricted presentation of exogenous antigen by thymic antigen-presenting cells in vitro and in vivo. J Immonul (1992) 148:13–18. 16. Harding CV, Song R. Phagocytic processing of exogenous particulate antigens by macrophages for presentation by class I MHC molecules. J Immunol (1994) 153:4925–33. 17. Kovacsovic-Bankowski M, Rock KL. A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science (Washington DC) (1995) 267:243–5. 18. Rock KL, Gamble S, Rothstein L. Presentation of exogenous antigen with class I major histocompatibility complex molecules. Science (Washington DC) (1990) 249:918–21.

Cancer vaccines 19. Rock KL, Rothstein L, Gamble S. Characterization of antigenpresenting cells that present exogenous antigens in association with calls I MHC molecules. J Immonol (1993) 150:438–46. 20. Tanaka H, Tanaka J, Kjaargaard J, Shu S. Depletion of CD4 + CD25 + regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes. J Immunother (2002) 25(3):207–17. 21. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4 + CD25- naïve T cells to CD4 + CD25 + regulatory T cells by TGF-β induction of transcription factor Foxp3. J exp Med (2003) 198(12):1875–86. 22. Pawelec G, Koch S, Griesemann H, Rehbein A, Hahnel K, Gouttefangeas C. Immunosenescence, suppression and tumour progression. Cancer Immunol Immunother (2006) 55:981–86. 23. Garrido F, Carera T, Concha A, Glew S, Ruiz-Cabello F, Stern PL. Natural history of HLA expression during tumour development. Immunonol Today (1993) 14(1):491–99. 24. Garrido F, Ruiz-Cabello F, Cabrera T, Perez-Villar JJ, Lopez-Botet M, Duggan-Keen M, Stern PL. Implications for immunosurveillance of altered HLA class I phenotypes in human tumours. Immunol Today (1997) 18(2):89–95. 25. Speiser DE, Lienard D, Rufer N, Rubio-Gudoy V, Rimoldi D, Lejeune F, Krieg AM, Cerottini JC, Romero P. Rapid and strong human CD8 + T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest (2005) 115(3):739–746. 26. Peng G, Guo Z, Kiniwa Y, Voo KS, Peng W, Fu T, Wang DY, Li Y, Wang HY, Wang RF. Toll-like receptor 8-mediated reversal of CD4 + regulatory T cells. J Clin Invest (2005) 309(5739):1380–84. 27. Dannull J, Su Z, Rizzieri D, Yang BK, Coleman D, Yancey D, Zhang A, Dahm P, Chao N, Gilboa E, Vieweg J. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest (2005) 115(12):3623–33. 28. Attia P, Maker AV, Haworth LR, Rogers-Freezeer L, Rosenberg SA. Inability of a fusion protein of IL-2 and diptheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lyphocytes in patients with melanoma. J Immunother (2005) 28(6):582–92. 29. Phan GQ, Yang JC, Sherry RM, Hwu P, Topalian SL, Schwartzentruber DJ, Restifo NP, Haworth LR, Seipp CA, Freezer LJ, Morton KE et al. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci USA (2003) 100(14):8372–77. 30. MacLean GD, Miles DW, Rubens RD, Reddish MA, Longenecker BM. Enhancing the effect of THERATOPE STn-KLH cancer vaccine in patients with metastatic breast cancer by pretreatment with low-dose intravenous cyclophosphamide. J Immunother with Emphasis Tumor Immunol (1996) 19(4):309–316. 31. Glaser M. Regulation of specific cell-mediated cytotoxic response against SV40-induced tumor associated antigens by depletion of suppressor T cells with cyclophosphamide in mice. J Exp Med (1979) 149(3):774–779. 32. Mitchell MS, Kan-Mitchell J, Kempf RA, Harel W, Shau HY, Lind S. Active specific immunotherapy for melanoma: phase 1 trial of allogeneic lysates and a novel adjuvant. Cancer Res (1988) 48(20):5883–93. 33. Velu T, Ramlau R, Quoix E, Pawlicki M, Pless M, Lena H, Levy E, Krzakowski M, Limacher J-M, Bizouarne N. A phase II study evaluating the clinical efficacy of TG4010 (MVA-MUC1-IL2) in association with chemotherapy in patients with non small cell

Kenneth A. Foon and Malek M. Safa

34.

35.

36.

37. 38.

39.

40.

41.

lung cancer. American Society of Clinical Oncology, Orlando, FL (2005) Abs 7132. Nowak AK, Robinson BW, Lake RA. Synergy between chemotherapy and immunotherapy in the treatment of established murine solid tumors. Cancer Res (2003) 63(15):4490–4496. Munoz N, Bosch FX, Castellsague X, Diaz M, de Sanjose S, Hammouda D, et al. Against which human papillomavirus types shall we vaccinate and screen? The international perspective. Int J Cancer (2004) 111:278–85. Giannimi SL, Hanon E, Moris P, Van Mechelen M, Morel S, Dessy F, et al. Enhanced humoral and memory B cellular immunity using HPV 16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine (2006) 24:5937–49. Hilleman MR. Overview of vaccinology with special reference to papillomavirus vaccines. J Clin Virol (2000) 19:79–90. Harper DM, Franco EL, Wheeler CM, Moscicki AB, Romanowski B, Rotelli-Martins CM, et al. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow up from a randomized control trial. Lancet (2006) 376:1247–55. Harper DM, Franco EL, Wheeler C, Ferris DG, Jenkins D, Schuind A, et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomized controlled trial. Lancet (2004) 364:757–65. Villa LL, Cost RL, Petta CA, Andrade RP, Ault KA, Giuliano AR, et al. Prophylactic quadrivalent human papillomavirus (types 6,11, 16, and 18) L1 virus-like particle vaccine in young women: a randomized double-blind placebo-controlled multicentre phase II efficacy trial. Lancet Oncol (2005) 6:271–8. Mitchell MS, Abrams J, Thompson JA, Kashan-Sabet M, DeConti RC, Hwu W, Atkins MB, Whitman E, Ernstoff MS, Haluska FG, Jakowatz JG, Das Gupta TK, Richards JM, Samlowski WE, Costanzi JJ, Aronson FR, Deisseroth AB, Dudek AZ, Jones VE. 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 (2007) 25(15):2078–85.

153 42. Small EJ, Fratesi P, Reese DM, Strang G, Laus R, Peshwa MV, Valone FH. Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells. J Clin Oncol (2000) 18(23):3894–3903. 43. Knutson KL. Technology evaluation: DCVax, Northwest biotherapeutics. Curr Opin Mol Ther (2002) 4(4):403–407. 44. Small EJ, Schellhammer PF, Higano CS, et al. Placebo-controlled phase III trial of immunologic therapy with Sipuleucel-T (ACP8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer. J Clin Oncol (2006) 24:3089–94. 45. Simons JW, Sacks N. Granulocyte-macrophage colony-stimulating factor-transduced allogeneic cancer cellular immunotherapy: the GVAX vaccine for prostate cancer. Urologic Oncology (2006) 24:419–24. 46. Michael A, Ball G, Quatan N, et al. Delayed disease progression after allogeneic cell vaccination in hormone-resistant prostate cancer and correlation with immunologic variables. Clin Cancer Res (2005) 11:4469–78. 47. Weng W-K, Czerwinski D, Timmerman J, Hsu FJ, Levy R. Clinical outcome of lymphoma patients after idiotype vaccination is correlated with humoral immune response and immunoglobulin G Fc receptor genotype. J Clin Oncol (2004) 22:4717–24. 48. Inoges S, Rodriguez-Calivillo M, Zabalegui N, Lopez-Diaz de Cerio A, Villanueava H, Soria E, et al. Clinical benefit associated with idiotype vaccination in patients with follicular lymphoma. J Natl Cancer Inst (2006) 98:1292–301. 49. Tokunaga N, Murakami T, Endo Y, Nishizaki M, Kagawa S, Tanaka N, Fujiwara T. Human monocyte-derived dendritic cells pulsed with wild-type p53 protein efficiently induced CTLs against p53 overexpressing human cancer cells. Clin Cancer Res (2005) 11(3):1312–18. 50. Knutson KL. GMK(progenics pharmaceuticals). Curr Opin Invest Drugs (2002) 3(1):159–164. 51. Chapman PB, Morrisey D, Panageas KS, Willimas L, Lewis JJ, Isreal RJ, Hamilton WB, Livingston PO. Vaccination with bivalent G(M2) and G(D2) ganglioside conjugate vaccine: a trial comparing doses of G(D2)-keyhole limpet hemocyanin. Clin Cancer Res (2000) 6(12):4658–62.

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

(continued)

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

(continued)

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

(continued)

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+

(continued)

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

(continued)

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].

216

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].

References 1. Aarvak T, Chaubaud M, Miossec P, Natvig JB. IL-17 is produced by some proinflammatory Th1/Th0 cells but not by Th2 cells. J Immunol 2000; 162: 1246–1251. 2. Abehsira-Amar O, Gilbert M, Joliy M, et al. IL-4 plays a dominant role in the differential development of Th0 into Th1 and Th2 cells. J Immunol 1992; 148: 3820–3829. 3. Abraham JA, Mergia A, Whang JL, et al. Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science 1986; 233: 545. 4. Acres B, Gantzer M, Remy C, et al. Fusokine interleukin-2/interleukin-18, a novel potent innate and adaptive immune stimulator with decreased toxicity. Cancer Res 2005; 65: 9536–9546. 5. Addison CL, Daniel TO, Burdick MD, et al. The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokineinduced angiogenic activity. J Immunol 2000; 165: 5269–5277.

217 6. Adorini L, Guery JC, Trembleau S. Manipulation of the Th1/Th2 cell balance: an approach to treat human autoimmune diseases? Autoimmunity 1996; 23: 53. 7. Agematsu K, Kobata T, Sugita K, et al. Direct cellular communications between CD45RO and CD45RA T cell subsets via CD27/ CD70. J Immunol 1995; 154: 3627–3635. 8. Agematsu K, Kobata T, Sugita K, et al. Role of CD27 in T cell immune response: analysis by recombinant soluble CD27. J Immunol 1994; 153: 1421. 9. Aggarwal BB, Hanzel WJ, Moffat B, Kohr WJ, Harkins RN. Primary structure of human lymphotoxin derived from 1788 lymphoblastoid cell line. J Biol Chem 1985; 260: 2334–2344. 10. Aggarwal S, Ghilardi N, Xie MH, et al. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by production of Interleukin-17. J Biol Chem 2003; 278: 1910–1914. 11. Aggarwal S, Xie MH, Maruoka M, et al. Acinar cell of pancreas are a target of interleukin-22. J Interferon Cyt Res 2001; 21: 1047. 12. Agostini C, Trentin L, Facco M, et al. Role of IL-15, IL-2 and their receptors in the development of T cell alveolitis in pulmonary sarcoidosis. J Immunol 1996; 157: 910. 13. Airoldi I, Giri G, Marshall JD, et al. Expression and function of IL-12 and IL-18 receptors on human tonsillar B cells. J Immunol 2000; 165: 6880–6888. 14. Akahoski T, Oppenheim JJ, and Matsushima K. Induction of high affinity interleukin 1 receptor on human peripheral blood lymphocytes by glucocorticosteroid hormones. J Exp Med 1988; 167: 924–936. 15. Akashi K, Kondo M, Von Freeden-Jeffry R, Murray R, Weissman IL. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor deficient mice. Cell 1997; 89: 1033. 16. Akashi K. Role of interleukin 4 in the negative regulation of leukemia cell growth. Leukemia and Lymphoma 1993; 9: 205–209. 17. Al Jabaari B, Ladyman HM, Larche M, et al. Elevated expression of interleukin 4 receptor in carcinoma. A target for immunotherapy? British J Cancer 1989; 59: 910–914. 18. Albanesi C, Scarponi C, Cavani A, et al. Interleukin 17 is produced by both Th1 and Th2 lymphocytes, modulates interferon-γ and interleukin-4-induced activation of human keratinocytes. J Invest Dermatol 2000; 115: 81. 19. Albanesi C, Scarponi C, Sebastiani S, et al. IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines. J Immunol 2000; 165: 1395–1402. 20. Albert ML, Sauter B, BharDwar N. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 1998; 392: 86. 21. Alderson MR, Armitage RJ, Tough TW, et al. CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligation for CD40. J Exp Med 1993; 178: 669. 22. Alderson MR, Sassenfeld HM, Widmer MB. Interleukin 7 enhances cytotoxic T lymphocyte generation and induces lymphokine activated killer cells from peripheral blood. J Exp Med 1990; 172: 577–587. 23. Alderson MR, Smith CA, Tough TW, et al. Molecular and biological characterization of human 4-1BB and its ligand. Eur J Immunol 1994; 24: 2219–2227. 24. Alderson MR, Tough TW, Ziegler SF, Grabstein KW. Interleukin 7 induces cytokine secretion and tumoricidal activity by human peripheral blood monocytes. J Exp Med 1991; 173: 923–930. 25. Alexander HR, Bartlett DL, Libutti SK, et al. Isolated hepatic perfusion with tumor necrosis factor and melphalan for unresectable cancers confined to the liver. J Clin Oncol 1998; 16: 1479–1486. 26. Alexander HR, Wong GGH, Doherty GM, et al. Differentiation factor/leukemia inhibitory factor protection against lethal endotoxemia in mice: synergistic effect with interleukin-1 and tumor necrosis factor. J Exp Med 1992; 175: 1139. 27. Ali S, Ahmad M, Lynam J, et al. Anti-tumor efficacy of OX40L in murine tumor model. Vaccine 2004; 22: 3585–3594. 28. Allakhverdi Z, Smith DE, Comeau MR, et al. The ST2 ligand IL-33 potently activates and drives maturation of human mast cells. J Immunol 2007; 2051–2054.

218 29. Allevena P, Giardina G, Bianchi G, Mantovani A. IL-15 is chemotactic for natural killer cells and stimulates their adhesion to vascular endothelial cells. J Leukocyte Biol 1997; 61: 729. 30. Allione A, Consalvo M, Nanni P, et al. Immunizing and curative potential of replicating and nonreplicating murine mammary adenocarcinoma cells engineered with interleukin (IL)-2, IL-4, IL-6, IL-7, IL-10, Tumor Necrosis Factor and γ-interferon gene or admixed with conventional adjuvants. Cancer Res 1994; 54: 6022–6026. 31. Allison J, Georgiou HM, Strasser A, Vaux DL. Transgenic expression of CD95 ligand on islet β cells induces a granulocytic infiltration but does not confer immune privilege upon islet allograft. Proc Natl Acad Sci USA 1997; 94: 3943. 32. Altenburg A, Baldus SE, Smola H, Pfister H, Hess S. CD40 ligand– CD40 interaction induces chemokines in cervical carcinoma cells in synergism with IFN-γ. J Immunol 1999; 162: 4140–4147. 33. Alters SE, Gadea JR, Holm B, Lebkowski J, Philip R. IL-13 can substitute for IL-4 in the generation of dendritic cells for the induction of cytotoxic T lymphocytes and gene therapy. J Immunother 1999; 22: 229–236. 34. Alves NL, Arosa FA, and van Lier RA. IL-21 sustains CD28 expression on IL-15 activated human naive CD8+ T cells. J Immunol 2005; 175: 755–762. 35. Alzona M, H-M Jack, Fisher RI, Ellis TM. CD30 defines a subset of activated human T cells that produce IFN-γ and IL-5 and exhibit enhanced B cell helper activity. J Immunol 1994; 153: 2861–2867. 36. Amakawa R, Hakem A, Kundig KM, et al. Impaired negative selection of T cells in Hodgkin’s disease antigen CD30 deficient mice. Cell 1996; 84: 551. 37. Amar S, Van Dyke TE, Eugster HP, Schultze N, Koebel P, Bluethmann H. TNF-induced cutaneous necrosis is mediated by tumor necrosis factor receptor R1. J Inflamm 1997; 47: 180. 38. Amaral MC, Miles CS, Kumar G, Nel AE. Oncostatin M stimulates tyrosine protein phosphorylation in parallel with activation of p42MAPK/ERK-2 in Kaposi’s cells: evidence that this pathway is important in Kaposi’s cell growth. J Clin Invest 1993; 93: 848. 39. Ambrus JL and Fauci AS. Human B cell lymphoma cell line producing B cell growth factor. J Clin Invest 1985; 75: 732–730. 40. Ambrus JL Chesky L, Stephany D, et al. Functional studies examining the subpopulation of human B lymphocytes responding to high molecular weight B cell growth factor. J Immunol 1990; 145: 3949–3955. 41. Ambrus JL, Jurgenson CH, Broom EJ, Fauci AS. Purification to homology of a high molecular weight human B cell growth factor. J Exp Med 1985; 162: 1319–1335. 42. Ambrus JL, Pippin J, Joseph A, et al. Identification of a cDNA for a human high molecular weight B cell growth factor. Proc Nat Acad Sci USA 1993; 90: 6330–6334. 43. Amiel C, Darcissac E, Truong MJ, et al. Interleukin 16 (IL-16) inhibits human immunodeficiency virus replication in cells from infected subjects and serum IL-16 levels drop with disease progression. J Infect Dis 1999; 179: 83–91. 44. Andarini S, Kikuchi T, Nukiwa M, et al. Adenovirus vector mediated in vivo gene transfer of OX40 ligand to tumor cells enhances antitumor immunity to tumor-bearing hosts. Cancer Res 2004; 64: 3281–3287. 45. Anders RA, Shubudhi SK, Wang J, et al. Contribution of the lymphotoxin β receptor to liver regeneration. J Immunol 2005; 175: 1295–1300. 46. Anderson DM, Kumaki S, Abdieh M, et al. Functional characterization of the human interleukin 15 receptor α chain and close linkage of the IL-15RA and IL-2RA genes. J Biol Chem 1995; 270: 29862. 47. Anderson DM, Lyman S, Baird A, et al. Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 1990; 63: 235–243. 48. Anderson DM, Marakovsky E, Billingsley WL, et al. A homolog of the tumor necrosis factor and its ligand enhance T cells growth and dendritic cell function. Nature 1997; 390: 175.

Cytokines 49. Andoh A, Zhang Z, Inatomi O, et al. Interleukin-22, a member of the IL-10 subfamily induces inflammatory responses in colonic subepithelial myofibroblasts. Gastroenterology 2005; 129: 969–984. 50. Andrews JVR, Schoof DD, Bertagnolli MM, et al. Immunomodulatory effects of Interleukin 12 on human tumor infiltrating lymphocytes. J Immunotherapy 1993; 14: 1–10. 51. Anegon I, Moreau J-F, Godard A, et al. Production of human interleukin for DA cells (HILDA)/leukemia inhibitory factor (LIF) by activated monocytes. Cell Immunol 1990; 130: 50. 52. Angiolillo AL, Sqadari C, Tosato G. A role for the interferon-inducible protein 10 in inhibition of angiogenesis by interleukin-12. Ann N Y Acad Sci 1996; 795: 158–167. 53. Anguita J, Barthold SW, Samanta S, Ryan J, Fikrig E. Selective anti-inflammatory action of interleukin-11 in murine Lyme disease: arthritis decreases while carditis persists. J Infect Dis 1999; 179: 734. 54. Ank N, West H, Bartholdy C, et al. Lambda interferon (IFN-λ), a type III interferon, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo. J Virol 2006; 80: 4501–4509. 55. Annunziato F, Cosmi L, Santarlasci V, et al. Phenotypic and functional features of human Th17 cells. J Expl Med 2007; 204: 1849–1861. 56. Antony PA, Restifo NP. CD4+ CD25+ T regulatory cells, immunotherapy of cancer and interleukin-2. J Immunother 2005; 28: 120–128. 57. Antonysamy MA, Fanslow WC, et al. Evidence for a role of IL-17 in organ allograft rejection: IL-17 promotes functional differentiation of dendritic cell progenitors. J Immunol 1999; 162: 577–584. 58. Aoki T, Kikuchi H, Miyatake S-I, et al. Interleukin 5 enhances interleukin 2 mediated lymphokine activated killer activity. J Exp Med 1989; 23: 163–177. 59. Aoki T, Tashiro K, Miyatake S, et al. Expression of murine IL-7 in a murine glioma cell line results in reduced tumorigenicity in vivo. Proc Natl Acad Sci USA 1992; 89: 3850–3894. 60. Aoki Y, Jaffee ES, Chang Y, et al. Angiogenesis and hematopoiesis induced by Kaposi’s sarcoma associated herpes virus-encoded interleukin-6. Blood 1999; 93: 4034. 61. Arai H, Chan SY, Bishop DK, Nabel GJ. Induction of the alloantibody response by CD95 ligand. Nat Med 1997; 3: 843. 62. Arch RH, Thompson CB. 4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor subfamily that bind TNF receptor-associated factors and activate nuclear factor κB. Mol Cell Biol 1998; 19: 558. 63. Arenberg DA, Keane MP, DiGiovine B, et al. Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor in non-small cell lung carcinoma. J Clin Invest 1998; 102: 465. 64. Arenberg DA, Kunkel SL, Polverini PJ, et al. Interferon-γ-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human nonsmall cell lung cancer (NSCLC) tumorigenesis and spontaneous metastasis. J Exp Med 1996; 184: 981–992. 65. Arend WP, Malyak M, Smith Jr MF, et al. Binding of IL-1α, IL-1β and IL-1 receptor antagonist by soluble IL-1 receptors and levels of soluble IL-1 receptors in synovial fluids. J Immunol 1994; 153: 4766–4774. 66. Arens R, Schepers K, Nolte MA, et al. Tumor rejection induced by CD70-mediated quantitative and qualitative effects on effector CD8+ T cell formation. J Exp Med 2004; 199: 1595–1605. 67. Arienti F, Belli F, Napolitano F, Sule-Suso J, et al. Vaccination of melanoma patients with interleukin 4 gene-transduced allogeneic cells. Hum Gene Ther 1999; 10: 2907–2916. 68. Ariizumi K, Kitajima T, Bergstresser PR, Takashima A. Interleukin-1 β converting enzyme in murine Langerhans cells and epidermal derived dendritic cell lines. Eur J Immunol 1995; 25: 2137. 69. Ariizumi K, Meng Y, Bergstresser PR, Takashima A. INF-γDependent IL-7 gene regulation in keratinocytes. J Immunol 1995; 154: 6031–6039. 70. Arima M, Plitt J, Stelatto C, et al. Expression of interleukin-16 by human epithelial cells. Inhibition by dexamethasone. Am J Respir Cell Mol Biol 1999; 21: 684–692.

Walter M. Lewko and Robert K. Oldham 71. Armitage RJ, MacDuff BM, Eisenman J, et al. IL-15 has stimulatory activity in the induction of B cell proliferation and differentiation. J Immunol 1995; 154: 483–490. 72. Armitage RJ, Macduff BM, Spriggs MK, Fanslow WC. Human B cell proliferation and Ig secretion induced by recombinant CD40 ligand and modulated by soluble cytokines. J Immunol 1993; 150: 3671. 73. Armitage RJ, Namen AE, Sassenfeld HM, Grabstein KH. Regulation of T cell proliferation by IL-7. J Immunol 1990; 144: 938. 74. Armitage RJ, Zeigler SF, Friend DJ, et al. Identification of a novel low affinity receptor for human interleukin-7. Blood 1992; 7: 1738. 75. Armitage RJ. Tumor necrosis factor receptor superfamily members and their ligands. Curr Opinion Immunol 1994; 6: 407–413. 76. Aronson FR, Libby P, Brandon EP, et al. IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury. J Immunol 1988; 141: 158. 77. Asadullah K, Docke WD, Ebeling M, et al. Interleukin 10 treatment of psoriasis: clinical results of a phase 2 trial. Arch Dermatol 1999; 135: 187–192. 78. Asao H, Okuyama C, Kumaki S, et al. The common γ-chain is an indispensable subunit of the IL-21 receptor. J Immunol 2001; 167: 1–5. 79. Ashkar S, Weber GF, Panoutsakopoulou V, et al. ETA-1: an early component of type-1 immunity. Science 2000; 287: 860. 80. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Science 1998; 281: 1305. 81. Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant Apo2 ligand. J Clin Invest 1999; 104: 155. 82. Asou Y, Rittling H, Yoshitake K, et al. Osteopontin facilitates angiogenesis, accumulation of osteoclasts, and resorption of ectopic bone. Endocrinology 2001; 142: 1325–1332. 83. Assuma R, Oates T, Cochran D, Amar S, Graves DT. IL-1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis. J Immunol 1998; 160: 403–409. 84. Atkins MB, Robertson MJ, Gordon M, et al. Phase I evaluation of recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 1997; 3: 409–417. 85. Atkins MB, Vachino G, Tilg HJ, et al. Phase I evaluation of thrice-daily intravenous bolus interleukin-4 in patients with refractory malignancy. J Clin Oncol 1992; 10: 1802–1809. 86. Attia P, Maker AV, Haworth LR, et al. Inability of a fusion protein of IL-2 and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with melanoma. J Immunother 2005; 28: 582–592. 87. Attur MD, Dave MN, Clancey RM, et al. Functional genomic analysis in arthritis affected cartilage: yin-yang regulation of inflammatory mediators by integrins. J Immunol 2000; 164: 2684. 88. Attur MG, Patel RN, Abramson SB, Amin AR. Interleukin-17 upregulation of nitric oxide production in human osteoarthritis cartilage. Arthritis Rheum 1997; 40: 1050. 89. Auron PE, Webb AC, Rosenwasser LJ, et al. Nucleotide sequence of human monocyte interleukin 1 precursor cDNA. Proc Natl Acad Sci USA 1984; 81: 7907. 90. Avanzi GC, Brizzi MF, Gianotti J, et al. M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3. J Cell Physiol 1990; 145: 458–464. 91. Aversa G, Punnonen J, de Vries JE. The 26 kDa transmembrane form of tumor necrosis factor α on activated CD4+ T cell clones provides a costimulatory signal for human B cell activation. J Exp Med 1993; 177: 1575. 92. Avice MN, Demeure CE, Delespesse G, Rubio M, Armant M, Sarfati M. IL-15 promotes IL-15 production by human monocytes via T cell-dependent contact and may contribute to IL-12 mediated IFN-γ secretion by CD4+ T cells in the absence of TCR ligation. J Immunol 1998; 161: 3408. 93. Awane M, Andres PG, Li DJ, Reinecker HC. NF-κB-inducing kinase is a common mediator of IL-17-, TNF-α- and IL-1β-induced

219

94.

95.

96. 97.

98.

99.

100. 101. 102. 103. 104. 105. 106. 107.

108.

109. 110. 111. 112. 113.

chemokine promoter activation in intestinal epithelial cells. J Immunol 1999; 162: 5337. Azimi N, Brown K, Bamford RN, et al. Human T cell lymphotropic virus type I Tax protein trans-activates interleukin-15 gene transcription through an NF-κB site. Proc Natl Acad Sci USA 1998; 95: 2452. Azimi N, Jacobsen S, Leist T, Waldmann TA. Involvement of IL-15 in the pathogenesis of human T lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis: implications for therapy with a monoclonal antibody directed to the IL-2/ IL-15 receptor. J Immunol 1999; 163: 4064. Azuma C, Tanabe T, Konishi M, et al. Cloning of cDNA for human T-cell replacing factor (interleukin-5) and comparison with the murine homologue. Nucleic Acid Res 1986; 14: 9149–9158. Bacon CM, McVicar DW, Ortaldo JR, et al. Interleukin-12 induces tyrosine phosphorylation of JAK2 and TYK2: differential use of tyrosine kinases by IL-2 and IL-12. J Exp Med 1995; 181: 399–404. Bacon KB, Westwick J, Camp RDR. Potent and specific inhibition of IL-8, IL-1α and β induced in vitro human lymphocyte migration by calcium channel antagonists. Biochem Biophys Res Commun 1989; 165: 349. Badache A, Hynes NE. Interleukin 6 inhibits proliferation and, in cooperation with epidermal growth factor receptor autocrine loop, increases migration of T47D breast cancer cells. Cancer Res 2001; 61: 383–391. Bagby GC Jr, Dinarello CA, Wallace P, et al. Interleukin 1 stimulates granulocyte macrophage colony-stimulating activity release by vascular endothelial cells. J Clin Invest 1985; 78: 1316. Baggiolini M, Kernan P. Neutrophil activation: control of shape change exocytosis and respiratory burst. News Physiol Sci 1992; 7: 215. Baier M, Bannert N, Werner A, Lang K, Kurth R. Molecular cloning, sequence, expression and processing of the interleukin 16 precursor. Proc Natl Acad Sci USA 1997; 94: 5273–5277. Baier M, Werner A, Bannert N, Metzner K, Kurth R. HIV suppression by interleukin-16. Nature 1995; 378: 563. Bakker TR, Reed T, Renno T, Jongeneel CV. Efficient adenovirus transfer of NF-κB inhibitor sensitizes melanoma cells to tumor necrosis factor-mediated apoptosis. Int J Cancer 1999; 80: 320. Balasa B, Krahl T, Patstone G, et al. CD40 ligand–CD40 ligation interactions are necessary for the initiation of insulinitis and diabetes in nonobese diabetic mice. J Immunol 1997; 159: 4260. Baluna R, Vitetta ES. Vascular leak syndrome: a side effect of immunotherapy. Immunopharmacology 1997; 37: 117–132. Bamford RN, DeFilippis AP, Azimi N, Kurys G, Waldmann TA. The 5′ untranslated region, signal peptide, and the coding sequence of the carboxyl terminus of IL-15 participate in its multifaceted translational control. J Immunol 1998; 160: 4418–4426. Bamford RN, Grant AJ, Burton JD, et al. The interleukin (IL) 2β chain is shared by IL-2 and a cytokine, provisionally designated IL-T that stimulates T cell proliferation and the induction of lymphokine activated killer cells. Proc Nat Acad Sci USA 1994; 91: 4940–4944. Banchereau J, Bazan D, Blanchard D, et al. The CD40 antigen and its ligand. Annual Rev Immunol 1994; 12: 881. Banks TA, Richert S, Benedict CA, et al. A lymphotoxin-IFN-β axis essential for lymphocyte survival revealed during cytomegalovirus infection. J Immunol 2005; 174: 7217–7225. Banks TA, Rouse BT, Kerley MK, et al. Lymphotoxin-α-deficient mice: effects on secondary lymphoid organ development and humoral immune response. J Immunol 1995; 44: 1685. Bansal-Pakala P, Jember AG, Croft M, et al. Signaling through OX40 (CD134) breaks peripheral T cell tolerance. Nat Med 2001; 7: 607–912. Barker BR, Parvani JG, Meyer D, et al. IL-21 induces apoptosis of antigen-specific T lymphocytes. J Immunol 2007; 179: 3596–3603.

220 114. Battaile R, Barlogie B, Lu ZY, et al. Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma. Blood 1995; 86: 685–691. 115. Batten M, Li J, Yi S, et al. Interleukin 27 limits autoimmune encephalitis by suppressing the development of interleukin 17-producing T cells. Nat Immunol 2006; 7: 929–936. 116. Bauer JH, Liu KD, You Y, Lai SY, Goldsmith MA. Heteromerization of the γ c chain with the interleukin-9 receptor α subunit leads to STAT activation and prevention of apoptosis. J Biol Chem 1998; 273: 9255–9260. 117. Baum PR, Gayle RB III, Ramsdell RF, et al. Molecular characterization of murine and human OX40/OX40 ligand systems: identification of a human OX40 ligand as the HTLV-1-regulated protein gp34. EMBO J 1994; 13: 3992. 118. Baxevanis CN, Gritzapis AD, Papamichail M. In vivo antitumor activity of NKT cells activated by the combination of IL-12 and IL-18. J Immunol 2003; 171: 2953–2959. 119. Bazan JF, Bacon CB, Hardiman G, et al. A new class of membranebound chemokine with CX3C motif. Nature 1997; 385: 640. 120. Bazan JF, Timans JC, Kastelein RA. A newly defined interleukin-1? Nature 1996; 379: 591. 121. Beatty PR, Krams SM, Martinez OM. Involvement of IL-10 in the autonomous growth of EBV-transformed B cell lines. J Immunol 1997; 158: 4045–4051. 122. Beckman MP, Cosman D, Fanslow W, et al. The interleukin 4 receptor: structure function and signal transduction. Chem Immunol 1992; 51: 107. 123. Bedard M, Mclure CD, Schiller NL, et al. Release of interleukin 8, interleukin 6, and colony stimulating factors by upper airway epithelial cells: implications for cystic fibrosis. Am J Respir Cell Mol Biol 1993; 9: 455. 124. Behrens CK, Igney FH, Arnold B, Moller P, Krammer PH. CD95 ligand-expressing tumors are rejected in anti-tumor TCR transgenic perforin knockout mice. J Immunol 2001; 166: 3240–3247. 125. Bejarano MT, de Waal Malefyt R, Abrams JS, et al. Interleukin 10 inhibits allogeneic proliferative and cytotoxic T cell responses generated in primary mixed lymphocyte culture. Int Immunol 1992; 4: 1389. 126. Bekker L-G, Freeman S, Murray PJ, Ryffel B, Kaplan G. TNF-α controls intracellular mycobacterial growth by both inducible nitric oxide synthetase dependent and inducible nitric oxide synthetaseindependent pathways. J Immunol 2001; 166: 6728–6734. 127. Bell MC, Carrol GJ, Chapman HM, Mills JN, Hui W. Oncostatin M induces leukocyte infiltration and cartilage proteoglycan degradation in vivo in goat joints. Arth Rheu 1999; 42: 2543. 128. Belladonna ML, Renauld JC, Bianchi R, et al. IL-23 and IL-12 have overlapping but distinct effects on murine dendritic cells. J Immunol 2002; 168: 5448–5454. 129. Bellone G, Carbone A, Smire C, et al. Cooperative induction of a tolerogenic dendritic cells phenotype by cytokines secreted by pancreatic carcinoma cells. J Immunol 2006; 177: 3448–3460. 130. Bellone G, Trinchieri G. Dual stimulatory and inhibitory effect of NK cell stimulatory factor/IL-12 on human hematopoiesis. J Immunol 1994; 153: 930–937. 131. Bemelmans MH, Abramowicz D, Gouma DJ, Goldman M, Buurman WA. In vivo T cell activation by anti-CD3 monoclonal antibody induces soluble TNF receptor release in mice: effects of pentoxifylline, methylprednisone and anti-TNF and anti-γ antibodies. J Immunol 1994 153; 499. 132. Ben-Sasson SZ, Le Gros G, Conrad DH, et al. IL-4 production by T cells from naive donors. J Immunol 1990; 145: 1127–1136. 133. Benchetrit F, Ciree A, Vives V, et al. Interleukin-17 inhibits tumor cell growth by means of a T-cell-dependent mechanism. Blood 2002; 99: 2114–2121. 134. Benjamin D, Sharma V, Knobloch TJ, et al. B cell IL-7. Human B cell lines constitutively secrete IL-7 and express IL-7 receptors. J Immunol 1994; 152: 4749–4757. 135. Bennett MW, O’Connell J, O’Sullivan GC, et al. The Fas counterattack in vivo: Apoptotic depletion of tumor infiltrating lymphocytes associated with Fas ligand expression by human esophageal carcinoma. J Immunol 1998; 160: 5669–5675.

Cytokines 136. Bennett SRM, Carbone FR, Karamalis F, et al. Help for cytotoxic T-cell responses is mediated by CD40 signaling. Nature 1998; 393: 478. 137. Benveniste EN, Tang LP, Law RM. Differential regulation of astrocyte TNF-α expression by cytokines TGF-β, IL-6, and IL-10. Int J Dev Neurosci 1995; 13: 341. 138. Berent-Maoz B, Philiponsky AM, Daigle I, et al. Human mast cells undergo TRAIL-induced apoptosis. J Immunol 2006; 176: 2272–2278. 139. Berkman N, Robichaud A, Robbins RA, et al. Inhibition of inducible nitric oxide synthase expression by interleukin-4 and interleukin-13 in human lung epithelial cells. Immunol 1996; 89: 363. 140. Bermudez LE, Parker A, Petrofsky M. Apoptosis of Mycobacterium avium-infected macrophages is mediated by both tumor necrosis factor (TNF) and Fas, and involves the activation of caspases. Clin Exp Immunol 1999; 116: 94. 141. Bernstein HB, Plasterer MC, Schiff SE, et al. CD4 expression on NK cells: ligation of CD4 induces cytokine expression and cell migration. J Immunol 2006; 177: 3669–3676. 142. Berro AI, Perry GA, Agarwal DK. Increased expression and activation of CD30 induce apoptosis in human eosinophils. J Immunol 2004; 173: 2173–2183. 143. Bertagnolli M and Herrmann S. IL-7 supports the generation of cytotoxic T lymphocytes from thymocytes. J Immunol 1990; 145: 1706–1712. 144. Bertini R, Bianchi M, Ghezzi P. Adrenalectomy sensitizes mice to the lethal effects of interleukin 1 and tumor necrosis factor. J Exp Med 1988; 167: 1708. 145. Bertoglio S, Melioli G, Baldini E, et al. Intraperitoneal infusion of recombinant interleukin-2 in malignant ascites in patients with gastrointestinal and ovarian cancer. Acta Med Austriaca 1989; 16: 81–83. 146. Bertram EM, Dawicki W, Sedgmen B, et al. A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 cell response to influenza in vivo. J Immunol 2004; 172: 981–988. 147. Bertram EM, Lau P, Watts TH. Temporal segregation of 4-1BB versus CD28-mediated costimulation: 4-1BB ligand influences T cell numbers late in primary response and regulates the size of the T cell memory response following influenza infection. J Immunol 2002; 168: 3777–3785. 148. Besedovsky H, del Rey A, Sorkin E, Dinarello CA. Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. Science 1986; 233: 652–654. 149. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441: 235–238. 150. Beutler B, Cerami A. The biology of cachectin/TNF: a primary mediator of the host immune response. Annual Rev Immunol 1989; 7: 625. 151. Beutler B, Greenwald D, Hulmes JD, et al. Identity of tumour necrosis factor and the macrophage secreted factor cachectin. Nature 1985; 316: 552–554. 152. Beutler B, Mahoney J, Le Trang N, et al. Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 2264.7 cells. J Exp Med 1985; 161: 984. 153. Beutler B, Milsark I, Cerami A. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science 1985; 229: 869. 154. Beutler BA, Cerami A. Recombinant interleukin 1 suppresses lipoprotein lipase activity in 3T3-L1 cells. J Immunol 1985; 135: 3969–3971. 155. Bigler RD, Bushkin Y, Chiorazzi N. S152 (CD27). A modulating dissulfide-linked activation antigen. J Immunol 1988; 141: 21. 156. Bird TA, Davies T, Balswin SA, Saklatvala J. Interleukin 1 stimulates hexose transport in fibroblasts by increasing the expression of glucose transporters. J Biol Chem 1990; 265: 13578. 157. Birg F, Courcoul M, Rosnet O, et al. Expression of FMS/KIT-like gene FLT3 in human leukemias of the myeloid and lymphoid lineages. Blood 1992; 80: 2584–2593.

Walter M. Lewko and Robert K. Oldham 158. Bissonnette EY, Chin B, Befus AD. Interferons differentially regulate histamine and TNF-α in rat intestinal mucosal mast cells. Immunol 1995; 86: 12–17. 159. Bjorek P, Bamchereua J, Flores-Romo L. CD40 ligation counteracts Fas-induced apoptosis of human dendritic cells. Int Immunol 1997; 9: 365. 160. Black RA, Kronheim SR, Cantrell M, et al. Generation of biologically active interleukin-1β by proteolytic cleavage of an inactive precursor. J Biol Chem 1988; 263: 9437. 161. Black RA, Rauch CT, Kozlosky CJ, et al. A metalloproteinase disintegrin that releases tumor necrosis factor α from cells. Nature 1997; 385: 729–733. 162. Blankenstein T, Qin Z, Uberla K, et al. Tumor suppression after cell targeted tumor necrosis factor α gene transfer. J Exp Med 1991; 173: 1047–1052. 163. Blazar BR, Taylor PA, Smith S, Vallera DA. Interleukin-10 decreases survival in murine recipients of major histocompatibility complex disparate donor bone marrow grafts. Blood 1995; 85: 842. 164. Blevin MP, Anderson KV. A conserved signaling pathway: the Drosophila Toll-dorsal pathway. Ann Rev Cell Dev Biol 1996; 12: 393. 165. Blin N, Stafford DW. Isolation of high molecular weight DNA. Nucleic Acid Res 1976; 3: 2303. 166. Block MI, Berg M, McNamara MJ, et al. Passive immunization of mice against D factor blocks lethality and cytokine release during endotoxemia. J Exp Med 1993; 178: 1085. 167. Blotta MH, Marshall JD, De Kruyff RH, Umetsu DT. Crosslinking of the CD40 ligand on human CD4+ lymphocytes generates a costimulatory signal that upregulates IL-4 synthesis. J Immunol 1996; 156: 3133. 168. Blum-Degen D, Muller T, et al. Interleukin-1β and interleukin-6 are elevated in cerebrospinal fluid of Alzheimer’s and de novo Parkinson’s disease patients. Neurosci Lett 1995; 202: 17. 169. Blumberg H, Conklin D, Xu WF, et al. Interleukin 20: discovery, receptor identification, and role in epidermal function. Cell 2001; 104: 9–19. 170. Blumenthal RD, Lew W, Juweid M, et al. Plasma FLT-3 ligand levels predict bone marrow recovery from myelosuppressive therapy. Cancer 2000; 88: 333–343. 171. Boehme SA, Lio FM, Maciejewski-Lenoir D, Bacon KB, Conlon PJ. The chemokine fractalkine inhibits Fas-mediated cell death in brain microglia. J Immunol 2000; 165: 397–403. 172. Bogdan C, Thuring H, Dlaska M, Rollinghoff M, Weiss G. Mechanism of suppression of nitric oxide release by IL-13: influence of the macrophage population. J Immunol 1998; 159: 4506. 173. Bogdan C, Vodovotz Y, Nathan CF. Macrophage inactivation by interleukin 10. J Exp Med 1991; 174: 1549. 174. Bohlen H, Kessler M, Sextro M, Diehl V, Tesch H. Poor clinical outcome of patients with Hodgkin’s disease and elevated interleukin-10 serum levels. Clinical significance of interleukin 10 serum levels for Hodgkin’s disease. Ann Hematol 2000; 79: 110–113. 175. Boileau C, Houde M, Dulude G, et al. Regulation of extrathymic T cell development and turnover by oncostatin M. J Immunol 2000; 164: 5713–5720. 176. Boldt DH, Mills BJ, Gemlo BT, et al. Laboratory correlates of adoptive immunotherapy with recombinant interleukin-2 and lymphokine-activated killer cells in human. Cancer Res 1988; 48: 4409. 177. Bolesta E, Kowalczyk A, Wierzbicki A, et al. Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 env glycoprotein when combined with IL-21 and IL-15. J Immunol 2006; 177: 177–191. 178. Boniface K, Bernard F-X, Garcia M, et al. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration in human keratinocytes. J Immunol 2005; 174: 3695–3702. 179. Borges L, Miller RE, Jones J, Ariail K, et al. Synergistic action of fms-like tyrosine kinase and CD40 ligand in the induction of

221

180. 181. 182.

183.

184.

185.

186. 187. 188.

189. 190.

191.

192. 193.

194. 195.

196. 197. 198.

dendritic cells and generation of antitumor immunity in vivo. J Immunol 1999; 163: 1289–1297. Born TL, Tomassen T, Bird TA, Sims Je. Cloning of a novel receptor subunit, AcPL, required for interleukin-18 signaling. J Biol Chem 1998; 273: 29445. Borrow PA, Tishon A, Lee S, et al. CD40 ligand-deficient mice show deficits in antiviral immunity and have impaired memory CD8+ CTL response. J Exp Med 1996; 183: 2129. Boskey AL, Maresca M, Ullrich W, et al. Osteopontin– hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatin-gel. Bone Miner 1993; 22: 147–159. Boulton TG, Stahl N, Yancopoulos GD. Ciliary neurotrophic factor/leukemia inhibitory factor/interleukin 6/oncostatin M family of cytokines induces tyrosine phosphorylation of a common set of proteins overlapping those induced by other cytokines and growth factors. J Biol Chem 1994; 269: 11648. Boussaud V, Soler P, Morreau J, Goodwin RG, Hance AJ. Expression of three members of the TNF-R family of receptors (4-1BB, lymphotoxin-β and Fas) in human lung. Eur Respir J 1998; 12: 926–931. Bover LC, Cardo-vila M, Kiniyasu A, et al. A previously unrecognized protein–protein interaction between Tweak and CD163: potential biological implications. J Immunol 2007; 178: 8183–8194. Bowen MA, Lee RK, Miragliotta G, Nam SY, Podack ER. Structure and expression of murine CD30 and its role in cytokine production. J Immunol 1996; 156: 442–449. Bowman MR, Crimmins AV, Yetz-Aldape J, et al. The cloning of CD70 and its identification as the ligand for CD27. J Immunol 1994; 152: 1756. Bracho F, Krailo MD, Shen V, et al. A phase I clinical, pharmacological, and biological trial of interleukin 6 plus granulocytecolony stimulating factor after ifosamide, carboplatin, and etoposide in children with recurrent/refractory solid tumors: enhanced hematologic responses but a high incidence of grade III/IV constitutional toxicities. Clin Cancer Res 2001; 7: 58–67. Bradding P, Feather IH, Howarth PH, et al. Interleukin 4 is localized and released by human mast cells. J Exp Med 1992; 176: 1381. Bradley EC, Louis AC, Paradise CM, et al. Antitumor response in patients with metastatic renal cell carcinoma is dependent upon regimen intensity (meeting abstract). Proc Am Soc Clin Oncol 1989; 8: A519. Bradley TR, Williams N, Kriegler AB, Fawcett J, Hodgson GS. In vivo effects of interleukin-1α on regenerating marrow myeloid colony forming cells after treatment with S-fluorouracil. Leukemia 1989; 3: 893. Brandt K, Bufone-Paus S, Foster DC, et al. Interleukin-21 inhibits dendritic cell activation and maturation. Blood 2003; 102: 4090–4098. Brasel K, McKenna HJ, Charrier K, et al. Flt3 ligand synergizes with granulocyte-macrophage colony stimulating factor or granulocyte colony stimulating factor to mobilize hematopoietic progenitor cells into the peripheral blood of mice. Blood 1997; 90: 3781–3788. Brasel K, McKenna HJ, Morrisey PJ, et al. Hematologic effects of flt3 ligand in vivo in mice. Blood 1996; 88: 2004–2012. Brazel CY, Ducceschi MH, Pytowski B, Levison SW. The flt3 tyrosine kinase receptor inhibits neural stem/progenitor cell proliferation and collaborates with NGF to promote neuronal survival. Mol Cell Neurosci 2001; 18: 381–393. Breder CD, Dinarello CA, Saper CB. Interleukin-1 immunoreactive innervation of the hypothalamus. Science 1988; 240: 321. Brennan FM, Chantry D, Jackson A, et al. Inhibitory effects of TNF α antibodies on synovial cell interleukin 1 production in rheumatoid arthritis. Lancet 1989; 11: 244. Brenne AT, Ro TB, Waage A, et al. Interleukin-21 is a growth and survival factor for human myeloma cells. Blood 2002; 99: 3756–3762.

222 199. Bresnihan B, Alvaro-Garcia JM, Cobby M, et al. Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rheum 1998; 41: 2196–2204. 200. Brodie SJ, Lewisohn DA, Patterson BK, et al. In vivo migration and function of transferred HIV-1-specific cytotoxic T cells. Nat Med 1999; 5: 34–41. 201. Brody DT, Durum SK. Membrane IL-1: IL-1α precursor binds to the plasma membrane via a lectin-like interaction. J Immunol 1989; 143: 1183. 202. Broide DH, Paine MM, Firestein GS. Eosinophils express interleukin 5 and granulocyte macrophage colony stimulating factor mRNA at sites of allergic inflammation in asthmatics. J Clin Invest 1992; 90: 1414. 203. Brooks B, Rees RC. Human recombinant IL-4 suppresses the induction of human IL-2 induced lymphokine activated killer (LAK) activity. Clin Exp Immunol 1988; 74: 162. 204. Brown MA, Pierce JH, Watson CJ, et al. B cell stimulatory factor-1/Interleukin-4 is expressed by normal and transformed mast cells. Cell 1987; 50: 809–818. 205. Brown PM, Tagari P, Rowan KR, et al. Epitope labeled soluble human interleukin-5 (IL-5) receptors: affinity cross-link labeling, IL-5 binding and biological activity. J Biol Chem 1995; 270: 29236. 206. Brown SA, Richards CM, Hanscom HN, et al. The Fn14 cytoplasmic tail binds tumor necrosis factor receptor associated factors 1, 2, 3, and 5 and mediates nuclear factor-κB activation. Biochemical J 2003; 371: 395–403. 207. Brown TJ, Lioubin MN, Marquardt H. Purification and characterization of cytostatic lymphokines produced by activated human T lymphocytes: synergistic antiproliferative activity of transforming growth factor-β1, interferon-γ, and oncostatin M for human melanoma cells. J Immunol 1987; 139: 2977–2983. 208. Brown TJ, Rowe JM, Shoyab H. Regulation of IL-6 expression by oncostatin M. J Immunol 1991; 147: 2175. 209. Browning JL, Douglas I, Ngam-ek A, et al. Characterization of surface lymphotoxin forms. J Immunol 1996; 154: 33. 210. Browning JL, Mattaliano RJ, Pinchang CE, et al. Disulfide scrambling of interleukin-2: HPLC resolution of the three possible isomers. Anal Biochem 1986; 155: 123. 211. Browning JL, Miatkowski K, Sizing I, et al. Signaling through the lymphotoxin β receptor induces the death of some adenocarcinoma tumor cell lines. J Exp Med 1996; 183: 867. 212. Browning JL, Ngam-ek A, Lawton P, et al. Lymphotoxin β, a novel member of the TNF family that forms a heteromeric complex with lymphotoxin on the cell surface. Cell 1993; 72: 847. 213. Bruce AG, Hoggart IH, Rose TM. Oncostatin M is a differentiation factor for myeloid leukemia cells. J Immunol 1992; 142: 1271–1275. 214. Brugger W, Heimfeld S, Berenson RJ, Mertlesman R, Kanz L. Reconstitution of hematopoiesis after high-dose chemotherapy by autologous progenitor cells generated ex vivo. N Eng J Med 1995; 333: 283–287. 215. Brugnoni D, Bettinardi A, Malacarne F, Airo P, Cattaneo R. CD134/OX40 expression by synovial fluid CD4+ T lymphocytes in chronic synovitis. Br J Rheumatol 1998; 37: 584. 216. Brunda MJ, Luistro L, Warrier RR, et al. Antitumor and antimetastatic activity of interleukin-12 against murine tumors. J Exp Med 1993; 178: 1223–1230. 217. Brunner T, Yoo NJ, LaFace D, Ware CF, Green DR. Activationinduced cell death in murine T cell hybridomas: differential regulation of Fas (CD95) versus Fas ligand expression by cyclosporin A and FK506. Int Immunol 1996; 8: 1017. 218. Bucay N, Sarosi I, Dunstan CR, Morony S, et al. Osteoprotegerin deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998; 12: 1260. 219. Budagian V, Bulanova E, Orinska Z, et al. Reverse signaling through membrane bound interleukin-15. J Biol Chem 2004; 279: 42192–42201. 220. Budd GT, Green S, Baker LH, et al. A Southwest Oncology Group phase II trial of recombinant tumor necrosis factor in metastatic breast cancer. Cancer 1992; 68: 1694–1695.

Cytokines 221. Buhlman JE, Foy TM, Aruffo A, et al. In the absence of a CD40 signal, B cells are tolerogenic. Immunity 1995; 2: 645. 222. Bukowski RM, Raymond P, Molto L, et al. Interferon-γ and CXC chemokine induction by interleukin 12 in renal cell carcinoma. Clin Cancer Res 1999; 5: 2780–2789. 223. Bulfone-Paus S, Ungureanu D, Pohl T, et al. Interleukin-15 protects from lethal apoptosis in vivo. Nat Med 1997; 3: 1124. 224. Bullock TN, Yagita H. Induction of CD70 on dendritic cells through CD40 or TCR stimulation contributes to the development of CD8+ T cell responses in the absence of CD4+ T cells. J Immunol 2005; 174: 710–717. 225. Burd PR, Thompson WC, Max EE, Mills FC. Activated mast cells produce interleukin-13. J Exp Med 1995; 181: 1373. 226. Burger R, Neipel F, Fleckenstein B, Savino R, et al. Human herpesvirus type 8 interleukin-6 homologue is functionally active on human myeloma cells. Blood 1998; 91: 1858. 227. Burgess SJ, Marusina AI Pathmanathan I, et al. IL-21 downregulates NKG2D/DAP10 expression on human NK and CD8+ T cells. J Immunol 2006; 176: 1490–1497. 228. Burton JD, Bamford RM, Peters C, et al. A lymphokine provisionally designated interleukin T and produced by a human adult T cell leukemia line stimulates T cell proliferation and the induction of lymphokine activated killer cells. Proc Nat Acad Sci USA 1994; 91: 4935–4939. 229. Cagnard N, Letourneur F, Essabani A, et al. Interleukin-32, CCL2, PF4F1 and GFD10 are the only cytokine/chemokine genes differentially expressed by in vitro cultured rheumatoid and osteoarthritis fibroblast-like synoviocytes. Eur Cytokine Netw 2005; 16: 289–292. 230. Cai L, Starovasnik MA, Hoque DA, et al. Pathways by which interleukin 17 induces cartilage breakdown in vitro and in vivo. Cytokine 2001; 16: 10–21. 231. Cai X, Gommoll-CP J, Justice L, Narula SK, Fine JS. Regulation of granulocyte colony stimulating factor gene expression by interleukin-17. Immunol Lett 1998; 62: 51. 232. Calderhead DM, Buhman JE, van den Eertwegh AJ, et al. Cloning of mouse Ox40: a T cell activation marker that may mediate T–B cell interactions. J Immunol 1993; 151: 5261. 233. Camerini D, Walz G, Loenen WAM, Borst J, Seed B. The activation antigen CD27 is a member of the nerve growth factor/ tumor necrosis factor receptor gene family. J Immunol 1991; 147: 3165. 234. Cameron LA, Taha RA, Tsicopoulos A, et al. Airway epithelium express interleukin-18. Eur Respir J 1999; 14: 553–559. 235. Campbell IL, Abraham CR, Masliah E, et al. Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin-6. Proc Natl Acad Sci USA 1993; 90: 10061. 236. Campbell KA, Ovendale PJ, Kennedy MK, et al. CD40L is required for protective cell-mediated immunity to Leishmania major. Immunity 1996; 4: 283. 237. Campbell S, Burkly LC, Gao H-X, et al. Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells. J Immunol 2006; 176: 1889–1898. 238. Cannons JL, Lau P, Ghumman B, et al. 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J Immunol 2001; 167: 1313–1324. 239. Cao H, Wolff RG, Meltzer MS, Crawford RM. Differential regulation of class II determinants on macrophages by IFN-γ and IL-4. J Immunol 143; 1989: 3524. 240. Cao R, Farnebo J, Kurimoto M, Cao Y. Interleukin-18 acts as an angiogenesis and tumor suppressor. FASEB J 1999; 13: 2195. 241. Cao X, Zhang W, He L, Xie Z, et al. Lymphotactin-gene-modified bone marrow dendritic cells act as more potent adjuvants for peptide delivery to induce specific immunity. J Immunol 1998; 161: 6238–6244. 242. Cao XX, Mohuiddin I, Chada S. Adenovirus transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells and is abrogated by over expression of BCL-XL. Mol Med 2002; 8: 869–876.

Walter M. Lewko and Robert K. Oldham 243. Cao Z, Henzel WJ, Gao X. IRAK: a kinase associated with the interleukin-1 receptor. Science 1996; 271: 1128. 244. Caput D, Laurent P, Kaghad M, et al. Cloning and characterization of a specific Interleukin (IL)-13 binding protein structurally related to the IL-5 receptor α chain. J Biol Chem 1996; 271: 16921–16926. 245. Carbone E, Ruggiero G, Terrazzano G, et al. A new mechanism of NK cytotoxicity activation: the CD40:CD40 ligand interaction. J Exp Med 1997; 183: 2053. 246. Carrascal MT, Mendoza L, Valcaracel M. Interleukin-18 binding protein reduces B16 melanoma hepatic metastasis by neutralizing adhesiveness and growth factors of sinusoidal endothelium. Cancer Res 2003; 63: 491–497. 247. Carriere V, Roussel L, Ortega N, et al. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo. Proc Natl Acad Sci USA 2007; 104: 282–287. 248. Carroll GJ, Bell MC, Chapman HM, Mills JN, Robinson WF. Leukemia inhibitory factor induces leukocyte infiltration and cartilage proteoglycan degradation in goat joints. J Interferon Cytokine Res 1995; 15: 567. 249. Carson WE, Giri JG, Lindenmann MJ, et al. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 1994; 180: 1395. 250. Carswell EA, Old LJ, Kassel RL, et al. An endotoxin induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci USA 1975: 72: 3666–3670. 251. Carter LL, Dutton RW. Relative perforin and Fas-mediated lysis in T1 and T2 CD8 effector populations. J Immunol 1995; 155: 1028. 252. Carveth HJ, Bohnsack JF, McIntyre TM, et al. Neutrophil activating factor (NAF) induces polymorphonuclear leukocyte adherence to endothelial cells and to subendothelial matrix proteins. Biochem Biophys Res Commun 1989; 162: 387. 253. Cascino I, Fiucci G, Papoff G, and Ruberti G. Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing. J Immunol 1995; 154: 2706. 254. Casey LS, Lichtman AH, Boothby M. IL-4 induced IL-2 receptor p75 β chain gene expression and IL-2 dependent proliferation in mouse T lymphocytes. J Immunol 1992; 148: 3418–3426. 255. Cassatella M, Bazzoni F, Ceska M, et al. IL-8 production by human polymorphonuclear leukocytes: the chemoattractant formylmethionyl leucylphenylalanine induces the pathway. J Immunol 1992; 148: 3216. 256. Cassatella MA, Gasperini S, Bovolenta C, et al. Interleukin-10 (IL-10) selectively enhances CIS3/SOCS3 mRNA expression in human neutrophils: evidence for and IL-10 induced pathway that is independent of STAT protein activation. Blood 1999; 94: 2880. 257. Cassatella MA, Meda L, Gasperini S, Calzetti F, Bonora S. Interleukin-10 (IL-10) upregulates IL-1 receptor antagonist production from lipopolysaccharide stimulated human polymorphonuclear leukocytes by delaying mRNA degradation. J Exp Med 1994; 179: 1695. 258. Castell JV, Gomez-Lechnon MJ, David M, et al. Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes. FEBS Lett 1989; 242: 237. 259. Castigli E, Pahwa R, Good RA, Geha RS, Chatila TA. Molecular basis of a common lymphokine deficiency in a patient with severe combined immunodeficiency. Proc Natl Acad Sci USA 1993; 90: 4728. 260. Castle BE, Kishimoto K, Stearns C, Brown ML, Kehry MR. Regulation of expression of ligand for CD40 on T helper lymphocytes. J Immunol 1993; 151: 1777. 261. Caudell EG, Mumm JB, Poindexter N, et al. The protein product of tumor suppressor gene, melanoma differentiation associated gene 7, exhibits immunostimulatory activity and is designated IL-24. J Immunol 2002; 168: 6041–6046. 262. Caux C, Dezutter-Dambuyant D, Schmitt D, Banchereau J. GM-CSF and TNF cooperate in the generation of dendritic Langerhans cells. Nature 1992; 360, 258.

223 263. Caux C, Vanbervliet B, Massactier C, et al. Activation of human dendritic cells by CD40 crosslinking. J Exp Med 1994; 180: 1263. 264. Cavallo F, Signorelli P, Giovarelli M, et al. Antitumor efficacy of adenocarcinoma cells engineered to produce interleukin 12 (IL-12) or other cytokines compared to exogenous IL-12. J Natl Canc Inst 1997; 89: 1049. 265. Cavender DE, Edelbaum D, Ziff M. Endothelial cell activation induced by tumor necrosis factor and lymphotoxin. Am J Pathol 1986; 134: 551. 266. Cayabyab M, Phillips JH, Lanier LL. CD40 preferentially costimulates activation of CD4+ T lymphocytes. J Immunol 1994; 152: 1523. 267. Cella M, Scheidegger D, Palmer-Lehman K, et al. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J Exp Med 1996; 184: 747. 268. Center DM, Berman JS, Kornfeld H, Theodore AC, Cruikshank WW. The lymphocyte chemoattractant factor. J Lab Clin Med 1995; 125: 167–172. 269. Center DM, Cruikshank WW, Zhang Y. Pro-IL-16 regulation of T cell proliferation: p27Kip1-dependent G0/G1 arrest mediated by inhibition of Skp2 transcription. J Immunol 2004; 172: 1654–1660. 270. Center DM, Cruikshank WW. Modulation of lymphocyte migration by human lymphokines. I. Identification and characterization of a chemoattractant activity for lymphocytes from mitogen stimulated mononuclear cells. J Immunol 1982; 128: 2563. 271. Center DM, Kornfeld H, Cruikshank WW. Interleukin-16 and its function as a CD4 ligand. Immunol Today 1996; 17: 476. 272. Cerretti DP, Kozlowsky CJ, Mosely B, et al. Molecular cloning of the IL-1 β converting enzyme. Science 1992; 256: 97. 273. Cerutti A, Schaffer A, Goodwin RG, et al. Engagement of CD153 (CD30 Ligand) by CD30+ T cells inhibits class switch DNA recombination and antibody production in human IgD+ IgM+ B cells. J Immunol 2000; 165: 786–794. 274. Cerutti A, Schaffer A, Zan H, et al. CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching. Immunity 1998; 9: 247. 275. Chabaud M, Durand JM, Buchs N, et al. Human Interleukin-17: a T cell derived proinflammatory cytokine produced by the rheumatoid synovium. Arthritis Rheum 1999; 42: 963. 276. Chabaud M, Fossiez F, Taupin JL, Miossec P. Enhancing effect of IL-17 on IL-1 induced IL-6 and leukemia inhibitory factor production by rheumatoid arthritis synoviocytes and its regulation by Th2 cytokines. J Immunol 1998; 161: 409. 277. Chada S, Bocangel D, Ramesh R, et al. mda-7/IL-24 kills pancreatic cancer cells by inhibition of the Wnt/PI3K signaling pathways: identification of IL-20 receptor-mediated bystander activity against pancreatic cancer. Mol Ther 2005; 11: 724–733. 278. Chada S, Sutton RB, Ekmekcioglu S, et al. MDA-7/IL-24 is a unique cytokine-tumor suppressor in the IL-10 family. Int Immunopharmacol 2004; 4: 649–667. 279. Chakir J, Shannon J, Molet S, et al. Airway remodeling-associated mediators in moderate to severe asthma: effect of steroids on TGF-β, IL-11, IL-17 and type I and type III collagen expression. J Allergy Clin Immunol 2003; 111: 1293–1298. 280. Chakrabarty S, Nagata M, Yasuda H, et al. Critical roles of CD30/ CD30L interactions in murine autoimmune diabetes. Clin Exp Immunol 2003; 133: 318–325. 281. Chakraborty M, Abrams SI, Camphausen K, et al. Irradiation of tumor cells upregulates Fas and enhances CTL lytic activity and CTL adoptive immunotherapy. J Immunol 2003; 170: 6338–6347. 282. Chakraborty NG, Li L, Sporn JR, et al. Emergence of regulatory CD4+ T cell response to repetitive stimulation with antigenpresenting cells in vitro: implications in designing antigen presenting cell-based tumor vaccines. J Immunol 1999; 162: 5576–5583. 283. Chakravarty PK, Alfieri A, Thomas EK, et al. FLT-3 ligand administration after radiation therapy prolongs survival in a

224

284.

285.

286.

287. 288. 289. 290. 291.

292. 293. 294. 295. 296.

297. 298. 299.

300.

301. 302.

303.

Cytokines murine model of metastatic lung cancer. Cancer Res 1999; 59: 6028–6032. Chan SH, Perussia B, Gupta JW, et al. Induction of interferon γ production by natural killer cell stimulatory factor: characterization of responding cells and synergy with other inducers. J Exp Med 1991; 173: 869–879. Chan WL, Pejnovic N, Lee CA, Al-Ali NA. Human IL-18 receptor and ST2L are stable and selective markers for the respective type 1 and type 2 circulating lymphocytes. J Immunol 2001; 167: 1238–1244. Chang C-I, Zoghi B, Liao JC, Kuo L. The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13 mediated arginase I induction in macrophages: its implications in IL-13 inhibited nitric oxide production. J Immunol 2000; 165: 2134–2141. Chang H, Hanawa H, Liu H, et al. Hydrodynamic-based delivery of an interleukin-22-Ig fusion gene ameliorates experimental autoimmune myocarditis in rats. J Immunol 2006; 3635–3643. Chao T-C, Van Alter PJ, Greager JA, Walter RJ. Sex steroid hormones regulate the release of tumor necrosis factor by macrophages. Cell Immunol 1995; 160: 43. Chapoval AI, Tamda K, Chen L. In vitro inhibition of a broad spectrum of tumor cell lines by activated human dendritic cells. Blood 2000; 95: 2346. Charlton B, Lafferty KJ. The Th1/Th2 balance in autoimmunity. Curr Opinion Immunol 1995; 7: 793–798. Chattopadhyay S, Tracy E, Liang P, et al. Interleukin-31 and oncostatin-M mediate distinct signaling reactions and response patterns in lung epithelial cells. J Biol Chem 2007; 282: 3014–3026. Chaudhari U, Romano P, Mulcahy LD, et al. Efficacy and safety of infliximab monotherapy for plaque-type psoriasis: a randomized trial. Lancet 2001; 357: 1842–1847. Chazen GD, Pereira GMB, Le Gross G, et al. Interleukin 7 is a T cell growth factor. Proc Nat Acad Sci USA 1989; 86: 5923–5927. Chen IA, McAdam AJ Buhlman JE, et al. Ox40-ligand has a critical costimulatory role in dendritic cell:T Cell interactions. Immunity 1999; 11: 689. Chen J, Chada S, Mhashilkar A, Miano JM. Tumor suppressor MDA-7/IL-24 selectively inhibits vascular smooth muscle cell growth and migration. Mol Ther 2003; 8: 220–229. Chen L, Mory Y, Zilberstein A, Revel M. Growth inhibition of human breast carcinoma and leukemia/lymphoma cell lines by recombinant interferon-β2. Proc Natl Acad Sci USA 1988; 85: 8037. Chen WF, Zlotnik A. IL-10: a novel cytotoxic T cell differentiation factor. J Immunol 1991; 147: 528. Chen Y, Langrish CL, McKenzie B, et al. Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalitis. J Clin Invest 2006; 116: 1317–1326. Cheneval D, Ramage P, Kastelic T, et al. Increased mature interleukin-1β (IL-1β) secretion form THP-1 cells induced by nigericin is a result of activation of p45 IL-1β-converting enzyme processing. J Biol Chem 1998; 273: 18846. Chernoff AE, Granowitz EV, Shapiro L, et al. A randomized controlled trial of IL-10 in humans: inhibition of inflammatory cytokine production and immune response. J Immunol 1995; 154: 5492–5499. Cheung PF, Wong CK, Ip WK and Lam CW. IL-25 regulates the expression of adhesion molecules on eosinophils: mechanism of eosinophilia in allergic inflammation. Allergy 2006; 61: 878–885. Chiaramonte MG, Mentink-Kane M, Jacobson BA, et al. Regulation and function of the interleukin-13 receptor α2 during a T helper cell type 2-dominant response. J Exp Med 2003; 197: 687–701. Chiarle R, Podda A, Prolla G, et al. CD30 overexpression enhances negative selection in the thymus and mediates programmed cell death via a Bcl-2-sensitive pathway. J Immunol 1999; 163: 194.

304. Chicheportiche Y, Bourbon PR, Xu H, et al. TWEAK, a new secreted ligand in the tumor necrosis family that weakly induces apoptosis. J Biol Chem 1997; 272: 32401–32410. 305. Chicheprotiche Y, Chicheportiche R, Sizing I, et al. Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies. Arthritis research 2001; 4: 126–133. 306. Chien Y, Gascoigne N, Kavaler J, et al. Somatic recombination in a murine T-cell-receptor gene. Nature 1984; 309: 322. 307. Chilton PM, Fernandez-Botran R. Production of soluble IL-4 receptors by murine spleen cells is regulated by activation and IL-4. J Immunol 1993; 151: 5907–5917. 308. Chiu C-P, Moulds C, Coffman RL, et al. Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells. Proc Nat Acad Sci USA 1988; 85: 7099–7103. 309. Chiyo M, Shimozato O, Yu Y, et al. Expression of IL-27 in murine carcinoma cells produces antitumor effects and induces protective immunity in inoculated host animals. Int J Cancer 2005; 115: 437–442. 310. Chizzonite R, Truitt T, Kilian PL, et al. Two high affinity interleukin 1 receptors represent separate gene products. Proc Natl Acad Sci USA 1989; 86: 8029. 311. Cho D, Song H, Kim YM, et al. Endogenous interleukin-18 modulates immune escape of murine melanoma cells by regulating the expression of Fas ligand and reactive oxygen intermediates. Cancer Res 2000; 60: 2703–2709. 312. Choi BK, Asai T, Vinay DS, et al. 4-1BB-mediated amelioration of experimental autoimmune uveoretinitis is caused by indoleamine 2,3-diogygenase-dependent mechanisms. Cytokine 2006; 34: 233–242. 313. Chouat G, Milani AA, Martal J, et al. IL-10 prevents naturally occurring fetal loss in the CBA × DBA/2 mating combination and local defect in IL-10 production in the abortion prone combination is corrected by in vivo injection of IFN-γ. J Immunol 1995; 154: 4261. 314. Chowers Y, Cahalon L, Lahav M, et al. Somatostatin through its specific receptor inhibits spontaneous and TNF-α and bacterialinduced IL-8 and IL-1β secretion from intestinal epithelial cells. J Immunol 2000; 165: 2955–2961. 315. Christen U, Wolfe T, Mohrle U, et al. A dual role of TNF-α in type 1 diabetes: Islet-specific expression abrogates the ongoing autoimmune process when induced late but not early during pathogenesis. J Immunol 2001; 166: 7023–7032. 316. Chtanova T, Tangye SG, Newton R, et al. T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J Immunol 2004; 173: 68–78. 317. Chu NR, DeBenedette MA, Stiernholm BJN, Barber BH, Watts TH. Role of IL-12 and 4-1BB ligand in cytokine production by CD28+ and CD28− T cells. J Immunol 1997; 158: 3081–3089. 318. Chua AO, Chizzonite R, Desai BB, et al. Expression cloning of a human IL-12 receptor component: a new member of the cytokine superfamily with strong homology with gp130. J Immunol 1994; 153: 128. 319. Chuntarapai A, Dodge K, et al. Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4. J Immunol 2001; 166: 4891–4898. 320. Ciavarra RP, Somers KD, et al. Flt3 ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer. Cancer Res 2000; 60: 2081–2084. 321. Claasen JL, Levine AD, Buckley RH. Recombinant human IL-4 induces IgE and IgG synthesis by normal and atopic donor mononuclear cells. J Immunol 1991; 144: 123–130. 322. Clamin H. Mast cells, T-cells, and abnormal fibrosis. Immunol Today 1985; 6: 192. 323. Clark-Lewis I, Kent SBH, Schrader JW. Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells. J Biol Chem 1984; 259: 7488.

Walter M. Lewko and Robert K. Oldham 324. Clark-Lewis I, Schrader JW. P-cell stimulating factor: biochemical characterization of a new T-cell-derived factor. J Immunol 1981; 127: 1941. 325. Clegg CH, Haugen HS, Rulffes JT, Friend SL, Farr AG. Oncostatin M transforms lymphoid tissue function in transgenic mice by stimulating lymphnode T cell development and thymus autoantibody production. Exp Haematology 1999; 27: 712. 326. Coccia EM, Passini N, Battistini A, et al. Interleukin-12 induces expression of interferon regulatory factor-1 via signal transducer and activator of transcription-4 in human T helper type 1 cells. J Biol Chem 1999; 274: 6698–6703. 327. Coceani F, Lees J, Reford J, Bishai I. Interleukin-1 receptor antagonist: effectiveness against interleukin-1 fever. Can J Physiol Pharmacol 1992; 7: 1590. 328. Cocks BG, de Waal Malefyt R, Galizzi JP, de Vries JE. IL-13 induces proliferation and differentiation of human B cells activated by the CD40 ligand. Int Immunol 1993; 5: 657. 329. Coffman RL, Bond MW, Carty J, et al. B cell stimulatory factor 1 enhances the IgE response of lipopolysaccharide-activated B cells. J Immunol 1986; 136: 4538–4541. 330. Cohen PL and Eisenberg RA. Lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative diseases. Ann Rev Immunol 1991; 9: 243. 331. Cohen SBA, Katsikis PD, Feldman M, Londei E. IL-10 enhances expression of the IL-2 receptor α chain on T cells. Immunol 1994, 83: 329. 332. Cohen SBA, Parry SL, Feldman M, Foxwell B. Autocrine and paracrine regulation of human T cell IL-10 production. J Immunol 1997; 158: 5596–5602. 333. Cohney SJ, Sanden D, Cacalano NA, et al. SOCS-3 is tyrosine phosphorylated in response to interleukin-2 and suppresses STAT5 phosphorylation and lymphocyte proliferation. Mol Cell Biol 1999; 19: 4980. 334. Colotta F, Re F, Muzio M, Bertini R, et al. Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science 1993; 261: 472. 335. Collision LW, Workman CI, Kuo TT, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007; 450: 566–569. 336. Colombel JF, Rutgeerts P, Malchow H, et al. Interleukin-10 (Tenovil) in the prevention of post operative Crohn’s disease. Gut 2001; 49: 42–46. 337. Colotta F, Re F, Muzio M, Polentarutti N, et al. Interleukin-13 induces expression and release of interleukin-1 decoy receptor in human polymorphonuclear cells. J Biol Chem 1994; 269: 12403. 338. Comes A, Rosso O, Orengo AM, et al. CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine. J Immunol 2006; 176: 1750–1758. 339. Consentino G, Soprana E, Thienes CP, et al. IL-13 down-regulates CD14 expression and TNF-α secretion in normal human monocytes. J Immunol 1995; 155: 3145–3151. 340. Conti B, Jahng JW, Tinti C, et al. Induction of IFN-γ inducing factor in the adrenal cortex. J Biol Chem 1997; 272: 2025–2037. 341. Contractor NV, Bassiri H, et al. Lymphoid hyperplasia, autoimmunity, and compromised intestinal intraepithelial lymphocyte development in colitis-free gnotobiotic IL-2 – deficient mice. J Immunol 1998; 160: 385–394. 342. Contractor V, Pippin J, Russell J, et al. Identification of cDNA and genomic localization for IL-14. FASEB J 1994; 8: A506. 343. Conway JG, Andrews RC, Beaudet B, Bickett DM, et al. Inhibition of tumor necrosis factor-α (TNF-α) production and arthritis in the rat by GW3333, a dual inhibitor of TNF-α converting enzyme and matrix metalloproteinases. J Pharmacol Exp Ther 2001; 298: 900–908. 344. Cope AP, Aderka D, Wallach D, et al. Soluble TNF receptor production by activated T lymphocytes: differential effects of acute and chronic exposure to TNF. Immunology1995: 84: 21. 345. Copeland NG, Gilgert DJ, Cho BC, et al. Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles. Cell 1990; 63: 175–183.

225 346. Costa JJ, Demetri GD, Harrist TJ, et al. Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo. J Exp Med 1996; 2681–2686. 347. Costello R, Brailly H, Mallet F, et al. Interleukin-7 is a potent costimulus of the adhesion pathway involving CD2 and CD28 molecules. Immunology 1993; 80: 451–457. 348. Couderc B, Zitvogel L, Douin-Echinard V, et al. Enhancement of antitumor immunity by expression of CD70 (CD27 ligand) or CD154 (CD40 ligand) costimulatory molecules in tumor cells. Cancer Gene Ther 1998; 5: 163–175. 349. Coughlin CM, Salany KE, Wysocka M, et al. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression, which involves inhibition of angiogenesis. J Clin Invest 1998; 101: 1441. 350. Coughlin CM, Salhany KE, Gee MS, et al. Tumor cell responses to IFNγ affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity 1998; 9: 25. 351. Crawford R, Finboom D, Ohara J, et al. B cell stimulatory factor 1 (Interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens. J Immunol 1987; 139: 135. 352. Cretney E, Takeda K, Yagita H, et al. Increased susceptibility to tumor initiation and metastasis in TNF-related apoptosis-inducing ligand-deficient mice. J Immunol 2002; 168: 1356–1361. 353. Crooks GM, Hao QL, Peterson D, Barsky LW, Brockstoce D. IL-3 increases production of B lymphoid progenitors from human CD34+ CD38−cells. J Immunol 2000; 165: 2382–2389. 354. Croston GE, Cao Z, Goeddel DV. NF-κB activation by interleukin-1 (IL-1) requires an IL-1 receptor-associated protein kinase activity. J Biol Chem 1995; 270: 16514. 355. Crowe PD, van Arsdale TL, Walter BN, et al. A lymphotoxin-β specific receptor. Science 1994; 264: 707. 356. Crowell S, Knauper V, Stewart ML, et al. Induction of martix metalloproteinase activation cascades based on membrane-type matrix metalloproteinase associated activation of gelatinase A, gelatinase B, and collagenase 3. Biochem J 1998, 331: 453. 357. Crown J, Jakubowski A, Kemeny N, et al. A phase I trial of recombinant human interleukin-1β alone and in combination with myelosuppressive doses of 4-fluorouracil in patients with gastrointestinal cancer. Blood 1991; 78: 1420–1427. 358. Cruikshank WW, Berman JS, Theodore AC, Bernardo J, Center DM. Lymphokine activation of T4+ lymphocytes and monocytes. J Immunol 1987; 138: 3817. 359. Cruikshank WW, Center DM, Nisar N, et al. Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function and CD4 expression. Proc Natl Acad Sci USA 1994; 91: 5109. 360. Cruikshank WW, Center DM. Modulation of lymphocyte migration by human lymphokines. II. Purification of a lymphotactic factor (LCF). J Immunol 1982; 128: 2569. 361. Cruikshank WW, Greenstein JL, Theodore AC, Center DM. Lymphocyte attractant factor induces CD-4 dependent intracytoplasmic signaling in lymphocytes. J Immunol 1991; 146: 2928. 362. Cruikshank WW, Long A, Tarpy RE, et al. Early identification of interleukin-16 (Lymphocyte chemoattractant factor) and macrophage inflammatory protein 1α (MIP1α) in bronchoalveolar lavage fluid of antigen-challenged asthmatics. Am J Respir Cell Mol Biol 1995; 13: 738–747. 363. Cua DJ, Sherlock J, Chen Y, et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 2003; 421: 744–748. 364. Cui X, Yang SC, Sharma S, et al. IL-4 regulates COX-2 and PGE2 production in human non-small-cell lung cancer. Biochem Biophys Res Commun 2006; 343: 995–1001. 365. Cumberbatch M, Dearman RJ, Kimber I. Constitutive and inducible expression of interleukin-6 by Langerhans cells and lymph node dendritic cells. Immunology 1996; 87: 513–518. 366. Cunningham CC, Chada S, Merritt JA, et al. Clinical and biological effects of an intratumoral injection of mda-7 (IL-24; INGN 241)

226

367.

368. 369.

370. 371. 372. 373. 374. 375.

376. 377.

378. 379. 380. 381.

382. 383. 384. 385. 386. 387.

Cytokines in patients with advancer carcinoma: a phase I study. Mol Ther 2005; 11: 149–159. Curti A, Fogli M, Ratta M, Tura S, Lemoli RM. Stem cell factor and FLT3 ligand are strictly required to sustain the long-term expansion of primitive CD34+ DR− dendritic cell precursors. J Immunol 2001; 166: 848–854. D’Andrea A, Rangaraju M, Valiante NM, et al. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J Exp Med 1992; 176: 1387–1398. D’Andrea AM, Aste-Amezaga M, Valiante NM, et al. Interleukin-10 (IL-10) inhibits human interferon-γ production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med 1993; 178: 1041. Da Silva N, Hu ZB, Ma W, et al. Expression of the Flt3 gene in human leukemia-lymphoma cell lines. Leukemia 1994; 8: 885–888. Dadi KH and Roifman CM. Activation of phosphatidylinositol-3 kinase by ligation of the interleukin 7 receptor on human thymocytes. J Clin Invest 1993; 92: 1559–1563. Dadi KH and Roifman CM. Interleukin 7 receptor mediates the activation of phosphatidyl-3 inositol kinase in human B-cell precursors. Biochem Biophys Res Commun 1993; 192: 450–464. Dai CH, Krantz SB, Zsebo KM. Human burst forming units erythroid need direct interaction with stem cell factor for further development. Blood 1991; 78: 2493–2497. Dalloul A, Laroche L, Bagot M, et al. Interleukin-7 is a growth factor for Sezary lymphoma cells. J Clin Invest 1992; 90: 1054–1060. Damle NK, Doyle LV, Bender JR, Bradley EC. Interleukin 2-activated human lymphocytes exhibit enhanced adhesion to normal vascular endothelial cells and cause their lysis. J Immunol 1987; 138: 1779. Dandona P, Aljada A, Garg R, Mohanty P. Increase in plasma interleukin-10 following hydrocortisone injection. J Clin Endocrinol metal 1999; 84: 1141–1144. Danese S, de la Motte C, Reyes BM, et al. T cells trigger CD40dependent platelet activation and granular RANTES release: novel pathway for immune response amplification. J Immunol 2004; 172: 2011–2015. Danforth DN Jr, Sgagias MK. Interleukin-1α and interleukin-6 act additively to inhibit growth of MCF-7 breast cancer cells in vitro. Cancer Res 1993; 53: 1538–1545. Dannull J, Nair S, Su Z, et al. Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand. Blood 2005; 105: 3206–3213. Dannull J, Su Z, Rizzieri D. Enhancement of vaccine-mediated antitumor immunity in patients after depletion of regulatory T cells. J Clin Invest 2005; 115: 3623–3633. Dao T, Ohashi K, Kayako T, Kurimoto M, Okamura H. Interferon-γ inducing factor, a novel cytokine, enhances Fas ligand mediated cytotoxicity of murine T helper cells. Cell Immunol 1996; 173: 230. Dastych J, Metcalf DD. Stem cell factor induces mast cell adhesion to fibronectin. J Immunol 1994; 152: 213. Davis ID, Skrumsager BK, Cebon J, et al. An open-label, two arm, phase I trial of recombinant interleukin-21 in patients with metastatic melanoma. Clin Cancer Res 2007; 13: 3630–3636. Davis S, Aldrich TH, Stahl N, et al. LIFRβ and gp130 as heterodimerizing signal transducers of the tripartite CNTF receptor. Science 1993; 260: 1805. De Bie JJ, Hendricks PA, Cruikshank WW, et al. Effects of interleukin-16-blocking peptide on parameters of allergic asthma in a murine model. Eur J Pharmacol 1999; 383: 189–196. De Smedt T, Smith J, Baum P, et al. OX40 costimulation enhances the development of T cell responses induced by dendritic cells in vivo. J Immunol 2002; 168: 661–670. De Totero D, Meazza R, Zupo S, et al. Interleukin-21 (IL-21) receptor is upregulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells. Blood 2006; 107: 3708–3715.

388. De Waal Malefyt R, Abrams J, Bennett B, et al. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 by monocytes. J Exp Med 1991b; 174: 1209. 389. DeBenedette MA, Chu NR, Pollok KE, et al. Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 V lymphocytomas by cAMP. J Exp Med 1995; 181: 985. 390. DeBenedette MA, Wen T, Bachmann MF, et al. Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J Immunol 1999; 163: 4833–4841. 391. DeBenedette MA, Shahinian A, Mak TW, Watts TH. Costimulation of CD28− T lymphocytes by 4-1BB ligand. J Immunol 1997; 158: 551–559. 392. Debets R, Timens JC, Churakowa T, et al. IL-18 receptors, their role in ligand binding and function: Anti-IL-1RAcPL antibody, a potent antagaonist of IL-18. J Immunol 2000; 165: 4950–4956. 393. Debinski W, Obiri NI, Powers SK, Pastan I, Puri RK. Human glioma cells overexpress receptors for IL-13 and are extremely sensitive to a novel chimeric protein composed of IL-13 and Pseudomonas exotoxin. Clin Cancer Res 1995; 1: 1253. 394. DeBlaker-Hohe DF, Yamauchi A, Yu CR, et al. IL-12 synergizes with IL-2 to induce lymphokine activated cytotoxicity and granzyme gene expression in fresh human NK cells. Cell Immunol 1995; 165: 33. 395. Dechanet J, Taupin JL, Chomarat P, et al. Interleukin-4 but not interleukin-10 inhibits the production of leukemia inhibitory factor by rheumatoid synovium and synoviocytes. Eur J Immunol 1994; 24: 3222. 396. DeChiara TM, Vejsada R, Poueymirou WT, et al. Mice lacking CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth. Cell 1995; 83: 313. 397. Declercq W, Denecker G, Fiers W, Vandenabeele P. Cooperation of both TNF receptors in inducing apoptosis: involvement of the TNF receptor-associated factor binding domain of the TNF receptor 75. J Immunol 1998; 161: 390–399. 398. Defrance T, Carayon P, Billian G, et al. Interleukin 13 is a B cell stimulating factor. J Exp Med 1994; 179: 135. 399. Defrance T, Fluckinger AC, Rossi JF, et al. Antiproliferative effects of Interleukin-4 on freshly isolated non-Hodgkin malignant B cell lymphoma cells. Blood 1992; 79: 990–996. 400. Defrance T, Vanbervliet B, Briere F, et al. Interleukin-10 and transforming growth factor β cooperate to induce anti-CD40-activated naive human B cells to secrete immunoglobulin A. J Exp Med 1992; 175: 671. 401. Degli-Esposti M. To die or not to die: the quest of the trail receptors. J Leukocyte Biol 1999; 65: 535. 402. Degli-Esposti MA, Davis-Smith T, Din WS, et al. Activation of lymphotoxin-β receptor by cross-linking induces chemokine production and growth arrest in A375 melanoma cells. J Immunol 1997; 158: 1756. 403. Degli-Esposti MA, Dougall WC, Smolak PJ, et al. The novel receptor TRAIL-R4 induces NF-κB and protects against TRAILmediated apoptosis, yet contains an incomplete death domain. Immunity 1997; 7: 813. 404. Degli-Esposti MA, Smolak PJ, Walczak H, et al. Cloning and characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family. J Exp Med 1997; 186: 1165. 405. DeGoer de Herve MG, Durali D, Tran TA, et al. Differential effect of agonistic anti-CD40 on human mature and immature dendritic cells: the Janus face of anti-CD40. J Immunol Blood 2005; 106: 2806–2814. 406. Dejana E, Bertocci F, Bortolami MC, et al. Interleukin 1 promotes adhesion to cultured endothelial cells. J Clin Invest 1988; 82: 1466. 407. Del Peso L, Gonzalez-Garcia M, Page C, Herrara R, Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 1997; 278: 687–689.

Walter M. Lewko and Robert K. Oldham 408. Del Pozo V, De Andreas B, Martin E, et al. Eosinophil as antigen presenting cells: activation of T cell clones and T cell hybridoma by eosinophils after antigen processing. Eur J Immunol 1992; 22: 1919. 409. Del Prete G, Maggi E, Parronchi P, et al. IL-4 is an essential factor for the IgE synthesis induced in vitro by human T cell clones and their supernatants. J Immunol 1988; 140: 4193–4198. 410. Del Rey A, Besdovsky H, Sorkin E, Dinarello CA. Interleukin-1 and glucocorticoid hormones integrate an immunoregulatory feedback circuit. Ann NY Acad Sci 1987; 496: 85. 411. Della Bella s, Nicola S, Timofeeva I, et al. Are interleukin-16 and thrombopoietin new tool for the generation of dendritic cells? J Immunol 2004; 104: 4020–4028. 412. Demoulin JB, Uyttenhove C, Van Roost E, et al. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, anti-apoptotic activity, growth regulation by IL-9. Mol Cell Biol 1996; 16: 4710–4716. 413. Denhardt DT, Chambers AF. Overcoming obstacles to metastasis defenses to host defenses: osteopontin (OPN) as a shield against attack by cytotoxic host cells. J Cell Biochem 1994; 56: 48. 414. Dennig D, O’ Reilly RJ. L7 induces surface expression of B7/BB1 on Pre-B cells and an associated increase in their costimulatory effects on T cell proliferation. Cellular Immunol 1994; 153: 227–238. 415. Derocq J-M, Sequi M, Poinot-Chazel C, et al. Interleukin-13 stimulates interleukin-6 production by human keratinocytes. FEBS Lett 1994; 343: 32. 416. Desai BB, Quinn PM, Wolitzky AS, et al. IL-12 receptor II. Distribution and receptor expression. J Immunol 1992; 148: 3125–3132. 417. Desplat-Jego S, Varriale S, Creidy R, et al. TWEAK is expressed by glial cells, induces astrocyte proliferation and increases EAE severity. J Neuroimmunol 2002; 133: 116–123. 418. Desreumauz P, Janin A, Colombel JF, et al. Interleukin 5 messenger RNA expression by eosinophils in the intestinal mucosa of patients with coeliac disease. J Exp Med 1992; 175: 293. 419. DeTogni PD, Goellner J, Ruddle NH, et al. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science 1994; 264: 703. 420. Deveraux QL, Reed JC. IAP family proteins: suppressors of apoptosis. Genes Dev 1999; 113: 239. 421. Devos R, Guisez Y, Tavernier J, Plaetinck G. Recombinant soluble interleukin-5 (hIL-5) receptor molecules: cross-linking and stoichiometry of binding to IL-5. J Biol Chem 1993; 268: 6581. 422. DeWaal Malefyt R, Figdor C, Huijbens R, et al. Effects of IL-13 on phenotype, cytokine production and cytotoxic function of human monocytes. J Immunol 1993; 151: 6370–6381. 423. DeWaal Malefyt R, Haansen J, et al. IL-10 and viral IL-10 strongly reduce antigen specific T cell proliferation by diminishing the antigen presenting capacity of monocytes vial downregulation of class II MHC expression. J Exp Med 1991; 174: 915. 424. Dewhirst FE, Stashenko PP, Mole JE, Tsurumachi T. Purification and partial sequence of human osteoclast activating factor: identity with interleukin-1β. J Immunol 1985; 135: 2562–2568. 425. Dhingra K, Sahin A, Emani K, Hortobagyi GN, Estrov Z. Expression of leukemia inhibitory factor and its receptor in breast cancer: a potential autocrine and paracrine growth regulatory mechanism. Breast Cancer Res Treatment 1998; 48: 165–174. 426. Di Carlo E, Comes A, Basso S, et al. The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement. J Immunol 2000; 165: 3111–3118. 427. Di Carlo E, Comes A, Orengo AM, et al. IL-21 induces tumor rejection by specific CTL and IFN-γ-dependent CXC chemokines in syngeneic mice. J Immunol 2004; 172: 1540–1547. 428. Dickensheets HL, Venkataraman C, Schindler U, Donnelly RP. Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression. Proc Natl Acad Sci USA 1998; 96: 10800. 429. Diegmann J, Junker K, Loncarevic IF, et al. Immune escape from renal cell carcinoma: CD70 mediates apoptosis in lymphocytes. Neoplasia 2006; 8: 933–938.

227 430. Diehl L, Boer AT, Schoenberger SP, et al. CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte and augments anti-tumor vaccine efficiency. Nat Med 1999; 5: 774. 431. Dillon SR, Sprecher C, Hammond A, et al. Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice. Nat Immunol 2004; 5: 752–760. 432. Dilloo D, Bacon K, Holeden W, et al. Combined chemokine and cytokine gene transfer enhances antitumor immunity. Nat Med 1996; 2: 1090. 433. Dilloo D, Brown M, Roscrow M, et al. CD40 ligand induces and antileukemia response in vivo. Blood 1997; 90: 1927. 434. Dinarello CA. An IL-1 family member required caspase-1 processing and signals through the ST2 receptor. Immunity 2005; 23: 461–462. 435. Dinarello CA, Cannon JG, Wolff SM, et al. Tumor necrosis factor (Cachectin) is an endogenous pyrogen and induces production of interleukin 1. J Exp Med 1986; 163: 1433. 436. Dinarello CA, Goldin NP, Wolf SM. Demonstration and characterization of two distinct human leukocyte pyrogens. J Exp Med 1974; 139: 1369. 437. Dinarello CA, Ikejima T, Warner SJ, et al. Interleukin-1 induces interleukin-1. I. Induction of circulating interleukin-1 in rabbits in vivo and in human mononuclear cells in vitro. J Immunol 1987; 139: 1902. 438. Dinarello CA, Kim SH. IL-32, a novel cytokine with a possible role in disease. Ann Rheum Dis 2006; 65: Suppl 3: iii61–iii64. 439. Dinarello CA. IL18: A Th1-inducing proinflammatory cytokine and a member of the IL-1 family. J Allergy Clin Immunol 1999; 103: 11. 440. Ding O, Stewart J Jr, Prince CW, et al. Promotion of malignant astrocytoma cell migration by osteopontin expressed in the normal brain: differences in integrin signaling during cell adhesion to osteopontin versus vitronectin. Cancer Res 2002; 62: 5336–5343. 441. DiSanto JP, Bonnefoy JY, Gauchat JF, Fischer A, de Saint Badile G. CD40 ligand mutations in X-linked immunodeficiency with hyper-IgM. Nature 1993; 361: 541. 442. Dokter WH, Sierdsema SJ, Esselink MT, et al. IL-7 enhances expression of IL-3 and granulocyte-macrophage-CSF mRNA in activated human T cells by post-transcriptional mechanisms. J Immunol 1993; 150: 2584–2590. 443. Dokter WH, Sierdsema SJ, Esselink MT, et al. Interleukin 4 mRNA and protein in activated human T cells are enhanced by Interleukin 7. Exp Hematology 1994; 22: 74–79. 444. Domzig W, Stadler BM, Herberman RB. Interleukin 2 dependence of human natural killer (LAK) cell activity. J Immunol 1983; 130: 1970. 445. Donaldson DD, Whitters MJ, Fitz LJ, et al. The murine IL-13 receptor α2: molecular cloning, characterization and comparison with murine IL-13 receptor α. J Immunol 1998; 161: 2317–2324. 446. Donepudi M, Quach DD, Mokyr MB. Signaling through CD40 enhances cytotoxic T lymphocyte generation by CD8+ T cells from mice bearing large tumors. Cancer Immunol Immunother 1999; 48: 153. 447. Donohoe RE, Yang YC, Clark SC. Human P40 T cell growth factor (interleukin 9) supports erythroid colony formation. Blood 1990; 75: 2271–2275. 448. Donohue JH, Rosenberg SA. The fate of interleukin-2 after in vivo administration. J Immunol 1983; 130: 2203. 449. Donohue PJ, Richards CM, Brown SA, et al. TWEAK is an endothelial cell growth and chemotactic factor that also potentiates FGF-2 and VEGF-A mitogenic activity. Arterioscler Throm Vasc Biol 2003; 23: 594–600. 450. Dougall WC, Glaccum M, Charrier K, et al. RANK is essential for osteoclast and lymph node development. Genes Dev 1999; 13: 2412. 451. Doull IJ, Lawrence S, Watson M, et al. Allelic association of gene markers on chromosomes 5q and 11q with atopy and bronchial hyperresponsiveness. Am J Respir Crit Care Med 1996; 153: 1280.

228

452. Drakes ML, Lu L, Subbotin VM, Thomson AW. In vivo administration of Flt3 ligand markedly stimulates generation of dendritic cell progenitors from mouse liver. J Immunol 1997; 159: 4268. 453. Drexler HG. Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells. Leukemia 1996; 10: 588–599. 454. Dripps DJ, Brandhuber BJ, Thompson RC, Eisenberg SP. Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction. J Biol Chem 1991; 266: 10331–10336. 455. Du XX, Doershuk CM, Orazi A, Williams DA. A bone marrow stromal derived growth factor, interleukin-11, stimulates recovery of small intestinal mucosal cells after cytoablative therapy. Blood1994; 83: 33. 456. Du XX, Williams DA. Interleukin-11: review of molecular, cell biology and clinical use. Blood 1997; 89: 3897. 457. Duan L, Gan H, Arm J, Remold HG. Cytosolic phospholipase A2 participates with TNF-α in the induction of apoptosis of human macrophages infected with Mycobacterium tuberculosis H37Ra. J Immunol 2001; 166: 7469–7476. 458. Dubinett SM, Huang M, Lichtenstein A, et al. Tumor necrosis factor-α plays a central role in interleukin-2 induced pulmonary vascular leak and lymphocyte accumulation. Cell Immunol 1994; 157: 170. 459. Dubucquoi S, Desreumaux P, Janin A, et al. Interleukin 5 synthesis by eosinophils: association with granules and immunoglobulin-dependent secretion. J Exp Med 1994; 179: 703. 460. Duda D, Sunamura M, Lozonschi L, et al. Direct in vitro evidence and in vivo analysis of the antiangiogenesis effects of interleukin 12. Cancer Res 2000; 60: 1111. 461. Dugas B, Renauld J-C, Pene J, et al. Interleukine-9 potentiates the interleukine-4-induced immunoglobulin (IgG, IgM and IgE) production by normal B lymphocytes. Eur J Immunol 1993; 23: 1687. 462. Dumoutier L, Leemans C, Lejeune D, Kotenko SV, Renauld J-C. STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J Immunol 2001; 167: 3545–3549. 463. Dumoutier L, Lejeune D, Colau D, Renauld J-C. Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10 related T cell-derived inducible factor/IL-22. J Immunol 2001; 166: 7090–7095. 464. Dumoutier L, Louahed J, Renauld J-C. Cloning and characterization of IL-10 related T cell derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9. J Immunol 2000; 164: 1814. 465. Dumoutier L, Van Roost E, Ameye G, Michaux L, Renauld J-C. IL-TIL/IL-22: genomic organization and mapping of the human and mouse genes. Genes Immun 2000; 1: 488. 466. Dumoutier L, Van Roost E, Colau D, Renauld J-C. Human interleukin-10-related T cell-derived inducible factor: molecular cloning and functional characterization as a hepatocyte-stimulating factor. Proc Natl Acad Sci USA 2000c; 97: 10144–10149. 467. Dunbar CE, Kohn DB, Schiffman R, et al. Retroviral transfer of the cerebrosidase gene into CD34+ cells from patients with Gaucher disease: in vivo detection of transduced cells without myeloablation. Hum Gene Ther 1998; 9: 2629. 468. Duncan MR, Berman B. Stimulation of collagen and glycosaminoglycan production in cultured human adult dermal fibroblasts by recombinant human interleukin-6. Invest Dermatol 1991; 97: 686. 469. Duncan MR, Hasan A, Berman B. Oncostatin M stimulates collagen and glycosaminoglycan production by cultured normal dermal fibroblasts: insensitivity of scleroderma and keloid fibroblasts. J Invest Dermatol 1995; 104: 128. 470. Durie FH, Aruffo A, Ledbetter J, et al. Antibody to the ligand of CD40, gp39, blocks the occurrence of the acute and chronic forms of graft-vs-host disease. J Clin Invest 1994; 94: 1333. 471. Durie FH, Fava RA, Foy TM, Aruffo A, Ledbetter JA, Noelle RJ. Prevention of collagen-induced arthritis with one antibody to gp39, the ligand of CD40. Science 1993; 261: 1328. 472. Durkop H, Latza U, Himmelreich P, Stein H. Expression of the human OX40 (hOX40) antigen in normal and neoplastic tissues. Br J Haematol 1995; 91: 927.

Cytokines

473. Durkop H, Latza U, Hummel M, et al. Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin’s disease. Cell 1992; 68: 421. 474. Dutcher JP, Creekmore S, Weiss GR, et al. A Phase II study of interleukin-2 and lymphokine-activated killer cells in patients with metastatic malignant melanoma. J Clin Oncol 1989; 7: 477. 475. Dzierzak EA, Papayannopoulou T, Mulligan RG. Lineage specific expression of a peptide β-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature 1988; 331: 35–41. 476. Eckenberg R, Moreau J-L, Melnyk O, Theze J. IL-2Rβ Agonist P1–30 acts in synergy with IL-2, IL-4, IL-9 and IL-15: Biological and Molecular Effects. J Immunol 2000; 165: 4312–4318. 477. Eckenberg R, Rose T, Moreau JL, et al. The first helix of IL-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor β chain and induces lymphokine activated killer cells. J Exp Med 2000; 131: 529. 478. Economou JS, McBride WH, Essner R, et al. Tumour necrosis factor production by IL-2-activated macrophages in vitro and in vivo. Immunology 1989; 67: 514. 479. Eder M, Geissler G, Ganser A. IL-3 in the clinic. Stem Cells 1997; 15: 327–333. 480. Ehrhardt H, Fuda S, Schmid I. TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-κB. Oncogene 2003; 22: 3842–3852. 481. Einarrson O, Geba GP, Zhu Z, Landry M, Elias JA. Interleukin-11: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyper responsiveness. J Clin Invest 1996; 97: 915. 482. Eisenberg S, Brewer MT, Verderber E, et al. Interleukin-1 receptor antagonist is a member of the interleukin-1 gene family: evolution of a cytokine control mechanism. Proc Natl Acad Sci USA 1991; 88: 5232. 483. Elewaut D, Brossay L, Santee SM, et al. Membrane lymphotoxin is required for the development of different populations of NK T cells. J Immunol 2000; 165: 671–679. 484. Elias JA, Wu Y, Zheng T, Panettieri R. Cytokine and virus stimulated airway smooth muscle cells produce interleukin-11 and other IL-6-type cytokines. Am J Physiol (Lung Cell Mol Physiol) 1997; 273: L648. 485. Elias JA, Zheng T, Einerrson O, et al. Epithelial interleukin-11: regulation by cytokines, respiratory syncytial virus and retinoic acid. J Biol Chem 1994; 169: 22261. 486. Elias JA, Zheng T, Whiting NL, et al. Interleukin-1 and transforming growth factor β regulation of fibroblast derived interleukin-11. J Immunol 1994; 152: 2421. 487. Eliopoulos A, Stack GM, Dawson CW, et al. Epstein-Barr virus encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-κB pathway involving tumor necrosis factor receptor-associated factors. Oncogene 1997; 14: 2899. 488. Ellerhorst JA, Prieto VG, Ekmekcioglu S, et al. Loss of MDA-7 expression with progression of melanoma. J Clin Oncol 2002; 20: 1069–1074. 489. Ellis TM, Simms PE, Slivnick DJ, Jack H-M, Fisher RI. CD30 is a signal-transducing molecule which defines a subset of human activated CD45RO+ T cells. J Immunol 1993; 151: 2380. 490. Emery JG, McDonnell P, Burke MB, et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 1998; 273: 14363. 491. Emmel EA, Verweij CL, Durand DB, et al. Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science 1989; 246: 1617. 492. Endo TA, Mashuhara M, Yokuchi M, et al. A new protein containing as SF2 domain that inhibits JAK kinases. Nature 1997; 387: 921. 493. Engelman H, Novick D, Wallach D. Two tumor necrosis factor binding proteins purified from human urine. J Biol Chem 1990; 265: 1531. 494. Enk CD, Sredni D, Blauvelt A, Katz SI. Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro. J Immunol 1995; 154: 4851–4856.

Walter M. Lewko and Robert K. Oldham

495. Erickson SL, Sauvage FJD, Kikly K, et al. Decreased sensitivity to tumor necrosis factor but normal T cell development in TNFreceptor-2-deficient mice. Nature 1994; 372: 560. 496. Ericson SG, Zhao Y, Gao H, et al. Interleukin-6 production by human neutrophils after Fc receptor cross-linking or exposure to granulocyte colony stimulating factor. Blood 1998; 91: 2099–2107. 497. Ernst E. Plasma fibrinogen-an independent cardiovascular risk factor. J Int Med 1990; 227: 365. 498. Escary J-L, Perreau J, Dumenil D, Ezine S, Brulet P. Leukemia inhibitory factor is necessary for maintenance of hematopoietic stem cells and thymic stimulation. Nature 1993; 363: 361. 499. Esche C, Subbotin VM, Maliszewski C, Lotze MT, Shurin MR. FLT3 ligand administration inhibits tumor growth in murine melanoma and lymphoma. Cancer Res 1998; 58: 380–383. 500. Estrov Z, Samal B, Lapushin R, et al. Leukemia inhibitory factor binds to breast cancer cells and stimulates their proliferation. J Interferon Cytokine Res 1995; 15: 905–913. 501. Ettinger R, Sims GP, Fairhurst AM, et al. IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. J Immunol 2005; 175: 7867–7879. 502. Eugster H, Muller M, Karrer U, et al. Multiple immune abnormalities in tumor necrosis factor and lymphotoxin-α double deficient mice. Int Immunol 1996; 8: 23. 503. Eustace D, Han X, Gooding R, Rowbottom A, Riches P, Heyderman E. Interleukin-6 functions as an autocrine growth factor in cervical carcinomas in vitro. Gynecol Oncol 1993, 50: 15. 504. Evans CH, Watkins SC, Stefanovic-Racic M. Nitric oxide and cartilage metabolism. Methods Enzymol 1996; 269: 75. 505. Evans CH. Lymphotoxin – An immunologic hormone with anti-carcinogenic and anti-tumor activity. Cancer Immunol Immunother 1982; 12: 181. 506. Facon T, Harousseau JL, Maloisel F, et al. Stem cell factor in combination with filgastim after chemotherapy improves peripheral blood progenitor cell yield and reduces apheresis requirements in multiple myeloma patients: a ramdomized controlled trial. Blood 1999; 94: 1218–1225. 507. Falcone M, Rajan AJ, Bloom BR, Brosman CF. A critical role for IL-4 in regulating disease severity in experimental allergic encephalitis as demonstrated in IL-4-deficient C57BL/6 mice and BALB/c mice. J Immunol 1998; 160: 4822–4830. 508. Fallon PG, Ballantyne SJ, Mangan NE, et al. Identification of an Interleukin-25-dependent cell population that provides IL-4, IL-5, and IL-13 at the onset of helminth expulsion. J Exp Med 2006; 203: 1105–1116. 509. Fan Z, Yu P, Wang Y, et al. NK-cell activation by LIGHT triggers tumor specific CD8+ T cell immunity to reject established tumors. Blood 2006; 107: 1342–1351. 510. Fanger NA, Maliszewski CR, Schooley K, Griffith TS. Human dendritic cells mediate apoptosis via tumor necrosis factor-related apoptosis inducing ligand (TRAIL). J Exp Med 1999; 190: 1155. 511. Fanslow WC, Clifford KN, Seaman M, et al. Recombinant CD40 ligand exerts potent biologic effects on T cells. J Immunol 1994; 152: 4262. 512. Fantuzzi G, Reed DA, Dinarello CA. IL-12-induced IFNγ is dependent on caspase-1 processing of the IL-18 precursor. J Clin Invest 1999; 104: 761. 513. Faour WH, Mancini A, He OW Di Batista JA. T-cell derived IL-17 regulates the levels and stability of cyclooxygenase-2 (COX-2) mRNA through restricted activation of the p38 mitogen-activated protein kinase cascade. J Biol Chem 2003; 278: 26897–26907. 514. Farrar J, Hilfiker M, Johnson B, et al. Identification of a T-cell derived B-cell growth factor distinct from interleukin-2. J Exp Med 1982; 155: 914–923. 515. Faulkner H, Humphries N, Renauld J-C, Van Snick J, Grencis R. Interleukin-9 is involved in host protective immunity to intestinal nematode infection. Eur J Immunol 1997; 27: 2536. 516. Faure E, Thomas L, Xu H, et al. Bacterial lipopolysaccharide and IFN-γ induce Toll-like receptor 2 and Toll-like receptor 4 expression in endothelial cells: role of NF-κB activation. J Immunol 2001; 166: 2018–2024.

229

517. Faust J, Lacey DL, Hunt P, et al. Osteoclast markers accumulate on cells developing from the peripheral blood mononuclear precursors. J Cell Biochem 1999; 72: 67. 518. Favre C, Saeland S, Caux C, et al. Interleukin-4 has growth promoting activity on cord blood cells. Blood 1990; 75: 67–73. 519. Favre-Felix N, Fromentin A, Hammann A, et al. The tumor counterattack revisited: colon cancer cells do not induce T cell apoptosis via the Fas (CD95, Apo-1) pathway. J Immunol 2000; 164: 5023–5027. 520. Fearon DT, Locksley RM. The instructive role of innate immunity in the acquired immune response. Science 1996; 272: 50. 521. Fehninger TA, Shah MH, et al. Differential cytokine and chemokine expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response. J Immunology 1999; 162: 4511. 522. Feldman ER, Creagan ET, Schaid DJ, Ahmann DL. Phase II trial of recombinant tumor necrosis factor in disseminated malignant melanoma. Am J Clin Oncol 1992; 15: 256–259. 523. Feng SY, Guo Y, Factor VM, et al. The Fn14 intermediate-early response gene is induced during liver regeneration and highly expressed in both human and murine hepatocellular carcinomas. Am J Pathol 2000; 156: 1253–1261. 524. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV entry cofactor: functional cDNA cloning of a seven-transmembrane, G proteincoupled receptor. Science 1996; 272: 872. 525. Fenton MJ, Buras JA, Donnelly RP. IL-4 reciprocally regulates IL-1 and IL-1 receptor antagonist expression in human monocytes. J Immunol 1992; 149: 1283. 526. Fernandez Botran R, Sanders VM, Oliver KG, Vitetta ES. Evidence that soluble interleukin-4 receptors may act as transport proteins. J Exp Me 1991; 174: 673–681. 527. Fernandez-Botran R. Soluble cytokine receptors and their role in immunoregulation. FASEB 1991; 5: 2567. 528. Ferrari D, Chiozzi P, Falzoni S, et al. Extracellular ATP triggers IL-1β release by activating the purinergic P2Z receptor of human macrophages. J Immunol 1997; 159: 1451. 529. Ferrari D, Chiozzi P, Falzoni S, et al. Purinergic modulation of IL-1β release from microglial cells stimulated with bacterial endotoxin. J Exp Med 1997; 185: 579. 530. Fibbe WE, van der Meer JWM, et al. A single low dose of human recombinant interleukin 1 accelerates the recovery of neutrophils in mice with cyclophosphamide-induced neutropenia. Exp Hematol 1989; 17: 805. 531. Fichtner-Feigl S, Strober W, Kawakami K, et al. IL-13 signaling through the IL-13α(2) receptor is involved in induction of transforming growth factor β(1) production and fibrosis. Nature Med 2006; 12: 99–106. 532. Fickenscher H, Pirzer H. Interleukin-26. Int Immunopharmacol 2004; 4: 609–613. 533. Finbloom DS and Winestock KD. IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1α and STAT3 complexes in human T cells and monocytes. J Immunol 1995; 155: 1079–1090. 534. Finke JH, Rayman P, Hart L, et al. Characterization of tumor infiltrating lymphocyte subsets from human renal cell carcinoma: specific reactivity defined by cytotoxicity interferon γ secretion and proliferation. J Immunother with Emphasis on Tumor Immunology 1994; 15: 91–104. 535. Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper IV: Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 1989; 170: 2081–2095. 536. Fiorentino DF, Zlotnik A, Mosmann TR, et al. IL-10 inhibits cytokine production by activated macrophages. J Immunol 1991; 147: 3815. 537. Fiorentino DF, Zlotnik A, Viera P, et al. IL-10 acts on the antigen presenting cell to inhibit cytokine production by TH1 cells. J Immunol 1991; 146: 3444–3451. 538. Firestein GS, Boyle DL, Yu C, et al. Synovial interleukin-1 receptor antagonist and interleukin-1 balance in rheumatoid arthritis. Arthritis Rheum 1994; 37: 644–652.

230 539. Fischer M, Harvima IT, Carvalho RF, et al. Mast cell CD30 ligand is upregulated in cutaneous inflammation and mediates degranulation-independent chemokine secretion. J Clin Invest 2006; 116: 2748–2756. 540. Fisher PB. Is mda-7/IL-24 a “magic bullet” for cancer? Cancer Res 2005; 65: 10128–10138. 541. Fisher RI, Coltman CA, Doroshow JH, et al. Metastatic renal cancer treated with interleukin-2 and lymphokine activated killer (LAK) cells. A phase II clinical trial. Ann Intern Med 1988; 108: 518–523. 542. Fitzpatrick LA, Severson A, Edwards WD, et al. Diffuse calcification in human coronary arteries. Association of osteopontin with atherosclerosis. J Clin Invest 1994; 94: 1597–1604. 543. Flanagan JG, Chan DC, Leder P. Transmembrane form of the kit growth factor is determined by alternative splicing and is missing in the S1d mutant. Cell 1991; 64: 1025–1035. 544. Flanagan WM, Corthesy B, Bram RJ, Crabtree GR. Nuclear association of a T cell transcription factor blocked by FK-506 and cyclosporin A. Nature 1991; 352: 803. 545. Flesch IEA and Kaufman SHE. Activation of bacteriostatic macrophage functions by γ interferon, interleukin-4 and tumor necrosis factor. Infect Immun 1990; 58: 2675. 546. Fluckiger A-C, Durand I, Banchereau J. Interleukin-10 induces apoptotic death of B-chronic lymphocytic leukemia cells. J Exp Med 1994; 179: 91. 547. Fluckiger A-C, Garrone P, Durand I, Galizi J-P, Banchereau J. Interleukin-10 (IL-10) upregulates functional high affinity IL-2 receptors on normal and leukemic B lymphocytes. J Exp Med 1993; 178: 1473. 548. Foey AD, Parry SL, Williams LM, et al. Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-α: Role of the p38 and p42/44 mitogen-activated protein kinases. J Immunol 1998; 160: 920–928. 549. Fong L, Hou Y, Rivas A, Benike C, et al. Altered peptide ligand vaccination with Flt3 ligand expanded cells for tumor immunotherapy. Proc Natl Acad Sci USA 2001; 98: 8809–8814. 550. Fong Y, Tracey KJ, Moldawer LL, et al. Antibodies to cachectin/ tumor necrosis factor reduce interleukin-1B and interleukin 6 appearance during lethal bacteremia. J Exp Med 1989; 170: 1627. 551. Fontana A, Kristensen F, Dubs R, et al. Production of prostaglandin E and interleukin-1 like factor by cultured astrocytes and CG glioma cells. J Immunol 1982; 129: 2413–2419. 552. Ford R, Tamayo A, Martin B, et al. Identification of B-cell growth factors (interleukin-14; high molecular weight-B-cell growth factors) in patients with aggressive B-cell lymphomas. Blood 1995; 86: 283–293. 553. Ford RJ, Tamayo A, Marker B, Ambrus JL. IL-14 and growth control in human B cell lymphomas. FASEB J 1992; 8: A1021. 554. Foss FM, Koc Y, Stetler-Stevenson MA, et al. Costimulation of cutaneous T cell lymphoma cells by Interleukin 7 and interleukin 2: potential autocrine and paracrine effectors in the Sezary syndrome. J Clin Oncol 1994; 12: 326–335. 555. Fossiez F, Djossou O, Chomarat P, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med 1996; 183: 2593. 556. Foxwell BM, Beadling C, Gushin D, Kerr I, Cantrell D. Interleukin-7 can induce the activation of Jak1, Jak3, and STAT5 proteins in murine T cells. Eur J Immunol 1995; 25: 3041–3046. 557. Foxwell BM, Willcocks JL, Taylor-Fishwick DA, et al. Inhibition of activation induced changed in the structure of the T cell interleukin-7 receptor by cyclosporin A and FK506. Eur J Immunol 1993; 23: 85–89. 558. Foxwell BML, Taylor-Fishwick DA, Simon JL, et al. Activation induced changes in expression and structure of the IL-7 receptor in human T cells. Int Immunol 1992; 4: 277. 559. Fraker DL, Alexander HR, Andrich M, Rosenberg SA. Treatment of patients with melanoma of the extremity using hyperthermic isolated limb perfusion with melphalan, tumor necrosis factor, and interferon γ: results of a tumor necrosis factor dose escalation study. J Clin Oncol 1996; 26: 404–410.

Cytokines 560. Franco AV, Zhang XD, Van Berkel E, et al. The role of NF-κB in TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of melanoma cells. J Immunol 2001; 166: 5337–5345. 561. Frank MB, Watson J, Gillis S. Biochemical and biologic characterization of lymphocyte regulatory molecules. VIII. Purification of interleukin-2 from a human T-cell leukemia. J Immunol 1981; 127: 2361. 562. Franklin WA, Glaspy J, Pflaumer SM, et al. Incidence of tumor cell contamination in leukapherisis products of breast cancer patients mobilized with stem cell factor and granulocyte colony stimulating factor (G-CSF) or G-CSF alone. Blood 1999; 94: 340–347. 563. Fransen L, Van de Hayden J, Ruysshaert R, et al. Recombinant tumor necrosis factor: its effects and its synergism with interferon γ on a variety of normal and transformed human cell lines. Eur J Cancer and Clin Oncol 1986; 22: 419–426. 564. Fraser CC, Thacker JD, Hogge DE, et al. Alterations in lymphopoiesis after hematopoietic reconstitution with IL-7 virus infected bone marrow. J Immunol 1993; 151: 2409–2418. 565. Freeman GJ, Boussiotis VA, Anunmanthan A, et al. B7–1 and B7–2 do not deliver identical costimulatory signals, since B7–2 but not B7–1 preferentially costimulates initial production of IL-4. Immunity 1995; 2: 523. 566. Freisen C, Herr I, Krammer PH, Debatin KM. Involvement of CD95 (APO-1/Fas) receptor/ligand system in drug-induced apoptosis in leukemia cells. Nature Med 1996. 567. French RR, Chan HTC, Tutt AL, Glennie MJ. CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat Med 1999; 5: 548. 568. Friend SL, Hosier S, Nelson A, Foxworthe D, Williams DE, Farr A. A thymic stromal cell line supports in vitro development of surface IgM+ B cells and produces a novel growth factor affecting B and T lineage cells. Exp Hematol 1994; 22: 321. 569. Frishman J, Long B, Knospe W, et al. Genes for interleukin 7 are translated in leukemia cell subsets of individuals with chronic lymphocytic leukemia. J Exp Med 1993; 177: 955–964. 570. Frohlich A, Marsland BJ, Sonderegger I, et al. IL-2 receptor signaling is integral to the development of Th2 effector responses in vitro. Blood 2006. 571. Fu Y-X, Huang G, Wang Y, Chaplin DD. Lymphotoxin-α dependent spleen microenvironment supports the generation of memory B cells and is required for their subsequent antigen-induced activation. J Immunol 2000; 164: 2508–2514. 572. Fujita T, Matsumoto Y, Hirai I, et al. Immunosuppressive effect on T cell activation by interleukin 16-cDNA-transfected human squamous cell line. Cell Immunol 2000; 202: 54–60. 573. Fukao T, Matsuda S, Koyasu S. Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-γ production by dendritic cells. J Immunol 2000; 164: 64. 574. Fukushi J, Ono M, Morikawa W, Iwamoto Y, Kuwano M. The activity of soluble VCAM-1 in angiogenesis stimulated by IL-4 and IL-13. J Immunol 2000; 165: 2818–2823. 575. Funakoshi S, Longo DL, Beckwith M, et al. Inhibition of human B cell lymphoma growth by CD40 stimulation. Blood 1994; 83: 2787. 576. Furman WL, Luo X, Fairclough D, et al. Phase I trial of subcutaneous interleukin-1α in children with malignant solid tumors. Med Pediatr Oncol 1997; 28: 444–450. 577. Furukawa J, Hara I, Nagai H, et al. Interleukin-21 gene transfection into mouse bladder cancer cells, results in tumor rejection through the cytotoxic T lymphocyte response. J Urol 2006; 176: 1198–1203. 578. Gabbianelli M, Pelosi E, Montesoro E, et al. Multilevel effects of Flt-3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors. Blood 1995; 86: 1661–1670. 579. Gadient R, Patterson PH. Leukemia inhibitory factor, interleukin-6 and other cytokines using the GP130 transducing receptor: roles in inflammation and injury. Stem Cells 1999; 17: 127.

Walter M. Lewko and Robert K. Oldham 580. Gagari E, Tsai M, Lantz CS, Fox LG, Galli SJ. Differential release of mast cell interleukin-6 via c-kit. Blood 1997; 89: 2654. 581. Gajewski TF, Fallarino F, Ashikari A, Sherman M. Immunization of HLA-A2+ patients with MAGE-3 or Melan A peptide-pulsed autologous peripheral blood mononuclear cells plus recombinant human interleukin 12. Clin Cancer Res 2001; 7: 895s–901s. 582. Gallagher G, Dickensheets H, Eskdale J, et al. Cloning, expression and initial characterization of interleukin-19, a novel homologue of human interleukin-10 (IL-10). Genes Immun 2000; 1: 442–450. 583. Gallagher G, Eskdale J, Jordan W, et al. Human interleukin-19 and its receptor: a potential role in the induction of Th2 responses. Int Immunopharmacol 2004; 4: 615–626. 584. Gallagher G, Stimson WH, Findlay J, et al. Interleukin 6 enhances the induction of human lymphokine activated killer cells. Cancer Immunol Immunother 1990; 31: 49–52. 585. Galy AHM and Spits H. CD40 is functionally expressed on thymic epithelial cells. J Immunol 1992; 149: 775. 586. Gamble JR, Harlan JM, Klebamoff SJ, Vadas MA. Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc Natl Acad Sci USA 1985; 82: 8667. 587. Gansbacher B, Bannerji R, Daniels B, et al. Retroviral vector mediated γ-interferon gene transfer into tumor cells generates potent and long lasting antitumor immunity. Cancer Res 1990; 50: 7820–7825. 588. Gansbacher B, Zier K, Daniels B, et al. Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity. Exp Med 1990; 172: 1217–1224. 589. Ganser A, Lindemann A, Spielt G, et al. Effects of recombinant human interleukin-3 in patients with normal hematopoiesis and in patients with bone marrow failure. Blood 1990; 76: 666–676. 590. Gao JJ, Xue Q, Papasian CJ, Morrison DC. Bacterial DNA and lipopolysaccharide induce synergistic production of TNF-α through a post-transcriptional mechanism. J Immunol 2001; 166: 6855–6860. 591. Garcia VE, Jullien ED, Song M, et al. IL-15 enhances the response of human γδ T cells to non-peptide microbial antigens. J Immunol 1998; 160: 4322. 592. Garvy BA Riley RL. IFN γ abrogates IL-7 dependent proliferation in per-B cells coinciding with onset of apoptosis. Immunology 1994; 81: 381–388. 593. Gaspal FM, Kim MY, McConnell FM, et al. Mice deficient in OX40 and CD30 signals lack memory antibody responses because of deficient CD4 T cell memory. J Immunol 2005; 174: 3891–3896. 594. Gasparetto C, Laver J, Abboud M, et al. Effect of interleukin-1 on hematopoietic progenitors: evidence of stimulatory and inhibitory activities in a primate model. Blood 1989; 74: 547. 595. Gately MK, Caravajal DM, Connaughton SE, et al. Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo. Ann NY Acad Sci 1996; 795: 1. 596. Gately MK, Wilson DE, Wong HL. Synergy between recombinant Interleukin 2 and interleukin 2 depleted lymphokine-containing supernatants in facilitating allogeneic human cytolytic T lymphocyte responses in vitro. J Immunol 1986; 136: 1274. 597. Gately MK, Wolitzky AG, Quinn PM, Chizzonite R. Regulation of human cytolytic lymphocyte responses by interleukin 12. Cellular Immunology 1992; 143: 127–142. 598. Gattei V, Aldinucci D, Rossi FM, et al. CD30 ligand (CD30L)expressing acute myeloid leukemias: a new model of paracrine interactions for the regulation of blast cell proliferation. Leuk Lymphoma 1999; 35: 21. 599. Gattei V, Degan M, Glogini A, et al. CD30 ligand is frequently expressed in hematopoietic malignancies of myeloid and lymphoid origin. Blood 1997; 89: 2048–2059. 600. Gazzinelli RT, Oswald IP, James SL, Sher A. IL-10 inhibits parasite killing and nitrogen oxide production by IFNγ activated macrophages. J Immunol 1992; 148: 1792. 601. Gearing DP, Bruce AG. Oncostatin M binds the high affinity leukemia inhibitory factor receptor. New Biol 1992; 4: 61–65.

231 602. Gearing DP, Comeau MR, Friend DJ, et al. The IL-6 signal transducer gp130: an oncostatin M receptor and an affinity converter for the LIF receptor. Science 1992; 225: 1434. 603. Gearing DP, Cosman D. Homology of the p40 subunit of natural killer cell stimulatory factor (NKSF) with the extracellular domain of the interleukin-6 receptor. Cell 1991; 66: 9. 604. Gearing DP, Gough NM, King JA, et al. Molecular cloning and expression of cDNA encoding a murine myeloid leukemia inhibitory factor (LIF). EMBO 1987: 6: 3995–4002. 605. Gedrich RW, Gilfillan MC, Druckett CS, Van Dongen LW, Thompson CB. CD30 contains two binding sites with different specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins. Biol Chem 1996; 271: 12852. 606. Gee MS, Koch CJ, Evans SM, et al. Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12. Cancer Res 1999; 59: 4882. 607. Geiger T, Tobin H, Consenti-Vargas A, et al. Neutralization of interleukin-1β in vivo with a monoclonal antibody alleviates collagen-induced arthritis in DBA/1 mice and prevents the associated acute-phase response. Clin Exp Rheumatol 1993; 11: 515. 608. Geissinger E, Weisser C, Fisher P, et al. Autocrine stimulation by osteopontin contributes to antiapoptotic signaling of melanocytes in dermal collagen. Cancer Res 2002; 62: 4802–4828. 609. Geissler EN, Ryan MA, Housman DE. The dominant-white spotting (W) locus of the mouse encodes the c-kit protooncogene. Cell 1988; 55: 185. 610. Geisterfer M, Richards C, Baumann M, et al. Regulation of IL-6 and the hepatic IL-6 receptor in acute inflammation in vivo. Cytokine 1993; 5: 1–7. 611. Gemlo BT, Palladino MA Jr, Jaffe HS, et al. Circulating cytokines in patients with metastatic cancer treated with recombinant interleukin 2 and lymphokine-activated killer cells. Cancer Res 1988; 48: 5864. 612. Georganas C, Liu H, Perlman H, et al. Regulation of IL-6 and IL-8 expression in rheumatoid arthritis synovial fibroblasts: the dominant role for NF-κB but not C/EBPβ or c-Jun. J Immunol 2000; 165: 7199–7206. 613. Gerard C, Bruyns C, Marchant A, et al. Interleukin-10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia. J Exp Med 1993; 177: 547. 614. Gerli R, Pitzalis C, Bistoni O, et al. CD30+ T cells in rheumatoid synovitis: mechanisms of recruitment and functional role. J Immunol 2000; 164: 4399. 615. Gerritse K, Laman JD, Noelle RJ, et al. CD40–CD40 ligand interactions in allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci USA 1996; 93: 2499. 616. Gershenwald JE, Fong YM, Fahey TJ, et al. Interleukin 1 receptor blockade attenuates the host inflammatory response. Proc Natl Acad Sci USA 1990; 87: 4966. 617. Gery I, Gershon RK, Waksman BH. Potentiation of the T-lymphocyte response to mitogens. II. The cellular source of potentiating mediators. J Exp Med 1972; 136: 143. 618. Ghayur T, Banerjee S, Hugunin M, et al. Caspase-1 processes IFN-γ inducing factor and regulates LPS-induced IFN γ production. Nature 1997: 386: 619. 619. Ghezzi P, Saccardo B, Villa P, et al. Role of interleukin-1 in the depression of liver drug metabolism by endotoxin. Infect Immun 1986; 54: 837–840. 620. Giavazzi R, Garofalo A, Bani MR, et al. Interleukin 1-induced augmentation of experimental metastases from a human melanoma in nude mice. Cancer Res 1991; 50: 4771. 621. Gilfillan MC, Noel PJ, Podack ER, Reiner SL, Thompson CB. Expression of the costimulatory receptor CD30 is regulated by both CD28 and cytokines. J Immunol 1998; 160: 2180–2187. 622. Gimbaud R, Abitbol V, Bertrand V, et al. Leukemia inhibitory factor involvement in human ulcerative colitis and its potential role in malignant course. Eur Cytokine Netw 1998; 9: 607–612. 623. Gimbrone MA, Obin MS, Brock AF, et al. Endothelial interleukin 8. a novel inhibitor of leukocyte endothelial interactions. Science 1989; 246: 1601.

232 624. Giovarelli M, Musiani P, Modesti A, et al. Local release of IL-10 by transfected mouse mammary adenocarcinoma cells does not suppress but enhances antitumor reaction and elicits a strong cytotoxic lymphocyte and antibody dependent immune memory. J Immunol 1995; 155: 3112–3123. 625. Girasole G, Passeri G, Jilka RL, Manolagas SC. Interleukin-11: a new cytokine critical for osteoclast development. J Clin Invest 1994; 93: 1516. 626. Giri JG, Adhieh M, Eisenman J, et al. Utilization of the β and γ chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J 1994; 13: 2822–2830. 627. Giri JG, Kumaki S, Ahdieh M, et al. Identification and cloning of a novel IL-15 binding protein that is structurally related to the α chain of the IL-2 receptor. EMBO J 1995; 14: 2354–3663. 628. Giri JG, Newton RC, Horuk R. Identification of a soluble interleukin-1 binding protein in cell free supernatants: evidence for soluble interleukin-1 receptor. J Biol Chem 1990; 265: 17416. 629. Go NF, Castle BE, Barrett R, et al. Interleukin 10 (IL-10) a novel B cell stimulatory factor: unresponsiveness of X chromosome linked immunodeficiency B cells. J Exp Med 1990; 172: 1625–1631. 630. Goda C, Kanaji T, Kanaji S, et al. Involvement of IL-32 in activation-induced cells death in T cells. Int Immunol 2006; 18: 233–240. 631. Godfraind C, Louahed J, Faulkner H, et al. Intraepithelial infiltration by mast cells with both connective tissue-type and mucosaltype characteristics in gut, trachea, and kidneys of IL-9 transgenic mice. J Immunol 1998; 160: 3989. 632. Godfrey WR, Fagnoni FF, Harara MA, et al. Identification of a human Ox-40 ligand, a costimulator of CD4+ T cells with homology to tumor necrosis factor. J Exp Med 1994; 180: 757. 633. Goebeler M, Schnarr A, Toksoy A, et al. Interleukin-13 selectively induces monocyte chemoattractant protein-1 synthesis and secretion by human endothelial cells: involvement of IL-4Rα and stat6 phosphorylation. Immunobiology 1997; 91: 450. 634. Goldfeld AE, McCaffrey PG, Strominger JL, Rao A. Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor α gene promoter. J Exp Med 1993; 178: 1365. 635. Gollob JA, Mier JW, Veenstra K, et al. Phase I trial of twiceweekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain IFN-γ induction is associated with clinical response. Clin Cancer Res 2000; 6: 1678–1692. 636. Golumbek PT, Lazenby AJ, Levitsky HI, et al. Treatment of established renal caner by tumor cells engineered to secrete interleukin-4. Science 1991; 254: 713–716. 637. Gonzalez-Hernandez JA, Ehrhart-Bornstein M, et al. Human adrenal cells express tumor necrosis factor messenger ribonucleic acid: evidence for paracrine control of adrenal function. J Clin Endo Metab 1996; 81: 807. 638. Goodwin RG, Alderson MR, Smith CA, et al. Molecular and biochemical characterization of a ligand for CD27 defines a new family of cytokines with homology to tumor necrosis factor. Cell 1993; 73: 447. 639. Goodwin RG, Din WS, Davis-Smith T, et al. Molecular cloning of a ligand for the inducible T cell gene 4-1BB: a member of an emerging family of cytokines with homology to tumor necrosis factor. Eur J Immunol 1993; 23: 2631–2641. 640. Goodwin RG, Friend D, Ziegler SF, et al. Cloning of the human and murine interleukin-7 receptors: Demonstration of a soluble form and homology to a new receptor superfamily. Cell 1990; 60: 941. 641. Goodwin RG, Lupton GS, Schmierer A, et al. Human interleukin 7: molecular cloning and growth factor activity on human and murine B-lineage cells. Proc Nat Acad Sci USA 1989; 86: 302. 642. Gordon C, Ranges GE, Greenspan JS, Wolfsy D. Chronic therapy with tumor necrosis factor-α in autoimmune NZB/NZW F1 mice. Clin Immunol Immunopathol 1989; 52: 421. 643. Gordon J, MacLean LD. A lymphocyte-stimulating factor produced in vitro. Nature 1965; 208: 795–796.

Cytokines 644. Gordon JR, Galli SJ. Promotion of mouse collagen gene expression by mast cells stimulated via the Fc epsilon RI: role of mast cell derived transforming growth factor β and tumor necrosis factor α. J Exp Med 1994; 180: 2027–2037. 645. Gordon MS, McCaskill-Stevens WJ, et al. A phase I trial of recombinant human interleukin-11 (neumega rhIL-11 growth factor) in women with breast cancer receiving chemotherapy. Blood 1996; 87: 3615–3624. 646. Gorierly S, Vincart B, Stordeur P, et al. Deficient IL-12 (p35) gene expression by dendritic cells derived from neonate monocytes. J Immunol 2001; 166: 2141–2146. 647. Grabstein K, Eisenman J, Shanebeck K, et al. Cloning of a T cell growth factor that interacts with the B-chain of the interleukin 2 receptor. Science 1994; 264: 965–968. 648. Grabstein KH, Namen AE, Shanebeck K, et al. Regulation of human T cell proliferation by IL-7. J Immunol 1990; 144: 3015–3020. 649. Grabstein KH, Park LS, Morrissey JP, et al. Regulation of T cell proliferation by T cell stimulating factor-1.J Immunol 1987; 139: 1148–1153. 650. Grabstein KH, Waldschmidt TJ, Finkelman FD, et al. Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody. J Exp Med 1993; 178: 257. 651. Graca L, Honye K, Adams E, Cobbold SP, Waldmann H. AntiCD154 therapeutic antibodies induce infectious transplant tolerance. J Immunol 2000; 165: 4783–4786. 652. Gracie JA, Forsey RJ, Chan WL, et al. A proinflammatory role for IL-18 in rheumatoid arthritis. J Clin Invest 1999; 104: 1393. 653. Graf D, Muller S, Korthauer U, et al. A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation. Eur J Immunol 1995; 25: 1749. 654. Gramaglia I, Jember A, Pippig SD, et al. Ox40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion. J Immunol 2000; 165: 3043–3050. 655. Gramaglia I, Weinberg AD, Lemon M, Croft M. OX-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J Immunol 1998; 161: 6510–6517. 656. Granelli-Piperno A, Nolan P Inaba K, Steinman RM. The effect of immunosuppressive agents on the induction of nuclear factors that bind to sites on the interleukin-2 promoter. J Exp Med 1990; 172: 1869. 657. Granger GA, Williams TW. Lymphocyte cytotoxicity in vitro: Activation and release of a cytotoxic factor. Nature 1968; 218: 1253. 658. Granowitz EV, Clark BD, Mancilla J, Dinarello CA. Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to type II interleukin-1 receptor. J Biol Chem 1991; 266: 14147–14150. 659. Gray PW, Barret K, Chantry D, Turner M, Feldman M. Cloning of human tumor necrosis factor (TNF) receptor cDNA: expression of recombinant soluble TNF-binding protein. Proc Natl Acad Sci USA 1990; 87: 7380–7384. 660. Green PM, Noel PJ, Sperling AI, et al. Absence of B7-dependent responses in CD28-deficient mice. Immunity1994; 1: 501. 661. Green S, Dobrjansky A, Chiasson MA, et al. Corynebacterium parvum as the priming agent in the production of tumor necrosis factor in the mouse. J Natl Cancer Inst 1977; 59: 1519. 662. Greene CM, Meachery G, Taggart CC, et al. Role of IL-18 in CD4+ T lymphocyte activation in sarcoidosis. J Immunol 2000, 165; 4718. 663. Greenfelder S, Nunes AP, Kwee L, Labow M, Chizzonite RA, Ju G. Molecular cloning and characterization of a second subunit of the interleukin-1 receptor complex. J Biol Chem 1995; 270: 13757. 664. Greil J, Gramatzki M, Burger R, et al. The acute lymphoblastic leukemia cell line SEM with t(4;11) chromosomal rearrangement is biphenotypic and responsive to interleukin-7. British J Haematology 1994; 86: 275–283. 665. Grell M, Douni E, Wajant H, Lohden M, et al. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 1995; 83: 793.

Walter M. Lewko and Robert K. Oldham 666. Grenier A, Dehoux M, Boutten A, et al. Oncostatin M production and regulation by human polymorphonuclear neutrophils. Blood 1999; 93: 1413. 667. Grewal IS, Foellmer HG, Grewell KD, et al. Requirement for CD40 ligation in costimulation induction, T cell activation and experimental allergic encephalomyelitis. Science 1996; 273: 1864. 668. Grewal IS, Grewell KD, Wong FS, et al. Local expression of transgene encoded TNF α in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto reactive islet-specific T cells. J Exp Med 1996; 184: 1963. 669. Gri G, Gallo E, Di Carlo E, et al. OX40-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-APC signaling to boost the host T cell antitumor response. J Immunol 2003; 170: 99–106. 670. Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995; 270: 1189. 671. Griffith TS, Chin WA, Jackson GC, et al. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol 1998; 161: 2833. 672. Griffith TS, Wiley SR, Kubin MZ, et al. Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL. J Exp Med 1999; 189: 1343. 673. Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA. Lymphokine activated killer cell phenomenon. J Exp Med 1982; 155: 1823. 674. Grimm EA, Smid CM, Lee JJ, et al. Unexpected cytokines in serum of malignant melanoma patients during sequential biochemotherapy. Clin Cancer Res 2000; 6: 3895–3903. 675. Grohmann U, Bianchi R, Belladonna ML, et al. IL-12 acts selectively on CD8α-dendritic cells to enhance presentation of a tumor peptide. J Immunol 1999; 163: 3100. 676. Gross V, Andrus T, Caesar I, Roth M, Scholmerich J. Evidence for continuous stimulation of interleukin-6 production in Crohn’s disease. Gastroenterology 1992; 102: 514. 677. Groux H, Bigler M, de Vries JE, Roncarolo M-G. Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells. J Immunol 1998; 160: 3188. 678. Groux H, Cottrez F, Rouleau M, et al. A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigenpresenting cells. J Immunol 1999; 162: 1723–1729. 679. Grove RI, Eberhardy C, Abid S, et al. Oncostatin M is a mitogen for vascular smooth muscle cells. Proc Natl Acad Sci USA 1993; 90: 823–827. 680. Grubler U, Chua AO, Schoenhaut DS, et al. Coexpression of two distinct genes required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc Nat Acad Sci USA 1991; 88: 4143–4147. 681. Grunig G, Warnock M, Wakil AE, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998; 282: 2261–2263. 682. Gruol DL, Nelson TE. Physiological and pathological roles of interleukin-6 in the central nervous system. Mol Neurobiol 1997; 15: 307. 683. Gruss H-J, Boiani N, Williams DE, et al. Pleiotrophic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines. Blood 1994; 83: 2045. 684. Gruss HJ, Brach MA, Drexler HG, Bross KJ, Herrmann F. Interleukin 9 is expressed by primary and cultured Hodgkin and Reed-Sternberg cells. Cancer Res 1992; 52: 1026. 685. Gruss HJ, Dower SK. Tumor necrosis factor ligand superfamily. Blood 1995; 85: 3378–3404. 686. Grzegorzewski K, Komschlies KL, et al. Administration of recombinant human interleukin-7 to mice induces the exportation of myeloid progenitor cells from the bone marrow to peripheral sites. Blood 1994; 83: 377. 687. Gu L, Tseng S, Horner RM, Tam C, Loda M, Rollins BJ. Control of TH2 by the chemokine monocyte chemoattractant protein-1. Nature 2000; 404: 407. 688. Gu Y, Kuida K, Tsutui H, et al. Activation of interferon γ inducing factor mediated by interleukin-1β converting enzyme. Science 1997; 275: 206.

233 689. Gu ZJ, Costes V, Lu ZY, Zhang XG, et al. Interleukin-10 is a growth factor for human myeloma cells by induction of an oncostatin autocrine loop. Blood 1996; 88: 3972–3986. 690. Gu ZJ, Zhang XG, Hallet MM, et al. A ciliary neurotrophic factor-sensitive human myeloma cell line. Exp Hematol 1996; 24: 1195–1200. 691. Guesdon F, Freshney N, Waller RJ, Rawlinson L, Saklatvala J. Interleukin-1 and tumor necrosis factor stimulate two novel protein kinases that phosphorylate hsp27 and β-casein. J Biol Chem 1993; 268: 4236. 692. Guimbaud R, Abitbol V, Bertrand V, et al. Leukemia inhibitory factor involvement in human ulcerative colitis and its potential role in malignant course. Eur Cytokine Netw 1998; 9: 607–612. 693. Guinn BA, Bertram EM, DeBenedette MA, et al. 4–1 BBL enhances anti-tumor responses in the presence or absence of CD28 but CD28 is required for protective immunity against parental tumors. Cell Immunol 2001; 210: 56–65. 694. Guo H, Cai CQ, Schroeder RA, Kuo PC. Osteopontin is a negative feedback inhibitor of nitrogen oxide synthetase in murine macrophages. J Immunol 2001; 166: 1079–1086. 695. Gurunathan S, Irvine KR, Wu C-Y, et al. CD40 ligand/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and immune challenge. J Immunol 1998; 161: 4563. 696. Gustavson LE, Nadeau RW, Oldfield NF. Pharmacokinetics of Teceleukin (recombinant human interleukin-2) after intravenous or subcutaneous administration to patients with cancer. J Biol Response Modif 1989; 8: 440. 697. Guthridge MA, Stomski FC, Thomas D, et al. Mechanism of activation of GM-CSF, IL-3, IL-5 family of receptors. Stem Cells 1998; 16: 301–313. 698. Guy GR, Chua SP, Wong NS, et al. Interleukin 1 and tumor necrosis factor activate common multiple protein kinases in human fibroblasts. J Biol Chem 1991; 266: 14343–14352. 699. Habib T, Senadheera S, Weinberg K, et al. The common γ chain (γC) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3. Biochemistry 2002; 41: 8725–8731. 700. Hahne M, Rimoldi D, Schroter M, et al. Melanoma cell expression of FAS (Apo-1/CD95) ligand: implications for tumor immune escape. Science 1996; 274: 1363–1366. 701. Halfter H, Kremerskothen, Weber J, et al. Growth inhibition of newly established human glioma cell lines by leukemia inhibitory factor. J Neurooncol 1998; 39: 1–18. 702. Halfter H, Stogbauer F, Friedrich M, et al. Oncostatin M-mediated growth inhibition of human glioblastoma cells does not depend on stat 3 or on mitogen-activated protein kinase activation. J Neurochem 2000; 75: 973–981. 703. Halloran MM, Haskel CJ, Woods JM, et al. Interleukin-13 is an endothelial chemotactin. Pathobiology 1997; 65: 287. 704. Hamann D, Hilkens CMU, Grogan JL, et al. CD30 expression does not discriminate between human Th1- and Th2-type T cells. J Immunol 1996; 156: 1387–1391. 705. Hamilton JA, Leizer T, Piccoli DS, et al. Oncostatin M stimulates urokinase-type plasminogen activator activity in human synovial fibroblasts. Biochem Biophys Res Commun 1991; 180: 652. 706. Hammonds P, Beggs M, Beresford G, et al. Insulin secreting β cells possess specific receptors for interleukin-1β. FEBS Lett 1990; 261: 97. Biochem Biophys Res Commun 1991; 180: 652. 707. Han X, Itoh K, Balch CM, Pellis JH. Recombinant interluekin-4 (RIL-4) inhibits interleukin 2 induced activation of peripheral blood lymphocytes. Lymphokine Cytokine Res 1988; 7: 227–234. 708. Hanavadi S, Martin TA, Watkins G, et al. Expression of interleukin 11 and its receptor and their prognostic value in breast cancer. Ann Surg Oncol 2006; 13: 802–808. 709. Hanna N, Hanna I, Hleb M, et al. Gestational age dependent expression of IL-10 and its receptor in human placental tissues and isolated cytotrophoblasts. J Immunol 2000; 164: 5721–5728. 710. Hannum C, Culpepper J, Campbell D, et al. Ligand for FLT3/ FLT2 receptor tyrosine kinase regulates growth of hematopoietic

234

711. 712. 713.

714.

715.

716.

717. 718.

719.

720.

721. 722. 723. 724. 725.

726.

727. 728. 729.

Cytokines stem cells and is coded by variant RNAs. Nature 1994; 368: 643–648. Hansen HP, Diedrich Sebastian, et al. CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-α-converting enzyme. J Immunol 2000; 165: 6703–6709. Happel KI, Zheng M, Young E. Roles of Toll-like receptor 4 and IL-23 in IL-17 expression in response to Klebsiella pneumonia infection. J Immunol 2003; 170: 4432–4436. Harada N, Nakayama N, Nakano H, et al. Pro-inflammatory effect of TWEAK/Fn14 interaction on human umbilical vein endothelial cells. Biochem Biophys Res Commun 2002; 299: 488–493. Harashima S, Horiuchi T, Hatta N, et al. Outside to inside signal through the membrane TNF-α induces E selectin (CD62E) expression on activated human CD4+ T cells. J Immunol 2001; 166: 130–136. Harding FA, McArthur JG, Gross JA, Gaulet DH, Allison JP. CD-28-mediated signaling costimulates murine T cells and prevents induction of anergy in T cell clones. Nature 1992; 356: 607. Hargreaves PG, Wang F, Antcliff J, et al. Human myeloma cells shed the interleukin-6 receptor: inhibition of tissue inhibitor of metalloproteinase-3 and a hydroxamate-bases metalloproteinase inhibitor. Br J Haematol 1998; 101: 694. Harlan JM, Killen PD, Harker LA, Striker GE, Wright DG. Neutrophil-mediated endothelial injury in vitro mechanisms of cellular detachment. J Clin Invest 1981; 68: 1394. Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005; 6: 1123–1132. Harrop JA, McDonnell PC, Brigham-Burke M, et al. Herpes virus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T cells and inhibits HT29 cell growth. J Biol Chem 1998; 273: 27548. Harrop JA, Reddy M, Dede K, et al. Antibodies to TR2 (herpesvirus entry mediator), a new member of the TNF receptor superfamily, block T cell proliferation, expression of activation markers, and the production of cytokines. J Immunol 1998; 161: 1786–1794. Hartmann F, Renner C, Jung W, Pfreundschuh M. Anti-CD16/ CD30 bispecific antibodies as possible treatment for refactory Hodgkin’s disease. Leuk Lymphoma 1998; 31: 385–392. Haselmayer P, Tenzer S, Kwon BS, et al. Herpes virus entry mediator synergizes with toll-like receptor mediated neutrophil inflammatory responses. Immunology 2006; 119: 404–411. Haskill S, Martin M, Van Le L, et al. cDNA cloning of an intracellular form of the interleukin-1 receptor antagonist associated with epithelium. Proc Natl Acad Sci USA 1991; 88: 3681. Hawley RG, Berger LC. Growth control mechanisms in multiple myeloma. Leuk Lymphoma 1998; 29: 465–475. Hayakawa F, Towatari M, Kiyoi H, et al. Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene 2000; 19: 624–631. Hayashida K, Kitamura T, Gorman DM, et al. Molecular cloning of a second subunit for the receptor for human granulocyte-macrophage colony stimulating factor GM-CSF: reconstitution of a high affinity GM-CSF receptor. Proc Natl Acad Sci USA 1990; 87: 9655–9659. Hayward AR, Levy J, Facchetti F, et al. Cholangiopathy and tumors of the pancreas liver, and biliary tree in boys with X-linked immunodeficiency with hyper-IgM. J Immunol 1997; 158: 977. He Y-W, Malek TR. Interleukin-7 receptor α is essential for the development of γδ+ T cells, but not natural killer cells. J Exp Med 1996; 184: 289. Heath WR, Kurts C, Caminschi I, Carbone FR, Miller JF. CD30 prevents T cell responses to non-lymphoid tissues. Immunol Rev 1999; 169: 23.

730. Hedrick SM, Cohen D, Nieson E, et al. Isolation of cDNA clones encoding T-cell specific membrane association proteins. Nature 1984; 308: 149. 731. Heinzel FP, Hujer AM, Ahmed FN, Rerko RM. In vivo production and function of IL-12 p40 homodimers. J Immunol 1997; 158: 4381. 732. Held-Feindt J, Mentlein R. CD70/CD27 Ligand, a member of the TNF family, is expressed in brain tumors. Int J Cancer 2002; 98: 352–356. 733. Hellerstein MK, Meydani SN, Meydani M, Wu K, Dinarello CA. Interleukin-1-induced anorexia in rat. Influence of prostaglandins. J Clin Invest 1989; 84: 228. 734. Henney CS, Kuribayashi K, Kern DE, Gillis S. Interleukin-2 augments natural killer cell activity. Nature 1981; 291: 335. 735. Herbelin A, Machzvoine F, Schneider E, et al. IL-7 is requisite for IL-1 induced thymocyte proliferation. Involvement of IL-7 in the synergistic effects of granulocyte macrophage colony stimulating factor or tumor necrosis factor with IL-1. J Immunol 1992; 148: 99–105. 736. Herbert JM, Savi P, Laplace M-C, et al. IL-4 and IL-13 exhibit comparable abilities to produce pyrogen induced expression of procoagulant activity in endothelial cells and monocytes. FEBS Lett 1993; 328: 268. 737. Hercend T, Meuer SC, Reinherz EL, et al. Generation of a cloned NK cell line from the null cell fraction of human peripheral blood. J Immunol 1982; 129: 1299. 738. Herman C, Chernajovsky Y. Mutation of proline 211 reduces shedding of the human 75 TNF receptor. J Immunol 1998; 160: 2478–2487. 739. Hermann E, Darcissac E, Idziorek T, Capron A, Bahr GM. Recombinant interleukin-16 selectively modulates surface receptor expression and cytokine release in macrophages and dendritic cells. Immunology 1999; 97: 241–248. 740. Hermans IF, Ritchie DS, Daisch A, et al. Impaired ability of MHC class II−/− dendritic cells to provide protection is rescued by CD40 ligation. J Immunol 999; 163: 77. 741. Hershey GK. IL-13 receptors and signaling pathways: an evolving web. J Allergy Clin Immunol 2003; 111: 677–691. 742. Herx L, Rivest S, Yong VW. Central nervous system-initiated neurotrophism in trauma: IL-1β is required for production of ciliary neurotrophic factor. J Immunol 2000; 165: 2232–2239. 743. Hess SA, Rensing-Ehl A, Schwabe R, Bufler P, Engelman H. CD40 functions in non-hematopoietic cells: mobilization of NF-κB and induction of IL-6 production. J Immunol 1995; 155: 4588. 744. Heufler C, Koch F, Schuler G. Granulocyte macrophage colony stimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells. J Exp Med 1988; 167: 700. 745. Heufler C, Topar G, Grasseger A, et al. Interleukin 7 is produced by murine and human keratinocytes. J Exp Med 1993; 178: 1109–1114. 746. Heuze-Vourc’h N, Liu M, Dalwadi H, et al. IL-20, an anti-angiogenic cytokine that inhibits COX-2 expression. Biochem Biophys Res Commun 2005; 333: 470–475. 747. Hibbert L, Pflanz S, De Waal Malefyt R, et al. IL-27 and INF-α signal via Stat 1 and Stat 3 and induce T-bet and IL-12Rβ2 in naive T cells. J Interferon Cytokine Res 2003; 23: 513–522. 748. Hibi K, Takahashe T, Sekido Y, et al. Coexpression of the stem cell factor and the c-kit genes in small cell lung cancer. Oncogene 1991; 6: 2291–2296. 749. Hickman CJ, Crim JA, Mostowski HS, Siegel JP. Regulation of human cytotoxic T lymphocyte development by interleukin-7. J Immunol 1990; 145: 2415–2420. 750. Hidi R, Riches V, Al-Ali M, et al. Role of B7-CD28/CTLA-4 costimulation and KF-kappa B in allergen-induced T cell chemotaxis by IL-16 and RANTES. J Immunol 2000; 164: 266–271. 751. Higgins LM, McDonald SA, et al. Regulation of T cell activation in vitro and in vivo by targeting the OX40–OX40ligand interaction:

Walter M. Lewko and Robert K. Oldham

752.

753.

754. 755. 756. 757. 758.

759. 760. 761. 762. 763. 764.

765. 766. 767. 768. 769. 770.

771.

772.

amelioration of ongoing inflammatory bowel disease with an OX40-IgG fusion protein. J Immunol 1999; 162: 486. Higuchi CM, Thompson JA, Lindgren CG, et al. Induction of lymphokine-activated killer activity by interleukin 4 in human lymphocytes preactivated by interleukin 2 in vitro or in vivo. Cancer Res 1989; 49: 6487–6492. Higuchi M, Nagasawa K, Horiuchi T, et al. Membrane tumor necrosis factor-α (TNF-α) expressed on HTLV-I infected T cells mediates a costimulatory signal for B cell activation: characterization of membrane TNF-α. Clin Immunol Immunopathol 1997, 82: 133. Hilbert DM, Kopf M, Mock BA, Kohler G, Rudikoff S. Interleukin-6 is essential for in vivo development of B lineage neoplasms. J Exp Med 1995; 182: 243–248. Hill RJ, Warren MK, Levin J. Stimulation of thrombopoiesis in mice by human recombinant interleukin 6. J Clin Invest 1990; 85: 1242. Hill S, Youde SJ, Man S, et al. Activation of CD40 in cervical carcinoma cells facilitates CTL responses and augments chemotherapy-induced apoptosis. J Immunol 2005; 174: 41–50. Hilliard B, Wilmen A, Seidel C, et al. Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalitis. J Immunol 2001; 166: 1314–1319. Hilton DJ, Zhang J-G, Metcalf D, et al. Cloning and characterization of a binding subunit of the interleukin-13 receptor that is also a component of the interleukin-4 receptor. Proc Natl Acad Sci USA 1996; 93: 497. Hilton DJ. LIF: lots of interesting functions. Trends Biochem Sci 1991: 17: 72. Hinrichs CS, Spolski R, Paulos CM, et al. IL-2 and IL-21 confer opposing differentiation programs too CD8+ T cells for adoptive immunotherapy. Blood; 00: 000–000. Hintzen RQ, de Jong R, Hack CE, et al. A soluble from of the human T cell differentiation antigen CD27 is released after triggering of the TCR/CD3 complex. J Immunol 1991; 147: 29–35. Hintzen RQ, Lens SM, Lammers K, et al. Engagement of CD27 with its ligand CD70 provides a second signal for T cell activation. J Immunol 1995; 154: 2612–2623. Hintzen RQ, Lens SMA, Koopman G, Paris ST, Spitz H, van Lier RAW. CD70 represents the human ligand for CD27. Int Immunol 1994; 6: 477. Hirahara N, Nio Y, Sasaki S, Takamura M, et al. Reduced invasiveness and metastasis of Chinese hamster ovary cells transfected with human interleukin-17 gene. Anticancer Res 2000; 20: 3137. Hirano A, Longo DL, Taub DD, et al. Inhibition of human breast carcinoma growth by a soluble recombinant human CD40 ligand. Blood 1999; 93: 2999. Hirano T, Yasukawa K, Harada H, et al. Complementary DNA for a novel interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature 1986; 324: 73–76. Hirayama F, Clark SC, Ogawa M. Negative regulation of early B lymphopoiesis by interleukin 3 and interleukin 1a. Proc Natl Acad Sci 1994; 91: 469. Hirayama F, Lyman SD, Clark SC, Ogawa M. The flt3 ligand supports proliferation of lymphohematopoietic progenitors and early B-lymphoid progenitors. Blood 1995; 85: 1762. Hirota H, Kiyama H, Kishimoto T, Taga T. Accelerated nerve regeneration in mice by upregulated expression of interleukin (IL) 6 and IL-6 receptor after trauma. J Exp Med 1996; 183: 2627. Hirota S, Ito A, Nagoshi J, et al. Expression of bone protein messenger ribonucleic acids in human breast cancers. Possible involvement of osteopontin in development of calcifying foci. Lab Invest 1995; 72: 64–69. Hirte HW, Miller D, Tonkin K, et al. A randomized trial of paracentesis plus intraperitoneal tumor necrosis factor-α versus paracentesis alone in patients with symptomatic ascites from recurrent ovarian carcinoma. Gynecol Oncol 1997; 64: 80–87. Hisada M, Kamiya S, Fujita K, et al. Potent antitumor activity of interleukin-27. Cancer Res 2004; 64: 1152–1156.

235 773. Ho AS, Liu Y, Khan TA, et al. A receptor for interleukin-10 is structurally related to interferon receptors. Proc Natl Acad Sci USA 1993; 90: 11267–11271. 774. Ho DH, Vu H, Brown SAN, et al. Soluble tumor necrosis factorlike weak inducer of apoptosis overexpression in HEK293 cells promotes tumor growth and angiogenesis in athymic nude mice. Cancer Res 2004; 64: 8968–8972. 775. Hoch H, Dorsch M, Diamantstein T, Blankenstien T. Interleukin 7 induces CD4+ T cell dependent tumor rejection. J Exp Med 1991; 174: 1291–1298. 776. Hoch H, Dorsch M, Kunzendorf U, et al. Mechanism of the rejection induced by tumor cell targeted gene transfer of interleukin 2, interleukin 4, interleukin 7 tumor necrosis factor or interferon γ. Proc Nat Acad Sci USA 1993; 90: 2774–2778. 777. Hochman PS, Majeau GR, Mackey F, Browning JL. Proinflammatory responses are efficiently induced by homotrimeric but not heterotrimeric lymphotoxin. J Inflamm 1996; 46: 220. 778. Hofbauer LC, Khosla S, Dunstan CR, et al. Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 1999; 140: 4367. 779. Hoffman TK, Meidenbauer N, Dworacki G, Kanaya H, Whiteside TL. Generation of tumor specific T lymphocytes by cross priming with human dendritic cells ingesting apoptotic tumor cells. Cancer Res 2000; 60: 3542. 780. Hogaboam C, Kunkel SL, Strieter RM, et al. Novel role of transmembrane stem cell factor for mast cell activation and eotaxin production in mast cell–fibroblast interactions. J Immunol 1998; 160: 6166. 781. Hogge DE, Yalcintepe L, Wong SH, et al. Variant diphtheria toxin-interleukin-3 fusion proteins with increased receptor affinity have enhanced cytotoxicity against acute myeloid leukemia progenitors. Clin Cancer Res 2006; 12 1284–1291. 782. Hogquist KA, Unanue ER, Chaplin DD. Release of IL-1 from mononuclear phagocytes. J Immunol 1991; 147: 2181. 783. Hoidal JR. The eosinophils and acute lung injury. Am Rev Respir Dis 1990; 142: 1245. 784. Holbrook ST, Ohls RK, Schibler KR, et al. Effect of interleukin 9 on clonogenic maturation and cell cycle status of fetal and adult hematopoietic progenitors. Blood 1991; 77: 2129–2134. 785. Holen I, Croucher PI, Hamdy FC, et al. Osteoprotegerin (OPG) is a survival factor for human prostate cancer cells. Cancer Res 2002; 62: 1619–1623. 786. Hollenbaugh D, Mischel-Petty N, Edwards CP, et al. Expression of functional CD40 by vascular endothelial cells. J Exp Med 1995; 182: 33. 787. Holmes WE, Lee J, Kuang WJ, et al. Structure and functional expression of a human interleukin-8 receptor. Science 1991; 253: 1278. 788. Holtmann H, Wallach D. Downregulation of the receptors for tumor necrosis factor by interleukin 1 and 4 β-phorbol-12 myristate-13-acetate. J Immunol 1987; 139: 1161. 789. Hong HJ, Lee JW, Park SS, et al. Humanized anti-4-1BB monoclonal antibody suppresses antigen-induced humoral immune reaponse in nonhuman primates. J Immunother 2000; 23: 613–621. 790. Hoon DSB, Banex M, Okun E, et al. Modulation of human melanoma cells by interleukin-4 and in combination with γ-interferon or α-tumor necrosis factor. Cancer Res 1991; 51: 2002–2008. 791. Hoon DSB, Edward O, Banez M, et al. Interleukin 4 alone and with γ-interferon or tumor necrosis factor inhibits the growth and modulates cell surface antigens on human renal cell carcinoma. Cancer Res 1991; 51: 5687–5693. 792. Hor S, Pirzer H, Dumoutier L, et al. The T cell lymphokine interleukin-26 targets epithelial cells through the interleukin-20 receptor1 and interleukin-10 receptor 2 chains. J Biol Chem 2004; 279: 33343–33351. 793. Horn D, Fitzpatrick WC, Gompper PT, et al. Regulation of cell growth by recombinant oncostatin M. Growth Factors 1990; 2: 157. 794. Horton SA, Oldham RK, Yannelli JR. Generation of human lymphokine-activated killer cells following brief exposure to high dose interleukin-2. Cancer Res 1990; 50: 1686–1692.

236 795. Hoshino K, Tsutsui H, Kawai T, et al. Generation of IL-18 deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor. J Immunol 1999; 162: 5041. 796. Hoshino T, Kawase Y, Okamoto M, et al. IL-18 transgenic mice: In vivo evidence of a broad role for IL-18 in modulating immune function. J Immunol 2001; 166: 7014–7018. 797. Hoshino T, Wiltrout RH, Young HA. IL-18 is a potent coinducer of IL-13 in NK and T cells: a new potential role for IL-18 in modulating the immune response. J Immunol 1999; 162: 5070–5077. 798. Hosken NA, Shibuya K, Heath AW, Murphy KM, O’Garra A. The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-transgenic model. J Exp Med 1995; 182: 1579. 799. Hosoi T, Wada S, Suzuki S, et al. Bacterial endotoxin induces IL-20 expression in the glial cells. Brain Res Mol Brain Res 2004; 130: 23–29. 800. Hossiau FA, Renauld JC, Stevens M, et al. Human T cell lines and clones respond to IL-9. J Immunol 1993; 150: 2634–2640. 801. Howard M, Hilfiker M, Johnson B, et al. Identification of a T cell derived B cell growth factor distinct from Interleukin-2. J Exp Med 1982; 155: 914–923. 802. Howard M, Muchamual S, Andrade S, Menon S. Interleukin 10 protects mice from lethal endotoxemia. J Exp Med 1993; 177: 1205. 803. Howells G, Pham P, Taylor D, Foxwell B, Feldman M. Interleukin 4 induces interleukin 6 production by endothelial cells: synergy with interferon-γ. Eur J Immunol 1991; 21: 97. 804. Hsieh CS, Macatonia SE, Tripp CS, et al. Development of TH1 CD4+ T cells through IL-12 produced by Listerial-induced macrophages. Science 1993; 260: 547–549. 805. Hsieh MY, Chen WY, Jiang MJ, et al. Interleukin-20 promotes angiogenesis in a direct and indirect manner. Genes Immun 2006; 7: 234–242. 806. Hsu D-H, de Waal Malefyt R, Fiorentino DF, et al. Expression of interleukin 10 activity by Epstein Barr virus protein BCRFI. Science 1990; 250: 830–832. 807. Hsu H, Lacey DL, Dunstan C R, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci USA 1999; 96: 3540. 808. Hsu H, Solovyev I, Colombero A, Elliott G, Kelley JM, Boyle WJ. ATAR, a novel TNF receptor family member, signals through TRAF2 and TRAF5. J Biol Chem 1997; 272: 13471–13474. 809. Hsu YH, Li HH, Hsieh MY, et al. Function of interleukin-20 as a proinflammatory molecule in rheumatoid and experimental arthritis. Arthritis Rheum 2006; 54: 2722–2733. 810. Huang EY, Madireddi MT, Gopalkrishnan RV, et al. Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties. Oncogene 2001; 20: 7051–7063. 811. Huang J, Kerstan KW, Ahmadzadeh M, et al. Modulation by IL-2 of CD70 and CD27 expression on CD8+ T cells: Importance for the therapeutic effectiveness of cell transfer immunotherapy. J Immunol 2006; 176: 7726–7735. 812. Huang S, Mills L, Mian B. Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma. Am J Pathol 2002; 161: 125–134. 813. Huang SK, Xiao H-Q, Kleine-Tebbe J, et al. IL-13 expression at the sites of allergic challenge in patients with asthma. J Immunol 1995; 155: 2688–2694. 814. Huang SM, Lopez CA, Heck DE, et al. Osteopontin inhibits induction of nitric oxide synthase gene expression by inflammation mediators in mouse kidney epithelial cells. J Biol Chem 1994; 2712: 5414. 815. Huber AR, Kunkel SL, Todd RF, Weiss SJ. Regulation of transendothelial neutrophil migration by endogenous interleukin-8. Science 1991; 254: 99.

Cytokines 816. Hudkins KL, Giacelli CM, Eitner F, Couser WG, Johnson RJ, Alpers CE. Osteopontin expression in human cresentic glomerulonephritis. Kid Int 2000; 57: 105–116. 817. Hui W, Bell M, Carrol G. Detection of oncostatin M in synovial fluid from patients with rheumatoid arthritis. Ann Rheu Dis 1997, 56: 184. 818. Huland E, Huland H, Heinzer H. Interleukin-2 by inhalation: Local therapy for metastatic renal cell carcinoma. J Urology 1992; 147: 344–348. 819. Hultner L, Druez C, Moeller J, et al. Mast cell growth enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGF1II. Eur J Immunol 1990; 20: 1413–1416. 820. Hultner L, Kolsch S, Stassen M, et al. In activated mast cells, IL-1 upregulates the production of several Th2 related cytokines including IL-9.J Immunol 2000; 164: 5556–5563. 821. Humphreys NE, Xu D, Hepworth MR, et al. IL-33, a potent inducer of adaptive immunity to intestinal nematodes. I Immunol 2008; 180: 2443–2449. 822. Hunninghake GW, Glazier MM, Monick MM, Dinarello CR. Interleukin-1 is a chemotactic factor for T lymphocytes. Am Rev Respir Dis 1987; 135: 66. 823. Hunte BE, Hudak D, Campbell D, Xu Y, Rennick D. flk2/flt3 ligand is a potent cofactor for the growth of primitive B cell progenitors. J Immunol 1995; 156: 486. 824. Hunter CA, Chizzonite R, Remmington JS. IL-1β is required for IL-12 to induce production of IFNγ by NK cells. J Immunol 1995; 155: 4347–4354. 825. Hurst SD, Muchamuel T, Gorman DM, et al. New IL-17 family members promote Th1 and Th2 responses in the lung: in vivo function of the novel cytokine IL-25. J Immunol 2002; 169: 443–453. 826. Hurst, SM, McLoughlin RM, Monslow J, et al. Secretion of oncostatin M by infiltrating neutrophils: regulation of IL-6 and chemokine expression in mesothelial cells. J Immunol 2002; 169: 5244–5251. 827. Hurtado JC, Kim SH, Pollok KE, Kwon BS. Potential role of 4-1BB in T cell activation: comparison with costimulatory molecule CD28. J Immunol 1995; 155: 3360. 828. Hurtado JC, Kim Y-J, Kwon BS. Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death. J Immunol 1997; 158: 2600–2609. 829. Hurteau JA, Blessing JA, DeCesare SL, Creasman WT. Evaluation of recombinant human interleukin-12 in patients with recurrent or refactory ovarian cancer: a gynecologic cncology group study. Gynecol Oncol 2001; 82: 7–10. 830. Husain SR, Puri RK. Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside. J Neurooncol 2330; 65: 37–48. 831. Hutchins D, Steel CM. Regulation of ICAM-1 (CD54) expression in human breast cancer cell lines by interleukin 6 and fibroblast derived factors. Int J Cancer 1994; 58: 80–84. 832. Hutson TE, Mekhail T, Molto L, et al. Phase I trial of subcutaneously administered rhuIL-12 and rhuIFN-α2b in patients with metastatic renal cell carcinoma or malignant melanoma. Proc ASCO 2001; 20: 1030. 833. Hymowitz SG, Filvaroff EH, Yin JP, et al. IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding. The EMBO Journal 2001; 20: 5332–5341. 834. Iemura A, Tsai M, Ando A, Wershil BK, Galli SJ. The c-kit ligand, stem cell factor, promotes mast cell survival by suppressing apoptosis. Am J Pathol 1994, 144: 321. 835. Ihle JN, Keller J, Oroszlan S, et al. Biological properties of interleukin-3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, P cell-stimulating activity, colonystimulating activity, and histamine producing cell-stimulating factor activity. J Immunol 1983; 131: 282.

Walter M. Lewko and Robert K. Oldham 836. Ihn H, LeRoy EC, Trojanowska M. Oncostatin M stimulates transcription of the human α2(I) collagen gene via the Sp1/Sp3binding site. J Biol Chem 1997; 272: 24666. 837. Ihn H, Tamaki K. Oncostatin M stimulates the growth of dermal fibroblasts via a mitogen-activated protein kinase-dependent pathway. J Immunol 2000; 165: 2149–2155. 838. Iho S, Shau HY, Golub SH. Characteristics of Interleukin 6 enhanced lymphokine activated killer cell function. Cell Immunol 1991; 135: 66–77. 839. Iikura M, Suto H, Kajiwara N, et al. IL-33 can promote survival, adhesion, and cytokine production in human mast cells. Lab. Invest 2007; 87: 971–978. 840. Iinuma H, Okinaga K, Fukushima R, et al. Superior protection and therapeutic effect of IL-12 and IL-18 gene-transduced dendritic neuroblastoma fusion cells on liver metastasis of murine neuroblastoma. J Immunol 2006, 176: 3461–3469. 841. Iizuka K, Chaplin DD, Wang Y, Wu Q, et al. Requirement for membrane lymphotoxin in natural killer cell development. Proc Natl Acad Sci USA 1999; 96: 6336. 842. Ikebuchi K, Clark SC, Ihle JN, Souza L, Ogawa M. Granulocytecolony stimulating factor enhances interleukin-3 dependent proliferation of multipotential hematopoietic progenitors. Proc Natl Acad Sci USA 1988; 85: 2445. 843. Ikebuchi K, Wong GG, Clark SC, et al. Interleukin-6 enhancement of interleukin-3 dependent proliferation of multipotential hematopoietic progenitors. Proc Natl Acad Sci USA 1987; 84: 9035. 844. Ikeda K, Nakajima H, Suzuki K, et al. Mast cells produce interleukin-25 upon FcεRI-mediated activation. Blood 2003; 101: 3594–3596. 845. Ikejima T, Okusawa S, Ghezzi P, van der Meer JWM, Dinarello CA. IL-1 induces TNF in human PBMC in vitro and a circulating TNF-like activity in rabbits. J Inf Dis 1990; 162: 215. 846. Ikejima T, Okusawa S, van der Meer JW, Dinarello CA. Induction by toxic shock syndrome toxin-1 of a circulating tumor necrosis factor-like substance in rabbits and of immunoreactive tumor necrosis factor and interleukin-1 from human mononuclear cells. J Infect Dis 1988; 158: 1017. 847. Ikeuchi H, Kuroiwa T, Hiramatsu N, et al. Expression of interleukin-22 in rheumatoid arthritis: potential role as a proinflammatory cytokine. Arthritis Rheum 2005; 52: 1037–1046. 848. Im D-H, Barchan D, Maiti PK, Fuchs S, Souroujon MC. Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Th1 differentiation and up-regulation of CTLA-4. J Immunol 2001; 166: 6893–6898. 849. Imai T, Hieshima K, Haskell C, et al. Identification and molecular characterization of fractalkine receptor CX3CR-1, which mediates both leukocyte migration and adhesion. Cell 1997; 91: 521. 850. Imai Y, Nara N, Tohda S, et al. Antiproliferative and differentiative effects of recombinant interleukin-4 on a G-CSF-stimulatingdependent myeloblastic leukemic cell line. Blood 1991; 78: 471–478. 851. Imura A, Hori T, Imada K, et al. The human OX40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells. J Exp Med 1996; 183: 2185–2195. 852. Infante-Duarte C, Horton HF, Byrne MC, Kamradt T. Microbial lipopeptides induce the production of IL-17 in Th Cells. J Immunol 2000; 165: 6107–6115. 853. Inoue M, Kyo S, Fujita M, et al. Coexpression of the c-kit receptor and the stem cell factor in gynecological tumors. Cancer Res 1994; 54: 3049–3053. 854. Inoue S, Shanker M, Miyahara R. MDA-7/IL-24-based cancer gene therapy: translation from the laboratory to the clinic. Curr Gene Ther 2006; 6: 73–91. 855. Ip WK, Wong CK, Li ML, et al. Interleukin-31 induces cytokine and chemokine production from human bronchial epithelial cells through activation of mitogen-activated protein kinase signaling pathways: implications for allergic response. Immunology 2007; 122: 532–541.

237 856. Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997; 388: 190. 857. Iscove NN. Erythropoietin independent stimulation of early erythropoiesis in adult marrow cultures by conditioned medium of lectin stimulated mouse spleen cells. In: Golde D, Kline MJ, Metcalf D, Fox CF, eds. Hematopoietic Cell Differentiation. Academic Press, New York, 1978; 37–52. 858. Ishida T, Kobayashi N, Tojo T, et al. CD40 signaling-mediated induction of Bcl-XL, Cdk4 and Cdk6. J Immunol 1995; 155: 5527. 859. Ishimi Y, Miyaura C, Jin CH, et al. IL-6 is produced by osteoblasts and induces bone resorption. J Immunol 1990; 145: 3297–3303. 860. Ishimitsu R, Nishimura H, Yajima T, et al. Overexpression of interleukin 15 in vivo enhances Tc1 response, which inhibits allergic inflammation in a murine model of asthma. J Immunol 2001; 166: 1991–2001. 861. Ito D, Back TC, Shakov AN, Wiltrout RH, Nedospasov SA. Mice with targeted mutation in lymphotoxin-α exhibit enhanced tumor growth and metastasis: impaired NK development and recruitment. J Immunol 1999; 163: 2809. 862. Ito F, Li O, Shreiner AB, et al. Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendriticbased vaccines. Cancer Res 2004; 64: 8411–8419. 863. Ito H, Kurtz J, Shaffer J, Sykes M. CD4 T cell-mediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40–CD40 ligand interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway. J Immunol 2001; 166: 2970–2981. 864. Ito R, Kitadai Y, Kyo E, et al. Interleukin 1 acts as an autocrine growth stimulator for human gastric carcinoma cells. Cancer Res 1994; 54: 4102–41206. 865. Ito T, Wang YH, Duramad O, et al. OX40 ligand shuts down IL-10-producing regulatory T cells. Proc Natl Acad Sci USA 2006; 103: 13138–13143. 866. Itoh K, Hirohata S. The role of IL-10 in human B cell activation, proliferation and differentiation. J Immunol 1995; 154: 4341–4350. 867. Itoh K, Inoue T, Ito K, Hirohata S. The interplay of IL-10 and IL-2 in humoral immune responses: IL-10 synergizes with IL-2 to enhance responses of human B lymphocytes in a mechanism which is different from up-regulation of CD25 expression. Cell Immunol 1994; 157: 478. 868. Iwagaki H, Hizuta A, Koha H, et al. Circulating levels of soluble CD30 and other markers in colorectal cancer. J Med 1999; 30: 111–121. 869. Iwakura Y, Ishigame H. The IL-23/IL-17 axis of inflammation. J Clin Invest 2006; 116: 1218–1222. 870. Jacob CO, Aiso S, Michie SA, McDevitt HO, Acha-Orbea H. Prevention of diabetes in nonobese diabetic mice by tumor necrosis factor (TNF): similarities between TNF-α and interleukin-1. Proc Natl Acad Sci USA 1990; 87: 968. 871. Jacobsen FW, Veiby OP Jacobsen SE. IL-7 stimulates CSFinduced proliferation of murine bone marrow macrophages and Mac-1+ myeloid progenitors in vitro. J Immunol 1994; 153: 270–276. 872. Jacobsen SEW, Okkenhaug C, Myklebust J, Veiby OP, Lyman SD. The flt3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interaction with interleukin (IL)-11, IL-12, and the other hematopoietic growth factors. J Exp Med 1995; 181: 1357–1363. 873. Jacobson NG, Szabo SJ, Weber-Nordt RM, et al. Interleukin-12 signaling in the T helper Th1 cells involves tyrosine phosphorylation of signal transducer and activator of transcription STAT3 and STAT4. J Exp Med 1995; 181: 1755–1762. 874. Jadus MR, Good RW, Crumpacker DB, Yannelli JR. Effects of Interleukin 4 upon human tumoricidal cells obtained from

238

875. 876.

877.

878.

879.

880. 881. 882.

883.

884. 885. 886. 887. 888. 889.

890. 891. 892.

893.

894.

Cytokines patients bearing solid tumors. J Leukocyte Biol 1991; 49: 139–151. Jadus MR, Parkman R. The selective growth of murine newbornderived suppressor cells and their probable mode of action. J Immunol 1986; 136: 783–792. Jadus MR, Schmunk G, Djeu JY, Parkman RJ. Morphology and lytic mechanism of interleukin-3-dependent natural cytotoxic cells: tumor necrosis factor as a possible mediator. Immunol 1986; 137: 2774–2783. Jain S, Chakraborty G, Kundu GC. The crucial role of cyclooxygenase-2 in osteopontin-induced protein kinase c α/c-src/IκB kinase α/β-dependent prostate tumor progression and angiogenesis. Cancer Res 2006; 66: 6638–6648. Janovic DV, Di Battista JA, Martel-Pellitier J, et al. IL-17 stimulates production and expression of proinflammatory and hematopoietic cytokines, IL-1β, and TNF-α, by human macrophages. J Immunol 1998; 160: 3513. Janovic DV, Martel-Pellitier J, et al. Stimulation of a 92-kd gelatinase (matrix metalloproteinase 9) production by interleukin-17 in human monocyte/macrophages: a possible role in rheumatoid arthritis. Arthritis Rheum 2000; 43: 1134. Jansen PM, de Jong IW, Hart M, et al. Release of Leukemia inhibitory factor in primate sepsis. Analysis of the role of TNF-α. J Immunol 1996; 156: 4401–4407. Jansson M, Panoutsakopoulou V, Baker J, et al. Attenuated experimental autoimmune encephalitis in eta-1/osteopontin-deficient mice. J Immunol 2002; 168: 2096–2099. Jarnicki AG, Lysaght J, Todryk S, et al. Suppression of antitumor immunity by IL-10 and TGF-β-producing T cells infiltrating the growing tumor: influence of tumor environment on the induction of CD4+ and CD8+ regulatory T cells. J Immunol 2006; 177: 896–904. Jarsdorfer B, Blackwell SE, Wolldridge JE, et al. B-chronic lymphocytic leukemia cells and other B cells can produce granzyme B and gain cytotoxic potential after IL-21-based activation. Blood 2006; 108: 2712–2719. Jaye M, Howk R, Burgess W, et al. Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome location. Science 1986; 233: 541. Jelinek DF and Braten JK. Role of IL-12 in human B lymphocyte proliferation and differentiation. J Immunol 1995; 154: 1606. Jelinek DF, Splawski JB, Lipsky PE. The roles of IL-2 and IFNγ in human B cell activation, growth and differentiation. Eur J Immunol 1986; 16: 925. Jenkins MK, Taylor PS, Norton SD, Urdahl KB. CD28 delivers a costimulatory signal involved in antigen specific IL-2 production by human T cells. J Immunol 1991; 147: 2461. Jeremias I, Herr I, Boehler T, Debatin KM. TRAIL/Apo-2 ligand in activated mouse T and B cells. Eur J Immunol 1998; 28: 143. Jiang H, Lin JJ, Su ZZ, et al. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 1995; 11; 2477–2486. Jiang H, Su ZZ, Lin JJ, et al. Melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc Natl Acad Sci USA 1996; 93: 9160–9165. Jin L, Nakao A, Nakayama M, et al. Induction of RANTES by TWEAK/Fn14 interaction in human keratinocytes. J Invest Dermatol 2004; 122: 1175–1179. Jin L, Raymond DP, Crabtree TD, Pelletier SJ, Houlgrave CW, Pruett TL, Sawyer RG. Enhanced murine macrophage TNF receptor shedding by cytosine guanine sequences in oligodeoxynucleotides. J Immunol 2000; 165: 5153–5160. Jinquan T, Deleuran B, Gesser B, et al. Regulation of human T lymphocyte chemotaxis in vitro by T cell derived cytokines IL-2, IFN-λ, IL-4, IL-10, and IL-13. J Immunol 1995; 154: 3742–3752. Jinquan T, Jing C, Jacobi HH, et al. CXCR3 expression and activation of eosinophils: role of IFN-γ-inducible protein-10 and monokine-induced by IFN-γ. J Immunol 2000; 165: 1548–1566.

895. Jinquan T, Larsen CG, Gesser B, Matsushima K, ThestrupPedersen K. Human IL-10 is a chemoattractant for CD8+ T lymphocytes and an inhibitor of IL-8-induced CD4+ T lymphocyte migration. J Immunol 1993; 151: 4545–4551. 896. Jo M, Kim T-M, Soel D-W, Esplen J, et al. Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand. Nat Med 2000; 6: 564. 897. Johnson CS, Chang MJ, Braunschweiger PG, Furmanski P. Acute hemorrhagic necrosis of tumors induced by interleukin-1α: effects independent of tumor necrosis factor. J Natl Cancer Inst 1991; 83: 842–848. 898. Johnson CS, Keckler DJ, Topper MI, Braunschweiger PG, Furmanski P. In vivo hematopoietic effects of recombinant IL-1α in mice: stimulation of granulocytic, monocytic, megakaryocytic and early erythroid progenitors, suppression of late stage erythropoiesis and reversal of erythroid suppression with erythropoietin. Blood 1989; 73: 678. 899. Jones SE, Khandelwal P, McIntyre K, et al. Randomized, double blind, placebo controlled trial to evaluate the hematopoietic growth factor PIXY321 after moderate-dose fluorouracil, doxorubicin, and cyclophosphamide in stage II and III breast cancer. J Clin Oncol 1999; 17: 3025–3032. 900. Joosten LA, Netea MG, Kim SH, et al. IL-32, a proinflammatory cytokine in rheumatoid arthritis. Proc Natl Acad Sci USA 2006; 103: 3298–3303. 901. Joosten LAB, Helen MMA, Van de Loo FAJ, Van den Berg WB. Anti-cytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti-TNFα, anti-IL-1 α/β, and IL-1Ra. Arthritis Rheum 1996; 39: 797. 902. Jordan WJ, Eskdale J, Lemmon GP, et al. Human IL-19 regulates immunity through auto-induction of IL-19 and production of IL-10. Eur J Immunol 2005, 35: 1576–1582. 903. Josien R, Wong BR, Li HL, Steinman RM, Choi Y. TRANCE, a TNF family member, is differentially expressed on T cell subsets and induced cytokine production in dendritic cells. J Immunol 1999; 162: 2562. 904. Josimovic-Alasevic O, Durkop H, Schwarting R, et al. Ki-1 (CD30) antigen is released by Ki-1 positive tumor cells in vitro and in vivo. I. Partial characterization of soluble Ki-1 antigen and detection of the antigen in cell culture supernatants and in serum by an enzyme linked immunosorbent assay. Eur J Immunol 1989; 19: 157. 905. Joyce DA, Gibbons DP, Green P, Steer JH, Feldman M, Brennan FM. Two inhibitors of cytokine release, interleukin-10 and interleukin-4, have contrasting effects on release of soluble p75 tumor necrosis factor receptor by cultured monocytes. Eur J Immunol 1994; 24: 2699. 906. Ju SK, Lee SC, Kwon TH, et al. Immunity to melanoma mediated by 4-1BB is associated with enhanced activity of tumor infiltrating lymphocytes. Immunol Cell Biol 2005; 83: 344–351. 907. Jung HC, Eckmann L, Yang SK, et al. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 1995; 95: 55–65. 908. Jung T, Widjdeness J, Neumann C, de Vries JE, Yssl H. Interleukin-13 is produced by activated CD45RA+ and CD45RO+ T cells: modulation by interleukin-4 and interleukin-12. E J Immunol 1996; 26: 571. 909. Junn E, Lee Ju HR, Han SH, et al. Requirement for hydrogen peroxide generation in TGF-β1 signal transduction in human lung fibroblast cells: Involvement of hydrogen peroxide and Ca+ 2 in TGF-β1-induced IL-6 expression. J Immunol 2000; 165: 2190–2197. 910. Kaashoek JG, Mout JR, Falkenburg JHF, et al. Cytokine production by the bladder carcinoma cell line 5637. Lymphokine Cytokine Res 1991; 10: 231. 911. Kalinski P, Smits HH, Schuitemaker JHN, et al. IL-4 is a mediator of IL-12p70 induction by human Th2 cells: reversal of polarized Th2 phenotype by dendritic cells. J Immunol 2000; 165: 1877–1881.

Walter M. Lewko and Robert K. Oldham 912. Kamanaka M, Yu P, Yasui T, et al. Protective role of CD40 in Leishmania major infected at two distinct phases of cell mediated immunity. Immunity 1996; 4: 275. 913. Kaneda T, Nojima T, Nakagawa M, et al. Endogenous production of TGF-β is essential for osteoclastogenesis induced by a combination of receptor activator of NF-κB ligand and macrophagecolony stimulating factor. J Immunol 2000; 165: 4254–4263. 914. Kaneko H, Hori T, Yamagita S, et al. Introduction of OX40 ligand into lymphoma cells elicits anti-lymphoma immunity in vivo. Exp Hematol 2005; 33: 336–343. 915. Kang CM, Jang AS, Ahn MH, et al. Interleukin-25 and interleukin-13 production by alveolar macrophages in response to particles. Am J Respir Cell Mol Biol 2005; 33: 290–296. 916. Kang SM, Le D, Springer ML, Stock PG, Blau HM. Immune response and myoblasts that express Fas ligand. Science 1997; 278: 1322. 917. Kang WK, Park C, Yoon HL, et al. Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study. Hum Gene Ther 2001; 10: 671–684. 918. Kang Y, He W, Tully S. Breast cancer bone metastasis mediated by the Smad pathway. Proc Natl Acad Sci USA 2005; 102: 13909–13914. 919. Kasaian MT, Whitters MJ, Carter LL, et al. IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition form innate to adaptive immunity. Immunity 2002; 16: 559. 920. Kasama T, Strieter RM, Lukacs NW, Burdick MD, Kunkel SL. Regulation of neutrophil-derived chemokine expression by IL-10. J Immunol 994; 152: 3559. 921. Kaser A, Dunzendorfer S, Offner FA, et al. A role of IL-16 in the cross-talk between dendritic and T cells. J Immunol 1999; 163: 3232–3238. 922. Kaser A, Dunzendorfer S, Offner FA, et al. B-lymphocytederived IL-16 attracts dendritic cells and Th cells. J Immunol 2000; 165: 2474–2480. 923. Kaspenberg ML, Wierenga EA, Bos JD, Jansen HM. Functional subsets of allergen-reactive human CD4+ T cells. Immunol Today 1991; 12: 392. 924. Katayama N, Clark SC, Ogawa M. Growth factor requirement for survival in cell-cycle dormancy of primitive murine lymphohematopoietic progenitors. Blood 1993; 81: 610. 925. Katsura K, Park M, Gatanaga M, et al. Identification of the proteolytic enzyme which cleaves human p75 TNF receptor in vitro. Biochem Biophys Res Commun 1996; 222: 298–302. 926. Katz A, Shulman LM, Porgador A, et al. Abrogation of B16 melanoma metastases by long term low dose interleukin 6 therapy. J Immunother Emphasis Tumor Immunol 1993; 13: 98–109. 927. Katz A, Shulman LM, Revel M, et al. Combined therapy with IL-6 and inactivated tumor cell suppressed metastasis in mice bearing 3LL lung carcinomas. Int J Cancer 1993; 53: 812–818. 928. Kaufmann Y, Armiglio N, Rosenthal E, et al. Proliferation response of leukemic cells to CD70 ligation oscillates with recurrent remission and relapse in a low-grade lymphoma. J Immunol 2005; 175: 6940–6947. 929. Kawaguchi M, Kokubu F, Odaka M, et al. Induction of granulocyte-macrophage colony stimulating factor by a new cytokine, ML-1 (IL-17F), via Raf I-MEK-ERK pathway. J Allergy Clin Immunol 2004; 114: 444–450. 930. Kawaguchi M, Onuchic LF, Li X-D, et al. Identification of a novel cytokine, ML-1, and its expression in subjects with asthma. J Immunol 2001; 167: 4430–4435. 931. Kawaguchi M, Takahashi D, Hizawa N, et al. IL-17F sequence variant (His161Arg) is associated with protection against asthma and antagonizes wild-type IL-17 activity. J Allergy Clin Immunol 2006: 117: 795–801. 932. Kawakami Y, Custer M, Rosenberg SA, Lotze M. Interleukin-4 regulates Interleukin-2 induction of lymphokine activated killer activity from human leukocytes. J Immunol 1989; 142: 3452. 933. Kawakami Y, Haas, GP, Lotze MT. Expansion of tumor infiltrating lymphocytes from human tumors using the T cell growth fac-

239

934. 935.

936. 937. 938. 939.

940.

941. 942. 943.

944. 945. 946. 947.

948.

949. 950. 951. 952. 953. 954.

tors interleukin-2 and interleukin-4. J Immunotherapy 1993; 14; 336–347. Kawakami Y, Rosenberg SA, Lotze MT. Interleukin 4 promotes the growth of tumor infiltrating lymphocytes cytotoxic for human autologous melanoma. J Exp Med 1988; 168: 2183–2191. Kawano M, Matsushima K, Masuda A, et al. A major 50 kDa human B cell growth factor II induces both Tac antigen expression and proliferation by several lymphocytes. Cell Immunol 1988; 111: 273–286. Kawasakura S, Lowenstein L. A factor stimulating DNA synthesis derived from the medium of leucocyte cultures. Nature 1965; 208: 794–795. Kawashima I, Ohsumi J, Mita-Honjo K, et al. Molecular cloning of the cDNA encoding adipogenesis inhibitory factor and identity with interleukin 11. FEBS Lett 1991; 283: 199–202. Kay AB. Inflammatory blood cells in bronchial asthma. J Asthma 1989; 26: 335. Kayagaki N, Yamiguchi N, Nakayama M, et al. Type I interferons (IFNs) regulate tumor necrosis factor related apoptosis-inducing ligand (TRAIL) expression on human T cells: a novel mechanism for the antitumor effects of type I interferons. J Exp Med 1999; 189: 1451. Kaye J, Gillis S, Mizel SB, et al. Growth of a cloned helper T cell line induced by a monoclonal antibody specific for the antigen receptor: interleukin 1 is required for the expression of receptors for interleukin 2. J Immunol 1984; 133: 1339–1345. Keane J, Nicoll J, Kim S, et al. Conservation of structure and function between human and murine IL-16. J Immunol 1998; 160: 5945–5954. Keates AC, Castagliuolo I, Cruickshank WW, et al. Interleukin-16 is upregulated in Crohn’s disease and participates in TNBS colitis in mice. Gastroenterology 2000; 119: 972–982. Keegan AD, Johnson JA, Tortolani PJ, et al. Similarities and differences in signal transduction by interleukin-4 and interleukin-13: analysis of Janus kinase activation. Proc Natl Acad Sci USA 1995; 92: 7681–7685. Keegan AD, Pierce JH. The interleukin-4 receptor: signal transduction by a hematopoietic receptor. J Leuk Biol 1994; 5: 272–279. Kehri JH, Dukovich M, Whalen G, et al. Novel interleukin 2 receptor appears to mediate IL-2-induced activation of natural killer cells. J Clin Invest 1988; 81: 779. Kehry MR. CD40-mediated signaling in B cells: balancing cell survival, growth and death. J Immunol 1996; 156: 2345–2348. Kelleher K, Bean K, Clark SC, et al. Human intertleukin-9: genomic sequence, chromosomal location, sequences essential for its expression in human T-cell leukemia virus HTLV-1 transformed human T cells. Blood 1991; 77: 1436–1441. Kellokumpu-Lehtinen P, Talpaz M, et al. Leukemia inhibitory factor stimulates breast, kidney and prostate cancer cell proliferation by paracrine and autocrine pathways. Int J Cancer 1996; 66: 515–519. Kelner GS, Kennedy SJ, Bacon KB, et al. Lymphotactin: a cytokine that represents a new class of chemokine. Science 1994; 266: 1395. Kemeny L, Ruzicka T, Dobozy A, Michel G. Role of Interleukin 8 receptor in skin. International Arch Allergy and Immunology 1994; 104: 317–322. Kennedy J, Rossi DL, Zuraski SM, et al. Mouse IL-17: a cytokine preferentially expressed by αβTCR+ CD4−CD8− T cells. J Interferon Cyto Res 1996; 16: 611–617. Kennedy MK, Mohler KM, Shanebeck KD, et al. Induction of B cell costimulatory function of recombinant murine CD40 ligand. E J Immunol 1994, 24: 116. Kenyon BM, Browne F, D’Amato RJ. Effects of thalidomide and related metabolites on a mouse corneal model for neovascularization. Exp Eye Res 1997; 64: 971–978. Kern PA Saghizadeh M, Ong JM, et al. The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss and relationship to lipoprotein lipase. J Clin Invest 1995; 95: 2111–2119.

240 955. Khan SY, Kelher MR, Heal JM, et al. Soluble CD40L accumulates in stored blood components, primes neutrophils through CD40, and is a potential cofactor in the development of transfusion-related acute lung injury. Blood 2006; 108: 2455–2562. 956. Khapli SM, Mangashetti LS, Yogesha SD, et al. IL-3 acts directly on osteoclast precursors and irreversibly receptor activation of NF-κB ligand-induced osteoclast differentiation by diverting the cells to macrophage lineage. J Immunol 2003; 171: 142–151. 957. Khatri VP, Caligiuri MA. A review of the association between interleukin-10 and human B-cell malignancies. Cancer Immunol Immunother 1998; 46: 239–244. 958. Kieth JC, Albert L, Sonis ST, Pfieffer CJ, Schaub RG. IL-11, a plieotropic cytokine: exciting new effects on gastrointestinal mucosal biology. Stem Cells 1994; 12: (Suppl 1): 79–89. 959. Kilian PL, Kaffka KL, Biondi KA, et al. Antiproliferative effect of interleukin-1 on human ovarian carcinoma cell line (NIH: OVCAR-3). Cancer Res 1991; 51: 1823–1828. 960. Kilinc MO, Aulakh KS, Nair RE, et al. Reversing tumor immune suppression with intratumoral IL-12: Activation of memory associated T effector/memory cells, induction of T suppressor apoptosis, and infiltration of CD8+ T effectors. J Immunol 2006; 177: 6962–6973. 961. Kim J, Sanders SP, Sierkierski ES, Casolaro V, Proud D. Role of NF-κB in cytokine production induced from human airway epithelial cells by rhinovirus infection. J Im-munol 2000; 165: 3384–3392. 962. Kim KE, Song H, Kim TS, et al. Interleukin-18 is a critical factor for vascular endothelial growth factor-induced migration in gastric cancer cell lines. Oncogene 2006; 9: 1480–1486. 963. Kim S-H, Eisenstein M, Resnikov L, et al. Structural requirements of six naturally occurring isoforms of the interleukin 18 binding protein to inhibit interleukin 18. Proc Natl Acad Sci USA 2000; 97: 1190. 964. Kim SH, Han SY, Azam T, et al. Interleukin-32: a cytokine and inducer of TNFα. Immunity 2005; 22: 131–142. 965. Kim SH, Reznikov LL, Stuyt RJL, et al. Functional reconstitution and regulation of IL-18 activity by the IL-18Rβ Chain. J Immunol 2001; 166: 148–154. 966. Kim SW, Hayashi M, Lo JF, et al. Tid 1 negatively regulates the migratory potential of cancer cells by inhibiting the production of interleukin-8. Cancer Res 2005; 65: 8784–8791. 967. Kim SW, Kang YJ, Kim WJ, et al. TWEAK can induce proinflammatory cytokines and matrix metalloproteinase-9 in macrophages. Circ J 2004; 68: 396–399. 968. Kimura Y, Takeshita T, Kondo M, et al. Sharing of the IL-2 receptor γ chain with the functional IL-9 receptor complex. Int Immunol 1995; 7: 115–120. 969. Kinashi T, Harada N, Severinson E, et al. Cloning of complementary DNA encoding T-cell replacing factor and the identity of B-cell growth factor II. Nature 1986; 324: 70–73. 970. Kincaid PW, Medina KL, Smithson G. Sex hormones are negative regulators of lymphopoiesis. Immunol Rev 1994; 137: 119. 971. Kirkwood JM, Wilson J, Whiteside TL, Donnelly S, Herberman RB. Phase IB trial of picibanil (OK-432) as an immunomodulator in patients with resected high risk melanoma. Cancer Immunol Immunother 1997; 44: 137–149. 972. Kirman I, Nielsen OH. Increased numbers of interleukin-15-expressing cells in active ulcerative colitis. Am J Gastroenterology 1996; 91: 1789. 973. Kirnbauer R, Charvat B, Schauer E, et al. Modulation of intracellular adhesion molecule 1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7 TNFβ, and UVB light. J Invest Dermat 1992; 98: 320–326. 974. Kishimoto T, Akira S, Taga T. Interleukin 6 and its receptor: a paradigm for cytokines. Science 1992; 258: 593–597. 975. Kishimoto T, Hirano T. Molecular regulation of B lymphocyte response. Ann Rev Immunol 1988; 6: 485–512. 976. Kitadai Y, Haruma K, Sumii K, et al. Expression of interleukin-8 correlates with vascularity in human gastric carcinomas. Am J Pathol 1998; 152: 93.

Cytokines 977. Kitamura T, Sato N, Arai K, Miyajima A. Expression cloning of the human IL-3 receptor cDNA reveals a shared β subunit for human IL-3, and GM-CSF receptors. Cell 1991; 66: 1165–1174. 978. Kitamura Y, Go S, Hatanaka K. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood 1978; 52: 447. 979. Kitamura Y, Go S. Decreased production of mast cells in S1/S1d anemic mice. Blood 1979; 53: 492. 980. Kitazawa H, Muegge K, Badolato R, et al. IL-7 activates α4β1 integrin in murine lymphocytes. J Immunol 1997; 159: 2259–2264. 981. Kituchi T, Joki T, Saitoh S, et al. Antitumor activity of interleukin-2-producing tumor cells and recombinant interleukin-12 against mouse glioma cells located in the central nervous system. Int J Cancer 1999; 425–430. 982.Kivisakk P, Matusevicius B, He B, et al. IL-15 mRNA expression is up-regulated in blood and cerebrospinal fluid mononuclear cells in multiple sclerosis (MS). Clin Exp Immunol 1998; 111: 193. 983. Klebanoff SJ, Vadas MA, Harlam JM, et al. Stimulation of neutrophils by tumor necrosis factor. J Immunol 1986; 136: 4220. 984. Klein U, Rajewsky K, Kuppers R. Human immunoglobulin (Ig) M+ IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J Exp Med 1998; 188: 1679. 985. Kleinschek MA, Owyang AM, Joyce-Shaikh B, et al. IL-25 regulates Th17 function in autoimmune inflammation. J Exp Med 2007; 204: 161–170. 986. Klimiuk PA, Goronzy JJ, Weyland CM. IL-16 as an anti-inflammatory cytokine in rheumatoid arthritis. J Immunol 1999; 162: 4293–4299. 987. Klouche M, Bhakdi S, Hemmes M, Rose-John S. Novel path of activation of primary smooth muscle cells: up-regulation of gp130 creates an autocrine activation loop by IL-6 and its soluble receptor. J Immunol 1999; 163: 4583. 988. Knappe A, Hor S, Wittmann S, Flickenscher H. Induction of a normal cellular homolog of IL-10, AK155, by transformation of T lymphocytes with herpesvirus saimiri. J Virol 2000; 74: 3881–3887. 989. Knapper S, Burnett AK, Littlewood T, et al. A phase 2 trial of the flt3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood 2006; 108: 3262–3270. 990. Kneitz B, Herrmann T, Yonehara S, Schimpl A. Normal clonal expansion but impaired Fas-mediated cell death and anergy induction in interleukin-2 deficient mice. Eur J Immunol 1995; 25: 2572. 991. Knopf M, Baumann H, Freer G, et al. Impaired immune and acute phase responses in interleukin-6-deficient mice. Nature 1994; 368: 339–342. 992. Kobata T, Agematsu K, Kameoka J, Schlossman SF, Morimoto C. CD27 is a signal transducing molecule involved in CD45RA+ naive T cell costimulation. J Immunol 1994; 153: 5422. 993. Kobayashi M, Fitz L, Ryan M, et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biological effects on human lymphocytes. J Exp Med 1989; 170: 827–845. 994. Koch AE, Polverini PK, Kunkel SL, et al. Interleukin 8 as a macrophage-derived mediator of angiogenesis. Science 1992; 258: 1798–1801. 995. Kodama T, Matsuyama T, Kuribashi K, et al. IL-18 deficiency selectively enhances allergy induced eosinophilia in mice. J Allergy Clin Immunol 2000; 105: 45. 996. Kohase M, May LM, Tamm I, Vilcek J, Sehgal PB. A cytokine network in human diploid fibroblasts: interactions of β interferons, tumor necrosis factor, platelet-derived growth factor and interleukin 1. Mol Cell Biol 1987; 7: 273. 997. Kohn DB, Weinberg KI, Nolta JA, et al. Engraftment of genemodified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat Med 1995; 1: 1017.

Walter M. Lewko and Robert K. Oldham 998. Kohno K, Kataoka T, Ohtsuki Y, et al. IFN-γ-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J Immunol 1997; 158: 1541. 999. Koi A. The role of interleukin 6 as the hepatocyte-stimulating factor in the network of inflammatory cytokines. Ann NY Acad Sci 1989; 557: 1–8. 1000. Kojima H, Takeuchi M, Ohta T, et al. Interleukin-18 activates the IRAK-TRAF6 pathway in mouse EL-4 cells. Biochem Biophys Res Communic 1998; 244: 183. 1001. Komschlies KL, Back TT, Gregorio TA, et al. Effects of rhIL-7 on leukocyte subsets in mice: implications for antitumor activity. Immunology 1994; 61: 95–104. 1002. Kondo M, Takeshita T, Higuchi M, et al. Functional participation of the IL-2 receptor γ chain in IL-7 receptor complexes. Science 1994; 263: 1453–1454. 1003. Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphnode development and lymph node organogenesis. Nature 1999; 397: 315. 1004. Koni PA, Sacca R, Lawton P, et al. Distinct roles of lymphoid organogenesis for lymphotoxins α and β revealed in lymphotoxin β-deficient mice. Immunity 1997; 6: 491. 1005. Koo AS, Armstrong C, Bochner B, et al. Interleukin-6 and renal cell cancer: production, regulation, and growth effects. Cancer Immunol Immunother 1992; 35: 97–105. 1006. Kopf M, Ruedl C, Schmitz M, et al. OX40-deficient mice are defective in Th cell proliferation but are competent in generating B cell and CTL responses after virus infection. Immunity 1999; 11: 699. 1007. Kopp WC, Urba WJ, Rager HC, et al. Induction of IL-1R antagonist after interleukin 1 therapy in patients with cancer. Clin Cancer Res 1996; 2: 501–506. 1008. Korherr C, Hofmeister R, Wesche H, Falk W. A critical role for the interleukin-1 receptor accessory protein in interleukin-1 signaling. Eur J Immunol 1997; 27: 262. 1009. Korzus E, Nagase H, Rydell R, Travis J. The mitogen-activated protein kinase and JAK-STAT signaling pathways are required for an oncostatin M-responsive element-mediated activation of matrix metalloproteinase 1 gene expression. J Biol Chem 1997; 272: 1188. 1010. Kosugi H, Nakagawa Y, Hotta T, et al. Structure of the gene encoding the α subunit of the human interleukin-3 receptor. Biochem Biophys Res Commun 1995; 208: 360. 1011. Kotake S, Udagawa N, Takahashi N, et al. IL-17 in synovial fluid from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 1999; 103: 1345. 1012. Kotenko SV, Izotova LS, et al. Identification of the functional IL-TIF (IL-22) receptor complexes: the IL-10R2 chain (IL-10Rβ) is a shared component of both IL-10 and IL-TIF (IL-22) receptor complexes. J Biol Chem 2000; 276: 2725. 1013. Kotenko SV, Izotova LS, Mirochnitchenko OV, et al. Identification, cloning and characterization of a novel soluble receptor that binds IL-22 and neutralizes its activity. J Immunol 2001; 166: 7096–7103. 1014. Kotenko SV, Krause CD, et al. Identification and functional characterization of a second chain of the interleukin-10 receptor complex. EMBO J 1997; 16: 5894. 1015. Kotowicz K, Callard RE. Human immunoglobulin class and IgG subclass regulation: dual action of interleukin 4. Eur J Immunol 1993; 23: 2250. 1016. Kovacs EJ, Beckner SK, Longo DL, et al. Cytokine gene expression during the generation of human lymphokine-

241

1017.

1018.

1019. 1020.

1021. 1022.

1023. 1024.

1025.

1026.

1027.

1028.

1029.

1030.

1031.

1032. 1033.

1034.

1035.

1036.

activated killer cells: early induction of interleukin 1β by interleukin 2. Cancer Res 1989; 49: 940. Kovacs JA, Vogel S, Albert JM, et al. Controlled trial of interleukin-2 infusions in patients infected with the human immunodeficiency virus. N Eng J Med 1996; 335: 1350–1356. Kradin RL, Kurnick JT, Lazarus DS, et al. Tumor-infiltrating lymphocytes and interleukin-2 in treatment of advanced cancer. Lancet 1989; 1: 577–580. Kramer PH. CD90 (Apo/Fas)-mediated apoptosis: live and let die. Adv Immunol 1999; 71: 163. Kratz A, Campos-Neto A, Hanson MS, Ruddle NH. Chronic inflammation caused by lymphotoxin is lymphoid neogenesis. J Exp Med 1996; 183: 1461. Krautwald S. IL-16 activates the SPAK signaling pathway in CD4+ macrophages. J Immunol 1998; 160: 5874–5879. Krenger W, Snyder K, Smith S, Ferrara JL. Effects of exogenous IL-10 in a murine model of graft-versus-host disease to minor histocompatibility antigens. Transplantation 1994; 58: 1251. Kreuger J, Ray A, Tamm I, et al. Expression and function of IL-6 in epithelial cells. J Cell Biol 1991; 45: 327. Kroncke R, Loppnow H, Flad HD, Gerdes J. Human follicular dendritic cells and vascular cells produce interleukin 7: a potential role for interleukin-7 in the germinal center reaction. Eur J Immunol 1996; 26: 2541–2544. Kryczek I, Wei S, Zou L, et al. Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment. J Immunol 2007; 178: 6730–6733. Ku C, Murakami M, Sakamoto A, Kappler J, Marrack P. Control of homeostasis of CD8+ memory T cells by opposing cytokines. Science 2000; 288: 675. Ku H, Yonemura Y, Kaushansky K, Ogawa M. Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice. Blood 1996; 87: 4544. Kubin M, Kamoun M, Trinchieri G. Interleukin 12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells. J Exp Med 1994; 180: 211–222. Kudo-Sato C, Hodge JW, Kwak H, et al. 4-1BB ligand enhances tumor specific immunity of poxvirus vaccines. Vaccine 2006; 24: 4975–4986. Kuestner RE, Taft DW, Haran A, et al. Identification of the IL-17 receptor related molecule IL-17RC as a receptor for IL-17F. J Immunol 2007; 179: 5462–5473. Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W. Interleukin-10 deficient mice develop chronic enterocolitis. Cell 1993; 75: 263–274. Kuhn R, Rajewski K, Muller W. Generation and analysis of Interleukin-4 deficient mice. Science 1991; 254: 707–709. Kumano K, Nakao A, Nakajima H, et al. Interleukin-18 enhances antigen induced eosinophilia recruitment into the mouse airways. Am J Respir Care Med 1999; 160: 873. Kumanogoh A, Wang X, Lee I, et al. Increased T-cell autoreactivity in the absence of CD40–CD40 ligand interactions: A role of CD40 in regulatory T cell development. J Immunol 2001; 166: 353–360. Kumaraguru U, Davis IA, Deshpande S, Tevethia SS, Rouse BT. Lymphotoxin α−/−mice develop functionally impaired CD8+ T cell responses and fail to contain virus infection of the central nervous system. J Immunol 2001; 166: 1066–1077. Kupper T, Horowitz M, Lee F, et al. Autocrine growth of T cells independent of growth factor for a cloned antigen specific helper T cell. J Immunol 1987; 138: 4280.

242 1037. Kurek B, Austin L, Cheema SS, Bartlett PF, Murphy M. Upregulation of leukemia inhibitory factor and interleukin-6 in transected sciatic nerve and muscle following-denervation. Neuromusc Disord 1996; 6: 105. 1038. Kurschner C and Yusaki M. Neuronal interleukin-16: A dual function PZP domain protein. J Neuroscience 1999; 19: 7770–7780. 1039. Kurt-Jones EA, Beller DI, Mizel SB, Unanui ER. Identification of a membrane-associated interleukin 1. Proc Natl Acad Sci (USA) 1985; 82: 1204–1208. 1040. Kurt-Jones EA, Hamburg S, Ohara J, et al. Heterogeneity of helper/inducer T lymphocytes. J Exp Med 1987; 166: 1774–1787. 1041. Kurts C, Carbone FR, Krummel MF, et al. Signaling through CD30 protects against autoimmune diabetes mediated by CD8 T cells. Nature 1999; 398: 341. 1042. Kurzrock RZ, Estrov M, Wetzler M, Gutterman JU, Talpaz M. LIF: not just a leukemia inhibitory factor. Endocrinol Rev 1991; 12: 208. 1043. Kuwana M, Medsger TA, Wright TM. Analysis of soluble and cell surface factors regulating anti-DNA topoisomerase I autoantibody production demonstrates synergy between Th1 and Th2 autoreactive cells. J Immunol 2000; 164: 6138–6146. 1044. Kwon BS, Tan KB, Ni J, Lee NO, et al. A newly identified member of the tumor necrosis factor superfamily with a wide tissue distribution and involvement in lymphocyte activation. J Biol Chem 1997; 272: 14272. 1045. Kwon BS, Weissman SM. cDNA sequences of two inducible T-cell genes. Proc Natl Acad Sci USA 1989; 86: 1993–1993. 1046. Laan M, Cui Z-H, Hoshino H, et al. Neutrophil recruitment by human IL-17 via C-X-C chemokine release in the airways. J Immunol 1999; 162: 2347–2352. 1047. Labarge S, Cruikshank WW, Beer DJ, Center DM. Secretion of IL-16 (Lymphocyte chemoattractant factor) from serotoninstimulated CD8+ T cells in vitro. J Immunol 1996; 156: 310–315. 1048. Labarge S, Cruikshank WW, Kornfeld H, Center DM. Histamine induced secretion of lymphocyte chemoattractant factor from CD8+ T cells is independent of transcription and translation: evidence for constitutive protein synthesis and storage. J Immunol 1995; 155: 2902–2910. 1049. Laberge S, Pinsonneault S, Varga EM, et al. Increased expression of IL-16 immunoreactivity in bronchial mucosa after segmental allergen challenge in patients with asthma. J Allergy Clin Immunol 2000; 106: 293–301. 1050. Lacey DL, Tims E, Tan H-L, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93: 165. 1051. Lachman LB, Dinarello CA, Llanska ND, Fidler IJ. Natural and recombinant human interleukin-1β is cytotoxic for human melanoma cells. J Immunol 1986; 136: 3098–3103. 1052. Lacraz S, Nicod LP, Chicheportiche R, Welgus HG, Dayer JM. IL-10 inhibits metalloproteinase and stimulates TIMP-1 production in human mononuclear phagocytes. J Clin Invest 1995; 96: 2304. 1053. Lahn M, Kalaharadi H, Mittlestadt P, et al. Early preferential stimulation of γδ T cells by TNF-α. J Immunol 1998; 160: 5221–5230. 1054. Laky K, Lefrancois L, Puddington L. Age-dependent intestinal lymphoproliferative disorder due to stem cell factor receptor deficiency: Parameters in small and large intestine. J Immunol 1997; 158: 1417–1427.

Cytokines 1055. Lane P. Brocker T, Hubele S, et al. Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand to signal to B cells in T cell-dependent activation. J Exp Med 1993; 177: 1209. 1056. Lang D, Knop J, Wesche H, et al. The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness. J Immunol 1998; 161: 6871. 1057. Langowski JL, Zhang X, Wu L, et al. IL-23 promotes tumor incidence and growth. Nature 2006; 442: 461–465. 1058. Langrish CL, Chen Y, Blumenschein WM, et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005; 210: 233–240. 1059. Langstein J, Michel J, Fritsche J, et al. CD137 (ILA/4-1BB), a member of the TNF receptor family, induces monocyte activation via bidirectional signaling. J Immunol 1998; 160: 2488–2494. 1060. Lantz M, Gullberg U, Nilsson E, Olsson I. Characterization in vitro of a human tumor necrosis factor binding protein, a soluble form of the tumor necrosis factor receptor. J Clin Invest 1990; 86: 1396. 1061. Lanz CS, Boesiger J, Song CH, et al. Role of interleukin-3 in mast cell and basophil development and in immunity to parasites. Nature 1998; 392: 90. 1062. Larmann K, Hughes CCW, Schechner J, Fanslow WC, Pober JS. CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression. Proc Natl Acad Sci USA 1995; 92: 4342. 1063. Larsen CG, Anderson AO, Oppenheim JJ, Matsushima K. Production of Interleukin 8 by human dermal fibroblasts and keratinocytes in response to interleukin 1 or tumor necrosis factor. Immunology 1989; 68: 31. 1064. Larsen CP, Alexander DZ, Hollenbaugh D, et al. CD40–gp39 interactions play a critical role during allograft rejection. Transplantation 1996; 61: 4. 1065. Lasfar A, Lewis-Antes A, Smirnov SV, et al. Characterization of the mouse interferon-λ Ligand–receptor system: IFN-λs exhibit antitumor activity against B16 melanoma. Cancer Res 2006; 66: 4468–4477. 1066. Lauw FN, Pajkrt D, Hack CE, et al. Proinflammatory effect of IL-10 during human endotoxemia. J Immunol 2000; 165: 2783–2789. 1067. Le PT, Lazorick S, Whichard LP, et al. Human thymic epithelial cells produce IL-6, granulocyte-monocyte-CSF and leukemia inhibitory factor. J Immunol 1990; 145: 3310. 1068. Leali D, Dell’Era P, Stabile H, et al. Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis. J Immunol 2003; 171: 1085–1093. 1069. Lean JM, Fuller K, Chambers TJ. FLT3 ligand can substitute for macrophage-colony stimulating factor in support of osteoclast differentiation and function. Blood 2001; 98: 2707–2713. 1070. Lebedeva IV, Sauane M, Gopalkrishnan RV, et al. mda-7/IL-24: exploiting cancer’s Achilles’ heal. Mol Ther 2005; 11: 4–18. 1071. Lebedeva IV, Su ZZ, Chang Y, et al. The cancer suppressor gene mda-7 induces apoptosis in human melanoma cells. Oncogene 2002; 21: 708–718. 1072. Lechan RM, Toni R, Clark BC, et al. Immunoreactive interleukin-1β localization in the rat forebrain. Brain Res 1990; 514: 135. 1073. Lee J, Ho W-H, Maruoka M, et al. IL-17E, a novel proinflammatory ligand for the IL-17 receptor homolog IL-17Rh1. J Biol Chem 2001; 276: 1660–1664.

Walter M. Lewko and Robert K. Oldham 1074. Lee J, Horuk R, Rice GC, et al. Characterization of two high affinity human interleukin-8 receptors. J Biol Chem 1992; 267: 16283. 1075. Lee P, Wang F, Kuniyoshi J, et al. Effects of interleukin-12 on the immune response to a multipeptide vaccine for resected metastatic melanoma. J Clin Oncol 2001, 19: 3836–3847. 1076. Lee S, Kaneko H, Sekigawa I, et al. Circulating interleukin-16 in systemic lupus erythematosus. Br J Rheumatol 1998; 37: 1334–1337. 1077. Lee SC, Liu W, Dickson DW, Bronson CF, Berman JW. Cytokine production by human microglia and astrocytes. Differential induction by lipopolysaccharide and IL-1 β. J Immunol 1993; 150: 2659–2667. 1078. Lee SH, Aggarwal BB, Rinderknecht E, et al. The synergistic antiproliferative effect of γ-interferon and human lymphotoxin. J Immunol 1984; 133: 1083. 1079. Lee SJ, Drabik K, Van Wagner NJ, et al. ICAM-induced expression of pro-inflammatory cytokines in astrocytes: Involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways. J Immunol 2000, 165: 4658–4666. 1080. Lee SW, Tsou AP, Chan H, et al. Glucocorticoids selectively inhibit the transcription of the interleukin 1β gene and decrease the stability of interleukin 1 beat mRNA. Proc Natl Acad Sci (USA) 1988; 85: 1204–1208. 1081. Lee SY, Park CG, Choi Y. T cell receptor-dependent cell death of T cell hybridomas mediated by the CD30 cytoplasmic domain in association with tumor necrosis factor reeptor-associated factors. J Exp Med 1996; 183: 669. 1082. Lee TY, Koo AS, Peyret C, et al. The effect of IL-6 on TIL derived from human renal cell carcinoma. J Urol 1991; 145: 663–667. 1083. Leeuenberg JF, Dententer MA, Buurman WA. Lipopolysaccharide LPS-mediated soluble TNF release and TNF receptor expression by monocytes: role of CD14, LPS binding protein and bactericidal/permeability increasing protein. J Immunol 1994; 152: 5070. 1084. Leibovich SJ, Polverini PJ, Shepard HM, et al. Macrophage induced angiogenesis is mediated by tumor necrosis factor-α. Nature 1987; 329: 630. 1085. Leite-de-Moraes MC, Hameg A, et al. IL-18 enhances IL-4 production by ligand-activated NKT lymphocytes: a pro-Th2 effect of IL-18 exerted through NK cells. J Immunol 2001: 166: 945–951. 1086. Leite-de-Moraes MC, Herbelin A, et al. MHC class I selected CD4-CD8-TCRαβ+ T cells are a potential source of IL-4 during primary immune response. J Immunol 1995; 155: 4544. 1087. Leite-de-Moraes MC, Moreau G, et al. The IL-4 producing NKT cells are biased towards IFN-γ production by IL-12: influence of the microenvironment on the functional capacities of NKT cells. Eur J Immunol 1998; 28: 1507. 1088. Leleivre E, Plun-Favreau H, Chevalier S, et al. Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1composite cytokine. J Biol Chem 2001; 276: 22476. 1089. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996; 86: 973. 1090. LeMay RG, Otterness IG, Vander AJ, Kluger MJ. In vivo evidence that the rise in plasma IL-6 following injection of a fever-inducing dose of LPS is mediated by IL-1β. Cytokine 1990; 2: 199.

243 1091. Lembersky B, Baldisseri M, Kunschner A, et al. Phase I-II study of IP low dose interleukin-2 in refractory stage III ovarian cancer (meeting abstract). Proc Annu Meet Am Soc Clin Oncol 1989; 8: A636. 1092. Lenardo M. Interleukin-2 programs mouse αβ T lymphocytes for apoptosis. Nature 1991; 353: 858. 1093. Lens SM, Drillenburg P, den Drijver BF, et al. Aberrant expression and reverse signaling of CD70 on malignant B cells. Brit J Haematol 1999; 106: 491–503. 1094. Lens SMA, de Jong R, Hooibrink B, et al. Phenotype and function of human B cells expressing CD70 (CD27 ligand). Eur J Immunol 1996; 26: 2964–2971. 1095. Lentsch A, Crouch LD, Jordan JA, et al. Regulatory effects of interleukin-11 during acute lung inflammatory injury. J Leukocyte Biol 1999; 66: 151. 1096. Lenzi R, Kudelka MP, Verschraegen C, Nash M, et al. Recombinant human interleukin-12 (RHIL-12) in patients with ovarian and gastrointestinal cancers. Evidence of biological activity and lack of significant toxicity with low-dose intraperitoneal (IP) administration. Proc ASCO 1999; 18: 1721. 1097. Leonard WJ, Noguchi M, Russel SM, McBride OW. The molecular basis of severe combined immunodeficiency: the role of the interleukin-2 receptor γ chain as a common γ chain, γc. Immunological Reviews 1994; 138: 61–86. 1098. Lesback ME, McKenna HJ, Hoek JA, et al. Safety of FLT3 ligand in healthy volunteers. Blood 1997; 90: 751a. 1099. Lesser SS, Lo D. CNTFII, I presume? Nat Neurosci 2000; 3: 851. 1100. Lesslauer W, Tabuchi H, Gentz R, et al. Recombinant soluble tumor necrosis factor receptor protects mice from lipopolysaccharide lethality. Eur J Immunol 1991; 21: 2883. 1101. Lester MR, Hofer, MF, Gately M, Trumble A, Leung DYM. Down-regulating effects of IL-4 and IL-10 on the IFN-γ response in atopic dermatitis. J Immunol 1995; 154: 6174–6181. 1102. Leung BP, Culshaw S, Gracie JA, et al. A role for IL-18 in neutrophil activation. J Immunol 2001; 167: 2879–2886. 1103. Leung BP, McInnes IB, Esfandiari E, Wei X-Q, Liew FY. Combined effects of IL-12 and IL-18 on the induction of collagen-induced arthritis. J Immunol 2000: 164: 6495–6502. 1104. Leung SX, Elias JA. Interleukin-11 inhibits macrophage interleukin-12 production. J Immunol 1997; 159: 2161. 1105. Levi-Schaffer F, Rubinchik E. Activated mast cells are fibrogenic for 3T3 fibroblasts. J Invest Dermatol 1995; 104: 999. 1106. Levitsky JW, Simons H, Vogelstein B, Frost P. Interleukin 2 produced by tumor cell bypasses T helper function in the generation of an antitumor response. Cell 1990; 60: 397–403. 1107. Levy Y, Brouet JC. Interleukin-10 prevents spontaneous death of germinal center B cells by induction of the Bcl-2 protein. J Clin Invest 1994; 93: 424. 1108. Lewko WM, Good, RW, Bowman D, et al. Growth of tumor derived activated T cells for the treatment of cancer. Cancer Biotherapy 1994; 9(3): 211–224. 1109. Lewko WM, Hall PB, Oldham RK. Growth of tumor derived activated T cells for the treatment of advanced cancer. Cancer Biotherapy and Radiopharmaceuticals 2000; 15: 357–366. 1110. Lewko WM, Smalley RV, Oldham RK. Lymphokines and Cytokines. In: Oldham RK, ed. Principles of Cancer Biotherapy. Kluwer Academic Press, Dordrecht, The Netherlands, 1998: 211–265. 1111. Lewko WM, Smith TL, Bowman DJ, et al. Interleukin-15 and the growth of tumor derived activated T-cells. Cancer Biotherapy 1995; 10: 13–20. 1112. Li C-B, Gray PW, Lin PF, et al. Cloning and expression of lymphotoxin cDNA. J Immunol 1987; 138: 4496–4501.

244 1113. Li H, Chen J, Hung A, Stinson J, et al. Cloning and characterization of IL-17B and IL-17C, two new members of the IL-17 cytokine family. Proc Natl Acad Sci USA 2000; 97: 773–778. 1114. Li HH, Lin YC, Chen PJ, et al. Interleukin-19 upregulates keratinocyte growth factor and its association with psoriasis. Brit J Dermatol 2005; 153: 591–595. 1115. Li L, Elliott J, Mosmann T. IL-10 inhibits cytokine production, vascular leakage and swelling during T helper 1 cell induced delayed-type hypersensensitivity. J Immunol 1994; 153: 3967. 1116. Li Q, Carr A, Ito F, et al. Polarization effects of 4-1BB during CD28 costimulation in generating tumor-reactive T cells for cancer immunotherapy. Cancer Res 2003; 63: 2546–2552. 1117. Li Q, Carr AL, Donald EJ, et al. Synergistic effects of IL-12 and IL-18 in skewing tumor-reactive T-cell responses towards a type 1 pattern. Cancer Res 2005; 65: 1063–1070. 1118. Li WY, Lusheng S, Kanbour A, et al. Lymphocytes infiltrating human ovarian tumors: synergy between tumor necrosis factor and interleukin 2 in the generation of CD8+ effectors from tumor infiltrating lymphocytes. Cancer Res 1989; 49: 5979. 1119. Li XC, Roy-Chaudhury P, Hancock WW, et al. IL-2 and IL-4 double knockout mice reject islet allografts: a role for novel T cell growth factors in allograft rejection. J Immunol 1998; 161: 890–896. 1120. Li Y, Bleakley M, Yee C. IL-21 influences the frequency, phenotype and affinity of the antigen-specific CD8 T cell response. J Immunol 2005; 175: 2261–2269. 1121. Li Y, Yee C. IL-21 mediated Foxp3 suppression leads to enhanced generation of antigen-specific CD8+ T lymphocytes. Blood 2008; 111: 229–235. 1122. Liang SC, Long AJ, Bennett F, et al. An IL-17F/A heterodimer protein is produced by mouse Th17 cells and induces airway neutrophil recruitment. J Immunol 2007; 179: 7791–7799. 1123. Liang SC, Tan X-Y, Luxenberg DP, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006; 203: 2271–2279. 1124. Liao Y-C, Cheng Y-C, Wang Y-C, et al. IL-19 induces Th2 cytokines and was upregulated in asthma patients. J Immunol 2004; 173: 6712–6718. 1125. Liao Y-C, Liang W-G, Chen F-W, et al. IL-19 induces production of IL-6 and TNF-α and results in cell apoptosis through TNF-α. J Immunol 2002; 169: 4288–4297. 1126. Libby P. Warner SJ, Friedman GB, Interleukin1: a mitogen for vascular human smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest 1988; 81: 487–498. 1127. Lichtman A, Kurt-Jones E, Abbas A. B cell stimulatory factor 1 and not interleukin 2 is and autocrine growth factor for some helper T lymphocytes. Proc Nat Acad Sci 1987; 84: 824. 1128. Lienard D, Eggermont AM, Koops HS, et al. Isolated limb perfusion with tumor necrosis factor-α and melphalan with or without interferon-γ for the treatment of in-transit melanoma metastases: a multicentre ramdomized phase II study. Melanoma Res 1999; 9: 491–502. 1129. Lim KG, Wan HC, Bozza PT, et al. Human eosinophils elaborate the lymphocyte chemoattractants IL-16 (lymphocyte chemoattractant factor) and RANTES. J Immunol 1996; 156: 2566–2570. 1130. Lin T-J, Issekutz TB, Marshall JS. Human mast cells transmigrate through human umbilical vein endothelial monolayers and selectively produce IL-8 in response to stromal cell-derived factor-1α. J Immunol 2000; 165: 211–220.

Cytokines 1131. Lind M, Deleuran B, Yssl H, Fink-Eriksen E, Thestrup-Peterson K. IL-4 and IL-13 but not IL-10 are chemotactic factors for human osteoblasts. Cytokine 1995; 7: 78. 1132. Lindgren CG, Thompson JA, Higuchi CM, Fefer A. Growth and autologous tumor lysis by tumor infiltrating lymphocytes from metastatic melanoma expanded in interleukin 2 or interleukin 2 plus Interleukin 4.J Immunother 1993; 14: 322–328. 1133. Lindqvist C, Ostman AL, Okerblom C, Akerman K. Decreased interleukin-2β chain receptor expression by interleukin-4 on LGL: influence on the IL-2 induced cytotoxicity and proliferation. Cancer Lett 1992; 64: 43–49. 1134. Linsley PS, Ledbetter JA. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol 1993; 11: 191. 1135. Lisby S, Muller KM, Jongeneel CV, Saurat JH, Hauser C. Nickle and skin irritants upregulate tumor necrosis factor mRNA in keratinocytes by differential but potentially synergistic mechanisms. Int Immunol 1995; 7: 343–352. 1136. Liu C, Hart RP, Liu X-J, et al. Cloning and characterization of an alternately processed human type II interleukin-1 receptor mRNA. J Biol Chem 1996; 271: 20965. 1137. Liu J, Hadjokas N, Mosey B, et al. Oncostatin M-specific receptor expression and function in the regulation of cell proliferation of normal and malignant mammary epithelial cells. Cytokine 1998; 10: 295–302. 1138. Liu J, Modrell B, Aruffo A, et al. Interaction between oncostatin M and the signal transducer, gp130. Cytokine 1994; 6: 272–278. 1139. Liu L, Ding C, Heuer JG, et al. Selective enhancement of multipotential hematopoietic progenitors in vito and in vivo by IL-20. Blood 2005; 117: 3206–3209. 1140. Liu X, Yue P, Schonthal AH, et al. Cellular FLICE-inhibitory protein down regulation contributes to celecoxib-induced apoptosis in human lung cancer cells. Cancer Res 2006; 66: 11115–11119. 1141. Liu Y, Wu Y, Ramarathinam L, et al. Gene-targeted B-deficient mice reveal a critical role for B cells in the CD4 cell response. Int Immunol 1995; 7: 1353. 1142. Llorente L, Richard-Patin Y, Garcia-Padilla C, et al. Clinical and biological effects of anti-interleukin-10 monoclonal antibody administration in systemic lupus erythematosis. Arthritis Rheu 2000; 43: 1790–1800. 1143. Lo CH, Lee SC, Wu PY, et al. Antitumor and antimetastatic activity of IL-23. J Immunol 2003; 171: 600–607. 1144. Lockhart E, Green AM, Flnn JL. IL-17 production is dominated by γδ T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J Immunol 2006; 177: 4002–4009. 1145. Lodolce JP, Boone DL, Chai S, et al. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 1998; 9: 669. 1146. Loenen WAM, De Vries E, Gravestein LA, et al. The CD27 membrane receptor, a lymphocyte specific member of the nerve growth factor family, gives rise to a soluble form by protein processing that does not involve endocytosis. Eur J Immunol 1992; 22: 447–455. 1147. Loetscher M, Gerber B, Loetscher P, et al. Chemokine receptor specific for IP-10 and Mig: structure, function and expression in activated T-lymphocytes. J Exp Med 1996; 184: 963. 1148. Londei M, Verhoef A, Hawrylowicz C, et al. Interleukin 7 is a growth factor for mature human T cells. Eur J Immunol 1990; 20: 425–428. 1149. London L, Perussiia B, Trinchieri G. Induction and proliferation in vitro of resting human natural killer cells: expression of surface activation antigens. J Immunol 1985; 134: 718.

Walter M. Lewko and Robert K. Oldham 1150. Lonnemann G, Shapiro L, Engler-Blum G, et al. Cytokines in human renal interstitial fibrosis. I. Interleukin-1 is an autocrine factor for cultured fibrosis-derived kidney fibroblasts. Kidney Int 1995; 47: 845–854. 1151. Lopez AF, Sanderson CJ, Gamble JR, et al. Recombinant human interleukin 5 is a selective activator of human eosinophil function. J Exp Med 1988; 167: 219. 1152. Lopez MV, Adris SK, Bravo AI, et al. IL-12 and IL-10 expression synergize to induce immune-mediated eradication of established colon and mammary tumors and lung metastases. J Immunol 2005; 175: 5885–5894. 1153. Lorenz MG, Kantor JA, Schlom J, et al. Antitumor immunity elicited by a recombinant vaccinia virus expressing CD70 (CD27L). Human Gene Ther 1999; 10: 1095–1103. 1154. Lorenzen J, Lewis CE, McCracken D, et al. Human tumor associated NK cells secrete increased amounts of interferon-γ and interleukin-4. British J Cancer 1991; 64: 457–462. 1155. Lotze M, Jirik F, Kabouridis P, et al. B cell stimulating factor-2/ interleukin 6 is a co-stimulant for human thymocytes and T lymphocytes. J Exp Med 1988; 167: 1253–1258. 1156. Lotze M. Blanco FJ, Von Kempis J, et al. Cytokine regulation of chondrocyte functions. J Rheumatol 1995; 22: 104. 1157. Lotze M. Role of IL-4 in the antitumor response. In: Spits H, ed. IL-4: Structure and Function. CRC Press, Boca Raton, Florida, 1992; 111: 274–280. 1158. Lotze MT, Frana LW, Sharrow SO, et al. In vivo administration of purified human interleukin 2. I. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2. J Immunol 1985; 134: 157. 1159. Lotze MT, Hellerstedt B, Stolinski L, et al. The role of IL-2, IL-12 and dendritic cells in cancer therapy. Cancer J Sci Am 1997 3: 1: S109–114. 1160. Lotze MT, Matory YL, Ettinghausen SE, et al. In vivo administration of purified human interleukin 2. II. Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant interleukin 2. J Immunol 1985; 135: 2865–2875. 1161. Louahed J, Kermouni A, Van Snick J, Renauld J-C. IL-9 induces expression of granzymes and high affinity IgE receptor in murine T helper clones. J Immunol 1995; 154: 5061–5070. 1162. Louahed J, Renauld J-C, Demoulin J-B, et al. Differential activity of dexamethasone on IL-2, IL-4, or IL-9-induced proliferation of murine factor-dependent T cell lines. J Immunol 1996; 156: 3704–3710. 1163. Lowenthal JW, MacDonald HR. Binding and internalization of interleukin 1 by T cells. Direct evidence for high and low affinity classes of interleukin-1 receptors. J Exp Med 1986; 164: 1060. 1164. Lu C, Kerbel RS. Interleukin 6: undergoes transition from paracrine growth inhibitor to autocrine growth stimulator during human melanoma progression. J Cell Biol 1993; 120: 1281–1288. 1165. Lu C, Vickers MF, Kerbel RS. Interleukin 6: a fibroblast derived growth inhibitor of human melanoma cells from early but not advanced stages of tumor progression. Proc Nat Acad Sci USA 1992; 89: 9215–9219. 1166. Lu G, Janjic BM, Janjic J, et al. Innate direct anticancer effector function of human immature dendritic cells. II. Role of TNF, lymphotoxin-α1β2, Fas ligand and TNF-relalted apoptosis-inducing ligand. J Immunol 2002; 168: 1831–1839. 1167. Luan J, Shattuck-Brandt R, Hagnegahdar H, et al. Mechanism and biological significance of constitutive expression of MGSA/ GRO chemokines in malignant melanoma tumor progression. J Leukocyte Biol 1997; 62: 588.

245 1168. Lubbert M, Mantovani L, Lindemann A, Mertlesmann R, Herrmann F. Expression of leukemia inhibitory factor is regulated in human mesenchymal cells. Leukemia 1991; 5: 361. 1169. Lubberts E, Joosten LAB, Opers B, et al. IL-1-independent role of IL-17 in synovial inflammation and joint destruction during collagen-induced arthritis. J Immunol 2001; 167: 1004–1013. 1170. Luger TZ, Stadler BM, Katz SI, et al. Epidermal cell (keratinocyte)-derived thymocyte-activating factor (ETAF). J Immunol 1981; 127: 1493–1498. 1171. Lundin J, Kimby E, Bergman L, et al. Interleukin 4 therapy for patients with chronic lymphocytic leukemia: a phase I/II study. Br J Haematol 2001: 112: 155–160. 1172. Lundqvist A, Abrams SI, Schrump DS, et al. Bortezomib and depsipeptide sensitize tumors to tumor necrosis factor-related apoptosis-inducing ligand: a novel way to potentiate natural killer cell tumor cytotoxicity. Cancer Res 2006; 66: 7317–7325. 1173. Luscher U, Filgueira L, Juretic A, et al. The pattern of cytokine gene expression in freshly excised human metastatic melanoma suggests a state of reversible anergy of tumor infiltrating lymphocytes. Int J Cancer 1994; 57: 612. 1174. Luzza F, Parello T, Monteleone G, et al. Up-regulation of IL-17 is associated with bioactive IL-8 expression in Helicobacter pylori-infected human gastric mucosa. J Immunol 2000; 165: 5332–5337. 1175. Lyman SD, Jacobsen SEW. c-kit ligand and Flt-3 ligand: stem/ progenitor cell factors with overlapping yet distinct activities. Blood 1998; 91: 1101–1134. 1176. Lyman SD, James L, Escobar S, et al. Identification of soluble and membrane-bound isoforms of murine Flt-3 ligand generated by alternative splicing of mRNAs. Oncogene 1995; 10: 149–157. 1177. Lyman SD, James L, Vanden Bos T, et al. Molecular cloning of a ligand for primitive hematopoietic cells. Cell 1993; 75: 1157–1167. 1178. Lyman SD, James L, Zappone J, et al. Characterization of the protein encoded by the Flt3 (FLK2) receptor-like tyrosine kinase gene. Oncogene 1993; 6: 815–822. 1179. Lyman SD, Seaberg M, Hanna R, et al. Plasma/serum levels of Flt3 ligand are low in normal individuals and highly elevated in patients with Fanconi anemia and acquired aplastic anemia. Blood 1995; 86: 4091–4096. 1180. Lynch CN, Wang YC, Lund JK, et al. TWEAK induces angiogenesis and proliferation of endothelial cells. J Biol Chem 1999; 274: 8455–8459. 1181. Lynch DH and Miller RE. Induction of murine lymphokine activated killer cells by recombinant IL-7. J Immunol 1990; 145: 198. 1182. Lynch DH and Miller RE. Interleukin 7 promotes Long term in vitro growth and antitumor cytotoxic T lymphocytes with immunotherapeutic efficacy in vivo. J Exp Med 1994; 179: 31–42. 1183. Lynch DH, Andreasen A, Maraskovsky E, et al. Flt-3 ligand induces tumor regression and antitumor immune responses in vivo. Nat Med 1997; 3: 625–631. 1184. Lynch DH. The role of FasL and TNF in the homeostatic regulation of immune response. Adv Exp Med Biol 1996; 406: 135. 1185. Ma A, Datta M, Margosian E, Chen J, Horak I. T cells but not B-cells are required for bowel inflammation in Interleukin-2deficient mice. J Exp Med 1995; 182: 1567. 1186. Ma H-L, Liang S, Li J, et al. IL-22 is required for Th17 cellmediated pathology in a mouse model of psoriasis-like skin inflammation. J Clin Invest 2008; 118: 597–607. 1187. Ma HL, Whitters MJ, Konz R, et al. IL-21 activates both innate and adaptive immunity to generate potent antitumor responses

246

1188.

1189.

1190. 1191. 1192.

1193.

1194.

1195.

1196.

1197.

1198.

1199.

1200.

1201.

1202.

1203.

1204.

1205.

1206.

Cytokines that require perforin but are independent of IFN-γ. J Immunol 2003; 171: 608–615. Macatonia SE, Doherty TM, Knight SC, et al. Differential effect of IL-10 on dendritic cell induced T cell proliferation and IFN-γ production. J Immunol 1993; 150: 3755. Macatonia SE, Hosken NA, Litton M, et al. Dendritic cells produce IL-12 and direct the development of Th1 cells form naive CD4+ T cells. J Immunol 1995; 154: 5071–5079. MacGlashan D Jr, White JM, Huang S, et al. Secretion of IL-4 from human basophils. J Immunol 1994; 152: 3006. Maciaszek JW, Parada NA, et al. IL-16 represses HIV-1 promoter activity. J Immunol 1997; 158: 5–8. Mackarehtschian K, Hardin JD, Moore KA, et al. Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity 1995; 3: 147–161. Mackey F, Loetscher H, Steuber D, Gher G, Lesslauer W. Tumor necrosis factor-α (TNF-α)-induced cell adhesion to endothelial cells is under dominant control of one TNF receptor type, TNF-R55. J Exp Med 1993; 177: 1277. Mackey F, Majeau GR, Hochman PS, Browning JL. Lymphotoxin β receptor triggering induces activation of the nuclear factor κB transcription factor in some cell types. J Biol Chem 1996; 271: 24934–24938. Mackey MF, Gunn JR, Ting PP, et al. Dendritic cells require maturation via CD40 to generate protective anticancer immunity. J Immunol 1998; 161: 2094. Mackey MF, Gunn JR, Ting PP, et al. Protective immunity induced by tumor vaccines requires interaction between CD40 and its ligand CD154. Cancer Res 1994; 57: 2569. Mackiewicz A, Schooltink H, Heinrich PC, Rose-John S. Complex of soluble human IL-6-receptor/IL-6 up regulates expression of acute phase proteins. J Immunol 1992; 149: 2021. MacNeil IA, Suda T, Moore KW, et al. IL-10, a novel growth factor for mature and immature T cells. J Immunol 1990; 145: 4167. Maeker HT, Hansen G, Walter DM, DeKruyff RH, Levy S, Umetsu DT. Vaccination with allergen-IL-18 fusion DNA protects against and reverses established airway hyperreactivity in a murine asthma model. J Immunol 2001; 166: 959–965. Magazin M, Guillemot JC, Vita N, Ferra P. Interleukin13 is a monocyte chemoattractant. Eur Cytokine Network 1994; 5: 397. Mahasreshti PJ, Kataram M, Wu H. Ovarian cancer targeted adenoviral-mediated mda-7/IL-24 gene therapy. Gynecol Oncol 2006; 100: 521–532. Maier R, Ganu V, Lotz M. Interleukin 11, an inducible cytokine in human articular chondrocytes and synoviocytes, stimulates the production of tissue inhibitor of metalloproteinases. J Biol Chem 1993; 268: 21527. Maini R, Clair EW, Breedveld F, et al. Infliximab (chimeric anti-tumour necrosis factor monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomized phase III trial. ATTRACT study group. Lancet 1999; 354: 1932–1939. Maione TE, Gray GS, Petro J, et al. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science 1990; 247: 77. Maki K, Sunaga S, Ikuta K. The V-J recombination of T cell receptor-γ genes is blocked in interleukin-7 receptor deficient mice. J Exp Med 1996; 184: 2423. Maldonado-Lopez R, De Smedt T, et al. CD8α+ and CD8α−subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med 1999; 189: 587.

1207. Maleckar JR, Fridell CS, Lewko WM, et al. Tumor derived activated cells: cell culture conditions and characterization. In: Stevenson HC, ed. Adoptive cellular immunotherapy of cancer. Marcel Dekker, New York, 1989; 159–173. 1208. Maleckar JR, Fridell CS, Sferruzza A, et al. Activation and Expansion of tumor derived activated cells (TDAC). J Nat Cancer Inst 1989; 1: 1655–1660. 1209. Malik N, Haugen HS, Mondrell B, Shoyab M, Clegg CH. Developmental abnormalities in mice transgenic for bovine oncostatin M. Mol Cell Biol 1995; 15: 2349. 1210. Malik N, Kallestad JC, Gunderson NL, et al. Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M. Mol Cell Biol 1989, 9: 2847. 1211. Maling HM, Webster ME. Williams WA, Saul W, Anderson W. Inflammation induced by histamine, serotonin, bradykinin and compound 48/80 in the rat: antagonists and mechanism of action. J Pharmacol Exp Ther 1973; 191: 300. 1212. Maliszewski CR, Sato TA, Vanden Bos T, et al. Cytokine Receptors and B cell functions. I. Recombinant soluble receptors specifically inhibit IL-4 and IL-4 induced B cell activities in vitro. J Immunol 1990; 144: 3028–3033. 1213. Mallett S, Fossum S, Barclay AN. Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes: a molecule related to nerve growth factor receptor. EMBO J 1990; 9: 1063. 1214. Manabe A, Cousan-Smith E, Kumagai M, et al. Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia. Blood 1994; 83: 1731–1737. 1215. Manabe N, Kawaguchi H, Chicuda H, et al. Connection between B-lymphocyte and osteoclast differentiation pathways. J Immunol 2001; 167: 2625–2653. 1216. Mandrup-Poulsen T, Bendtzen K, Dinarello CA, Nerup J. Human tumor necrosis factor potentiates human interleukin 1-mediated rat pancreatic β-cell cytotoxicity. J Immunol 1987; 139: 4077. 1217. Manfield L, Jang D, Murrell AC. Nitric oxide enhances cyclooxygenase activity in articular cartilage. Inflamm Res 1996; 45: 254. 1218. Mangan PR, Harrington LE, O’Quinn DH, et al. Transforming growth factor-β induces development of the TH17 lineage. Nature 2006; 41: 231–234. 1219. Manganini M, Maier JA. Transforming growth factor β2 inhibition of hepatocyte growth factor-induced endothelial proliferation and migration. Oncogene 2000, 19: 124. 1220. Manolagas SC, Bellido T, Jilka PL. New insights into the cellular, biochemical and molecular basis of postmenopausal and senile osteoporosis: roles of IL-6 and pg130. Int J Immunopharm 1996; 17: 109. 1221. Marakowsky E, Daro E, Roux E, et al. In vivo generation of human dendritic cell subsets by FLT3 ligand. Blood 2000; 96: 878. 1222. Maraskovsky E, Brasel K, Teepe M, et al. Dramatic increase in the numbers of functionally mature dendritic cells in FLT3 ligand-treated mice: multiple dendritic cell populations identified. J Exp Med 1996; 184: 1953. 1223. Maraskovsky E, O’Reilly LA, Teepe M, et al. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1−/−mice. Cell 1997; 89: 1011. 1224. Maraskovsky E, Teepe M, Morrissey PJ, et al. Impaired survival and proliferation in IL-7 receptor deficient peripheral blood T cells. J Immunol 1996; 157: 5315. 1225. March CJ, Mosely B, Larsen A, et al. Cloning, sequence and expression of two distinct human interleukin-1 complementary cDNA’s. Nature 1985; 315: 641–647.

Walter M. Lewko and Robert K. Oldham 1226. Margolin K, Aronson FR, Sznol M, et al. Phase II studies of recombinant human IL-4 in advanced renal cancer and malignant melanoma. J Immunother 1994; 15: 147–153. 1227. Mariner JM, Lantz V, Waldmann TA, Azimi N. Human T cell lymphotrophic virus type I Tax activates IL-15Rα gene expression through NF-κB site. J Immunol 2001; 166: 2602–2609. 1228. Marshall DJ, Rudnick KA, McCarthy SG, et al. Interleukin-18 enhances Th1 immunity and tumor protection of a DNA vaccine. Vaccine 2006; 24: 244–253. 1229. Marshall E, Chakir J, Laviolette M, et al. IL-11 expression is increased in severe asthma: association with epithelial cells and eosinophils. J Allergy Clin Immunol 2000; 105: 232. 1230. Marshall JS, and Bienenstock J. The role of mast cells in inflammatory reactions of the airways, skin and intestine. Curr Opin Immunol 1990; 6: 853. 1231. Marsters SA, Ayres TM, Skubatch M, et al. Herpesvirus entry mediator, a member of the tumor necrosis factor receptor (TNFR) family, interacts with members of the TNFR-associated factor family and activates the transcription factors NF-κB and AP-1. J Biol Chem 1997; 272: 14029. 1232. Marsters SA, Sheridan JP, Pitti RM, et al. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Current Biol 1997; 7: 1003. 1233. Martinet O, Divino CM, Zang Y, et al. T cell activation with systemic agonistic antibody versus 4-1BB ligand gene delivery combined with interleukin-12 eradicate liver metastases of breast cancer. Gene Ther 2002; 9: 786–792. 1234. Marz P, Cheng J-C, Gadient RA, et al. Sympathetic neurons can produce and respond to interleukin-6. Proc Natl Acad Sci USA 1998; 95: 3251. 1235. Mashikian MV, Ryan TC, Seman A, et al. Reciprocal desensitization of CCR5 and CD4 is mediated by IL-16 and macrophageinflammatory protein-1β, respectively. J Immunol 1999; 163: 3123–3130. 1236. Mashikian MV, Tarpy RE, Saukkonen JJ, et al. Identification of IL-16 as the lymphocyte chemotactic activity in the bronchoalveolar lavage of fluid of histamine challenged asthmatic patients. J Allergy Clin Immunol 1998; 101: 786–792. 1237. Mastorakos G, Chrousos GP and Weber JS. Recombinant interleukin-6 activates the hypothalamic-pituitary-adrenal axis in humans. J Clin Endocrinol Metab 1993; 77: 1690–1694. 1238. Mateo RB, Reichner JS, and Albina JE. Interleukin-6 activity in wounds. Am J Physiol 1994; 226 (Suppl): R1840. 1239. Mathy NL, Bannert N, Norley SG, Kurth R. CD4 is not required for functional activity of IL-16. J Immunology 2000; 164: 4429–4432. 1240. Mathy NL, Scheuer W, Lanzendoerfer M, et al. IL-16 stimulates the expression and production of proinflammatory cytokines by human monocytes. Immunology 2000; 100: 63–69. 1241. Matikainen S, Siren J, Tissari J, et al. Tumor necrosis factor α enhances influenza A virus-induced expression of antiviral cytokines by activating RIG-I gene expression. J Virol 2006; 80: 3515–3522. 1242. Matsuda M, Motoji T, Oshimi K, Mizoguchi H. Effects of interleukin 7 on proliferation of hematopoietic malignant cells. Exp Hematol 1990; 18: 965–967. 1243. Matsuda M, Salazar F, Peterson M, et al. Interleukin 10 pretreatment protects target cells from tumor and allo-specific cytotoxic T cells and downregulates HLA class 1 expression. J Exp Med 1994; 180: 2371. 1244. Matsuda R, Takahashi T, Nakamura S, et al. Expression of the c-kit protein in human solid tumors and in corresponding fetal and adult normal tissues. Am J Pathol 1993; 142: 339–349.

247 1245. Matsue H, Bergstresser PR, Takashima A. Keratinocyte derived IL-7 serves as a growth factor for dendritic epidermal T cells in mice. J Immunol 1993; 151: 6012–6019. 1246. Matsue H, Bergstresser PR, Takashima A. Reciprocal cytokine mediated cellular interactions in mouse epidermis: promotion of γδ T cell growth by IL-7 and TNFα and inhibition of keratinocyte growth by γ interferon. J Invest Dermatol 1993b; 101: 543. 1247. Matsuguchi T, Musikacharoroen T, Ogawara T, Yoshikai Y. Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4. is induced by LPS and inflammatory cytokines in mouse macrophages. J Immunol 2000; 165: 5767–5772. 1248. Matsui Y, Zsebo KM, Hogan LM. Embryonic expression of hematopoietic growth factor encoded by Sl locus and the ligand for c kit. Nature 1990; 343: 667–669. 1249. Matsumoto M, Mariathyasan S, Nahm HM, et al. Role of lymphotoxin and the type 1 TNF receptor in the formation of germinal centers. Science 1996; 271: 1289. 1250. Matsushima K, Oppenheimer JJ. Interleukin 8 and MCAF: Novel inflammatory cytokines inducible by IL-1 and TNF∞. Cytokine 1989; 1: 2–13. 1251. Matthews W, Jordan CT, Weigand GW, Pardoll D, Lemischka IR. A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 1991; 65: 1143–1152. 1252. Mattner F, Fisher S, Gluckes S, et al. The interleukin-12 subunit p40 specifically inhibits effects of the interleukin-12 heterodimer. Eur J Immunol 1993; 23: 2203–2208. 1253. Mauer D, Holter W, Majdic O, Fisher GF, Knapp W. CD27 expression by a distinct subpopulation of human B lymphocytes. Eur J Immunol 1999; 20: 2679. 1254. Mauri DN, Ebner R, Montgomery RI, et al. LIGHT, a new member of the TNF superfamily, and lymphotoxin α are ligands for herpesvirus entry mediator. Immunity 1998; 8: 21. 1255. Mavalia C, Scaletti C, Romagnani P, et al. Type 2 helper T cell predominance and high CD30 expression in systemic sclerosis. Am J Pathol 1997; 151: 1751. 1256. Maxwell JR, Campbell JD, Kim KH, Velli HT. CD40 activation boosts T cell immunity in vivo by enhancing T cell clonal expansion and delaying peripheral T cell deletion. J Immunol 1999; 162: 2024–2034. 1257. May LT, Helfgott DC, Sehgal PB. Anti-β-interferon antibodies inhibit the increased expression of HLA-B7 mRNA in tumor necrosis factor-treated human fibroblasts: structural studies of the β2 interferon involved. Proc Natl Acad Sci USA 1986; 83: 8957–8961. 1258. Mayer P, Giessler K, Ward M, Metcalf D. Recombinant human leukemia inhibitory factor induces acute phase proteins and raises blood platelet counts in nonhuman primates. Blood 1993; 81: 3226. 1259. Mayordoma JI, Zorina T, Storkus WJ, et al. Bone marrow derived dendritic cells pulsed with synthetic tumor peptides elicit protective and therapeutic antitumor immunity. Mature Medicine 1995; 1: 1297–1302. 1260. Mazodier K, Marin V, Novick D. Severe imbalance of IL-18/ IL-18BP in patients with secondary hemophagic syndrome. Blood 2005; 106: 3483–3489. 1261. McBride WH, Thacker JD, Comora S, et al. Genetic modification of a murine fibrosarcoma to produce Interleukin 7 stimulates host cell infiltration and tumor immunity. Cancer Res 1992; 52: 3931–3937. 1262. McCaffery PG, Perrino BA, Soderling TR, Rao A. NF-ATp, a T-lymphocyte DNA-binding protein that is a target for calcineurin and immunosuppressive drugs. J Biol Chem 1993; 269: 3747.

248 1263. McCaffrey PG, Luo C, Kerppola TK, et al. Isolation of the cyclosporin sensitive transcription factor NF-ATp. Science 1993; 262: 750. 1264. McCain RW, Dessypris EN, Christman JW. Granulocyte/macrophage colony stimulating factor stimulates human polymorphonuclear leukocytes to produce interleukin 8 in vitro. J Respir Cell Mol Biol 1993; 8: 28. 1265. McClanahan T, Culpepper J, Campbell D, et al. Biochemical and genetic characterization of multiple splice variants of the Flt3 ligand. Blood 1996; 88: 3371–3382. 1266. McDermott DF, Terhu EG, et al. A two part phase I trial of high dose interleukin 2 in combination with soluble (Chinese Hamster Ovary) interleukin 1 receptor. Clin Cancer Res 1998; 4: 1203–1213. 1267. McGeachy MJ, Bak-Jensen KS, Chen Y, et al. TGF-β and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain TH−17 cell-mediated pathology. Nat Immunol 2007; 8: 1390–1397. 1268. McInnes IB, Al-Mugales J, Field M, et al. The role of interleukin 15 in T cell migration and activation in rheumatoid arthritis. Nat Med 1996; 2: 175. 1269. McKenna HJ, de Vries P, Brasel K, Lyman SD, Williams DE. Effect of flt 3 ligand on the ex vivo expansion of human CD34+ hematopoietic progenitor cells. Blood 1995; 86: 3413. 1270. McKenna HJ, Morrisey PJ. Flt3 ligand plus IL-7 supports the expansion of murine thymic B bell progenitors that can mature intrathymically. J Immunol 1998; 160: 4801–4809. 1271. McKenna HJ, Smith OF, Brasel K, et al. Effects of Flt3 ligand on acute myeloid and lymphocytic leukemic blast cells from children. Exp Hematol 1996; 24: 378–385. 1272. McKenna HJ, Stocking KL, Miller RE, et al. Mice lacking FLT3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells and natural killer cells. Blood 2000; 95: 3489–3497. 1273. McKenna SD, Chen F, Lai L, Goldschneider I. Identification of an IL-7 associated pre-pro-B cell growth-stimulating factor (PPBSF). I. Production of the non-IL-7 component by bone marrow stromal cells from IL-7 gene deleted mice. J Immunology 1998; 160: 2272–2279. 1274. McKenzie ANJ, Culpepper JA, De Waal Malefyt R, et al. Interleukin 13, a novel T cell derived cytokine that regulates human monocyte and B cell function. Proc Nat Acad Sci USA 1993; 90: 3735–3739. 1275. McKenzie ANJ, Li X, Largaespada DA, et al. Structural comparison and chromosomal localization of the human and mouse IL-13 genes. J Immunol 1993; 150: 5436–5444. 1276. McKenzie GJ, Emson CL, Bell SE, et al. Impaired development of Th2 cells in IL-13-deficient mice. Immunity 1998; 8: 423–432. 1277. McKenzie RC, Paglia D, Kondo S, Sauder DN. A novel endogenous mediator of cutaneous inflammation: leukemia inhibitory factor. Acto Dermato-Venereol 1996; 76: 111. 1278. McKinnon M, Page K, Uings IJ, et al. An interleukin 5 mutant distinguishes between two functional responses in human eosinophils. J Exp Med 1997; 186: 121–129. 1279. McLane MP, Haczku A, van der Rijn M, et al. Interleukin-9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice. Am J Respir Cell Mol Biol 1998; 19: 713. 1280. Medico E, Gentile A, Lo Celso C, et al. Osteopontin is an autocrine mediator of hepatocyte growth factor-induced invasive growth. Cancer Res 2001; 61: 5861–5868. 1281. Medvedev AE, Espevik T, Ganges G, Sundan A. Distinct roles of the two tumor necrosis factor (TNF) receptors in modulating TNF and lymphotoxin α effects. J Biol Chem 1996; 271: 9778–9784.

Cytokines 1282. Medzhitov R, Preston-Hurlburt P, Janeway Jr. CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1996; 388: 394. 1283. Mehrota PT, Grant AJG and Siegel JP. Synergistic effects of IL-7 and IL-12 on Human T cell activation. J Immunol 1995; 154: 5093–5102. 1284. Mehrota PT, Wu D, Crim JA, et al. Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes. J Immunol 1993; 151: 2444–2452. 1285. Mehta DS, Wurster AL, Whitters MJ, et al. IL-21 induces the apoptosis of resting and activated primary B cells. J Immunol 2003; 170: 4111–4118. 1286. Meierhoff G, Dehmel U, Gruss H-J, et al. Expression of Flt3 receptor and Flt3-ligand in human leukemia-lymphoma cell lines. Leukemia 1995; 9: 1368–1372. 1287. Melani C, Mattia GF, Silvani A, et al. Interleukin-6 expression in human neutrophil and eosinophil peripheral blood granulocytes. Blood 1993; 81: 2744–2749. 1288. Melchjorsen J, Siren J, Julkunen I, et al. Induction of cytokine expression by herpes simplex virus in human monocyte-derived macrophages and dendritic cells is dependent on virus replication and is counteracted by ICP27 targeting NF-κB and IRF-3. J Gen Virol 2006; 87: 1099–1108. 1289. Melero I, Johnson JV, Shuford WW, et al. NK 1.1 cells express 4–1 BB (CDw137) and are required for tumor immunity elicited by anti4–1 BB monoclonal antibodies. Cell Immunol 1998; 190: 167. 1290. Melero I, Shuford LL, Newby SA, et al. Monoclonal antibodies against 4-1BB T cell activation molecule eradicate established tumors. Nat Med 1997; 3: 682. 1291. Mellin GW, Katsenstein M. The saga of thalidomide. N Eng J Med 1962; 264: 1184–1193. 1292. Mennechet FJ, Uze G. Interferon-λ-treated dendritic cells specifically induce proliferation of FOXP3-expressing dendritic cells. Blood 2006; 107: 4417–4423. 1293. Merberg DM, Wolf SF, Clark SC. Sequence similarity between NKSF and the IL-6/G-CSF family. Immunol Today 1992; 14: 77. 1294. Merino R, Grillot AM, Simonian PL, et al. Modulation of antiIgM-induced B cell apoptosis by Bcl-XL and CD40 in WEHI231 cells. J Immunol 1995; 155: 3830. 1295. Metcalf D, Nicola NA, Gearing DP. The effects of injected leukemia inhibitory factor on hematopoietic and other tissues in mice. Blood 1990; 77: 2150. 1296. Metcalf D. Control of granulocytes and macrophages: molecular, cellular and clinical aspects. Science 1991; 254: 529. 1297. Metcalf D. Leukemia inhibitory factor (LIF). Int J Cloning 1991; 9: 95. 1298. Meunch MO, Roncarolo MG, Menon S, et al. Flk-2/Flt-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver. Blood 1995; 85: 963–972. 1299. Mian BM, Dinney CP, Bermejo CE, et al. Fully human antiinterleukin-8 antibody inhibits tumor growth in orthotopic bladder cancer xenografts via down-regulation of matrix metalloproteases and nuclear factor-κB. Clin Cancer Res 2003; 9: 3167–3175. 1300. Micallef MJ, Ohtsuki T, Kohno K, et al. Interferon-γ-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-γ production. Eur J Immunol 1996; 26: 1647–1651. 1301. Micallef MJ, Tanimoto T, Kohno K, Ikeda M, Kurimoto M. Interleukin-18 induces the sequential activation of natural killer cells and cytotoxic T lymphocytes to protect syngeneic mice from transplantation with Meth A sarcoma. Cancer Res 1997b; 57: 4557–4563.

Walter M. Lewko and Robert K. Oldham 1302. Micallef MJ, Yoshida K, Kawai S, et al. In vivo antitumor effects of murine interferon-γ-inducing factor IL-18 in mice bearing syngeneic Meth A sarcoma malignant ascites. Cancer Immunol Immunother 1997a; 43: 361–367. 1303. Micham JL, Parnet P, Bonnert TP, et al. T1/ST2 signaling establishes it as a member of the expanding interleukin-1 receptor family. J Biol Chem 1996; 271: 5777. 1304. Michie HR, Manogue KR, Spriggs DR, et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 1988; 318: 1481. 1305. Mier JW, Gallo RC. Isolation and purification of human T-cell growth factor. Proc Natl Acad Sci (USA) 1980; 77: 6134. 1306. Migliorati G, Pagilacci C, Moraca R, et al. Interleukins modulate glucocorticoid induced thymocyte apoptosis. Int J Clin Lab Res 1992; 21: 300–303. 1307. Miki S, Iwano Y, Miki Y, et al. Interleukin 6 functions as an in vitro autocrine growth factor in renal cell carcinoma. FEBS Lett 1989; 250: 607–610. 1308. Miles SA, Martinez-Mara O, et al. Oncostatin M is a potent mitogen for AIDS-Kaposi’s sarcoma derived cells. Science 1992; 255: 1432. 1309. Miller AM, Lundberg K, Ozenci V, et al. CD4+ CD25 high T cells are enriched in the tumor and peripheral blood of prostate cancer patients. J Immunol 2006; 177: 7398–7405. 1310. Miller AM, Xu D, Asquith DL, et al. IL-33 reduces the development of atherosclerosis. J Exp Med 2008; 205: 339–346. 1311. Miller LL, Korn EL, Stevens DS, et al. Abrogation of the hematological and biological activities of the interleukin-1/granulocyte macrophage colony stimulating factor fusion protein PIXY321 by neutralizing antibodies in cancer patients receiving high dose carboplatin. Blood 1999; 93: 3250–3258. 1312. Miller RE, Jones J, Le T, et al. 4-1BB-specific monoclonal antibody promotes the generation of tumor-specific immune responses by direct activation of CD8 T cells in a CD40dependent manner. J Immunol 2002; 160: 1792–1800. 1313. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 Macrophages and the Th1/Th2 paradigm. J Immunol 2000; 164: 6166–6173. 1314. Miner KT and Croft M. Generation, persistence and modulation of Th0 effector cells: a role of autocrine IL-4 and IFN-γ. J Immunol 1998; 160: 5280–5287. 1315. Minty A, Chalon P, Derocq J-M, et al. Interleukin-13: a novel human cytokine regulating inflammatory and immune response. Nature 1993; 362: 248–250. 1316. Mire-Sluis A, Page LA, Wadhwa M, Thorpe R. Evidence for a signaling role for the α chains of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors: divergent signaling pathways between GM-CSF/ IL-3 and IL-5. Blood 1995; 86: 2679. 1317. Mitsuyama K, Andus T, Caesar I, Roth M, Scholmerich J. Soluble interleukin-6 receptors in inflammatory bowel disease: relation to circulating interleukin-6. Gut 1995; 36: 45. 1318. Mittleman A, Puccio C, Gafney E, et al. A phase I pharmacokinetic study of recombinant human tumor necrosis factor administered by a 5-day continuous infusion. Invest New Drugs 1992; 10: 183–190. 1319. Mittler RS, Bailey TS, Klussman K, et al. Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy. J Exp Med 1999; 190: 1535–1540. 1320. Miu AL, Wakao H, O’Farrell AM, Harada N, Miyajima A. Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J 1995; 14: 1166–1175.

249 1321. Miura S, Ohtani K, Numata M, et al. Molecular cloning and characterization of a novel glycoprotein gp34, that is specifically induced by the human T cell leukemia virus I transactivator p40tax. Mol Cell Biol 1991; 11: 1313. 1322. Miyahira Y, Akiba H, Katae M, et al. A potent adjuvant effect of ligand to receptor Activator of NF-κB gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination. J Immunol 2003; 171: 6344–6348. 1323. Miyajima A, Mui AL, Ogorouchi T, Sakamaki K. Receptors for granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5. Blood 1993; 82: 1960. 1324. Miyazawa K, Mori A, Yamamoto K, Okudaira H. Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes: spontaneous activation of NF-κB and CBF1. Am J Pathol 1998; 152: 793. 1325. Miyomoto M, Prause O, Sjostrand M. Endogenous IL-17 as a mediator of neutrophil recruitment caused by endotoxin exposure in mouse airways. J Immunol 2003; 170: 4665–4672. 1326. Mizel SB, Dawyer JM, Krane SM, et al. Stimulation of rheumatoid synovial cell collagenase and prostaglandin production by partially purified lymphocyte-activating factor (interleukin-1). Proc Natl Acad Sci (USA) 1981; 78: 2474. 1327. Mizel SB. The interleukins. FASEB J 1989; 3: 2379–2388. 1328. Mizuno A, Amizuka N, Irie K, et al. Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Comm 1998; 247: 610. 1329. Mochizuki DY, Eisenman JR, Conlon PJ, et al. Interleukin 1 regulates hematopoietic activity, a role previously ascribed to hemopoietin 1. Proc Natl Acad Sci (USA) 1987; 84: 5267–5271. 1330. Modrowski D, Godet D, Marie PJ. Involvement of interleukin-1 and tumor necrosis factor α as endogenous growth factors in human osteoblastic cells. Cytokine 1995; 7: 720–726. 1331. Modur V, Li Y, Zimmerman GA, Prescott SM, McIntyre TM. Retrograde inflammatory signaling from neutrophils to endothelial cells by soluble interleukin-6 receptor α. J Clin Invest 1997; 100: 2752–2756. 1332. Moeller J, Hultner L, Schmitt E, Dormer P. Partial purification of mast cell growth-enhancing activity and its separation from IL-3 and IL-4. J Immunol 1989; 142: 3447. 1333. Mogi S, Sakurai J, Kohsaka T, et al. Tumor rejection by gene transfer of 4-1BB ligand into a CD80+ murine squamous cell carcinoma and the requirements of costimulatory molecules on tumor and host cells. Immunology 2000; 101: 541–547. 1334. Mohamadzadeh M, Takashima A, Dougherty I, et al. Ultraviolet B radiation up regulates the expression of IL-15 in human skin. J Immunol 1995; 155: 4492–4496. 1335. Moissec P, Yu CL, Ziff M. Lymphocyte chemotactic activity of interleukin 1. J Immunol 1984; 133: 2007. 1336. Molin D, Fisher M, Xiang Z, et al. Mast cells express functional CD30 ligand and are the predominant CD30L-positive cells in Hodgkin’s disease. Br J Haematol 2001; 114: 616–623. 1337. Moller A, Lippert U, Lessmann D, et al. Human mast cells produce IL-8. J Immunol 1993; 151: 3261. 1338. Moller P, Moller H, Sun Y, Dorbic T, et al. Increased non-major histocompatibility complex-restricted lytic activity in melanoma patients vaccinated with cytokine gene-transfected autologous tumor cells. Cancer Gene Ther 2000; 7: 976–984. 1339. Moller P, Sun Y, Dorbic T, et al. Vaccination with IL-7 genemodified autologous melanoma cells can enhance the antimelanoma lytic activity in peripheral blood of patients with good clinical performance status: a clinical phase I study. Br J Cancer 1998; 77: 1907–1916.

250 1340. Molmenti EP, Ziambaras T, Perlmutter DH. Evidence for an acute phase response in human intestinal epithelial cells. J Biol Chem 1993; 268: 12116–14124. 1341. Mongkolsapaya J, Cowper AE, Screaton GR, et al. Lymphocyte inhibitor of TRAIL (TNF-Related Apoptosis-Inducing Ligand): a new receptor protecting lymphocytes from the death ligand TRAIL. J Immunol 1998; 160: 3–6. 1342. Monroe JG, Haldar S, Prystowsky MB, Lammie P. Lymphokine regulation of inflammatory process: IL-4 stimulates fibroblast proliferation. Clin Immunol Immunopathol 1988; 49: 292. 1343. Montaner L, Doyle AG, Collin M, et al. Interleukin-13 inhibits human immunodeficiency virus type 1 production in primary blood-derived human macrophages in vitro. J Exp Med 1993; 178: 743. 1344. Montgomery RI, Warner MS, Lum BJ, Spear PG. Herpes simplex virus-1 entry into cells mediated by a novel member of the tumor necrosis factor superfamily. Cell 1996; 87: 427–436. 1345. Moon C, Yoo J-Y, Matarazzo V, et al. Leukemia inhibitory factor inhibits neuronal terminal differentiation through STAT3 activation. Proc Natl Acad Sci USA 2003; 99: 9015–9020. 1346. Moore BB, Arenberg DA, Addison CL, Keane MP, Strieter RM. Tumor angiogenesis is regulated by CXC chemokines. J Lab Clin Med 1998; 132: 97–103. 1347. Moore KW, O’Garra A, de Waal Malefyt R, Viera P, Mosmann TR. Interleukin-10. Ann Rev Immunol 1993; 11: 165. 1348. Moore KW, Viera P, Fiorentino DF, et al. Homology of cytokine synthesis inhibitor factor (IL-10) to the Epstein Barr virus gene BCRFI. Science 1990; 248: 1230–1234. 1349. Moore MAS, Warren DJ. Synergy of interleukin-1 and granulocyte colony-stimulating factor: in vivo stimulation of stem-cell recovery and hematopoietic regeneration following 5-fluorouracil treatment of mice. Proc Natl Acad Sci (USA) 1987; 84: 7134–7138. 1350. Moore TA, Zlotnik A. Differential effects of Flt2/flt3 ligand and stem cell factor on murine thymic progenitor cells. J Immunol 1997; 158: 4187. 1351. Morel F, Delwail V, Brizard A, et al. Effects of sCD23 on proliferation of leukemic cells from a patient with chronic myelogenous leukemia during blast crisis. Amer J Hematology 1993; 44: 60–62. 1352. Morel Y, Schiano de Colella J-M, et al. Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down regulates its own receptor. J Immunol 2000; 165: 4397–4404. 1353. Morgan H, Tumber A, Hill PA. Breast cancer cells induce osteoclast formation by stimulating host IL-11 production and downregulating granulocyte/macrophage colony stimulating factor. Int Journal Cancer 2004; 109: 653–660. 1354. Mori A, Suko M, Kaminuma O, Inoue S, et al. IL-15 promotes cytokine production of human T helper cells. J Immunol 1996; 156: 2400–2405. 1355. Mori L, Iselin S, De Libero G, Lesslauer W. Attenuation of collagen-induced arthritis in 55-kDa TNF receptor type I (TNFR1)-IgG1-treated and TNFR1-deficient mice. J Immunol 1996; 157: 3178. 1356. Moriera AL, Sampaio EP, Zmuidzinas A, et al. Thalidomide exerts its inhibitory action on tumor necrosis factor α by enhancing mRNA degradation. J Exp Med 1993; 177: 1675–1680. 1357. Morinaga Y, Suzuki H, Takatsuki F, et al. Contribution of IL-6 to the antiproliferative effect of IL-1 and tumor necrosis factor on tumor cell lines. J Immunol 1989; 143: 3538. 1358. Morisaki T, Uchiyama A, Yuzuki D, et al. Interleukin 4 regulates G1 cell cycle progression in gastric carcinoma cells. Cancer Res 1994; 54: 1113–1118.

Cytokines 1359. Morisaki T, Yuzuki D, Lin R, et al. Interleukin-4 receptor expression and growth inhibition of gastric carcinoma cells by interleukin-4. Cancer Res 1992; 52: 6059–6065. 1360. Morisset S, Patry C, Lora M, deBrum-Fernandes AJ. Regulation of cyclooxygenase-2 expression in bovine chondrocytes in culture by interleukin-1, tumor necrosis factor-α, glucocorticoids and 17β-estradiol. J Rheumatol 1998; 25: 1146. 1361. Moroz A, Eppolito C, Li Q, et al. Interleukin-21 enhances and sustains CD8+ T cell responses to achieve durable tumor immunity: comparative evaluation of IL-2, IL-15 and IL-21. J Immunol 2004; 173: 900–909. 1362. Morris A, Vetto JT, Ramstad T, et al. Induction of anti-mammary cancer immunity by engaging the OX-40 receptor in vivo. Breast Cancer Res Treatment 2001; 67: 71–80. 1363. Morris JC, Janik JE, White JD, et al. Preclinical and phase I clinical trial of blockade of IL-15 using Mikbeta1 monoclonal antibody in T cell large granular lymphocyte leukemia. Proc Natl Acad Sci USA 2006; 103: 401–406. 1364. Morrisey PJ, McKenna HJ, Widmer MB, et al. Steel factor (c-kit ligand) stimulates the in vitro growth of immature CD3−/ CD4−/CD8−thymocytes: synergy with IL-7. Cell Immunol 1994; 22: 1037. 1365. Morrissey PJ, Goodwin RG, Nordan RP, et al. Recombinant interleukin 7, pre-B cell growth factor has costimulatory activity on purified mature T cells. J Exp Med 1989; 169: 707–716. 1366. Morse MA, Nair S, Fernandez-Casal M, et al. Preoperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastatic colon cancer. J Clin Oncol 2000; 18: 3879–3882. 1367. Mortarini R, Borri A, Tragni G, et al. Peripheral burst of tumorspecific cytotoxic T lymphocytes and infiltration of metastatic lesions by memory CD8+ T cells in melanoma patients receiving interleukin-12. Cancer Res 2000; 60: 3559–3568. 1368. Mortarini R, Scarito A, Nonaka D, et al. Constitutive expression and costimulatory function of LIGHT/TNFSF14 on human melanoma cells and melanoma-derived microvesicles. Cancer Res 2005; 65: 3428–3436. 1369. Mortier E, Bernard J, Plet A, Jacques Y. Natural, proteolytic release of a soluble form of human IL-15 receptor α-chain that behaves as a specific, high affinity IL-15 antagonist. J Immunol 2004; 173: 1681–1688. 1370. Moseley TA, Haudenschild DR, Rose L, Reddi AH. Interleukin-17 family and IL-17 receptors. Cytokine Growth Factor Rev 2003; 14: 155–174. 1371. Moser B, Schumacher C, von Tscharner V, et al. Neutrophil activating peptide 2 and melanoma growth stimulatory activity interact with neutrophil activating peptide 1/interleukin 8 receptors on human neutrophils. J Biol Chem 1991; 266: 10667. 1372. Mosley B, Beckman MP, March CJ, et al. The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms. Cell 1989; 59: 335–348. 1373. Mosley B, De Imus C, Friend D, et al. Dual oncostatin M (OSM) receptors: cloning and characterization of an alternative signaling subunit conferring OSM specific receptor activation. J Biol Chem 1996; 271: 32635–32643. 1374. Mosmann T, Bond M, Coffman R, et al. T cell and mast cell lines respond to B cell stimulatory factor 1. Proc Nat Acad Sci USA 1986; 83: 1857. 1375. Mosmann T, Cherwinski H, Bond M, et al. Two types of murine helper T cell clones. 1. Definition to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136: 2348. 1376. Mosmann TR. Cytokine secretion patterns and cross regulation of T cell subsets. Immunol Res 1991; 10: 183–188.

Walter M. Lewko and Robert K. Oldham 1377. Moss ML, Jin S-LC, Milla ME, et al. Cloning of a disintegrin metalloproteinase that processed precursor tumor necrosis factor-α. Nature 1997; 385: 733. 1378. Motro B, Itin A, Sachs L, et al. Pattern of interleukin 6 gene expression in vivo suggests a role for this cytokine in angiogenesis. Proc Natl Acad Sci USA 1990; 87: 3092–3096. 1379. Motzer RJ, Rakhit A, Thomson JA, et al. Randomized multicenter phase II trial of subcutaneous recombinant human interleukin-12 versus interferon-α 2a for patients with advanced renal cell carcinoma. J Interferon Cytokine Res 2001; 21: 257–263. 1380. Movat HZ, Burrowes CE, Cybulsky MI, Dinarello CA. Acute inflammation and a Shwartzman-like reaction induced by interleukin-1 and tumor necrosis factor. Synergistic action of the cytokines in the induction of inflammation and microvascular injury. Am J Pathol 1987; 129: 463. 1381. Mrozek E, Anderson P, Caliguri MA. Role of interleukin 15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells. Blood 1996; 87: 2632. 1382. Muchamuel T, Menon S, Piscane P, Howard MC, Cockayne DA. IL-13 protects mice from lipopolysaccharide-induced lethal endotoxemia: correlation with down-modulation of TNF-α, IFN-γ and IL-12 production. J Immunol 1997; 158: 2898–2903. 1383. Muegge K, Vial MP, Durum SK. Interleukin 7: a cofactor for V(D)J rearrangement of the T cell receptor β gene. Science 1993; 261: 93–95. 1384. Mukaida N, Harada A, Yasumoto K, et al. Properties of proinflammatory cell type specific leukocyte chemotactic cytokines interleukin 8 (IL-8) and monocyte chemotactic and activating factor (MCAF). Microbiol Immunol 1992; 36: 773–789. 1385. Mule JJ, Custer MC, Travis WD, Rosenberg SA. Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice. J Immunol 1992; 148: 2622. 1386. Mule JJ, Shu S, Rosenberg SA. The antitumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo. J Immunol 1985; 135: 646. 1387. Mule JJ, Shu S, Schwarz SL, et al. Adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant interleukin-2. Science 1984; 225: 1487. 1388. Mule JJ, Smith CA, Rosenberg SA. Interleukin-4 (B cell stimulatory factor) can mediate the induction of lymphokine activated killer cell activity directed against fresh tumor cells. J Exp Med 1987; 166: 792–797. 1389. Mullberg J, Durie FH, Otten-Evans C, et al. A metalloproteinase inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor. J Immunol 1995; 155: 5198–5205. 1390. Mullberg J, Geib T, Jostock T, et al. IL-6 receptor independent stimulation of human gp130 by viral IL-6. J Immunol 2000; 164: 4672–4677. 1391. Mullberg J, Schooltink H, Stoyan T, et al. The soluble interleukin-6 receptor is generated by shedding. Eur J Immunol 1993; 23: 473. 1392. Mummery RS, Rider CC. Characterization of the heparin binding properties of IL-6. J Immunol 2000; 165: 5671–5679. 1393. Munder M, Mallo M, Eichmann K, Modolell M. Murine macrophages secrete interferon-γ upon combined stimulation with interleukin-12 and IL-18: a novel pathway of autocrine macrophage activation. J Exp Med 1998; 187: 2103. 1394. Muraguchi A, Hirano T, Tang B, et al. The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells. J Exp Med 1988; 167: 332. 1395. Muranski P, Boni A, Antony PA, et al. Tumor specific Th17polarized cells eradicate large established melanoma. Blood 2008: 112: 362–373.

251 1396. Murata K, Ishii N, Takano H, et al. Impairment of antigenpresenting cell function in mice lacking expression of OX40 ligand. J Exp Med 2000; 191: 365. 1397. Murata S, Ladle BH, Kim PS, et al. OX40 costimulation synergizes with GM-CSF whole cell vaccination to overcome established CD8+ T cell tolerance to an endogenous tumor antigen. J Immunol 2006; 176: 974–983. 1398. Murata Y, Takaki S, Migita M, et al. Molecular cloning and expression of the human interleukin-5 gene. J Exp Med 1992; 175: 341. 1399. Murphy CA, Langrish CL, Chen Y, et al. Divergent roles of IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 2003; 198: 1951–1957. 1400. Murphy P, Tiffany H. Cloning of complimentary DNA encoding a functional human IL-8 receptor. Science 1991; 253: 1280. 1401. Murphy PA, Chesney PA, Wood WB. Further purification of rabbit leukocyte pyrogen. J Lab Clin Med 1974; 83: 310. 1402. Murphy WJ, Back TC, Conlon KC, et al. Antitumor effects of interleukin 7 and adoptive immunotherapy on human colon carcinoma xenografts. J Clinical Invest 1993; 92: 1918–1924. 1403. Murrell GA, Jang CD, Williams RJ. Nitric oxide activates metalloprotease in articular cartilage. Biochem Biophys Res Commun 1995; 206: 15. 1404. Musashi M, Clark SC, Sudo T, Urdal DL, Ogawa M. Synergistic interactions between interleukin-11 and interleukin-4 in support of proliferation of primitive hematopoietic progenitors in mice. Blood 991; 78: 1448–1451. 1405. Musashi M, Yang Y-C, Paul SR, et al. Direct and synergistic effects of interleukin 11 on murine hematopoiesis in culture. Proc Nat Acad Sci USA 1991; 88: 765–769. 1406. Musgrave BL, Phu T, Butler JJ, Makrigiannis AP, Hoskin DW. Murine TRAIL (TNF-related apoptosis-inducing ligand) expression induced by T cell activation is blocked by rampamycin, cyclosporin A, and inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and protein tyrosine kinases: evidence for TRAIL induction via the T cell receptor signaling pathway. Exp Cell Res 1999; 252: 96. 1407. Muta H, Boise LH, Fang L, Podack ER. CD30 signals integrate expression of cytotoxic effector molecules, lymphocyte trafficking signals, and signals for proliferation and apoptosis. J Immunol 2000; 165: 5105–5111. 1408. Muzio M, Re F, Sironi M, et al. Interleukin-13 induces the production of interleukin-1 receptor antagonist (IL-1ra) and the expression of the mRNA for the intracellular (keratinocyte) form of the IL1-ra in human myelomonocytic cells. Blood 1994; 83: 1738. 1409. Nagasawa T, Kikutani H, Kishimoto T. Molecular cloning and structure of a pre-B cell growth-stimulating factor. Proc Natl Acad Sci USA 1994; 91: 2305. 1410. Nagata S and Suda T. Fas and Fas ligand: lpr and gld mutations. Immunol Today 1995; 16: 39. 1411. Nagata S, Ise T, Onda M, et al. Cell membrane specific epitopes on CD30: Potentially superior targets for immunotherapy. Proc Natl Acad Sci USA 2005; 102: 7946–7951. 1412. Nagayama H, Sato K, Kawasaki H, et al. IL-12 responsiveness and expression of IL-12 receptor in human peripheral blood monocyte-derived dendritic cells. J Immunol 2000; 165: 59–66. 1413. Naka T, Narazaki M, Hirata M, et al. Structure and function of a new STAT-induced STAT inhibitor. Nature 1997; 387: 924. 1414. Nakae S, Saijo S, Horai R, et al. IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist. Proc Natl Acad Sci USA 2003; 100: 5986–5990.

252 1415. Nakajima A, Kodama T, Morimoto S, et al. Antitumor effect of CD40 ligand elicitation on focal and systemic antitumor responses by IL-12 and B7. J Immunol 1998; 1661: 1901. 1416. Nakajima H, Gleich GJ, Kita H. Constitutive production of IL-4 and IL-10 and stimulated production of IL-8 by normal peripheral blood eosinophils. J Immunol 1996; 156: 4859–4866. 1417. Nakamura K, Okamura H, Wada M, et al. Endotoxin-induced serum factor that stimulates γ interferon production. Infect Immunol 1989; 57: 590. 1418. Nakamura S, Kashimoto S, Kajikawa F, Nakata K. Combination effect on recombinant interleukin 1α with antitumor drugs on syngeneic tumors in mice. Cancer Res 1991; 51: 215–221. 1419. Nakanishi K, Malek TR, Smith KA, et al. Both interleukin 2 and a second T cell derived factor in EL-4 supernatant have activity as differentiation factors in IgM synthesis. J Exp Med 1984; 160: 1605. 1420. Nakano H, Kishida T, Asada H, et al. Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma. J Gene Med 2006; 8: 90–99. 1421. Naldini A, Leali D, Pucci A, et al. IL-1β mediates the proangiogenic activity of osteopontin-activated human monocytes. J Immunol 2006; 177: 4267–4270. 1422. Namen AE, Schierer AE, March CJ, et al. B cell precursor-growth promoting activity and characterization of a growth factor active on lymphocyte precursors. J Exp Med 1988; 167: 988–1002. 1423. Namen AE, Lupton S, Hjerrild K, et al. Stimulation of B cell progenitors by cloned murine IL-7. Nature 1988; 333: 571. 1424. Namikawa R, Muench MO, DeVries, JE, Roncarolo M-G. The flt2/flt3 ligand synergizes with interleukin-7 in promoting stromal-cell-independent expansion and differentiation of human fetal pro-B cells in vitro. Blood 1996; 87: 1881. 1425. Nastala CL, Edington HD, McKinney TG, et al. Recombinant IL-12 administration induced tumor regression in association with IFN-production. J Immunol 1994; 153: 1697–1706. 1426. Natali PG, Nicotra MR, Sures I, et al. Breast cancer is associated with loss of c-kit oncogene product. Int J Cancer 1992; 52: 713–717. 1427. Natali PG, Nicotra MR, Sures I, et al. Expression of c kit receptor in normal and transformed human nonlymphoid tissues. Cancer Res 1992; 52: 6139–6143. 1428. Natali PG, Nicotra MR, Winkler AB, et al. Progression of human cutaneous melanoma is associated with loss of expression of c-kit protooncogene receptor. Int J Cancer 1992; 52: 197–201. 1429. Nathan CF. Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest 1987; 80: 1550. 1430. Nau GJ, Guilfoile P, Chupp GL, et al. A chemoattractant cytokine associated with granulomas in tuberculosis and silicosis. Proc Natl Acad Sci USA 1997; 6414–6419. 1431. Naume B, Espevik T, Sundan A. Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin 2 interleukin 7 and interleukin 12. Eur J Immunol 1993b; 23: 1831–1838. 1432. Naume B, Gately M, Espevik T. A comparative study of IL-12 (cytotoxic lymphocyte maturation factor), IL-2, and IL-7 induced effects on immunomagnetically purified CD56+ NK cells. J Immunol 1992; 148: 2429–2436. 1433. Naume B, Gately MK, Desai BB, et al. Synergistic effects of interleukin 4 and interleukin 12 on NK cell proliferation. Cytokine 1993; 5: 38–46. 1434. Navikas V, Matusevicius D, Soderstom M, et al. Increased interleukin-6 mRNA expression in blood and cerebrospinal fluid

Cytokines

1435.

1436.

1437. 1438.

1439.

1440.

1441. 1442.

1443.

1444.

1445.

1446.

1447.

1448.

1449.

1450. 1451.

1452. 1453.

mononuclear cells in multiple sclerosis. J Neuroimmunol 1996, 64: 63. Nawroth P, Handley D, Matsueda G, et al. Tumor necrosis factor/cachechtin-induced intravascular fibrin formation in meth A fibrosarcomas. J Exp Med 1988; 168: 637–647. Ndhlovu LC, Ishii N, Murata K, Sato T, Sugamura K. Critical involvement of the OX40 ligand signals in the T cell priming events during experimental autoimmune encephalomyelitis. J Immunol 2001; 167: 2991–2999. Neipel F, Albrecht JC, Ensser A, et al. Human herpesvirus 8 encodes a homolog of interleukin-6. J Virol 1997; 71: 839. Neis MM, Peters B, Dreuw A, et al. Enhanced expression levels of IL-31 correlate with IL-4 and IL-13 in atopic and allergic contact dermatitis. J Allergy Clin Immunol 2006; 118: 930–937. Nelms K, Huang H, Ryan J, Keegan A, Paul WE. Interleukin-4 receptor signaling mechanisms and their biological significance. Adv Exp Med Biol 1998; 452: 37–43. Nelson DR, Lauwers GY, Lau JY, Davis GL. Interleukin 10 treatment reduces fibrosis in patients with chronic hepatitis C: a pilot trial of interferon non-responders. Gastroenterology 2000; 118: 655–660. Neta R, Douches SD, Oppenheim JJ. Interleukin-1 is a radioprotector. J Immunol 1986; 136: 2483–2488. Neta R, Oppenheim JJ, Douces SD. Interdependence of the radioprotective effects of human recombinant interleukin 1α, tumor necrosis factor α, granulocyte-monocyte colony-stimulating factor and murine recombinant granulocyte-macrophage colony stimulating factor. J Immunol 1988; 140: 108. Netea MG, Azam T, Ferwerda G, et al. IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL-1β and IL-6 production through a caspase 1-dependent mechanism. Proc Natl Acad Sci USA 2005; 102: 16309–16314. Netea MG, Lewis EC, Azam T, et al. Interleukin-32 induces the differentiation of monocytes into macrophage-like cells. Proc Natl Acad Sci USA 2008; 102: 16309–16314. Neuman B, Luz A, Pfeffer K, Holzamnn B. Defective Peyer’s patch organogenesis in mice lacking the 55-kD receptor for tumor necrosis factor. J Exp Med 1996; 184: 259. Neumann D, Kollewe C, Martin MU, Boraschi D. The membrane form of the type II IL-1 receptor accounts for inhibitory function. J Immunol 2000; 165: 3350–3357. Nicholls SE, Heyworth CM, Dexter TM, et al. IL-4 promotes macrophage development by rapidly stimulating lineage restriction of bipotent granulocyte macrophage colony forming cells. J Immunol 1995; 155: 845–853. Nicholls SE, Winter S, Mottram R, Miyan JA, Whetton AD. Flt3 ligand can promote survival and macrophage development without proliferation in myeloid progenitor cells. Exp Hematol 1999; 27: 663–672. Nichols J, Evans EP, Smith AG. Establishment of germ-cell-line competent embryonic stem cells using differentiation-inhibiting activity. Development 1990; 110: 1341–1348. Nickolaides NC, Holroyd KJ, Ewart SL, et al. Interleukin 9: a candidate gene for asthma. Proc Natl Acad Sci USA 1997; 94: 13175. Niedbala W, Wei XO, Cai B, et al. IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells. Eur J Immunol 2007; 37: 3021–3029. Niehans GA, Brunner T, Frizelle SP, et al. Human lung carcinomas express Fas ligand. Cancer Res 1997; 57: 1007. Nieland JD, Graus YF, Dortmans YE, et al. CD40 and CD70 co-stimulate a potent in vivo antitumor response. J Immunother 1998; 21: 225–236.

Walter M. Lewko and Robert K. Oldham 1454. Nikolaus S, Raedler A, Kuhbacker T, Sfikas N, Folsch UR, Schreiber S. Mechanisms in failure of infliximab for Crohn’s disease. Lancet 2000; 356: 1475–1479. 1455. Nilsen EM, Johansen FE, Jahnsen FL, et al. Cytokine profiles of cultured microvascular endothelial cells from human intestine. Gut 1998; 42: 635–642. 1456. Nilsson SK, Johnston HM, Whitty GA, et al. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells. Blood 2005; 106: 1232–1239. 1457. Nishimura H, Hirimatsu K, Kobayashi N, et al. IL-15 is a novel growth factor for murine γδ T cells induced by Salmonella infection. J Immunol 1996; 156: 663. 1458. Nishimura T, Uchiyama Y, Yagi H, Hashimoto Y. Administration of slowly released recombinant interleukin 2. J Immunol Methods 1986; 91: 21. 1459. Noelle R, Krammer P, Ohara J, et al. Increased expression of Ia antigens on resting B cells. A new role for B cell growth factor 1. Proc Nat Acad Sci USA 1984; 81: 6149. 1460. Noguchi E, Shibasaki M, Arinami T, et al. Evidence for a linkage between asthma/atopy in childhood and chromosome 5q31-q33 in a Japanese population. Am J Respir Crit Care Med 1997; 156: 1390. 1461. Noguchi M, Nakamura Y, Russel SM, et al. Interleukin receptor γ chain, a functional component of the interleukin 7 receptor. Science 1993; 262: 1877–1880. 1462. Nogueira AC, Neubert R, Helge H, Neubert D. Thalidomide and the immune system. 3. Simultaneous up- and down-regulation of different receptors on human white blood cells. Life Sci 1994; 55: 77–92. 1463. Nonaka M, Nonaka R, Wooley K, et al. Distinct immunohistochemical localization of IL-4 in human inflamed airway tissues. IL-4 is localized to eosinophils in vivo and is released by peripheral blood eosinophils. J Immunol 1995; 155: 3234–3244. 1464. Nophar Y, Kemper O, Brakebusch C, et al. Soluble forms of tumor necrosis factor receptors (TNF-R’s). The cDNA for the type I TNF-R cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor. EMBO J 1990; 9: 3269–3278. 1465. Novick D, Kim S-H, Fantuzzi G, et al. Interleukin 18 binding protein: a novel modulator of the Th1 cytokine response. Immunity 1999; 10: 127–136. 1466. Numasaki M, Tagawa M, Iwata F, et al. IL-28 elicits antitumor responses against murine fibrosarcoma. J Immunol 2007, 178: 5086–5098. 1467. Numasaki M, Watanabe M, Suzuki T, et al. IL-17 enhances the net angiogenic activity and the in vivo growth of human nonsmall cell lung cancer in SCID mice through promoting CXCR2-dependent angiogenesis. J Immunol 2005; 175: 6177–6189. 1468. O’Connell J, Bennett MW, O’Sullivan GC, Collins JK, Shannon F. Fas counterattack: a molecular mechanism of tumor privilege. Mol Med 1997; 3: 294. 1469. O’Connell J, O’Sullivan GC, Collins JK, Shannon F. Fas counterattack: Fas mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med 1996; 184: 1075. 1470. O’Garra A, Chang R, Hatings R, et al. Ly IB (B-1) cells are the main source of B-cell derived IL-10. Eur J Immunol 1992; 22: 711. 1471. O’Malley WE, Archinstein B, Shear MJ. Action of bacterial polysaccharide on tumors: II. Damage of sarcoma 37 by serum of mice treated with Serratia marcescens polysaccharide and induced tolerance. Nutr Rev 1986; 46: 389–391. 1472. O’Neal KD, Yu-Lee L. The proline rich motif (PRM): a novel feature of the cytokine/hematopoietin receptor superfamily. Lymphokine Cytokine Res 1993; 12: 309–312.

253 1473. Obiri N, Hillman GG, Haas GP, et al. Expression of high affinity interleukin-4 receptors on human renal cell carcinoma cells and inhibition of tumor growth in vitro by interleukin-4. J Clin Investigation 1993; 91: 88–93. 1474. Obiri NI and Puri RK. Characterization of interleukin-4 receptors expressed in human renal cell carcinoma cells. Oncol Res 1995; 6: 419. 1475. Obiri NI, Hussain SR, Debinski W, Puri RK. Interleukin 13 inhibits growth of human renal carcinoma cells independently of the p140 interleukin 4 receptor chain. Clin Cancer Res 1996; 2: 1743–1749. 1476. Ogasawara H, Takeda-Hirokawa N, Sekigawa I, et al. Inhibitory effects of interleukin-16 on interleukin-2 production by CD4+ T cells. Immunology 1999; 96: 215–219. 1477. Ogasawara K, Hida S, Azimi N, et al. Requirement for IRF-1 in the microenvironment supporting the development of natural killer cells. Nature 1998; 391: 700. 1478. Ogata A and Anderson KC. Therapeutic strategies in the inhibition of interleukin-6 mediated multiple myeloma-mediated cell growth. Leuk Res 1996; 20: 303. 1479. Ogawa N, Itoh M, Gately Y. Abnormal production of B cell growth factor in patients with systemic lupus erythematosis. Clin Exp Immunol 1992; 89: 26–38. 1480. Oh J-W, Van Wagner N, Rose-John S, Benveniste EN. Role of IL-6 and the soluble IL-6 receptor in inhibition of VCAM-1 gene expression. J Immunol 1998; 161: 4992. 1481. Ohkobo C, Bigos D, Jain R. Interleukin-2 inducted leukocyte adhesion to the normal and tumor microvascular endothelium in vivo and its inhibition by dextran sulfate: implications for vascular leak syndrome. Cancer Res 1991; 512: 1561. 1482. Ohlsson K, Bjork P, Bergenfeldt M, Hageman R, Thompson RC. Interleukin-1 receptor antagonist reduces mortality from endotoxin shock. Nature 1990; 348: 550. 1483. Ohm JE, Shurin MR, Esche C, et al. Effect of vascular endothelial growth factor on dendritic generation in vivo. J Immunol 1999; 163: 3260–3268. 1484. Ohno R, Yamaguchi Y, Tohge T, et al. A dose escalation and pharmacology study of subcutaneously administered recombinant human interleukin-12 and its biological effects in Japanese patients with advanced malignancies. Clin Cancer Res 2000; 6: 2661–2669. 1485. Okada H, Pollack IF, Lieberman F, et al. Gene therapy of malignant gliomas: a pilot study of vaccination with irradiated autologous glioma and dendritic cells admixed with IL-4 transduced fibroblasts to elicit an immune response. Hum Gene Ther 2001; 12: 575–595. 1486. Okahara H, Yagita H, Miyake K, Okumura K. Involvement of very late activation antigen 4 (VLA-4) and Vascular Cell Adhesion Molecule 1 (VCAM-1) in tumor necrosis factor α enhancement of experimental metastasis. Cancer Res 1994; 54: 3233–3236. 1487. Okamoto I, Kohno K, Tanimoto T, Ikegami H, Kurimoto M. Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12. J Immunol 1999; 162: 3202–3211. 1488. Okamura H, Tsutsui H, Kashiwamura S-I, Yashimoto T-, Nakanishi K. IL18: A novel cytokine that augments both innate and acquired immunity. Adv Immunol 1998; 70: 281. 1489. Okamura H, Tsutsui H, Komatsu T, et al. Cloning a new cytokine that induces IFN-γ production by T-cells. Nature 1995; 378: 88. 1490. Okano F, Merad M, Furumoto K, et al. In vivo manipulation of dendritic cells overcomes tolerance to unmodified tumor-associated self-antigens and induces potent antitumor immunity. J Immunol 2005; 174: 2645–2652.

254 1491. Okayashiki K, Miyauchi J, Ohyashiki JH, et al. Interleukin 7 enhances colony growth and induces CD 20 antigen of a Ph+ acute lymphoblastic leukemia cell line. Leukemia 1993; 7: 1034–1040. 1492. Okazaki H, Ito M, Sudo T, et al. IL-7 promotes thymocytes bearing α/β or γ/δ T cell receptors in vitro: Synergism with IL-2. J Immunol 1989; 143: 2917–2922. 1493. Oldham RK, Lewko W, Good R, et al. Growth of tumor derived activated T-cells for the treatment of cancer. Cancer Biotherapy 1994; 9(3): 211–224. 1494. Oldham RK, Lewko WM, Good RW, Sharp E. Cancer biotherapy with interferon, interleukin 2 and tumor derived activated cells (TDAC). In Vivo 1994; 8: 653–664. 1495. Oldham RK, Thurman GB. Effects of cytokines on recovery from radiation exposure. In: Rotman M, Rosenthal CJ, eds. Medical radiology – Diagnostic imaging and radiation oncology. Concomitant continuous infusion chemotherapy and radiation. Springer-Verlag, New York, 1990. 1496. Olieman AF, Lienard D, Eggermont AM, et al. Hyperthermic isolated limb perfusion with tumor necrosis factor α, interferon γ, and melphalan for locally advanced nonmelanoma skin tumors of the extremities: a multicenter study. Arch Surg 1999; 134: 303–307. 1497. Oniki S, Nagai H, Horikawa T, et al. Interleukin-23 and interleukin-27 exert quite different antitumor and vaccine effects on poorly immunogenic melanoma. Cancer Res 2006; 66: 6395–6404. 1498. Onozaki K, Matsushima K, Aggarwal BB, Oppenheim JJ. Human interleukin 1 is a cytocidal factor for several tumor cell lines. J Immunol 1985; 135: 3962–3968. 1499. Opal SM, Fisher CJ Jr, Dhainaut JF, et al. Confirmatory interleukin-1 receptor antagonist trial in severe sepsis: a phase III, randomized, double-blind, placebo controlled, multicenter trial. The interleukin-1 receptor antagonist sepsis investigator group. Crit Care Med 1997; 25: 1115–1124. 1500. Oppenheim JJ, Kovacs EJ, Matsushima K, Durum SK. There is more than one interleukin-1. Immunol Today 1986; 7: 45–56. 1501. Oppenheim JJ, Neta R, Tiberghien P, et al. Interleukin-1 enhances survival of lethally irradiated mice treated with allogeneic bone marrow cells. Blood 1989; 74: 2257. 1502. Oppmann B, Lesley R, Blom B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 2000; 13: 715. 1503. Orazi A, Cooper RJ, Tong J, et al. Effects of recombinant human inteleukin-11 (Neumega rhIL-11 growth factor) on megakaryocytopoiesis in human bone marrow. Exp Hematol 1966; 24: 1289–1297. 1504. Orencole SF, Dinarello CA. Characterization of a subclone (D10S) of D10.G4.1 helper T-cell line, which proliferates to attomolar concentrations of interleukin-1 in absence of mitogens. Cytokine 1989; 1: 14–22. 1505. Orengo AM, Cantoni C, Neglia F, Biassoni R, Ferrini S. Reciprocal expression of CD70 and of its receptor, CD27, in human long term-activated T and natural killer (NK) cells: inverse regulation by cytokines and role in induction of cytotoxicity. Clin Exp Immunol 1997; 107: 608–613. 1506. Orlando S, Sironi M, Bianchi G, et al. Role of metalloprotease in the release of IL-1 type II decoy receptor. J Biol Chem 1997: 272: 31764. 1507. Ortaldo JR, Mason A, Overton R. Lymphokine-activated killer cells. Analysis of the progenitors and effectors. J Exp Med 1986; 164: 1193.

Cytokines 1508. Ortaldo JR, Mason AT, Gerard JP, et al. Effects of natural and recombinant IL-2 on regulation of IFNγ production and natural killer activity: lack of involvement of Tac antigen for these immunoregulatory effects. J Immunol 1984; 133: 779. 1509. Osaki T, Peron J-M, Cai Q, Okamura H, et al. IFN-γ-inducing factor/IL-18 Administration mediates IFN-γ-and IL-12-independent antitumor effects. J Immunol 1998; 160: 1742–1749. 1510. Oshima S, Saeki Y, Mina T, et al. Interleukin 6 plays a key role in the development of antigen-induced arthritis. Proc Natl Acad Sci USA 1998; 95: 8222. 1511. Oshima Y, Tanaka Y, Tozawa H, Takahashi Y, Maliszewski C, Delepesse G. Expression and function of Ox 40 ligand on human dendritic cells. J Immunol 1997; 159: 3838–3848. 1512. Oshimi K, Oshimi Y, Akutsu M, et al. Cytotoxicity of interleukin 2-activated lymphocytes for leukemia and lymphoma cells. Blood 1986; 68: 938. 1513. Ostensen ME, Thiele DL, and Lipsky PE. Tumor necrosis factor enhances cytolytic activity of human natural killer cells. J Immunol 1987; 138: 4185. 1514. Osterlund P, Veckman V, Siren J, et al. Gene expression and antiviral activity of α/β interferons and interleukin 29 in virus infected human myeloid dendritic cells. J Virol 2005; 79: 9608–9617. 1515. Ouaaz F, Li M, Begg AA. A critical role for the RelA subunit of nuclear factor κB in regulation of multiple immune-response genes and Fas-induced cell death. J Exp Med 1999; 189: 999. 1516. Overwiik WW, de Visser KE, Tirion FH, et al. Immunologic and antitumor effects of IL-23 as a cancer vaccine adjuvant. J Immunol 2006; 176: 5213–5222. 1517. Owen-Schaub LB, Gutterman JU, Grimm EA. Synergy of tumor necrosis factor and interleukin 2 in the activation of human cytotoxic lymphocytes: Effect of tumor necrosis factor and Interleukin 2 in the generation of human lymphokine activated killer cell cytotoxicity. Cancer Res 1988; 48: 788–792. 1518. Owyang AM, Zaph C, Wilson EH, et al. Interleukin 25 regulates type 2 cytokine-dependent immunity and limits inflammation in the gastrointestinal tract. J Exp Me 2006; 203: 843–849. 1519. Owyang AM, Zaph C, Wilson EH, et al. Interleukin-25 regulates type 2 cytokine –dependent immunity and limits chronic inflammation in the gastrointestinal tract. J Exp Med 2006; 203: 843–849. 1520. Ozaki K, Kikly K, Michalovich D, Young PR, Leonard WJ. Cloning of a type 1 cytokine receptor most related to the IL-2 receptor β chain. Proc Nat Acad Sci USA 2000; 97: 11439. 1521. Ozaki K, Spolski R, Ettinger R, et al. Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6. Immunol 2004; 173: 5361–5371. 1522. Ozaki K, Spolski R, Feng CG, et al. A critical role for IL-21 in regulating immunoglobulin production. Science 2002; 298: 1630. 1523. Page TH, Willcocks JL, Taylor-Fishwick DA, Foxwell BMJ. Characterization of a novel high affinity human IL-7 receptor. Expression on T cells and association with IL-7 driven proliferation. J Immunol 1993; 151: 4753–4763. 1524. Paige CJ, Figarella EF, Cuttito M, et al. Natural cytotoxic cells against solid tumors in mice: II. Some characteristics of the effector cells. J Immunol 1978; 121: 1827. 1525. Pajkrt D, Camoglio L, Tiel-van Buul MCM, et al. Attenuation of pro-inflammatory response by recombinant human IL-10 in human endotoxemia: effect of timing of recombinant human IL-10 administration. J Immunol 1997; 158: 3971–3977. 1526. Pan G, French D, Mao W, et al. Forced expression of murine IL-17 E induces growth retardation, jaundice, a Th2-biased

Walter M. Lewko and Robert K. Oldham

1527.

1528. 1529.

1530.

1531.

1532.

1533.

1534.

1535.

1536.

1537.

1538.

1539.

1540.

1541. 1542.

1543.

1544. 1545.

1546.

response, and multiorgan inflammation in mice. J Immunol 2001; 167: 6559–6567. Pan G, Ni J, Yu G, Wei YF, Dixit VM. TRUNDD, a new member of the TRAIL receptor family that antagonizes TRAIL signaling. FEBS Lett 1998; 424: 41. Pan G, O’Roarke K, Chinnaian AM, et al. The receptor for the cytotoxic ligand TRAIL. Science 1997; 276: 111. Pan PY, Zhang Y, Weber K, et al. OX40 ligation enhances primary and memory cytotoxic T lymphocyte responses in an immunotherapy for hepatic colon metastases. Mol Ther 2002; 6: 528–536. Pan Y, Lloyd C, Zhou H, et al. Neurotactin, a membrane-anchored chemokine up regulated in brain inflammation. Nature 1997; 387: 611. Pandrau D, Frances V, Martinez Valdez H, et al. Characterization of a t(1;19) pre-B acute lymphoblastic leukemia (ALL) cell line, which proliferates in response to IL-7. Leukemia 1993; 7: 635–642. Parada NA, Center DM, Kornfeld H, et al. Synergistic activation of CD4+ T cells by IL-16 and IL-2. J Immunol 1998; 160: 2115–2120. Parham C, Chirica M, Timans J, et al. A receptor for the heterodimeric IL-23 is composed of IL-12Rβ1 and a novel cytokine receptor subunit, IL23 R. J Immunol 2002; 168: 5699–56708. Parihar R, Nadella P, Jensen R, et al. Phase I trial of Herceptin and Interleukin-12 in patients with HER2-overexpressing malignancies. Proc ASCO 2001; 20: 1031. Park CC, Morel JCM, Amin MA, et al. Evidence of IL-18 as a novel angiogenesis mediator. J Immunol 2001; 167: 1644–1653. Park H, Li Z, Yang XO, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17.Nat Immunol 2005; 6: 1133–1141. Park LS, Friend D, Sassenfeld HM, Urdal DL. Characterization of human B cell stimulatory factor 1 receptor. J Exp Med 1987; 166: 476–488. Parnet P, Garka KE, Bonnert TP, Dower SK, Sims JE. IL-1 Rrp is a novel receptor like molecule similar to the type I interleukin 1 receptor and its homologues T1/ST2 and IL-R AcP. J Biol Chem 1996; 271: 3967. Parrish-Novak J, Dillon SR, Nelson A, et al. Interleukin-21 and its receptor are involved in NK expansion and regulation of lymphocyte function. Nature 2000; 408: 57. Parrish-Novak J, Xu W, Brender T, et al. Interleukins 19, 20, and 24 signal through two distinct receptor complexes. Differences in receptor–ligand interactions mediate unique biological functions. J Biol Chem 2002; 277: 47517–47523. Parronchi P, Maggi E, Romagnani S. Redirecting Th2 responses in allergy. Curr Top Microbiol Immunol 1999; 238: 27–56. Paterson DJ, Jeffries WA, Green JR, et al. Antigens of activated rat T thymocytes including a molecule of 50,000 Mr detected on CD4 positive T blasts. Mol Immunol 1987; 24: 1281. Paul SR, Bennett F, Calvetti JA, et al. Molecular cloning of a cDNA encoding interleukin 11, a novel stromal cell derived lymphopoietic and hematopoietic cytokine. Proc Nat Acad Sci USA 1990; 87: 7512. Paul WE, Ohara J. B-cell stimulatory factor-1/interleukin 4. Annu Rev Immunol 1987; 5: 429. Paulukat J, Bosmann M, Nold M, et al. Expression and release of IL-18 binding protein in response to interferon-γ.J Immunol 2001; 167: 7038–7043. Pawlowska AB, Hashino S, McKenna H, Taylor PA, Blazar BR. In vitro tumor pulsed or in vivo Flt3 ligand-generated dendritic

255

1547. 1548.

1549.

1550.

1551.

1552.

1553.

1554.

1555.

1556. 1557. 1558.

1559.

1560.

1561.

1562.

1563.

1564.

1565.

1566.

cells provide protection against acute myelogenous leukemia in nontransplanted or syngeneic bone marrow-transplanted mice. Blood 2001; 97: 1474–1482. Peace DJ, Cheever MA. Toxicity and therapeutic efficacy of high dose interleukin-2. J Exp Med 1989; 169: 161. Pechon JJ, Morrisey PJ, Grabstein KH, et al. Early lymphocyte expansion is severly impaired in interleukin-7 receptor-deficient mice. J Exp Med 1994; 180: 1955–1960. Peck R, Brockhaus M, Frey JR. Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells. Cell Immunol 1989; 122: 1. Peluso I, Fantini MC, Fina D, et al. IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes. J Immunol 2007; 178: 732–739. Pene J, Gauchat JF, Lecart S, et al. IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells. J Immunol 2004; 172: 5142–5147. Penn J, Rousset F, Briere F, et al. IgE production by normal human lymphocytes is induced by interleukin-4 and suppressed by interferon-γ and α and prostaglandin E2. Proc Natl Acad Sci USA 1988; 85: 880. Pennica D, Nedwin GE, Hayflick JS, et al. Human tumor necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 1984; 312: 724. Peritt D, Robertson S, Gri G, Showe L, Aste-Amezaga M, Trinchieri G. Differentiation of human NK cells into NK1 and NK2 subsets. J Immunol 1998; 161: 5821–5824. Peron J-M, Esche C, Subbotin VM, et al. FLT3 ligand administration inhibits liver metastases: role of NK cells. J Immunol 1998; 161: 6164–6170. Perper SJ, Browning B, Burkly LC, et al. TWEAK is a novel arthritogenic mediator. J Immunol 2006; 177: 2610–2620. Perregaux D, Gabel CA. Interleukin-1β maturation and release in response to ATP and nigericin. J Biol Chem 1994; 269: 15195. Perrigoue JG, Li J, Zaph C, et al. IL-31–IL-31R interactions negatively regulate type 2 inflammation in the lung. J Exp Med 2007; 204: 481–487. Pertyl U, Luster AD, Varki NM, et al. IFN-γ-inducible protein is essential for the generation of a protective tumor-specific CD8 T cell response induced by single-chain IL-12 gene therapy. J Immunol 2001; 166: 6944–6951. Perussia B, Ramoni C, Anegon I, et al. Preferential proliferation of natural killer cells among peripheral blood mononuclear cells cocultured with B lymphoblastoid lines. Nat Immun Cell Growth Regul 1987; 6: 171. Pesce J, Kaviratne M, Ramalingam TR, et al. The IL-21 receptor augments Th2 effector function and alternative macrophage activation. J Clin Invest 2006; 116: 2044–2055. Peters M, Muller A, Rose-John S. Interleukin-6 and soluble interleukin-6 receptor: direct stimulation of gp130 and hematopoiesis. Blood 1998; 92: 3495. Peters M, Schirmacher P, Goldschmidtt J, et al. Extramedullary expansion of hematopoietic progenitor cells in interleukin (IL)6-sIL-6R double transgenic mice. J Exp Med 1997; 185: 755. Petzer AL, Hogge DE, Landsdorp PM, Reid DS, Eaves CJ. Selfrenewal of primitive human hematopoietic cells (long-termculture initiating cells) in vitro and their expansion in defined medium. Proc Natl Acad Sci USA 1996; 93: 1470–1474. Peveri P, Walz A, Dewald B, Baggiolini M. A neutrophil activating factor produced by human mononuclear phagocytes. J Exp Med 1988; 167: 1547. Pflanz S, Kurth I, Grotzinger J, Heinrich PC, Muller-Newen G. Two different epitopes of the signal transducer gp130 sequen-

256

1567.

1568.

1569.

1570.

1571.

1572.

1573.

1574.

1575.

1576.

1577.

1578.

1579.

1580.

1581.

1582.

1583.

1584.

1585.

Cytokines tially cooperate in IL-6-induced receptor activation. J Immunol 2000, 165: 7042–7049. Phillips JH, Lanier LL. Dissection of the lymphokine-activated killer phenomenon. Relative contribution of peripheral blood natural killer cells and T lymphocytes to cytolysis. J Exp Med 1986; 164: 814. Piacibello W, Sanavio F, Garetto L, et al. Extensive amplification and self-renewal of human primitive hematopoietic stem cells form cord blood. Blood 1997; 89: 2644–2653. Picarella D, Kratz A, Li C-B, Ruddle NH, Flavel RA. Insulinitis in transgenic mice expressing TNF-β (lymphotoxin) in the pancreas. Proc Natl Acad Sci USA 1992; 89: 10036. Pinto A, Aldinucci D, Gloghini A, et al. Human eosinophils express functional CD30 ligand and stimulate proliferation of a Hodgkin’s disease cell line. Blood 1996; 88: 3299–3305. Pippig SD, Pena-Rossi C, Long J, et al. Robust B cell immunity but impaired T cell proliferation in the absence of CD134 (Ox40). J Immunol 1999; 163: 6520. Pippin BA, Rosenstein M, Jacob WF, et al. Local IL-4 delivery enhances immune reactivity to murine tumors: Gene therapy in combination with IL-2. Cancer Gene Therapy 1994; 1: 35–42. Pirhonen J, Matikainen S, Julkunen I. Regulation of virusinduced IL-12 and IL-23 expression in human macrophages. J Immunol 2002; 169: 5673–5678. Pisa P, Halapi E, Pisa EK, et al. Selective expression of interleukin 10, interferon γ, and granulocyte macrophage colony stimulating factor in ovarian cancer biopsies. Proc Nat Acad Sci 1992; 89: 7708. Piskin G, Sylva-Steenland RM, Bos JD, et al. In vitro expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin. J Immunol 2006; 176: 1908–1915. Pitti RM, Marsters SA, Ruppert S, et al. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 1996; 271: 12687. Pizaro TT, Michie MH, Bentz M, et al. IL-18, a novel immunoregulatory cytokine, is upregulated in Crohn’s disease: expression and localization in intestinal mucosal cells. J Immunol 1999; 162: 6829. Plate JMD, Knospe WH, Harris JE, Gregory SA. Normal and aberrant expression of cytokines in neoplastic cells from lymphocytic leukemias. Human Immunol 1993; 36: 249–258. Platzer C, Meisel C, Vogt K, Platzer M, Volk HD. Up-regulation of IL-10 by tumor necrosis factor-α and cAMP elevating drugs. Int Immunol 1995; 7: 517. Plummer R, Attard G, Pacey S, et al. Phase I and pharmacokinetic study of lexatumumab in patients with advancer cancers. Clin Cancer Res 2007; 13: 6187–6194. Pober JS, Lapeirre LA, Stolpen AH. Activation of cultured human endothelial cells by recombinant lymphotoxin: comparison with tumor necrosis factor and interleukin-1 species. J Immunol 1987; 138: 319. Poindexter NJ, Walch ET, Chada S, Grimm EA. Cytokine induction of interleukin-24 in human peripheral blood mononuclear cells. J Leukocyte Biol 2005; 78: 745–752. Polek TC, Talpaz M, Darnay BG, et al. Tweak mediates signal transduction and differentiation of RAW264.7 cells in the absence of Fn/TweakR. J Biol Chem 2003; 278: 32317–32323. Pollok KE, Kim YJ, Hurtado J, et al. 4-1BB T cell antigen binds to mature B cells and macrophages, and costimulates antimu-primed B cells. Eur J Immunol 1994; 24: 367–374. Pollok KE, Kim YJ, Zhou Z, et al. Inducible T cell antigen 4-1BB: analysis of expression and function. J Immunol 1993; 150: 771–781.

1586. Poltorak A, He X, Smirnova I, et al. Defective LPS signaling in CeH/HeJ and C57BL/10ScCr mice: mutations in the Tlr gene. Science 1998; 282: 2085. 1587. Porteu F, Nathan C. Shedding of tumor necrosis factor receptors by activated human neutrophils. J Exp Med 1990; 172: 599. 1588. Portielje JE, Kruit WH, Schuler M, et al. Phase I study of subcutaneously administered recombinant human interleukin 12 in patients with advanced renal cell cancer. Clin Cancer Res 1999; 5: 3983–3989. 1589. Powell DJ Jr, Dudley ME, Robbins PF, Rosenberg SA. Transition of late stage effector T cells to CD27+ CD28+ tumorreactive effector T cells in humans after adoptive cell transfer therapy. Blood 2005; 105: 241–250. 1590. Powell DJ, Filipe-Silva A, Merino MJ, et al. Administration of a CD25-directed immunotoxin, LMB-2, to patients with metastatic melanoma induces a selective partial reduction in regulatory T cells in vivo. J Immunol 2007; 179: 4919–4928. 1591. Prasad KV, Ao Z, Yoon Y, et al. CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds Siva, a proapoptotic protein. Proc Natl Acad Sci USA 1997; 94: 6346–6351. 1592. Prieyl JA, LeBien TW. Interleukin 7 independent development of human B cells. Proc Natl Acad Sci USA 1006; 93: 10348–10353. 1593. Pruitt JH, Welborn MB, Edwards PD, et al. Increased soluble interleukin-1 type II receptor concentrations in postoperative patients and in patients with sepsis syndrome. Blood 1996: 87: 3282. 1594. Pulaski BA, Yeh KY, Shastri N, et al. Interleukin-3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex re-presentation of exogenous antigen by tumor-infiltrating antigen-presenting cells. Proc Natl Acad Sci USA 1996; 93: 3669. 1595. Pulendran B, Banchereau J, Burkeholder S, et al. Flt-3 ligand and granulocyte-macrophage colony stimulating factor mobilize distinct human dendritic cell subsets in vivo. J Immunol 2000; 165: 566–572. 1596. Pulendran B, Smith JL, Caspary G, et al. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc Natl Acad Sci USA 1999; 96: 1036. 1597. Punnonen J, Aversa G, Cocks BG, et al. Interleukin 13 induces interleukin 4 independent IgG4 and IgE synthesis and CD 23 expression by B cells. Proc Nat Acad Sci USA 1993; 90: 3730–3735. 1598. Punnonen J, de Vries JE. IL-13 induces proliferation, Ig isotype switching, and Ig synthesis by immature fetal B cells. J Immunol 1994; 152: 1094. 1599. Puolakkainen P, Twardzik D, Ranchalis J, et al. Increase of plasma transforming growth factor β (TGFβ) during immunotherapy with IL-2. Cancer Invest 1995; 13: 583–589. 1600. Puren AJ, Fantuzzi G, Dinarello CA. Gene expression, synthesis and secretion of IL-1β and IL-18 are differentially regulated in human blood mononuclear cells and mouse spleen cells. Proc Natl Acad Sci 1999; 96: 2556. 1601. Puren AJ, Fantuzzi G, Gu Y, Su M S-S, Dinarello CA. Interleukin-18 (INF-γ-inducing factor) induces IL-1β and IL-8 via TNFα production from non-CD14+ human blood mononuclear cells. J Clin Invest 1998; 101: 711. 1602. Puri RK, Ogata M, Leland GM, et al. Expression of high affinity Interleukin 4 receptors on murine sarcoma cells and receptormediated cytotoxicity of tumor cells to chimeric protein between Interleukin 4 and pseudomonas exotoxin. Cancer Res 1991; 51: 3011–3017. 1603. Qi J, Kreutzer DL. Fibrin activation of vascular endothelial cells. Induction of IL-8 secretion. J Immunol 1995; 155: 867–876.

Walter M. Lewko and Robert K. Oldham 1604. Qi JC, Wang J, Mandadi S, et al. Human and mouse mast cells use the tetraspanin CD9 as an alternate interleukin-16 receptor. Blood 2007; 107: 135–142. 1605. Queen MM, Ryan RE, Holzer RG, et al. Breast cancer cells stimulate neutrophils to produce oncostatin M: Potential implications for tumor progression. Cancer Res 2005; 65: 8896–8904. 1606. Quinn TD, Miller FN, Wilson MA, et al. Interleukin-2-induced lymphocyte infiltration of multiple organs is differentially suppressed by soluble tumor necrosis factor receptor. J Surg Res 1990; 56: 117. 1607. Quinn, Dejoy S, Jeyasellan R, Torley L, et al. Attenuation of interleukin-2 induced pulmonary vascular leak syndrome by low doses of oral methotrexate. Cancer Res 1995; 55: 4929. 1608. Rabin EM, Ohara J, and Paul WE. B cell stimulatory factor 1 activates resting B cells. Proc Nat Acad Sci USA 1985; 82: 2935–2939. 1609. Rabinovitch A, Suarez-Pinson O, Sorenson O, Rajotte RV, Power RF. TNF-α down-regulates type I cytokines and prolongs survival of syngeneic islet grafts in nonobese diabetic mice. J Immunol 1997; 159: 6298. 1610. Racadot E, Audhuy B, Duvernoy H, et al. Clinical and immunological follow-up of patients with AIDS-associated Kaposi’s sarcoma treated with an anti-IL-6 monoclonal antibody. Cytokines Mol Ther 1995; 1: 133–1338. 1611. Radaeva S, Sun R, Pan HN, et al. Interleukin 22 (IL-22) plays a protective role in T cell-mediated murine hepatitis: IL-22 is a survival factor for hepatocytes via STAT3 activation. Hepatology 2004; 39: 1332–1342. 1612. Rafi AQ, Zeytun A, Bradley MJ, et al. Evidence for involvement of Fas ligand and perforin in the induction of vascular leak syndrome. J Immunol 1998; 161: 3077–3086. 1613. Rahangdale S, Morgan R, Heijens C, et al. Chemokine receptor CXCR3 desensitization by IL-16/CD4 signaling is dependent on CCR5 and intact membrane cholesterol. J Immunol 2006; 176: 2337–2345. 1614. Ralston SH, Graham R, Russel G, Gowen M. Estrogen inhibits release of tumor necrosis factor from peripheral blood mononuclear cells in postmenopausal women. J Bone Miner Res 1990; 5: 983. 1615. Raman VS, Akondy RS, Rath S, et al. Ligation of CD27 on B cells in vivo during primary immunization enhances commitment to memory B cell responses. J Immunol 2003; 171: 5876–5881. 1616. Raman VS, Bal V, Rath S, George A. Ligation of CD27 on murine B cells responding to T-dependent and T-independent stimuli inhibits the generation of plasma cells. J Immunol 2000; 165: 6809–6815. 1617. Ramesh R, Stewart AL, Boqui A. Melanoma differentiation associated antigen7/interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor. Cancer Res 2003; 63: 5105–5113. 1618. Rand T, Cruikshank WW, Center DM, Weller PF. CD4-mediated stimulation of eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration. J Exp Med 1991; 173: 1521. 1619. Ranheim EK, Cantwell MJ, Kipps TJ. Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells. Blood 1995; 85: 3556–3565. 1620. Ray A. A SAF binding site in the promoter region of human γ-fibrinogen gene functions as an IL-6 response element. J Immunol 2000; 165: 3411–3417. 1621. Re F, Sironi M, Muzio M, et al. Inhibition of interleukin-1 responsiveness by type II receptor gene transfer: a surface “receptor” with anti-interleukin-1 function. J Exp Med 1996; 183: 1841.

257 1622. Reali C, Curto M, Sogos V, et al. Expression of CD137 and its ligand in neurons, astrocytes, and microglia: modulation by FGF-2. J Neurosci Res 2003; 74: 67–73. 1623. Redlich CA, Gao X, Rockwell S, Kelly M, Elias JA. IL-11 enhances survival and decreases TNF production after radiation-induced thoracic injury. J Immunol 1996; 157: 1705. 1624. Redlinger RE Jr., Maillard RB, Lotze MT, et al. Synergistic Interleukin-18 and low dose interleukin-2 promote regression of established murine neuroblastoma in vivo. J Pediatric Surg 2003; 38: 301–307. 1625. Redman BG, Abubakr Y, Chou T, Esper P, Flaherty LE. Phase II recombinant interleukin-1β in patients with metastatic renal cell carcinoma. J Immunother Emphasis Tumor Immunol 1994; 16: 211–215. 1626. Refaeli Y, Van Parijs L, London CA, Tschopp J, Abbas A. Biochemical mechanisms of IL-2 regulated Fas mediated T cell apoptosis. Immunity 1998; 9: 615. 1627. Reich K, Garbe C, Blaschke V, et al. Response of psoriasis to interleukin-10 is associated with suppression of cutaneous type I inflammation, downregulation of the epidermal interleukin-8/ CXCR2 pathway and normalization of keratinocyte maturation. J Invest Dermatol 2001; 116: 319–329. 1628. Reitamo SA, Remitz A, Uitto J. Interleukin-10 modulates type I collagen and matrix metalloprotease gene expression in cultured human skin fibroblasts. J Clin Invest 1994; 94: 2489. 1629. Reitschel E, Brade H, Holst O, et al. Bacterial endotoxin: chemical constitution, biological recognition, host response, and immunological detoxification. Curr Topics Microbial Immunol 1996; 216: 40. 1630. Reittie JE, Hoffbrand AV. Interleukin-4 (IL-4) inhibits proliferation and spontaneous cytokine release by chronic leukemia cells. Leukemia Res 1994; 18: 55–60. 1631. Ren F, Zhan X, Martens G, et al. Pro-IL-16 regulation in activated murine CD4+ lymphocytes. J Immunol 2005; 174: 2738–2745. 1632. Renauld J-C, van der Lugt, Vink A, et al. Thymic lymphomas in interleukin 9 transgenic mice. Oncogene 1994; 9: 1327. 1633. Renauld J-C, Vink A, Louahed A, Van Snick J. Interleukin-9 is a major anti-apoptotic factor for thymic lymphomas. Blood 1995; 85: 1300. 1634. Renauld JC, Druez C, Kermouni A, et al. Expression cloning of murine and human Interleukin 9 receptor DNAs. Proc Nat Acad Sci USA 1992; 89: 5690–5694. 1635. Renkl AC, Wussler J, Ahrens T, et al. Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarization phenotype. Blood 2005; 106: 946–955. 1636. Rennert PD, Browning JL, Mebius R, Mackey F, Hochman P. Surface lymphotoxin α/β complex is required for the development of peripheral lymphoid organs. J Exp Med 1996; 184: 1999. 1637. Rennick D, Yang G, Muler-Sieburg C, et al. Interleukin 4 (B cell stimulatory factor 1) can enhance or antagonize the factor dependent growth of hematopoietic progenitor cells. Proc Nat Acad Sci USA 1987; 84: 6889. 1638. Renz H, Domenico J, Gelfand EW. IL-4 dependent upregulation of IL-4 receptor expression in murine T and B cells. J Immunol 1991; 146: 3049. 1639. Revhaug A, Michie HR, Manson JM, et al. Inhibition of cyclooxygenase attenuates the metabolic response to endotoxin in humans. Arch Surg 1988; 123: 162. 1640. Reznikov LL, Kim SH, et al. IL-18 binding protein increases spontaneous and IL-1 induced prostaglandin production via inhibition of IFN-γ. Proc Natl Acad Sci USA 2000; 97: 2174.

258 1641. Rich BE, Kupper TS. Cytokines: IL-20 – a new effector in skin inflammation. Curr Biol 2001; 10: R531–534. 1642. Richards CD, Brown TJ, Shoyab M, Baumann H, Gaudie J. Recombinant oncostatin-M stimulates the production of acute phase proteins in hepatocytes in vitro. J Immunol 1992; 148: 1731. 1643. Richards CD, Langdon C, Botelho F, Brown TJ, Agro A. Oncostatin M inhibits IL-1-induced expression of IL-8 and granulocyte-macrophage colony-stimulating factor by synovial and lung fibroblasts. J Immunol 1996; 156: 343–349. 1644. Richards CD, Shoyab M, Brown TJ, Gauldie J. Selective regulation of metalloproteinase inhibitor (TIMP-1) by oncostatin M in fibroblasts in culture. J Immunol 1993; 150: 5596. 1645. Ridge JP, Rosa FD, Matsinger P. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 1998; 393: 474. 1646. Ringheim GE, Burgher KL, Heroux JA. Interleukin-6 mRNA expression by cortical neurons in culture: evidence for neuronal sources of interleukin-6 production in the brain. J Neuroimmunol 1995; 63: 113–123. 1647. Rissoan MC, Soumelis V, Kadowaki N, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science1999; 283: 1183. 1648. Roake JJ. Rao AS, Morris PJ, et al. Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor and interleukin 1. J Exp Med 1995; 181: 2237. 1649. Robb RJ. Human T-cell growth factor: purification and interaction with a cellular receptor. Lymphokine Res 1982; 1: 37. 1650. Robertson MJ, Cameron C, Atkins MB, et al. Immunological effects of interleukin-12 administration by bolus intravenous injection to patients with cancer. Clin Cancer Res 1999; 5: 9–16. 1651. Robertson MJ, Mier JW, Logan T, et al. Clinical and biological effects of recombinant human interleukin-18 administered by intravenous infusion to patients with advanced cancer. Clin Cancer Res 2006; 15: 4265–4273. 1652. Robinson D, Shibuya A, Mui A, et al. IGIF does not drive Th1 development but synergizes with IL-12 for interferon-γ production and activates IRAK and NFκB. Immunity 1997; 7: 571. 1653. Rochester CL, Ackerman SJ, Zheng T, Elial JA. Eosinophil– fibroblast interactions: granule major basic protein interacts with IL-1 and Transforming-growth factor-β in the stimulation of lung fibroblast interleukin-6-type cytokine production. J Immunol 1996; 156: 4449. 1654. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF. A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA 1998; 95: 588. 1655. Roda JM, Parihar R, Lehman A. Interleukin-21 enhances NK cell activation in response to antibody-coated targets. J Immunol 2006; 177: 120–129. 1656. Rollins BJ. Chemokines. Blood 997; 90: 909. 1657. Rollo E, Laskin DE, Denhardt DT. Osteopontin inhibits nitric oxide production and cytotoxicity by activated RAW264.7 macrophages. J Leukocyte Biol 1996; 60: 397. 1658. Rolph MS, Kaufman SHE. CD40 signaling converts a minimally immunogenic antigen into a potent vaccine against the intracellular pathogen Listeria monocytogenes. J Immunol 2001; 166: 5115–5121. 1659. Romagnani S. Human TH1 and TH2 subset: Regulation of differentiation and role in protection and immunopathology. Int Arch Allergy Immunopathology Int Arch Allergy Immunol 1992; 98: 279–285.

Cytokines 1660. Romer J, Hasselager E, Norby PL, et al. Epidermal overexpression of interleukin-19 and IL-20 mRNA in psoriatic skin dissappears after short-term treatment with cyclosporine or calcipotriol. J Invest Dermatol 2003; 121: 1306–1311. 1661. Rook AH, Zaki MH, Wysocka M, et al. The role for interleukin-12 therapy of cutaneous T cell lymphoma. Ann NYAS 2001; 941: 177–184. 1662. Rooney IA, Butrovich KD, Glass AA, et al. The lymphotoxin-β is necessary and sufficient for LIGHT-mediated apoptosis of tumor cells. J Biol Chem 2000; 275: 14307. 1663. Rosa LE, Satokar AA, Roth KM, et al. Interleukin-27R (WSX1/T-cell cytokine receptor) gene deficient mice display enhanced resistance to leishmania donovani infection but develop severe liver immunopathology. Am J Pathol 2006; 168: 158–169. 1664. Rose TM, Bruce AG. Oncostatin M is a member of the cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin-6. Proc Natl Acad Sci USA 1991; 88: 8641. 1665. Rose-John S, Heinrich PC. Soluble receptors for cytokines and growth factors: generation and biological function. Biochem J 1994; 300: 281. 1666. Rosenberg SA, Grimm EA, McGrogan M, et al. Biological activity of recombinant human interleukin-2 produced in Escherichia coli. Science 1984; 223: 1412. 1667. Rosenberg SA, Lotze MT, Muul LM, et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 1987; 316: 889. 1668. 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. 1669. Rosenberg SA, Lotze MT, Yang JC, et al. 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: 622–632. 1670. Rosenberg SA, Mule JJ, Speiss PJ, et al. Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2. J Exp Med 1985; 161: 1169. 1671. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of tumorinfiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. N Engl J Med 1988; 319: 1676. 1672. Rosenberg SA, Spiess P, LaFreniere R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 1986; 233: 1318. 1673. Rosenberg SA, Sportes C, Ahmadzadeh M, et al. IL-7 administration to humans leads to the expansion of CD8+ and CD4+ cells but a relative decrease of CD4+ T-regulatory cells. J Immunother 2006; 29: 313–319. 1674. Rosenberg SA, Yang JC, Topalian SL, et al. The treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin-2. JAMA 1994; 271: 907–913. 1675. Rosenberg SA, Yang JC, White DE, Steinberg SM. Durability of complete responses in patients with metastatic cancer treated with high dose interleukin-2: identification of antigens mediating the response. Ann Surg 1998; 228: 307–319. 1676. Rosenstein M, Ettinghausen SE, Rosenberg SA. Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin 2. J Immunol 1986; 137: 1735.

Walter M. Lewko and Robert K. Oldham 1677. Rosenstein M, Yron I, Kaufmann Y, Rosenberg SA. Lymphocyteactivated killer cells: lysis of fresh syngeneic natural killer-resistant murine tumor cells by lymphocytes cultured in interleukin 2. Cancer Res 1984; 44: 1946. 1678. Rosnet O, Buhring H-J, Marchetto S, et al. Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia 1996; 10: 238–248. 1679. Rosnet O, Marchetto S, de Lapeyriere O, Birnbaum D. FLT3, a gene encoding a novel tyrosine kinase receptor of the PDGFR/ CDF1R family. Oncogene 1991; 3: 1641–1650. 1680. Rosnet O, Mattel MG, Marchetto S, Birnbaum D. Isolation and chromosomal location of a novel FMS-like tyrosine kinase gene. Genomics 1991; 9: 380–385. 1681. Rossi V, Breviario F, Ghezzi P, Dejena E, Mantovani A. Prostacyclin synthesis induced in vascular cells by interleukin-1. Science 1985; 229: 174. 1682. Roth I and Fisher SJ. IL-10 is an autocrine inhibitor of human placental cytotrophoblast MMP-9 production and invasion. Dev Biol 1999; 205: 194. 1683. 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 CD38+ cells in cancer patients. Cancer Res 2000; 60: 1934–1941. 1684. Rothe H, Hausman A, Casteels K, et al. IL-18 inhibits diabetes development in non-obese diabetic mice by counter regulation of Th1-dependent destructive insulitis. J Immunol 1999; 163: 1230. 1685. Rothe J, Lesslauer W, Lotscher H, et al. Mice lacking the tumor necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 1993; 364: 798. 1686. Rothenberg EV, Diamond RA, Pepper KA, Yang JA. IL-2 gene inducibility in T cells before T cell receptor expression. Changes in signaling pathways and gene expression requirements during intrathymic maturation. J Immunol 1990; 144: 1614. 1687. Rousset, F, Garcia E, Peronne C, et al. Interleukin-10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc Nat Acad Sci USA 1992; 89: 1890. 1688. Rouvier E, Luciani M-F, Mattei M-G, Denizot F, Goldstein P. CTLA-8, cloned from an activated T cell, bearing AU rich mRNA instability sequences and homologous to a herpesvirus samiri gene. J Immunol 1993; 150: 5445. 1689. Rovere P, Vallinoto C, Bondanza A, et al. Bystander apoptosis triggers dendritic cell maturation and antigen presentation function. J Immunol 1998; 161: 4467. 1690. Roy EJ, Gawlick U, Orr BA, Rund LA, Webb AG, Kranz DM. IL-12 treatment of endogenously arising murine brain tumors. J Immunol 2000; 165: 7293–7299. 1691. Roy M, Waldschmidt T, Arrufo A, Ledbetter A, Noelle RJ. The regulation and the expression of gp39, the CD40 ligand, on normal and cloned CD4 T cells. J Immunol 1993; 151: 2497. 1692. Ruchatz H, Leung BP, Wei XQ, McInnes IB, Liew HY. Soluble IL-15 receptor α chain administration prevents murine collagen-induced arthritis: a role for IL-15 in antigen induced immunopathology. J Immunol 1998; 160: 5654. 1693. Ruchert R, Asadullah K, Seifert M, et al. Inhibition of keratinocyte apoptosis by IL-15: a new parameter in the pathogenesis of psoriasis? J Immunol 2000; 165: 2240–2250. 1694. Ruddle NH, Bergman CM, McGrath KM, et al. An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis. J Exp Med 1990; 172: 1193. 1695. Ruddle NH, Waksman BH. Cytotoxic effect of lymphocyte– antigen interaction in delayed hypersensitivity. Science 1967; 157: 1060–1062.

259 1696. Rudland PS, Platt-Higgins A, El-Tanani M, et al. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res 2002; 62: 3417–3427. 1697. Rumsaeng V, Cruikshank WW, Foster B, et al. Human mast cells produce the CD4+ lymphocyte chemoattractant factor, IL-16. J Immunol 1997; 159: 2904–2910. 1698. Russell JH, Rush B, Weaver C, Wand R. Mature T cells of autoimmune lpr/lpr mice have a defect in antigen stimulated suicide. Proc Natl Acad Sci USA 1993; 90: 4409. 1699. Russell SM, Johnson JA, Noguchi M, et al. Interaction of IL-2R and c chains with Jak1 and Jak3: Implications for XSCID and XCID. Science 1994; 266: 1042–1044. 1700. Ryan TC, Cruikshank WW, Kornfeld H, Collins TL, Center DM. The CD4 associated tyrosine kinase p56 lck is required for lymphocyte chemoattractant factor induced T-lymphocyte migration. J Biol Chem 1995; 270: 17081–17086. 1701. Rygard K, Nakamura T, Spang-Thompsen M. Expression of the protooncogene cmet and c kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor in SCLC cell lines and xenografts. British J Cancer 1993; 67: 37–46. 1702. Saas P, Boucraut J, Walker PR, et al. Tweak stimulation of astrocytes and the proinflammatory consequences. Glia 2000; 32: 102–107. 1703. Saas P, Walker PR, Hahne M, et al. Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain? J Clin Invest 1997; 99: 1173. 1704. Sabelko K, Blumenthal D, Ford R, Ambrus J. IL-14 producing and responsive cells are found in germinal centers. FASEB J 1994; 8: A999. 1705. Sacca R, Cuff C, Lesslauer W, Ruddle NH. Differential activities of secreted lymphotoxin-α1β2 in lymphotoxin-induced inflammation: critical role of TNF receptor 1 signaling. J Immunol 1998; 160: 485–491. 1706. Sacca R, Turley S, Soong L, Mellman I, Ruddle NH. Transgenic expression of lymphotoxin restores lympnodes to lymphotoxinα-deficient mice. J Immunol 1997; 159: 4252–4260. 1707. Sad S, Krishnan L, Bleackley RC, et al. Cytotoxicity and weak CD40 ligand expression of CD8+ type 2 cytotoxic T cells restricts their B helper activity. Eur J Immunol 1997; 27: 914. 1708. Sad S, Marcotte R, Mosmann TR. Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines. Immunity 1995; 2: 271–279. 1709. Sad S, Mosmann TR. Single IL-2 secreting precursor CD4 T cell can develop into either Th1 or Th2 cytokine secreting phenotype. J Immunol 1994; 153: 3514. 1710. Sadlack B, Lohler J, Schorle H, et al. Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD4+ T cells. Eur J Immunol 1995; 25: 3053. 1711. Sadlack B, Merz H, Schorle H, et al. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 1993; 75: 253. 1712. Saeki T, Mhashilkar A, Swanson X, et al. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene 2002; 21: 4558–4566. 1713. Saito F, Ohno Y, Morisawa K, et al. Role of IL-27-producing dendritic cells in Th1-immunity polarization in Lewis rats. Biochem Biophys Res Commun 2005; 338: 1773–1778. 1714. Sakaguchi S. The origin of FOXP3-expressing CD4+ regulatory T cells: thymus or periphery. J Clin Invest 2003; 112: 1310–1312. 1715. Sakai T, Kusugami K, Nishimura H, et al. Interleukin 15 activity in the rectal mucosa of inflammatory bowel disease. Gastroenterology 1998; 114: 1237.

260 1716. Sakata T, Iwagami S, Tsurata Y, et al. Constitutive expression of interleukin 7 mRNA and production of IL-7 by a cloned murine thymic stromal cell line. J Leukocyte Biol 1990; 144: 3015. 1717. Salih HR, Kososksi SG, Haluska VF, Trail PA, Kiener PA, et al. Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells. J Immunol 2000; 165: 2903–2910. 1718. Samoilova EB, Horton JL, Hilliard B, Liu TST, Chen Y. IL-6-deficient mice are resistant to experimental autoimmune encephalomyelitis: roles of IL-6 in the activation and differentiation of autoreactive T cells. J Immunol 1998; 161: 6480. 1719. Sampaio EP, Kaplan G, Miranda A, et al. The influence of thalidomide on the clinical and immunologic manifestation of erythema nodosum leprosum. J Infect Dis 1993; 168: 408–414. 1720. Sanborg CI, Imfeld KL, Zaldivar F Jr, et al. IL-4 expression in human T cells is selectively inhibited by IL-1α and IL-1β. J Immunol 1995; 155: 5206–5212. 1721. Sandborn WJ, Hanauer SB, Katz S, et al. Etanercept for active Crohn’s disease: a randomized double blind, placebo-controlled trial. Gastroenterology 2001; 121: 1241–1246. 1722. Sanderson CJ, Campbell HD, Young IG. Molecular and cellular biology of eosinophil differentiation factor (interleukin 5) and its effects on human and mouse B cells. Immunol Rev 1988; 109: 29–50. 1723. Sanderson CJ. Interleukin-5, eosinophils, and disease. Blood 1992; 79: 3101. 1724. Sareneva T, Julkunen I, Matikainen S. IFN-α and IL-12 Induce IL-18 receptor gene expression in human NK and T cells. J Immunol 2000; 165: 1933–1938. 1725. Sareneva T, Matikainen S, Kurimoto M, Julkunen I. Influenza A virus-induced IFN-α/β and IL-18 Synergistically enhance IFN-γ gene expression in human T cells. J Immunol 1998; 160: 6032. 1726. Sarin A, Conal-Cibotti M, Henkart PA. Cytotoxic effect of TNF and lymphotoxin in T lymphoblasts. J Immunol 1995; 155: 3716. 1727. Sarris AH, Kliche KO, Pethambaram O, et al. Interleukin-10 levels are often elevated in serum of adults with Hodgkin’s disease and are associated with inferior failure-free survival. Ann Oncol 1999; 10: 433–440. 1728. Sato A, Ohtsuki M, Hata M, et al. Antitumor activity of IFN-lambda in murine tumor models. J Immunol 2006; 176: 7686–7694. 1729. Sato K, Akaki T, Tomioka H. Differential potentiation of antimycobacterial activity and reactive nitrogen intermediateproducing ability of murine peritoneal macrophages activated by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Clin Exp Immunol 1998; 112: 63. 1730. Sato TA, Widmer MB, Finkelman FD, et al. Recombinant soluble murine IL-4 receptor can inhibit of enhance IgE responses in vivo. J Immunol 1991; 150: 2717. 1731. Sauane M, Gopalkrishnan RV, Lebedeva I, et al. MDA-7/IL-24 induces apoptosis if diverse cancer cell lines through JAK/STAT independent pathways. J Cell Physiol 2003; 196: 334–345. 1732. Sauder DN, Mounessa NL, Katz SI, et al. Chemotactic cytokines: the role of leukocyte pyrogen and epidermal cell thymocyte-activating factor in neutrophil chemotaxis. J Immunol 1984; 132: 828. 1733. Saura M, Martinez-Dalmau R, Minty A, Perez-Sala D, Lamas S. Interleukin-13 inhibits inducible nitric oxide synthase expression in human mesangial cells. Biochem J 1996; 313: 641. 1734. Scala E, D’Offizi G, Rosso R, et al. C-C chemokines, IL-16 and soluble antiviral factor activity are increased in cloned T cells from subjects with long term nonprogressive HIV infection. J Immunol 1997; 158: 4485–4492.

Cytokines 1735. Scala G, Kuang YD, Hall RE, et al. Accessory cell function of human B cells. I. Production of both interleukin-1 like activity and interleukin-1 inhibitory factor by an EBV-transformed B cell line. J Exp Med 1984; 159: 1637. 1736. Schadendorf D, Bohm M, Moller P, Grunewald T, Czarnetzki BM. Interleukin 7 induces differential lymphokine-activated killer cell activity against human melanoma cell, keratinocytes and endothelial cells. J Invest Dermatol 1994; 102: 838–842. 1737. Schadendorf D, Moller A, Algermissen B, et al. IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor. J Immunol 1993; 15: 2667–2675. 1738. Schaefer G, Venkataraman C, Schindler U. FISP (IL-4-Induced secreted protein), a novel cytokine-like molecule secreted by Th2 cells. J Immunol 2001; 166: 5859–5863. 1739. Scheu S, Alferink J, Potzel T, et al. Targeted disruption of LIGHT causes defects in costimulatory T cell activation and reveals cooperation with lymphotoxin β in mesenteric lymphnode genesis. J Exp Med 2002; 95: 161324. 1740. Schieferdecker HL, Schlaf G, Koleva M, Gotze O, Jungermann K. Induction of functional anaphylatoxin C5a receptors on hepatocytes by in vivo treatment of rats with IL-6. J Immunol 2000; 164: 5453–5458. 1741. Schindler H, Lutz MB, Rollinghoff M, Bogdan C. The production of interferon-γ by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4. J Immunol 2001: 166: 3075. 1742. Schindler R, Mancilla J, Endres S, et al. Collelations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 1990; 75: 40. 1743. Schluns KS, Cook JE, Le PL. TGF-β differentially modulates epidermal growth factor-mediated increases in leukemia-inhibitory factor, IL-6, IL-1α, and IL-1β in human thymic epithelial cells. J Immunol 1997; 158: 2704–2712. 1744. Schmidt JA, Mizel SB, Cohen D, et al. Interleukin-1: a potential regulator of fibroblast proliferation. J Immunol 1982; 128: 2177. 1745. Schmitt E, Beuscher HU, Huls C, et al. IL-1 serves as a secondary signal for IL-9 expression. J Immunol 1991; 147: 3848. 1746. Schmitt E, Germann T, Goedert P, et al. IL-9 production of naive CD4+ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-β and IL-4 and is inhibited by IFN-γ. J Immunol 1994; 153: 3989. 1747. Schmitt E, van Brandwijk R, Fischer H-G, Rude E. Establishment of different T cell sublines using either interleukin 2 of interleukin 4 as growth factors. Eur J Immunol 1990; 20: 1709. 1748. Schmitt E, van Brandwijk R, Van Snick J, Siebold B, Rude E. TCGFIII/P40 is produced by naive CD4+ T cells but is not a general T cell growth factor. Eur J Immunol 1989; 19: 2167. 1749. Schmitter D, Lauber B, Fagg B, Stahel RA. Hematopoietic growth factors secreted by seven human pleural mesothelioma cell lines: interleukin 6 production as a common feature. Int J Cancer 1992; 51: 296–301. 1750. Schmitz J, Owyang A, Oldham E, et al. IL-33 an interleukin-1 like cytokine that signals via IL-1 receptor-related proteiST2 and induces T helper type 2-associated cytokines. Immunity 2005; 23: 479–490. 1751. Schneider P, Bodmer JL, Thome M, Hoffmann K, Holler N, Tschopp J. Characterization of two receptors for TRAIL. FEBS Lett 1997; 416: 329. 1752. Schobitz B, de Kloet ER, Sutano W, Holsber F. Cellular localization of interleukin-6 mRNA and interleukin 6 receptor mRNA in rat brain. Eur J Neurosci 1993; 5: 1426–1435.

Walter M. Lewko and Robert K. Oldham 1753. Schoenberger SP, Toes REM, van der Voort EIH, Offringa R, Melief CJM. T cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions. Nature 1998; 393: 480. 1754. Schorle H, Holtschke T, Hunig T, Schimpl A, Horak I. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature 1991; 352: 621. 1755. Schroder J, Mrowietz U, Morita E, Christophers E. Purification and partial biochemical characterization of a human monocytederived neutrophil activating peptide that lacks interleukin 1 activity. J Immunol 1987; 139: 3474. 1756. Schroder JM, Christophers E. Secretion of novel and homogeneous neutrophil activating peptides by LPS stimulated human endothelial cells. J Immunol 1989; 142: 244. 1757. Schuerer-Maly CC, Eckmann L, Kagoff MF, et al. Colonic epithelial cell lines as a source of interleukin-8: stimulation by inflammatory cytokines and bacterial lipopolysaccharide. Immunology 1994; 81: 85–91. 1758. Schuler M, Bruntsch U, Spath-Schwalbe E, Schrezenmeier H, et al. Lack of efficacy of recombinant human interleukin-6 in patients with advanced renal cell cancer: results of a phase II study. Eur J Cancer 1998; 34: 754–756. 1759. Schwab U, Stein H, Gerdes J, Lemke H, Kirchner HH, Schaat M. Production of a monoclonal antibody specific for Hodgkin’s disease and a subset of normal lymphoid cells. Nature 1982; 299: 65. 1760. Schwalb DM, Han H-M, Marino M, et al. Identification of a new receptor subtype for tumor necrosis factor α. J Biol Chem 1993; 268: 9949–9954. 1761. Schwarz H, Valbracht J, Tuckwell J, Kempis J, Lotz M. ILA, the human 4-1BB homologue, is inducible in lymphoid and their cell lines. Blood 1995; 85: 1043. 1762. Schwarz GN, MacVittie TJ, Vignuelle RM, Patchen ML, Douches SD, Oppenheim JJ, Neta R. Enhanced hematopoietic recovery in irradiated mice pretreated with interleukin-1 (IL-1). Immunopharmacol Immunotoxicol 1987; 9: 371. 1763. Schwarz H, Arden K, Lotz M. CD137, a member of the tumor necrosis factor receptor family is located on chromosome 1p36, in a cluster of related genes, and co-localizes with several malignancies. Biochem Biophys Res Commun 1997; 235: 699. 1764. Schwarz H, Blanco F, Valbracht J, Kempis J, Lotz M. ILA, a member of the human NGF/TNF receptor family regulates T lymphocyte proliferation and survival. Blood 1996; 87: 2839. 1765. Schwarz H, Tuckwell J, Lotz M. A receptor induced by lymphocyte activation (ILA): a new member of the human nerve growth factor/ tumor necrosis factor receptor family. Gene 1993; 134: 295. 1766. Schwarzenberger P, Huang W, Oliver P, et al. IL-17 mobilizes peripheral blood stem cells with short- and long-term repopulating ability in mice. J Immunol 2001; 167: 2081–2086. 1767. Schwarzenberger P, Huang W, Ye P, et al. Requirement for endogenous stem cell factor and granulocyte-colony-stimulating factor for IL-17 mediated granulopoiesis. J Immunol 2000; 164: 4783–4789. 1768. Schwarzenberger P, La Russa V, Miller A, et al. IL-17 stimulates granulopoiesis in mice: Use of an alternate, novel gene therapy derived method for in vivo evaluation of cytokines. J Immunol 1998; 161: 6383–6389. 1769. Schweizer RC, Welmers BAC, Raaijmakers JAM, et al. RANTES and Interleukin 8 induced responses in normal human eosinophils: effects of priming with IL-5. Blood 1994; 83: 3697. 1770. Sciaky D, Brazer W, Center DM, Cruikshank WW, Smith TJ. Cultured human fibroblasts express constitutive IL-16 mRNA: Cytokine induction of active IL-16 protein synthesis through a

261

1771.

1772.

1773.

1774.

1775. 1776.

1777.

1778. 1779.

1780.

1781.

1782.

1783.

1784.

1785.

1786.

1787.

1788.

1789.

caspase-3-dependent mechanism. J Immunol 2000; 164: 3806–3814. Scott JM, Wier ML, Wilson SM, et al. Osteopontin inhibits inducible nitrogen oxide synthetase activity in rat vascular tissue. Am J Physiol 1998; 275: H2258. Screaton RG, Mongkolsapaya J, Xu XN, et al. TRICK2, a new alternatively spliced receptor that transduces the cytoplasmic signal from TRAIL. Curr Biol 1997; 7: 693. Sebire G, Delfraissy J-F, Demotes-Mainard A, et al. Interleukin-13 and interleukin-4 act as interleukin-6 inducers in human microglial cells. Cytokine 1996; 8: 636. Sebok K, Woodside D, al-Aoukaty A. IL-8 induces the locomotion on human IL-2 activated natural killer cells. Involvement of a guanine nucleotide binding protein. J Immunol 1993; 150: 1524–1534. Seder RA and Paul WE. Acquisition of lymphokine producing phenotype by CD4+ T cells. Annu Rev Immunol 1994; 12: 635. Sedger LM, Shows DM, Blanton RA, et al. IFN-γ mediates a novel antiviral activity through dynamic modulation of TRAIL and TRAIL receptor expression. J Immunol 1999; 163: 920. Sehmi R, Cromwell O, Wardlaw AJ, et al. Interleukin 8 is a chemoattractant for eosinophils purified from subjects with blood eosinophilia but not from normal healthy subjects. Clin Exp Allergy 1993; 23: 1027. Seino K, Kayagaki N, Okumura K, Yagita H. Antitumor effect of locally produced CD95 ligand. Nat Med 1997; 3: 165. Seki E, Tsutsui H, Nakano H, et al. Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1β.J Immunol 2001; 166: 2651–2657. Sekido N, Mukaida N, Harada A, et al. Prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8. Nature 1993; 365: 654–657. Sekido Y, Obata Y, Ueda R, et al. Preferential expression of c-kit protooncogene transcripts in small cell lung cancer. Cancer Res 1991; 51: 2416–2419. Selin LK, Santolucito PA, Pinto AK, Szomolanyi-Tsuda E, Welsh RM. Innate immunity to viruses: Control of vaccinia virus infection by γδ T cells. J Immunol 2001; 166: 6784–6794. Senaldi G, Stolina M, Guo J, et al. Regulatory effects of novel neurotrophin-1/B cell-stimulating factor-3 (Cardiotrophin-like cytokine) on B cell function. J Immunol 2002; 168: 5690–5698. Senaldi G, Varnum BC, Sarmiento U, et al. Novel neurotrophin1/B cell-stimulating factor-3: a cytokine of the IL-6 family. Proc Natl Acad Sci USA 1999; 96: 11458–11463. Senger, DR, Peruzzi CA. Secreted phosphoprotein markers for neoplastic transformation of human epithelial and fibroblast cells. Cancer Res 1985; 45: 5818–5823. Serretta V, Piazza B, Pavone C, Piazza S, Pavone-Macaluso M. Is there a role for recombinant tumor necrosis factor α in the intravesical treatment of superficial bladder tumors? A phase II study. Int J Urol 1995; 2: 100–103. Setareh M, Schwarz H, Lotz M. A mRNA variant encoding a soluble form of 4-1BB, a member of the murine NGF/TNF receptor family. Gene 1995; 164: 311–315. Sguadari C, Angiolillo AL, Cherney BW, et al. Interferoninducible protein-10 identified as a mediator of tumor necrosis in vivo. Proc Natl Acad Sci USA 1996; 93: 13791–13796. Shade RJ, Younes A. In vitro and in vivo biologic effects of interleukin-3 in follicular low-grade lymphoma. Leuk Lymphoma 1997; 26: 17–25.

262 1790. Shaefer G, Venkataraman C, Schindler U. FISP (IL-4 induced secreted protein), a novel cytokine-like molecule secreted by Th2 cells. J Immunol 2001; 166: 5859–5863. 1791. Shah AJ, Smogorzewska EM, Hannum C, Crooks GM. Flt-3 ligand induces proliferation of quiescent human bone marrow CD34+ CD38− cells and maintains progenitor cells in vivo. Blood 1996; 87: 3563. 1792. Shalaby MR, Aggarwall BB, Rinderknetch E, et al. Activation of human polymorphonuclear neutrophil function by interferon-γ and tumor necrosis factor. J Immunol 1985; 135: 2069. 1793. Shalhoub V, Faust J, Boyle WJ, et al. Osteoprotegerin and osteoprotegerin ligand effects on osteoclast formation from human peripheral blood mononuclear cell precursors. J Cell Biochem 1999; 72: 251. 1794. Shan B, Yu L, Shimazoto O, et al. Expression of interleukin-21 and IL-23 in human esophageal tumors produced antitumor effects in nude mice. Anticancer Res 2004; 24: 79–82. 1795. Shan BE, Hao JS, Li OX, et al. Antitumor activity and immune enhancement of murine interleukin-23 expressed in murine colon carcinoma cells. Cell Mol Immunol 2006; 3: 47–52. 1796. Shanker G, Sorci-Thomas M, Adams ME. Estrogen modulates the expression of tumor necrosis factor mRNA in phorbol esterstimulated human monocytic THP-1 cells. Lymphokine Cytokine Res 1994; 13: 377. 1797. Sharma V, Sparks JL Vail JD. Human B-cell lines constitutively express and secrete interleukin-16. Immunology 2000; 99: 266–271. 1798. Shaw J-P, Utz PJ, Durand DB, et al. Identification of a putative regulator of early T cell activation genes. Science 1988; 241: 202. 1799. Shaw J, Caplan B, Paetkau V, et al. Cellular origins of co-stimulator (IL-2) and its activity in cytotoxic T-lymphocyte responses. J Immunol 1980; 124: 2231. 1800. Shaw J, Monticone V, Paetkau V. Partial purification and molecular characterization of a lymphokine (co-stimulator) required for the mitogenic response of mouse thymocytes in vitro. J Immunol 1978; 120: 1967. 1801. Shaw SG, Maung AA, Steptoe RJ, et al. Expansion of functional NK cells in multiple tissue compartments of mice treated with Flt3-ligand: implications for anticancer and antiviral activity. J Immunol 1998; 161: 2817–2814. 1802. Sheikh F, Baurin VV, Lewis-Antes A, et al. IL-26 signals through a novel receptor complex composed of Il-20 receptor 1 and IL-10 receptor 2. J Immunol 2004; 172: 2006–2010. 1803. Shen L, Zhang C, Wang T, et al. Development of autoimmunity in IL-14α-transgenic mice. J Immunol 2006; 177: 5676–5686. 1804. Shen MM, Skoda RC, Cardiff RD, et al. Expression of LIF in transgenic mice results in altered thymic epithelium and apparent intraconversion of thymic and lymphnode morphologies. EMBO J 1994; 13: 1375. 1805. Sheridan JP, Marsters SA, Pitti RM, et al. Control of TRAILinduced apoptosis by a family of signaling and decoy receptors. Science 1997; 277: 818. 1806. Shevach EM. Certified professionals: CD4+ CD25+ suppressor T cells. J Exp Med 2001; 193: F41–46. 1807. Shi Y, Ullrich SJ, Zhang J, et al. A novel cytokine receptor-ligand pair. Identification, molecular characterization, and in vivo immunomodulatory activity. J Biol Chem 2000; 275: 19167–19176. 1808. Shi Y, Wang W, Youri PA, et al. Computational EST database analysis identifies a novel member of the neuropoietic cytokine family. Biochem Biophys Res Commun 1999; 262: 132.

Cytokines 1809. Shibuya K, Robinson D, Zonin F, et al. IL-1α and TNF-α are required for IL-12-induced development of Th1 cells producing high levels of IFN-γ in BALB/c but not C57BL/6 mice. J Immunol 1998; 160: 1708–1716. 1810. Shiloni E, Eisenthal A, Sachs D, Rosenberg SA. Antibodydependent cellular cytotoxicity mediated by murine lymphocytes activated in recombinant interleukin 2. J Immunol 1987; 138: 1992. 1811. Shimizu M, Shimamura M, Owaki T, et al. Antiangiogenic and antitumor activities of IL-27. J Immunol 2006; 176: 7317–7324. 1812. Shintani F, Nakati T, Kanba S, et al. Involvement of unterleukin-1 in immobilization stress-induced increase in plasma adrenocorticotropic hormone and in the release of hypothalmic monoamines in the rat. J Neurosci 1995; 15: 1961–1970. 1813. Shipman CM and Croucher PI. Osteoprotegerin is a soluble decoy receptor for tumor necrosis factor apoptosis-inducing ligand/apo2 ligand and can function as a paracrine survival factor for human myeloma cells. Cancer Res 2003; 63: 912–916. 1814. Shiraki K, Tsuji N, Shioda T, Isselbacher KJ, Takahashi H. Expression of Fas ligand in liver metastases of colon adenocarcinoma. Proc Natl Acad Sci USA 1997; 94: 6420. 1815. Shoham S, DavenneD, Cady AB, Dinarello CA, and Krueger JM. Recombinant tumor necrosis factor and IL-1 enhance slowwave sleep. Am J Physiol 1987; 253: 142. 1816. Shrikant P, Weber E, Jilling T, Beneviste EN. ICAM gene expression by glial cells: differential mechanisms of inhibition by IL-6 and IL-10. J Immunol 1995; 155: 1489. 1817. Shu U, Kiniwa M, Wu CY, et al. Activated T cells induce interleukin-12 production by monocytes via CD-40–CD-40L interaction. J Immunol 1995; 25: 1125–1128. 1818. Shuford WW, Klussman K, Trichler DD, et al. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell response. J Exp Med 1997; 186: 47. 1819. Shurin MR, Pandharipande PP, Zorina TD, et al. FLT3-ligand induces the generation of functionally active dendritic cells in mice. Cell Immunol 1997; 179: 174. 1820. Sica D, Rayman P, Edinger M, et al. Interleukin 7 enhances the proliferation and effector function of tumor infiltrating lymphocytes from renal cell carcinoma. International J Cancer 1993; 53: 941–947. 1821. Sideras P, Palacios R. Bone marrow pre-T and pre-B lymphocyte clones express functional receptors for interleukin (IL)-3 and interleukin 4/BSF-1 and non-functional receptors for IL-2. Eur J Immunol 1987; 17: 217. 1822. Siebruth D, Jabs EW, Warrington JA, et al. Assignment of NKSF/IL-12, a unique cytokine composed of two unrelated subunits to chromosomes 3 and 5. Genomics 1992; 14: 59–62. 1823. Siegel JP, Sharon M, Smith PL, Leonard WJ. The IL-2 receptor β chain (p70): role in mediating signals for LAK, NK, and proliferative activities. Science 1987; 238: 75. 1824. Sillaber C, Sperr WR, Agis H, et al. Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4. Int Arch Allergy Immunol 1994; 105: 264. 1825. Silvani A, Ferrari G, Paonessa G, et al. Down regulation of interleukin 6 receptor α chain in interleukin 6 transduced melanoma cells causes selective resistance to interleukin 6 but not to oncostatin M. Cancer Res 1995; 55: 2200–2205. 1826. Silver DF, Hempling RE, Piver MS, Repasky EA. Flt-3 ligand inhibits growth of human ovarian tumors engrafted in severe combined immunodeficient mice. Gynecol Oncol 2000, 77: 377–382.

Walter M. Lewko and Robert K. Oldham 1827. Simic MM, Stosic-Grujicic S. The dual role of interleukin 1 in lectin-induced proliferation of T cells. Folia Biol 1985; 31: 410. 1828. Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin, a novel secreted protein involved in the regulation of bone density. Cell 1997; 89: 309. 1829. Sims JE, March CJ, Cosman D, et al. cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily. Science 1988; 241: 585. 1830. Singh B, Berry JA, Shorer A, et al. COX-2 induces IL-11 productioin in breast cancer cells. J Surg Res 2006; 131: 276–275. 1831. Singh K, Sirokman G, Communal C, et al. Myocardial osteopontin expression coincides with the development of heart failure. Hypertension 1999; 33: 670. 1832. Singh N, Hong S, Scherer DC, et al. Activation of NK T cells by CD1d and α-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype. J Immunol 1999; 163: 2373. 1833. Singh RK, Gutman M, Radinsky R, et al. Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice. Cancer Res 1994; 54: 3232–3247. 1834. Singhal H, Bautista DS, Tonkin KS, et al. Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival. Clin Cancer Res 1997; 3: 605–611. 1835. Sivakumar PV, Foster DC, Clegg CH. Interleukin-21 is a T-helper cytokine that regulates humoral immunity and cell mediated anti-tumor responses. Immunology 2004; 112: 177–182. 1836. Skillings J, Wierzbicki R, Eisenhauer E, et al. A phase II study of recombinant tumor necrosis factor in renal cell carcinoma: a study of the National Cancer Institute of Canada Clinical Trials Group. J Immunother 1992; 11: 67–70. 1837. Small D, Levenstein M, Kim E, et al. STK-1, the human homolog of Flk-2/Flt3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells. Proc Natl Acad Sci USA 1994 91: 459–463. 1838. Smalley RV and Oldham RK. Biological response modifiers: preclinical evaluation and clinical activity. Crit Rev Oncol Hematol 1984; 1: 259–294. 1839. Smith CA, Farrah T, Goodwin RG. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation and death. Cell 1994; 76: 959. 1840. Smith CA, Gruss HJ, Davis G, et al. CD30 antigen, a marker for Hodgkin’s lymphoma, is a receptor whose ligand defines an emerging superfamily of cytokines with homology to TNF. Cell 1993; 73: 1349. 1841. Smith E, Zrabock, Stark MA, et al. IL-23 is required for neutrophil homeostasis in normal and neutrophilic mice. J Immunol 2007; 179: 8274–8279. 1842. Smith JW 2nd, Longo DL, Alvord WG, et al. The effect of treatment with interleukin-1α on platelet recovery after high-dose carboplatin. N Engl J Med 1993; 328: 756–761. 1843. Smith JW. Tolerability and side-effect profile of rhIL-11. Oncology 2000; 14: 41–47. 1844. Smith MR, Kung H, Durum SK, Colburn NH, Sun Y. TIMP-3 induces cell death by stabilizing TNF-α receptors on the surface of human colon carcinoma cells. Cytokine 1997; 9: 770. 1845. Smith XG, Bolton EM, Ruchatz H, et al. Selective blockade of IL-15 by soluble IL-15 receptor-chain enhances cardiac allograft survival. J Immunol 2000; 165: 3444.

263 1846. Smyth MJ, Jonstone RW, Cretney E, et al. Multiple deficiencies underlie NK cell inactivity in lymphotoxin-α gene targeted mice. J Immunol 1999; 163: 1350. 1847. Smyth MJ, Ortaldo JR. Comparison of the effect of IL-2 and IL-6 on the lytic activity of purified human peripheral blood large granular lymphocytes. J Immunol 1991; 146: 1380–1384. 1848. Smyth MJ, Taniguchi M, Street SEA. The antitumor activity of IL-12: Mechanisms of innate immunity that are model and dose dependent. J Immunol 2000; 165: 2665–2670. 1849. Smyth MJ, Wallace ME, Nutt SL, et al. Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer. J Exp Med 2005; 201: 1973–1985. 1850. Snapper CM, Finkelman FD, and Paul WE. Differential regulation of IgG1 and IgE synthesis by interleukin 4. J Exp Med 1988; 167: 183. 1851. Snapper CM, Paul WE. Interferon γ and B cell stimulatory factor I reciprocally regulate Ig isotope production. Science 1987; 236: 944–947. 1852. Snyder LA, Goletz TJ, Gunn GR, et al. A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice. Vaccine 2006; 24: 3340–3352. 1853. So E-Y, Park H-H, Lee S-E Lee CE. IFN-γ and IFN-α posttranscriptionally down-regulate the IL-4-induced IL-4 receptor gene expression. J Immunol 2000; 165: 5472–5479. 1854. Son YI, Dallal RM, Lotze MT. Combined treatment with interleukin-18 and low-dose interleukin-2 induced regression of a murine sarcoma and memory response. J Immunother 2003; 234–240. 1855. Sondel PM, Hank JA, Kohler PC, et al. Destruction of autologous human lymphocytes by interleukin 2-activated cytotoxic cells. J Immunol 1986; 137: 502. 1856. Song K, Chen Y, Goke R, et al. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J Exp Med 2000; 191: 1095. 1857. Song MM, Shaui K. the suppressor of cytokine signaling (SOCS) 1 and SOCS3 but not SOCS2 proteins inhibit interferon-mediated antiviral and antiproliferative activities. J Biol Chem 1998; 273: 35056. 1858. Song X, Voronov E, Dvorkin T, et al. Differential effects of IL-1α and IL-1β on tumorigenicity patterns and invasiveness. J Immunol 2003; 171: 6448–6456. 1859. Sonkoly E, Muller A, Lauerma AI, et al. IL-31: a new link between T cells and pruritis in atopic skin inflammation. J Allergy Clin Immunol 2006; 117: 411–417. 1860. Sonoda Y, Kimura T, Sakabe H, et al. Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c-kit protein. Eur J Haematol 1997; 58: 257–264. 1861. Soong L, Xu J-C, Grewall IS, Kima P, et al. Disruption of CD40–CD40L interactions results in a susceptibility to Leishmania amazonensis infection. Immunity 1996; 4: 263. 1862. Soroosh P, Ine S, Sugamura K, et al. OX40–OX40 ligand interaction through Tcell-Tcell contact contributes to CD4 T cell longevity. J Immunol 2006; 176: 5975–5987. 1863. Sotomayor EM, Borrello I, et al. Conversion of tumor-specific CD4+ T-cell tolerance to T-cells priming through in vivo ligation of CD40. Nat Med 1999; 5: 780. 1864. Southern E. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503. 1865. Sower LE, Froelich CJ, Carney DH, et al. Thrombin induces IL-6 production in Fibroblasts and Epithelial Cells. J Immunol 1995; 155: 895–901.

264 1866. Sparwasser T, Miethke T, Lipford G, et al. Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor mediated shock. Eur J Immunol 1997; 27: 1671. 1867. Speer MY, McKee MD, Guldberg RE, et al. Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo. J Exp Med 2002; 196: 1047–1055. 1868. Spencer NFL and Daynes RA. IL-12 directly stimulates expression of IL-10 by CD5+ B cells and IL-6 by both CD5+ and CD5− B cells: possible involvement in age-associated cytokine dysregulation. Int Immunol 1997; 9: 745. 1869. Spencer SD, Di Marco F, Hooley J, et al. The orphan receptor CRF2–4 is an essential subunit of the interleukin-10 receptor. J Exp Med 1998; 187: 571–578. 1870. Spits H, Yssel H, Paliard X, et al. IL-4 inhibits IL-2 mediated induction of human lymphokine activated killer cells but not the generation of antigen specific cytotoxic T lymphocytes in mixed leukocyte cultures. J Immunol 1988; 141: 29–36. 1871. Splawski JB, Jelinek DF, Lipsky PE. Immunomodulatory role of IL-4 on the secretion of Ig by human B cells. J Immunol 1989; 142: 1569. 1872. Splawski JB, McAnally, Lipsky PE. IL-2 dependence of the promotion of human B cell differentiation by IL-6 (BSF-2). J Immunol 1990; 144: 562. 1873. Spranger M, Lindholm D, Brandtlow C, et al. Regulation of nerve growth factor (NGF) synthesis in the rat central nervous system: comparison between the effects of interleukin-1 and various growth factors in astrocyte cultures in vitro. Eur J Neurosci 1990; 2: 69. 1874. Squadari C, Farber JM, Angiolillo AL, et al. Mig, the monokine induced by interferon-gammma, promotes tumor necrosis in vivo. Blood 1997; 89: 2635–2643. 1875. Srivatsa SS, Harrity PJ, Maercklein P, et al. Increased cellular expression of matrix proteins, which regulate mineralization is associated with calcification of human and porcine xenograft bioprosthetic heart valves. J Clin Invest 1997; 99: 996–1009. 1876. Stacey KJ, Sweet MJ, Hume DA. Macrophages ingest and are activated by bacterial DNA. J Immunol 1996; 157: 2116. 1877. Stam TC, Swaak AJ, Kruit WH, Stoter G, Eggermont AM. Intrapleural administration of tumor necrosis factor α in patients with mesothelioma: cytokine patterns and acute-phase protein response. Eur J Clin Invest 2000; 30: 336–343. 1878. Standiford T, Kunkel S, Basha M, et al. Interleukin 8 gene expression by a pulmonary epithelial cell line. J Clin Invest 1990; 86: 1945. 1879. Starnes HF, Hartman G, Torti F, et al. Recombinant human interleukin-1β (IL-1β) has antitumor activity and acceptable toxicity in metastatic malignant melanoma. Proc Am Soc Clin Oncol 1991; 10: 292. 1880. Starnes T, Broxmeyer HE, Robertson MJ, Hromas R. IL-17D, a novel member of the IL-17 family, stimulates cytokine production and inhibits hematopoiesis. J Immunol 2002; 169: 642–646. 1881. Starnes T, Robertson MJ, Sledge G, et al. IL-17F, a novel cytokine selectively expressed in activated T cells and monocytes, regulates angiogenesis and endothelial cytokine production. J Immunol 2001 167: 4137–4140. 1882. Starr R, Willson TA, Viney EM, et al. A family of cytokine-inducible inhibitors of signaling. Nature 1997; 387: 917. 1883. Stashenko P, Dewirst FE, Peros WJ, Kent RL. Synergistic interaction between IL-1, tumor necrosis factor, and lymphotoxin in bone resorption. J Immunol 1987; 138: 1464.

Cytokines 1884. Stefana B, Ray DW, Melmed S, et al. Leukemia inhibitory factor induces differentiation of pituitary corticotroph function: an immuno-neuroendocrine phenotype switch. Proc Natl Acad Sci USA 1996; 93: 12502–12506. 1885. Stefanovic-Racic M, Mollers MO, Miller LA, Evans CH. Nitric oxide and proteoglycan turnover in rabbit articular cartilage. J Orthopaed Res 1997; 15: 442. 1886. Steiger J, Nickerson PW, Steurer W, Moscovitch-Lopatin M, Strom RB. IL-2 knockout recipient mice reject islet cell isografts. J Immunol 1995; 155: 489–498. 1887. Steiner GE, Newman ME, Paikl D, et al. Expression and function of pro-inflammatory interleukin IL-17 and IL-17 receptor in normal, hyperplastic and malignant prostate. Prostate 2003; 56: 171–182. 1888. Steinman RM. The dendritic cell system and its role in immunogenicity. Ann Rev Immunol 1991; 9: 271–296. 1889. Steinshamn S, Brekke OL, Waage A. Soluble tumor necrosis factor receptors, tumor necrosis factor and interleukin-6 in serum in granulocytopenic patients with fever. Brit J Haematol 1995; 89: 719. 1890. Steptoe RJ, Fu F, Li W, Drakes ML, et al. Augmentation of dendritic cells in murine organ donors by Flt3 ligand alters the balance between transplant tolerance and immunity. J Immunol 1997; 159: 5483–5491. 1891. Stern AS, Podlaski FJ, Hulmes JD, et al. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphocyte cells. Proc Nat Acad Sci USA 1990; 87: 6808–6812. 1892. Stewart CL, Kaspar P, Brunet LJ, et al. Blastocyst implantation depends on maternal expression of leukemia inhibitory factor. Nature 1992; 359: 76–79. 1893. Stier S, Ko Y, Forkert R, et al. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. J Exp Med 2005; 201: 1781–1791. 1894. Stoll S, Jonuleit H, Schmitt E, Muller G, et al. Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18 enhances IL-12 dependent Th1 development. Eur J Immunol 1998; 28: 3231. 1895. Stoll SG, Mueller G, Kurimoto M, et al. Production of IL-18 (interferon-γ-inducing factor) messenger RNA and functional protein by murine keratinocytes. J Immunol 1997; 159: 298. 1896. Stotter H, Custer MC, Bolton ES, et al. IL-7 induced lymphokine activated killer cell activity and is regulated by IL-4. J Immunol 1991; 146: 150–155. 1897. Strand S, Hoffmann WJ, Hug H, et al. Lymphocyte apoptosis induced by CD95 (Apo-1/Fas) ligand-expressing tumor cells: a mechanism for immune evasion? Nat Med 1996; 2: 1361. 1898. Strasly M, Cavallo F, Geuna M, et al. IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross talk. J Immunol 2001; 166: 3890–3899. 1899. Strassen M, Arnold M, Hultner L, et al. Murine bone marrowderived mast cells as potent producers of IL-9: Costimulatory function of IL-10 and kit ligand in the presence of IL-1. J Immunol 2000; 164: 5549–5555. 1900. Strassman G, Jacob CO, Fong M, Bertolini DR. Mechanisms of paraneoplastic syndromes of colon-26: involvement of interleukin 6 in hypercalcemia. Cytokine 1993; 5: 463–468. 1901. Strassman G, Fong M, Freter CE, et al. Suramin interferes with Interleukin-6 receptor binding in vitro and inhibits colon 26 mediated experimental cachexia in vivo. J Clin Invest 1993; 92: 2152–2159.

Walter M. Lewko and Robert K. Oldham 1902. Strengell M, Lehtonen A, Matikainen S, Julkunen I. IL-21 enhances SOCS gene expression and inhibits LPS-induced cytokine production in human monocyte-derived dendritic cells. J Leukoc Biol 2006; 79: 1279–1285. 1903. Strengell M, Matikainen S, Siren J, et al. IL-21 in synergy with IL-15 or IL-19 enhances interferon-γ production in human NK and T cells. J Immunol 2003; 170: 5464–5469. 1904. Strieter R, Kunkel S, Showell H, et al. Endothelial cell gene expression of a neutrophil chemotactic factor by TNFα, LPS, and IL-1β. Science 1989; 243: 1467. 1905. Strieter RM, Kunkel SL, Elner VM, et al. Interleukin-8: a corneal factor that induces neovascularization. Am J Pathol 1992; 141: 1279–1284. 1906. Strieter RM, Poverini PJ, Kunkel SL, et al. The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol Chem 1995; 270: 27348. 1907. Strohmeyer T, Peter S, Hartman M, et al. Expression of the hst-1 and c-kit protooncogene in human testicular germ cell tumors. Cancer Res 1991; 51: 181–1816. 1908. Stuber E, Neurath M, Calderhead D, Fell HP, Strober W. Crosslinking of OX40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells. Immunity 1995; 2: 507. 1909. Stuber E, Strober W. The T cell–B cell interaction via OX40– OX40L is necessary for the T cell-dependent humoral immune response. J Exp Med 1996; 183: 979. 1910. Stuber E, Von Frier A, Marinescu D, Folsch UR. Involvement of OX40–OX40L in the intestinal manifestations of the murine acute graft versus host disease. Gastroenterology 1998; 115: 1205. 1911. Stumhofer JS, Laurence A, Wilson EH, et al. Interleukin 27 negatively regulates the development of interleukin 17-producing helper T cells during chronic inflammation on the central nervous system. Nat Immunol 2006; 7: 936–045. 1912. Stutman O, Paige CJ, Figarella EF. Natural cytotoxic cells against solid tumors in mice. I. Strain and age distribution and target cell susceptibility. J Immunol 1979; 121: 1819. 1913. Su Z, Emdad L, Sauane M, et al. Unique aspects of mda-7/IL-24 antitumor bystrander activity: establishing a role for secretion of MDA-7/IL-24 protein by normal cells. Oncogene 2005; 24: 7552–7566. 1914. Su ZZ, Lebedeva IV, Sarkar D, et al. Ionizing radiation enhances therapeutic efficiency of mda-7/IL-24: overcoming radiationand mda-7/IL-24-resistance in prostate cancer cells overexpressing the antiapoptotic proteins bcl-xl or bcl-2. Oncogene 2006 25: 2339–2348. 1915. Su ZZ, Lebedeva IV, Sarkar D, et al. Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in a p53-independent manner. Oncogene 2003; 22: 1164–1180. 1916. Su ZZ, Madiereddi MT, Lin JJ. Cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc Natl Acad Sci USA 1998; 95: 14400–144405. 1917. Suda T, Murray R, Fischer M, et al. Tumor necrosis factor α and P40 induce day 15 murine fetal thymocyte proliferation in combination with IL-2. J Immunol 1990; 144: 1783–1787. 1918. Sudo T, Nishikawa S, Ohno N, et al. Expression and function of interleukin 7 receptor on murine lymphocytes. Proc Natl Acad Sci USA 1993; 90: 9125. 1919. Sudo T, Suda J, Ogawa M, Ihle JN. Permissive role of interleukin-3 (IL-3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture. J Cell Physiol 1985; 124: 182.

265 1920. Sudo Y, Shimazaki C, Ashihara E, et al. Synergistic effect of FLT-3 ligand on the granulocyte colony stimulating factor induced mobilization of hematopoietic stem cells and progenitor cells into blood in mice. Blood 1997; 89: 3186–3191. 1921. Sugarman BJ, Aggarwal BB, Hass PE, et al. Recombinant human tumor necrosis factor α: effects on proliferation of normal and transformed cells in vitro. Science 1985; 230: 943–945. 1922. Sugimoto K, Ogawa A, Mizoguchi E, et al. IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. J Clin Invest 2008; 118: 534–544. 1923. Sugita K, Robertson MJ, Ritz J, Schlossman SF, Morimoto C. Participation of the CD27 antigen in the regulation of IL-2 activated human NK cells. J Immunol 1992; 149: 1199. 1924. Sugita K, Torimoto Y, Nojima Y, et al. The 1A4 molecule (CD27) is involved in T cell activation. J Immunol 1991; 147: 1477. 1925. Sugita M, Yue Y, Foskett JK. CFTR CL-channel and CFTRassociated ATP channel pores regulated by common gates. EMBO J 1998; 17: 898. 1926. Sugiura S, Lahav R, Han J, et al. Leukemia inhibitory factor is required for normal inflammatory responses to injury in the peripheral and central nervous systems in vivo and is chemotactic for macrophages in vitro. Eur J Neurosci 2000; 12: 457. 1927. Sun Y, Blink SE, Liu W, et al. Inhibition of Th2-mediated allergic airway inflammation disease by CD137 costimulation. J Immunol 2006; 177: 814–821. 1928. Sun Y, Jurgovsky K, Moller P, et al. Vaccination with IL-12 genemodified autologous melanoma cells: preclinical results and a first clinical phase I study. Gene Therapy 1998; 5: 481–490. 1929. Sun Z, Yeargeau DA, Tuypens T, et al. Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor α subunit gene. J Biol Chem 1995; 270: 1462. 1930. Sutherland CL, Rabinowitch B, Chalupny NJ, et al. ULBPs, human ligands for the NKG2D receptor, stimulate tumor immunity with enhancement by IL-15. Blood 2006; 108: 1313–1319. 1931. Suto A, Nakajima H, Hirose K, et al. Interleukin 21 prevents antigen-induced IgE production by inhibiting germ line Cε transcription of IL-4 stimulated B cells. Blood 2002; 100: 4565. 1932. Suto A, Wurster AL, Reiner SL, et al. IL-21 inhibits IFN-γ production in developing Th1 cells through the repression of eomesodermin expression. J Immunol 2006; 177: 3721–3727. 1933. Suzuki H, Kundig T, Furlonger C, Wakeman A, et al. Deregulated T cell activation in mice lacking IL-2 receptor β. Science 1995; 268: 1472. 1934. Suzuki T, Tahara H, Narula S, et al. Viral interleukin 10 (IL-10), the human herpes virus 4 cellular IL-10 homologue, induces local anergy to allogeneic and syngeneic tumors. J Exp Med 1995; 182: 477. 1935. Swain SL, McKenzie DT, Dutton RW, et al. The role of IL-2 and IL-5: characterization of a distinct helper T cell subset that makes IL-4 and IL-5 (Th2) and requires priming before induction of lymphokine secretion. Immunol Rev 1988; 102: 77–106. 1936. Swain SL, McKenzie DT, Weinberg AD, Hancock W. Characterization of T helper 1 and 2 cell subsets in normal mice: helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine secreting cells. J Immunol 1994; 141: 3445. 1937. Swain SL. The activation and differentiation of T cells. The Immunologist 1995; 3: 209–211. 1938. Symons JA, Young PR, Duff GW. Soluble type II interleukin I (IL-1) receptor binds and blocks processing of IL-1β precursor

266

1939.

1940.

1941.

1942. 1943.

1944. 1945.

1946.

1947.

1948. 1949.

1950.

1951.

1952.

1953. 1954.

1955.

1956.

1957.

1958.

1959.

Cytokines and loses affinity for the IL-1 receptor antagonist. Proc Natl Acad Sci USA 1995; 92: 1714. Szabo SJ, Dighe AS, Gubler U, Murphy KM. Regulation of the interleukin (IL)-12R β2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J Exp Med 1997; 185: 817–824. Tabata Y, Chen W, Warrier MR, et al. Allergy-driven alternative splicing of IL-13 receptor α2 yields distinct membrane and soluble forms. J Immunol 2006; 177: 7905–7912. Tacchini-Cottier F, Vesin C, Redard M, Buurman Piquet PF. Role of TNFR1 and TNFR2 in TNF-induced platelet consumption in mice. J Immunol 1998; 160: 6182–6186. Taga K, Mostowski H, Tosato G. Human interleukin-10 can directly inhibit T cell growth. Blood 1993; 81: 2964. Taga T, Hibi M, Yamasaki K, et al. Interleukin 6 triggers the association of its receptor with a possible signal transducer gp130. Cell 1989; 58: 573–581. Taga T, Kishimoto T. GP130 and the interleukin-6 family cytokines. Ann Rev Immunol 1997; 15: 797. Tagaya Y, Burton JD, Miyamoto Y, Waldmann TA. Identification of a novel receptor/signal transduction pathway for IL-15/T in mast cells. EMBO J 1996; 15: 4928–4939. Tajima S, Tsuji K, Ebihara Y, et al. Analysis of interleukin-6 receptor and gp130 expressions and proliferative capability of human CD34+ cells. J Exp Med 1996; 184: 1357–1364. Takagi M, Nakahata T, Kubo T, et al. Stimulation of mouse connective tissue-type mast cells by hematopoietic stem cell factor, a ligand for the c-kit receptor. J Immunol 1992; 148: 3446. Takahashi C, Mittler RS, Vella AT. 4–1 BB is a bona fide CD8 T cell survival signal. J Immunol 1999; 162: 5037. Takahashi GW, Andrews DF, Lilly MB, et al. Effect of granulocyte macrophage colony stimulating factor and interleukin 3 on interleukin 8 production by human neutrophils and monocytes. Blood 1993; 81: 357. Takahishi N, Udagawa N, Suda T. A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 1999; 256: 449. Takaki Y, Hayakawa Y, Nelson A, et al. IL-21 enhances tumor rejection through an NKGD2-dependent mechanism. J Immunol 2005; 175: 2167–2173. Takaoka A, Arai I, Sugimoto M, et al. Involvement of IL-31 on scratching behavior in NC/Nga mice with atopic-like dermatitis. Exp Dermatol 2006, 15 161–167. Takatsu T. Interleukin 5 and its receptor. Prog Growth Factor Res 1991; 3: 87. Takeda K, Tsutsui H, Yoshimoto T, et al. Defective NK activity and Th1 response in IL-18-deficient mice. Immunity 1998; 8: 383. Takenawa J, Kaneko Y, Fukumoto M. Enhanced expression of interleukin-6 in primary human renal cell carcinomas. J Nat Cancer Inst 1991; 83: 1668–1672. Takeuchi O, Hoshino K, Kawai T, et al. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 1999; 11: 443. Tamada K, Shimozaki K, Chapoval AI, et al. LIGHT, a TNFlike molecule, costimulates T cell proliferation and it is required for dendritic cell-mediated allogeneic T cell responses. J Immunol 2000; 164: 4105–4110. Tan JT, Whitmire JK, Murali-Krishna K, et al. 4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination. J Immunol 2000; 164: 2320–2325. Tan KB, Harrop J, Reddy M, et al. Characterization of a novel TNF-like ligand and recently described TNF ligand and TNF

1960.

1961.

1962.

1963.

1964.

1965.

1966.

1967.

1968.

1969.

1970.

1971.

1972.

1973.

1974.

1975.

1976.

1977.

receptor superfamily genes and their constitutive and inducible expression in hematopoietic and non-hematopoietic cells. Gene 1997; 204: 35. Tan PL, Farmiloe S, Yeoman S, Watson JD. Expression of the interleukin-6 gene in rheumatoid synovial fibroblasts. J Rheumatol 1990: 17; 1608. Tan W, Huang W, Zhong O, et al. IL-17 receptor knockout mice have enhanced myelotoxicity and impaired hemopoietic recovery following γ irradiation. J Immunol 2006; 176: 6186–6193. Tanaka F, Hashimoto W, Okamura H, et al. Generation of potent and tumor-specific cytotoxic T lymphocytes by interleukin 18 using dendritic cells and natural killer cells. Cancer Res 2000; 60: 4838–4844. Taniguchi T, Matsui H, Fujita T, et al. Structure and expression of a cloned DNA for human interleukin-2. Nature 1983; 302: 305–310. Tannenbaum CS, Tubbs R, Armstrong D, et al. The CXC chemokines IP-10 and Mig are necessary for IL-12 mediated regression of the mouse RENCA tumor. J Immunol 1998; 161: 927–932. Tartaglia LA, Rothe M, Hu Y-H, Goeddel DV. Tumor necrosis factor’s cytotoxicity is signaled by the p55TNF receptor. Cell 1993; 73: 213. Tartour E, Dorval T, Mosseri V, et al. Serum interleukin 6 and C-reactive protein levels correlate with resistance to IL-2 therapy and poor survival in melanoma patients. Brit J Cancer 1994; 69: 911–913. Tartour E, Fossiez F, Joyeux I, et al. Interleukin-17, a T cell derived cytokine, promotes tumorigenicity of human cervical cancers in nude mice. Cancer Res 1999; 59: 3698. Taskiran D, Stefanovic-Racic, Georgescu H, Evans CH. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by IL-1. Biochem Biophys Res Commun 1994; 200: 142. Tatsumi T, Huang J, Gooding WE, et al. Intratumoral delivery of tumor cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broad Tc1-type immunity. Cancer Res 2003; 63: 6378–6386. Tavernier J, Tuypens T, Plaetinck G, et al. Molecular basis of the membrane-anchored and two soluble isoforms of the human interleukin-5 receptor α subunit. Proc Natl Acad Sci USA 1992; 89: 7041. Taylor CW, LeBlanc M, Fisher RI, et al. Phase II evaluation of interleukin-4 in patients with non-Hodgkin’s lymphoma: a Southwest Oncology Group trial. Anticancer Drugs 2000; 11: 695–700. Tchelingerian JL, Le Saux F, Jacque C. Identification and topography of neuronal cell populations expressing TNFα and IL-1α in response to hippocampal lesion. J Neurosci Res 1996; 43: 99–106. Te Velde AA, Huijbens RJF, Heije K, de Vries JE, Figor CG. Interleukin-4 inhibits the secretion of IL-1β, tumor necrosis factor α and IL-6 by human monocytes. Blood 1990; 76: 1392. Te Velde AA, Klomp JPG, Yard BA, et al. Modulation of phenotypic and functional properties of human peripheral blood monocytes by IL-4. J Immunol 1988; 140: 1548–1554. Teague RM, Sather BD, Sacks JA, et al. IL-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors. Nat Med 2006; 12: 335–341. Teh H-S, Seebaran A, Teh S-J. TNF receptor 2-deficient CD8T cells are resistant to Fas/Fas ligand-induced cell death. J Immunol 2000; 165: 4814–4821. Temann UA, Geba GB, Rankin JA, Flavell RA. Expression of interleukin 9 in the lungs of transgenic mice causes airway

Walter M. Lewko and Robert K. Oldham

1978.

1979. 1980.

1981.

1982.

1983.

1984.

1985.

1986.

1987.

1988.

1989.

1990.

1991.

1992.

1993.

1994.

inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J Exp Med 1998; 188: 1307. Tepler I, Elias L, Smith JW 2nd, Hussein M, et al. A randomized placebo-controlled trial of recombinant human interleukin-11 in cancer patients with severe thrombocytopenia due to chemotherapy. Blood 1996; 87: 3607–3614. Tepper RI, Pattengale PK, Leder P. Murine interleukin 4 displays potent antitumor activity in vivo. Cell 1989; 57: 503–512. Teunissen MB, Koomen CW, de Waal MR, Wierenga EA, Bos, JD. Interleukin-17 and Interferon-γ synergize in the enhancement of proinflammatory cytokine production by human keratinocytes. J Invest Dermat 1998; 111: 645. Theodore AC, Center DM, Nicoll J, et al. CD4 ligand IL-16 inhibits the mixed lymphocyte reaction. J Immunol 1996; 157: 1958–1964. Thijs LC, Hack CE, Strack van Schijndel RJ, et al. Activation of the complement system during immunotherapy with recombinant IL-2. J Immunol 1990; 144: 2419. Thoma-Uszynski S, Kiertscher SM, et al. Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10. J Immunol 2000; 165: 3804–3810. Thomas B, Bird TA, Friend DJ, et al. Oncostatin M and leukemia inhibitory factor overlapping and different signals through partially shared receptor complexes. J Biol Chem 1996; 271: 32635. Thomas WD, Hersey P. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas Ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells. J Immunol 1998; 161: 2195. Thomassen E, Bird TA, Renshaw RR, Kennedy KM, Sims JE. Binding of Interleukin-18 to the interleukin-1 receptor homologous receptor IL-1Rrp 1 leads to activation of signaling pathways similar to those used by interleukin-1. J Interferon Cytokine Res1998; 18: 1077. Thompsen MK, Larsen CG, Thompsen HK, et al. Recombinant human interleukin 8 is a potent activator of canine neutrophil aggregation migration and leukotriene B4 biosynthesis. J Invest Dermatol 1991; 96: 260. Thompson BM, Mundy GR, Chambers TJ. Tumor necrosis factors α and β induce osteoblastic cells to stimulate osteoclastic bone resorption. J Immunol 1987; 136: 775. Thompson JA, Curti BD, Redman BG, et al. Interleukin-21 (IL-21): Tolerability and anti-tumor activity following two 5-day cycles in patients with stage IV melanoma or renal cell carcinoma (RCC). Amer Soc Clin Oncol Proc 2006; 24: 2505. Thompson-Snipes LA, Dahr V, Bond MW, et al. Interleukin 10: a novel stimulatory factor for mast cells and their progenitors. J Exp Med 1991; 173: 507–510. Thorbecke GJ, Shah Leu CH, Kuruvilla AP, Hardison AM, Palladino MA. Involvement of endogenous tumor necrosis factor α and transforming growth factor β during the induction of collagen type II arthritis in mice. Proc Natl Acad Sci USA 1992; 89: 7375. Thornberry NA, Bull HG, Calaycay JR, et al. A novel heterodimeric cysteine protease is required for interleukin-1β processing in monocytes. Nature 1992; 356: 768. Tian J, Atkinson MA, Clare-Salzer M, et al. Nasal administration of glutaminate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. J Exp Med 1996; 183: 1561. Tian Z, Shen X, Feng H, Gao B. IL-1β attenuates IFN-αβInduced antiviral activity and STAT1 activation in the liver:

267

1995.

1996.

1997.

1998.

1999.

2000.

2001.

2002.

2003.

2004.

2005.

2006.

2007.

2008.

2009.

2010.

2011.

involvement of the proteosome pathway. J Immunol 2000; 165: 3959–3965. Tilden AB, Itoh K, Blach CM. Human lymphokine-activated killer (LAK) cells: identification of two types of effector cells. J Immunol 1987; 138: 1068. Tilg H, Atkins MB, Dinarello CA Mier JW. Induction of circulating IL-10 by interleukin 1 and interleukin 2, but not interleukin 6 immunotherapy. Cytokine 1995: 7: 734–739. Tilg H, Mier JW, Vogel W, et al. Induction of circulating IL-1 receptor antagonist by IFN treatment. J Immunol 1993; 150: 4687–4692. Tilg H, Trehu E, Atkins MB, Dinarello CA, Mier JW. Interleukin-6 (IL-6) as an anti-inflammatory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood 1994; 83: 113. Timeus F, Ricotti E, Crescenzio N, et al. FLT-3 and its ligand are expressed in neural crest derived tumors and promote survival and its proliferation of their cell lines. Lab Invest 2001; 81: 1025–1037. Tittle TV, Weinberg AD, Steinkeler CN, Marziarz RT. Expression of the T cell activation antigen, OX-40, identifies alloreactive T cells in acute graft-versus-host disease. Blood 1997; 89: 4652. Tocher AW, Mita M, Meropol NJ, et al. Phase I pharmacokinetic and biologic correlative study of mapatumumab, a fully human monpclonal antibody with agonist activity to tumor necrosis factor-related apoptosis-inducing ligand receptor-1. J Clin Oncol 2007; 25: 1390–1395. Toda A, Picarillo CA. Development and function of naturally occurring CD4+ CD25+ regulatory T cells. J Leukocyte Biol 2006; 80: 458–470. Tofaris GK, Patterson PH, Jessen KR, Mirsky R. Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by Interleukin-6 and LIF. J. Neurosci 2002; 22: 6696–6703. Toi M, Bicknel R, Harris AL. Inhibition of colon and breast carcinoma cell growth by interleukin-4. Cancer Res 1992; 52: 275–279. Toi M, Harris AL, Bicknell R. Interleukin 4 is a potent mitogen for capillary endothelium. Biochem Biophys Res Commun 1991; 174: 1287–1293. Toksoz D, Williams D, Smith KA, et al. Expression of two distinct DNAs of the steel gene (SCF) in deficient murine stromal cell lines. Exp Hematol 1992; 20: 138–139. Toksoz D, Zsebo KM, Smith KA, et al. Support of human hematopoiesis in the long term bone marrow cultures by murine stromal cells selectively expressing the membrane bound and secreted forms of the human analog of the steel gene product stem cell factor. Proc Nat Acad Sci USA 1992; 89: 7350–7354. Tomida MY, Yamamoto-Yamaguchi Y, Hozumi M. Purification of a factor inducing proliferation of mouse myeloid leukemia M1 cells from conditioned medium of mouse fibroblast L929 cells. J Biol Chem 1984; 259: 10978. Tomura M, Zhou X-Y, Maruo S, et al. A critical role for IL-18 in the proliferation and activation of NK1.1+ CD3− cells. J Immunol 1998; 160: 4738–4796. Tong AW, Nemunaitis J, Su D. Intratumoral injection of INGN 241, a non-replicating adenovector expressing the melanomadifferentiation associated gene-7 (mda-7/IL24): biologic outcome in advanced cancer patients. Mol Ther 2005; 11: 160–172. Topalian SL, Solomon D, Avis FP, et al. Immunotherapy of patients with advanced cancer using tumor-infiltrating lymphocytes and recombinant interleukin-2: a pilot study. J Clin Oncol 1988; 6: 839.

268 2012. Topp MS, Koenigsmann M, Mire-Sluis A, et al. Recombinant human interleukin-4 inhibits growth of some human lung tumor cell lines in vitro and in vivo. Blood 1993; 82: 2837–2844. 2013. Topp MS, Papadimitriou CA, Eitelbach F, et al. Recombinant human interleukin 4 has antiproliferative activity on human tumor cell lines derived from epithelial and nonepithelial histologies. Cancer Res 1995; 55: 2173–2176. 2014. Torigoe K, Ushio S, Okura T, et al. Purification and characterization of the human interleukin-18 receptor. J Biol Chem 1997; 272: 25737. 2015. Tortorella C, Schulze-Koops H, Thomas R, et al. Expression of CD45RB and CD27 identifies subsets of CD4+ memory T cells with different capacities to induce B cell differentiation. J Immunol 1995; 155: 149. 2016. Tosato G, Pike SE. Interferon β2/interleukin-6 is a co-stimulant for human T lymphocytes. J Immunol 1988; 141: 1556–1562. 2017. Touw I, van Agthoven T, van Gurp R, et al. Interleukin 7 is a growth factor of precursor B and T acute lymphoblastic leukemia. Blood 1990; 75: 2097–2101. 2018. Tran NL, McDonough WS, Donohue PJ, et al. The human Fn14 receptor gene is up regulated in migrating glioma cells in vitro and overexpressed in advanced glial tumors. Am J Pathol 2003; 162: 1313–1321. 2019. Tran NL, McDonough WS, Savitch BA, et al. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling system regulates glioma cell activation and BCL-XL/BCL-W expression. J Biol Chem 2005; 280: 3483–3492. 2020. Trehu EG, Mier JW, Dubois JS, et al. Phase I trial of interleukin 2 in combination with the soluble tumor necrosis factor receptor p75 UgG chimera. Clin Cancer Res 1996; 2: 1341–1351. 2021. Treisman J, Higuchi CM, Thompson JA, et al. Enhancement by interleukin 4 of interleukin 2 or antibody induced proliferation of lymphocytes from interleukin 2 treated cancer patients. Cancer Res 1990; 50: 1160. 2022. Trepicchio WL, Ozawa M, Walters IB, et al. Interleukin-11 therapy selectively downregulates type I cytokine proinflammatory pathways in psoriasis lesions. J Clin Invest 1999; 104: 1527. 2023. Trepicchio WL, Wand L, Bozza M, Dorner AJ. IL-11 regulates macrophage effector function through inhibition of nuclear factor-κB. J Immunol 1997; 159: 5661–5670. 2024. Trinchieri G, Matsumoto Kobayashi M, et al. Response of peripheral blood resting natural killer cells to interleukin-2. J Exp Med 1984; 160: 1147. 2025. Trinchieri G. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv Immunol 1998; 70: 83. 2026. Triozzi PL, Kin KA, Martin EW, et al. Phase I trial of escalating doses of interleukin-1β in combination with a fixed dose of interleukin-2. J Clin Oncol 1995; 13: 482–489. 2027. Tripp CS, Wolf SF, Unanue ER. Interleukin 12 and tumor necrosis factor α are costimulators of interferon γ production in severe combined immunodeficient mice with listeriosis and interleukin 10 is a physiologic antagonist. Proc Natl Acad Sci USA 1993; 90: 3725–3729. 2028. Truong MJ, Darcissac EC, Hermann E, et al. Interleukin-16 inhibits human immunodeficiency virus type entry and replication in macrophages and dendritic cells. J Virology 1999; 73: 7008–7013. 2029. Tsai EY, Yie D, Thanos D, Goldfeld AE. Cell type specific regulation of the human tumor necrosis factor α gene in B cells and T cells by NFATp and ATF-2/JUN. Mol Cell Biol 1996; 16: 5232.

Cytokines 2030. Tsou CL, Haskell CA, Charo IF. Tumor necrosis factor-αconverting enzyme mediates the inducible cleavage of fractalkine. J Biol Chem 2001; 276: 44622–44626. 2031. Tsuji K, Lyman SD, Sudo T, Clark SC, Ogawa M. Enhancement of murine hematopoiesis by synergistic interactions between steel factor (ligand for c-kit), interleukin-11, and other early acting factors in culture. Blood 1992; 79: 2855. 2032. Tsuji K, Nakahata T, Takagi M, et al. Effects of interleukin 3 and interleukin 4 on the development of connective tissue type mast cells: interleukin 3 supports their survival and interleukin 4 triggers and supports their proliferation synergistically with interleukin 3. Blood 1990; 75: 421–427. 2033. Tsuji K, Zsebo KM, Ogawa M. Murine mast cell colony formation supported by IL-3, IL-4 and recombinant stem cell factor, ligand from c-kit. J Cell Physiol 1991; 148: 362. 2034. Tsunoda T, Tanimura H, Yamaue H, et al. The promotive effect of interleukin-4 with interleukin-2 in the proliferation of tumor infiltrating lymphocytes from patients with malignant tumor. Biotherapy 1992; 4: 9–15. 2035. Tsutsui H, Nakanishi K, Matsui K, et al. IFN-γ-Inducing Factor Upregulates Fas ligand-mediated cytotoxic activity in murine natural killer cell clones. J Immunol 1996; 157: 3967–3973. 2036. Tuck AB, O’Malley FP, Singhal H, et al. Osteopontin expression in a group of lymph node negative breast cancer patients. Int J Cancer Predict Oncol 1998; 79: 502–508. 2037. Turley JM, Fu T, Ruscetti FW, et al. Vitamin E succinate induces Fas-mediated apoptosis in estrogen receptor-negative human breast cancer cells. Cancer Res 1997; 57: 881–890. 2038. Turner JG, Rakhmilevich AL, Burdelya L, et al. Anti-CD40 antibody induces antitumor and antimetastatic effects: The role of NK cells. J Immunol 2001; 166: 89–94. 2039. Tuting T, Wilson CC, Martin DM, et al. Autologous human monocyte-derived dendritic cells genetically modified to express melanoma antigens elicit primary cytotoxic T cell responses in vitro: enhancement by cotransfection of genes encoding the Th1-biasing cytokines IL-12 and IFN-α. J Immunol 1998; 160: 1139–1147. 2040. Tuypens T, Plaetinct G, Baker E, et al. Organization and chromosomal localization of the human interleukin-5 receptor α-chain gene. Eur Cytokine Network 1992; 3: 451. 2041. Udagawa N, Takahashi N, Katagiri T, et al. Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors. J Exp Med 1995: 182: 1461. 2042. Ueda M, Imada K, Imura A, et al. Expression of functional interleukin-21 receptor on adult T-cell leukemia cells. Br J Haematol 2005; 128: 169–176. 2043. Ugai S, Shimozato O, Yu L, et al. Transduction of IL-21 and IL-23 genes in human pancreatic carcinoma cells produces natural killer cell-dependent and cell-independent antitumor effects. Cancer Gene Ther 2003; 10: 771–778. 2044. Urba WJ, Clark JW, Steis RG, et al. Intraperitoneal lymphokineactivated killer cell/interleukin-2 therapy in patients with intraabdominal cancer: immunologic considerations. J Natl Cancer Inst 1989; 81: 602. 2045. Ushio S, Namba M, Okura T, et al. Cloning of the cDNA for human IFN-γ-inducing factor in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 1996; 156: 4274. 2046. Usui N, Mimnaugh EG, Sinha BK. A role for the interleukin 1 receptor in the synergistic antitumor effects of human interleukin 1α and etoposide against human melanoma cells. Cancer Res 1991; 51: 769–774.

Walter M. Lewko and Robert K. Oldham 2047. Vaccarello L, Wang YL and Whiteside TL. Sustained outgrowth of auto tumor reactive T lymphocytes from human ovarian carcinomas in the presence of tumor necrosis factor α and interleukin 2. Human Immunology 1990; 28: 216–227. 2048. Vagliani M, Rodolfo M, Cavallo F, et al. Interleukin 12 potentiates the curative effect of a vaccine based on interleukin 2-transduced tumor cells. Cancer Res 1996; 56: 467. 2049. Valera LM, Ip MM. Tumor necrosis factor-α: multifunctional regulator of mammary gland development. Endocrinol 1996, 137: 4915. 2050. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of TNF-α-induced apoptosis by NF-κB. Science 1996; 274: 787–789. 2051. Van Arsdale TL, Van Arsdale SL, et al. Lymphotoxin-β receptor signaling complex: role of tumor necrosis factor receptor associated factor 3 recruitment in cell death and activation of nuclear factor kappa B. Proc Natl Acad Sci USA 1997; 94: 2460–2465. 2052. Van Damme J, Opdenakker G, et al. Identification of the human 26 kD protein, interferon β2 (IFN-β2), as a B cell hybridoma/ plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor. J Exp Med 1987; 165: 914. 2053. Van Damme J, Schaafsma MR, Fibbe WE, et al. Simultaneous production of interleukin 6, interferon β2 and colony stimulating activity by fibroblasts after viral and bacterial infection. Eur J Immunol 1989; 19: 163. 2054. Van De Griend RJ, Krimpen BA, Ranfeltap CPM and Bolhuis RH. Rapidly expanded natural killer cell clones have strong antitumor cell activity and have surface phenotype of either T, non-T, or null cells. J Immunol 1984; 132: 3185. 2055. Van den Berg WB, Joosten LA, Helsen M, Van de Loo FA. Amelioration of established collagen-induced arthritis with anti-IL-1 treatment. Clin Exp Immunol 1994; 95: 237. 2056. Van der Burg SH, Piersma SJ, de Jong A, et al. Association of cervical cancer with the presence of CD4+ regulatory T cells specific for human papillomavirus antigens. Proc Natl Acad Sci USA 2007; 104: 12087–12092. 2057. Van Gameren MM, Willemse PH, Mulder NH, et al. Effects of recombinant human Interleukin-6 in cancer patients: a phase I-II trial. Blood 1994; 84: 1431–1441. 2058. Van Kooten C, Gaillard C, et al. B cells regulate expression of CD40 ligand on activated T cells by lowering the mRNA level and through the release of soluble CD40. E J Immunol 1994; 24: 787. 2059. van Leeuen EM, Gamadia LE, Baars PA, et al. Proliferation requirements of cytomegalovirus-specific effector-type human CD8+-T cells. J Immunol 2002; 169: 5838–5842. 2060. Van Lier RAW, Borst J, et al. Tissue distribution and biochemical functional properties of Tp55 (CD27), a novel T cell differentiation antigen. J Immunol 1987; 139: 1589. 2061. Van Oers HS, Pals ST, Evers LM, et al. Expression and release of CD27 in human B-cell malignancies. Blood 1993; 82: 3430. 2062. Van Parijs L, Buickians A, Ibragimov A, Alt F, Willerford D, Abbas A. Functional responses and apoptosis of CD25 (IL-2 R α)-deficient T cells expressing a transgenic antigen receptor. J Immunol 1997; 158: 3738. 2063. Van Snick J, Goethals A, Renauld J-C, et al. Cloning and characterization of cDNA for a new mouse T cell growth factor (P40). J Exp Med 1989; 169: 363–368. 2064. Van Snick J. Interleukin 6: an overview. Ann Rev Immunol 1990; 8: 253–280. 2065. Van Zaanen HC, Lokhorst HM, Aarden LA, et al. Blocking interleukin-6 activity with chimeric anti-IL-6 monoclonal anti-

269

2066.

2067. 2068.

2069.

2070.

2071.

2072.

2073.

2074.

2075.

2076.

2077.

2078.

2079.

2080.

2081.

2082.

bodies in multiple myeloma: effects on soluble IL6 receptor and soluble gp130. Leuk Lymphoma 1998; 31: 551–558. Varghese J, Chattopopadhaya S, Sarin A. Inhibition of p38 Kinase reveals TNF-α-mediated, caspase-dependent, apoptotic death pathway in a human myelomonocyte cell line. J Immunol 2001; 166: 6570–6577. Vassali P. The pathophysiology of Tumor Necrosis Factor. Ann Rev Immunol 1992; 10: 411. Veiby OP, Jacobsen FW, Cui L, Lyman SD, Jacobsen SEW. The flt-ligand promotes the survival of primitive hematopoietic progenitor cells with myeloid as well as B lymphoid potential. J Immunol 1996; 157: 2953–2960. Veiby OP, Lyman SD, Jacobsen SEW. Combined signaling through interleukin-7 receptors and flt3 but not c-kit potently and selectively promotes B-cells commitment and differentiation from uncommitted murine bone marrow progenitor cells. Blood 1996; 88: 1256. Veldhoen M, Hocking RJ, Atkins CJ, et al. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing cells. Immunity 2006; 24: 179–189. Vellenga E, Esselink MT, Straaten J, et al. The supportive effects of IL-7 on eosinophil progenitors from human bone marrow cells can be blocked by anti-IL-5. J Immunol 1992; 149: 2992. Verschraegen CF, Kudelka AP, Termrungruanglert W, et al. Effects of interleukin-1α on ovarian carcinoma in patients with recurrent disease. Eur J Cancer 1996; 32A: 1609–1611. Vetto JT, Lum S, Morris A, Sciotte M, et al. Presence of the T cell activation marker OX-40 on tumor infiltrating lymphocytes and draining lymph node cells from patients with melanoma and head and neck cancers. Am J Surg 1997; 174: 258. Vidal-Vanaclocha F, Amezaga C, Asumendi A, Kaplanski G, Dinarello CA. Interleukin 1 receptor blockade reduces the number and size of murine B16 melanoma hepatic metastases. Cancer Res 1994; 54: 2267. Vidal-Vanaclocha F, Mendoza L, Telleria N. Clinical and experimental approaches to the pathophysiology of interleukin-18 in cancer progression. Cancer Metastasis Rev 2006: 9: 1480–1486. Viera P, de Waal Malefyt R, Dang M-N, et al. Isolation and expression of human cytokine synthesis inhibitory factor (CSIF/ IL-10) cDNA clones: homology to Epstein Barr virus open reading frame BCRFI. Proc Nat Acad Sci USA 1991; 88: 1172–1176. Villers D, Dao T, Nguyen J-M, et al. Increased plasma levels of human interleukin for DA1.a cells/leukemia inhibitory factor in sepsis correlate with shock and poor prognosis. J Infect Dis 1995; 171: 232. Vinay DS, Choi BK, Bae JS, et al. CD137-deficient mice have reduced NK/NKT cell numbers and function, are resistant to lipopolysaccharide-induced shock syndromes and have lower IL-4 responses. J Immunol 2004; 173: 4218–4229. Vink A, Renauld JC, Warnier G, Van Snick J. Interleukin-9 stimulates in vitro growth of mouse thymic lymphomas. Eur J Immunol 1993; 23: 1134. Vissinga CS, Fatur-Saunders DJ and Takei F. Dual role of IL-7 in the growth and differentiation of immature thymocytes. Experimental Hematology 1992; 20: 998–1003. Vistintin A, Mazzoni A, Spitzer JH, et al. Regulation of Toll-like receptors in human monocytes and dendritic cells. J Immunol 2001; 166: 249–255. Vitetta ES, Ohara J, Myers CD, et al. Serological biochemical and functional identity of B cell stimulatory factor-1 and B cell

270

2083. 2084.

2085.

2086.

2087.

2088.

2089.

2090.

2091.

2092.

2093.

2094.

2095.

2096.

2097.

2098.

2099.

2100.

Cytokines differentiation factor for IgG1. J Exp Med 1985; 162: 1726–1731. Volpert OV, Fong T, Koch AE, et al. Inhibition of angiogenesis by interleukin-4. J Exp Med 1998; 88: 1039. Von Freeden-Jeffrey U, Viera P, Lucian LA, et al. Lymphopenia in interleukin (IL)-7 gene deleted mice identifies IL-7 and a nonredundant cytokine. J Exp Med 1995; 181: 1519–1526. Von Freeden-Jeffry R, Solvason N, Howard M, Murray R. The earliest T lineage-committed cells depend on interleukin-7 for Bcl-2 expression and normal cell cycle progression. Immunity 1997; 7: 147. Von Leoprechting A, van der Bruggen P, Pahl HL, Aruffo A, Simon JC. Stimulation of CD40 on immunogenic human malignant melanoma augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis. Cancer Res 1999; 59: 1287. Vonderheide RH, Dutcher JP, Anderson JE, et al. Phase I study of recombinant human CD40 ligand in cancer patients. J Clin Oncol 2001; 19: 3280–3287. Wahl AF, Klussman K, Thompson JD, et al. The anti-CD30 monoclonal antibody SGN-30 promotes growth arrest and DNA fragmentation in vitro and affects antitumor activity in models of Hodgkin’s lymphoma. Cancer Res 2002; 62: 3736–3742. Walczak H, Degli-Esposti MA, Johnson RS, et al. TRAIL-R2: a novel apoptosis mediating receptor for TRAIL. EMBO J 1997; 16: 5386. Walczak H, Miller RE, Arail K, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999; 5: 157. Waldmann T, Tagaya Y. The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host cell response to intracellular pathogens. Ann Rev Immunol 1999; 17: 19. Wallace PM, MacMaster JF, Rouleau KA, et al. Regulation of inflammatory responses by oncostatin M. J Immunol 1999; 162: 5547. Wallach D, Holtmann H, Aderka D, et al. Mechanisms which take part in the regulation of the response to tumor necrosis factor. Lymphokine Res 1989; 8: 359. Wallach D, Varfolomezv EE, Malinin NL, et al. Tumor necrosis factor and Fas signaling mechanisms. Ann Rev Immunol 1999; 17: 331. Walter EA, Greenberg PD, Gilbert MJ, et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Eng J Med 1995; 333: 1038–1044. Walz A, Peveri P, Aschauer H, Baggiolini M. Purification and amino acid sequencing of NAF, a novel neutrophil activating factor produced by monocytes. Biochem Biophys Res Commun 1987; 149: 755. Wang A, Braun SE, Sonpavde G, Cornetta K. Antileukemic activity of Flt3 ligand in murine leukemia. Cancer Res 2000; 60: 1895–1900. Wang C-Y, Cusack JC, Liu R, Baldwin AS. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-κB. Nat Med 1999; 5: 412. Wang C-Y, Mayo MW, Baldwin AS. TNF- and Cancer therapyinduced apoptosis: potentiation by inhibition of NF-κB. Science 1996; 274: 784–787. Wang C-Y, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS. NF-κB antiapoptosis: induction of TRAF1 and TRAF2 and the c-IAP1 and c-IAP2 to suppress caspace-8 activation. Science 1998; 281: 1680.

2101. Wang G, Tschoi M, Spolski R, et al. In vivo antitumor activity of interleukin 21 mediated by natural killer cells. Cancer Res 2003; 63: 9016–9022. 2102. Wang J, Homer RJ, Chen Q, Elias JA. Endogenous and exogenous IL-6 inhibit aeroallerogen-induced Th2 inflammation. J Immunol 2000; 165: 4051–4056. 2103. Wang J, Homer RJ, Hong L, et al. IL-11 selectively inhibits aeroallergen-induced pulmonary eosinophilia and Th2 cytokine production. J Immunol 2000; 165: 2222–2231. 2104. Wang M, Tan Z, Zhang R, et al. Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors IL22R1/IL-20R2 and IL-20R1/IL-20R2. J Biol Chem 2002; 277: 7341–7347. 2105. Wang P, Wu P, Cheewatrakoolpong B, et al. Selective inhibition of IL-5 receptor α-chain gene transcription by IL-5, IL-3 and granulocyte-macrophage colony stimulating factor in human blood eosinophils. J Immunol 1998; 160: 4427–4432. 2106. Wang R, Zhang L, Zhang X, et al. Differential regulation of the expression of CD95 ligand, receptor activator of nuclear factor-κB ligand (RANKL), TNF-related apoptosis-inducing ligand (TRAIL), and TNF-α during T cell activation. J Immunol 2001; 166: 1983–1990. 2107. Wang R, Zhang L, Zhang X, Yin D, Mufson RA, Shi Y. Protein kinase C regulates FAS (CD95/Apo1) expression. J Immunol 1998; 161: 2201. 2108. Wang X, Chen M, Wandinger KP, Williams G, Dhib-Jalbut S. IFN-β-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: Relevance to IFN-β-1b therapeutic effects in multiple sclerosis. J Immunol 2000; 165: 548–557. 2109. Wang Y-H, Angkasekwinai P, Lu N, et al. IL-25 augments type 2 immune responses by enhancing the expansion and functions of TSLP-DC-activated Th2 memory cells. J Exp Med 2007; 204: 1837–1847. 2110. Wang Y, Wang J, Sun Y, Wu Q, Fu Y-X. Complementary effects of TNF and lymphotoxin on the formation of germinal center and follicular dendritic cells. J Immunol 2001; 166: 330–337. 2111. Wang YL, Lusheng S, Kanbaul A, et al. Lymphocytes infiltrating human ovarian tumors: Synergy between tumor necrosis factor α and interleukin 2 in the generation of CD8+ effectors from tumorinfiltrating lymphocytes. Cancer Res 1989; 49: 5979–5985. 2112. Wang YO, Ugai S, Shimazato O, et al. Induction of systemic immunity by expression of interleukin-23 in murine colon carcinoma cells. Int J Cancer 2003; 105: 820–824. 2113. Wang Z, Karras JG, Howard RG, Rothstein TL. Induction of bcl-X by CD40 engagement rescues sIg-induced apoptosis in murine B cells. J Immunol 1995; 155: 3722–3725. 2114. Wanidworanum C and Strober W. Predominant role of tumor necrosis factor-α in human monocyte IL-10 synthesis. J Immunol 1993; 151: 6853. 2115. Wantanabe M, Ueno Y, Yajima T, et al. Interleukin-7 is produced by human intestinal epithelial cells and regulates the proliferation of intestinal mucosal lymphocytes. J Clin Invest 1995; 95: 2945. 2116. Ware CF, Crowe PD, Grayson MH, Androlewicz MJ, Browning JL. Expression of surface lymphotoxin and tumor necrosis factor on activated T, B, and natural killer cells. J Immunol 1992; 149: 3881–3888. 2117. Waring PM, Waring LJ, Billington T, Metcalf D. Leukemia inhibitory factor protects against lethal Escherichia coli septic shock in mice. Proc Natl Acad Sci USA 1995; 92: 1337. 2118. Waring PM, Waring LJ, Metcalf D. Circulating leukemia inhibitory factor levels correlate with disease severity in meningococcemia. J Infect Dis 1994; 170: 1224.

Walter M. Lewko and Robert K. Oldham 2119. Waring PM, Wycherly K, Cary D, Nicola N, Metcalf D. Leukemia inhibitory factor levels are elevated in septic shock and various inflammatory body fluids. J Clin Invest 1992; 90: 2031. 2120. Warren HS, Kinnear BF, Phillips JH, Lanier LL. Production of IL-5 by Human NK cells and regulation of IL-5 secretion by IL-4, IL-10, and IL-12. J Immunol 1995; 154: 5144–5152. 2121. Warringa RAJ, Koenderman L, Kok PTM, et al. Modulation and induction of eosinophil chemotaxis by granulocyte macrophage colony stimulating factor and interleukin 3. Blood 1991; 77: 2694. 2122. Wassink L, Veira PL, Smits HH, et al. ICOS expression by activated human Th cells is enhanced by IL-14 and IL-23: Increased ICOS expression enhances the effector function of both TH1 and Th2 cells. J Immunol 2004; 173: 1779–1786. 2123. Waxman AB, Einarrson O, Seres T, et al. Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation. J Clin Invest 1998; 101: 1970. 2124. Weaver A, Chang J, Wrigley E, et al. Randomized comparison of progenitor-cell mobilization using chemotherapy, stem cell factor, and filgrastrim or chemotherapy plus filgrastim alone in patients with ovarian cancer. J Clin Oncol 1998; 16: 2601–2612. 2125. Weber GF, Gaertner PC, Erl W, et al. IL-22-mediated tumor growth reduction correlates with inhibition of ERK1/2 and AKT phosphorylation and induction of cell cycle arrest in the G2-M phase. J Immunol 2006; 177: 8266–8272. 2126. Weber J, Yang JC, Topalian SL, et al. Phase I trial of subcutaneous interleukin 6 in patients with advanced cancer. J Clin Oncol 1993; 11: 499–506. 2127. Wegman TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal–fetal relationship: is successful pregnancy a Th2 phenomenon? Immunol Today 1993; 18: 479. 2128. Wei CC, Chen WY, Wang YC. Detection of IL-20 and its receptor on psoriatic skin. Clin Immunol 2005; 117: 65–72. 2129. Wei L, Lawrence A, Elias KM, et al. IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner. J Biol Chem 2007; 282: 34605–34610. 2130. Wei SJ, Liu JH, Blanchard DK, Djeu JY. Induction of IL-8 gene expression in polymorphonuclear leukocytes by recombinant IL-2. J Immunol 1994; 142: 244. 2131. Wei X-Q, Leung BP, Arthur HML, McInnes IB, Liew FY. Reduced incidence and severity of arthritis in mice lacking IL-18. J Immunol 2001; 166: 517–521. 2132. Weikowski MT, Leach MW, Evans EW, et al. Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility and premature death. J Immunol 2001; 166: 7563–7570. 2133. Weil-Hillman G, Fisch P, Prieve AF, et al. Lymphokine-activated killer activity induced by in vivo interleukin 2 therapy: predominant role for lymphocytes with increased expression of CD2 and Leu19 antigens but negative expression of CD16 antigens. Cancer Res 1989; 49: 3680. 2134. Weinberg AD, Bourdette DN, Sullivan TJ, et al. Selective depletion of myelin-reactive T cells with the anti-Ox-40 antibody ameliorates autoimmune encephalitis. Nat Med 1996; 2: 183. 2135. Weinberg K, Parkman R. Severe combined immunodeficiency due to a specific defect in the production of interleukin-2. N Eng J Med 1990; 322: 1718. 2136. Weiss GR, Margolin KA, Sznol M, et al. Phase II study of the continuous infusion of Interleukin 6 for metastatic renal cell carcinoma. J Immunother 1995; 18: 52–56.

271 2137. Weiss T, Grell M, Siemienski K, et al. TNFR80-dependent enhancement of TNFR60-induced cell death is mediated by TNFR-associated factor 2 and is specific for TNFR60. J Immunol 1998; 161: 3136–3142. 2138. Wiethe C, Dittmar K, Doan D, et al. Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumorassociated antigen. J Immunol 2003; 170: 2919–2922. 2139. Welch PA, Namen AE, Goodwin RG, et al. Human IL-7: a novel T cell growth factor. J Immunol 1989; 143(11): 3562–3567. 2140. Weller PF, Rand TH, Barrett T, et al. Accessory cell function of eosinophils: HLA DR dependent MHC restricted antigen presentation and interleukin 1α formation. J Immunol 1993; 150: 2554. 2141. Welte K, Chang YW, Mertelsmann R, et al. Purification of human interleukin-2 to apparent homogeneity and its molecular heterogeneity. J Exp Med 1982; 156: 435. 2142. Weng Y, Siciliano SJ, Waldburger KE, et al. Binding and functional properties of recombinant and endogenous CXCR3 chemokine receptors. J Biol Chem 1998; 273: 18288. 2143. Wershil BK, Tsai M, Geissler EN, Zsebo KM, Galli SJ. The rat c-kit ligand, stem cell factor, induces c-kit-dependent mouse mast cell activation in vivo. Evidence that signaling through the c-kit receptor can induce expression of cellular function. J Exp Med 1992; 175: 245. 2144. Wesche H, Korherr C, Kracht M, et al. The interleukin-1 accessory protein (IL-1RAcP) is essential for IL-1-induced activation of interleukin-1 receptor associated kinase (IRAK) and stressactivated protein kinases (SAP Kinases). J Biol Chem 1997; 272: 7727. 2145. Wesche H, Wenzel WJ, Shillinglaw W, Li S, Cao Z. MyD88: an adaptor that recruits IRAK to the IL-1 receptor complex. Immunity 1997; 7: 837. 2146. 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. 2147. Westendorf JJ, Jelinek DF. Growth regulatory pathways in myeloma. Eidence for autocrine oncostatin M expression. J Immunol 1996; 157: 3081–3088. 2148. Wetzler M, Kurzrock R, Estrov Z, et al. Altered levels of interleukin-1β and interleukin-1 receptor antagonist in chronic myelogenous leukemia: clinical and prognostic correlates. Blood 1994; 84: 3142–3147. 2149. Whitehead RP, Fleming T, MacDonald JS, et al. A phase II trial of recombinant tumor necrosis factor in patients with metastatic colorectal adenocarcinoma: a Southwest Oncology Group Study. J Biol Response Modifiers 1990; 9: 588–591. 2150. Whitehead RP, Unger JM, Goodwin JW, et al. Phase II trial of recombinant human interleukin-4 in patients with disseminated malignant melanoma: a Southwest Oncology Group study. J Immunother 1998; 21: 440–446. 2151. Whitman SP, Archer KJ, Feng L, et al. Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of Flt3: a cancer and leukemia group B study. Cancer Res 2001; 61: 7233–7239. 2152. Widmer M. Acres R, Sassenfeld H, Grabstein K. Regulation of cytolytic cell populations from human peripheral blood by B cell stimulatory factor 1 (interleukin 4). J Exp Med 1987; 166: 1447. 2153. Widmer MB, Morrissey PJ, Namen AE, et al. Interleukin 7 stimulates the growth of fetal thymic precursors of cytolytic cells: induction of effector function by interleukin 2 and inhibition by interleukin 4. Int Immunol 1990; 2: 1055–1061.

272 2154. Wierda WG, Cantwell ML, Woods SJ, Rassenti LZ, Prussak CE, Kipps TJ. CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia. Blood 2000; 96: 2917–2924. 2155. Wiethe C, Dittmar K, Doan T, et al. Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-κB (RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen. J Immunol 2003; 171: 4121–4130. 2156. Wiggington JM, Lee JK, Wiltrout TA, et al. Synergistic engagement of an ineffective endogenous anti-tumor response and induction of IFN-γ and Fas-ligand-dependent tumor eradication by combined administration of IL-18 and IL-2. J Immunol 2002; 169: 4467–4474. 2157. Wigginton JM, Kuhns DB, Back TC, et al. Interleukin 12 primes macrophages for nitric oxide production in vivo and restores depressed nitric oxide production by macrophages from tumor bearing mice: implications for antitumor activity of interleukin 12 and/or interleukin 2. Cancer Res 1996; 56: 1131. 2158. Wigginton JM, Park J-W, Gruys ME, et al. Complete regression of established spontaneous mammary carcinoma and the therapeutic prevention of genetically programmed neoplastic transition by IL-12/pulse IL-2: Induction of local T cell infiltration, Fas/Fas ligand gene expression and mammary epithelial apoptosis. J Immunol 2001; 166: 1156–1168. 2159. Wilcox RA, Chapoval AI, Gorski KS, et al. Expression of functional CD137 by murine dendritic cells. J Immunol 2002; 168: 4262–4267. 2160. Wilcox RA, Tamada K, Strome FE, et al. Signaling through NK cell-associated CD137 promotes both helper functions for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity. J Immunol 2002; 169: 4230–4236. 2161. Wilcoxen SC, Kirkman E, Dowdell KC, Stohlman SA. Genderdependent IL-12 secretion is regulated by IL-10. J Immunol 2000; 164: 6237–6243. 2162. Wild JS, Sigounas A, Sur N, Siddiqui MS, et al. IFN-γ inducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines and air way eosinophilia in a mouse model of allergic asthma. J Immunol 2000; 164: 2701. 2163. Wiley JA, Geha R, Harmsen AG. Exogenous CD40 ligand induces a pulmonary inflammation response. J Immunol 1997; 158: 2932–2938. 2164. Wiley SR, Cassiano L, Lofton T, et al. A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis. Immunity 2001; 15: 857–846. 2165. Wiley SR, Goodwin RG, Smith CA. Reverse signaling via CD30 ligand. J Immunol 1996; 157: 3635–3639. 2166. Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995; 3: 673. 2167. Willems F, Marchant A, Delville JP, et al. Interleukin-10 inhibits B7 and intracellular adhesion molecule-1 expression on human monocytes. Eur J Immunol 1994; 24: 1007. 2168. Willerford DM, Chen J, Ferry FA, Davidson L, Ma A, Alt FW. Interleukin-2 receptor α chain regulates the size and content of the peripheral lymphoid compartment. Immunity 1995; 3: 521. 2169. Williams DE, Eisenman J, Baird A, et al. Identification of a ligand for the c-kit proto-oncogene. Cell 1990; 63: 167–174. 2170. Williams DE, Morrissey PJ, Mochizuki DY, et al. T cell growth factor P40 promotes the proliferation of myeloid cell lines and enhances erythroid burst formation by normal murine bone marrow cells in vitro. Blood 1990; 76: 906–911. 2171. Williams N, Eger RR, Jackson HN, et al. Two factor requirement for murine megakaryocyte colony formation. J Cell Physiol 1982; 110: 101.

Cytokines 2172. Williams NS, Klem J, Puzanov IJ, et al. Natural killer cell differentiation: insights from knockout and transgenic mouse models and in vivo systems. Immunol Rev 1998; 165: 47–61. 2173. Williams RO, Martinova-Mutafchieva L, Feldman M, Maini RN. Evaluation of TNF-α and IL-1 blockade in collagen-induced arthritis and comparison with combined anti-TNF-α/anti-CD4 therapy. J Immunol 2000; 165: 7240–7245. 2174. Williamson BD, Carswell EA, Rubin BY, et al. Human tumor necrosis factor produced by human B cell lines. Synergistic cytotoxic interaction with human interferon γ. Proc Nat Acad Sci USA 1983; 80: 5397–5401. 2175. Wills-Karp M, Luyimbazi J, Xu X, et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282: 2258–2261. 2176. Wirtz S, Tubbe I, Galle PR, et al. Protection from lethal septic peritonitis by neutralizing the biological function of interleukin-27. J Exp Med 2006; 203: 1875–1881. 2177. Wischhusen J, Jung G, Radovanovic I, et al. Identification of CD70-mediated apoptosis of immune effector cells as a novel immune escape pathway of human glioblastoma. Cancer Res 2002; 62: 2592–2599. 2178. Witowski J, Pawlaczyk K, Breborowicz A, et al. IL-17 stimulates intraperitoneal neutrophil infiltration through the release of GROα chemokine from mesothelial cells. J Immunol 2000; 165: 5814–5821. 2179. Wittig B, Marten A, Dorbic T, Weineck S, et al. Therapeutic vaccination against metastatic melanoma by expression-modulated and immunomodified autologous tumor cells: a first clinical phaseI/II trial. Human Gene Therapy 2001; 12: 267–278. 2180. Woerly G, Roger N, Loiseau S, Capron M. Expression of Th1 and Th2 immunoregulatory cytokines by human eosinophils. Int Arch Allergy Immunol 1999; 118: 95. 2181. Wogensen L, Huang X, Sarvetnik N. Leukocyte extravasation into the pancreatic tissue in transgenic mice expressing interleukin 10 in the islets of Langerhans. J Exp Med 1993; 178: 175. 2182. Wogensen LD, Mandrup-Poulsen T, Markholst H, et al. Interleukin-1 potentiates glucose stimulated insulin release in the isolated perfused pancreas. Acta Endocrinol 1988; 117: 302. 2183. Wogun AW, van Gils FC, Wagemaker G. Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of human and rhesus monkeys. Blood 1993; 81: 2036–2043. 2184. Wolf D, Hallmann R, Sass G, et al. TNF-α-induced expression of adhesion molecules in the liver is under control of TNFRI. Relevance for Con A-induced Hepatitis. J Immunol 2001; 166: 1300–1307. 2185. Wolf SF, Sieburth D, Sypek J. Interleukin 12: A key modulator of immune response. Stem Cells 1994; 12: 154–168. 2186. Wolf SF, Temple PA, Kobayashi M, et al. Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. J Immunol 1991; 146: 3074–3081. 2187. Wolk K, Kunz S, Asadullah K, et al. Immune cells as sources and targets of the IL-10 family members. J Immunol 2002; 168: 5397–5402. 2188. Wolk K, Sabat R. Interleukin-22: a novel T- and NK-derived cytokine that regulates the biology of tissue cells. Cytokine Growth Factor Rev 2006; 17: 267–380. 2189. Wolk K, Witte E, Hoffmann U, et al. IL-22 induces lipopolysaccharide-binding protein in hepatocytes: a potential systemic role of IL-22 in disease. J Immunol 2007; 178: 5973–5981. 2190. Wolk K, Witte K, Witte E, et al. Maturing dendritic cells are an important source of IL-29 and IL-20 that may cooperatively increase the innate immunity of keratinocytes. J Leukocyte Biol 2008; 83: 000–000.

Walter M. Lewko and Robert K. Oldham 2191. Wolk K, Witte E, Wallace E, et al. Il-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentialtion, and mobility in keratinocytes: a potential role in psoriasis. Eur J Immunol 2006; 36: 1309–1323. 2192. Wong BR, Josien R, Choi Y. TRANCE is a TNF family member that regulates dendritic cell and osteoclast function. J Leukocyte Biol 1999; 65: 715. 2193. Wong BR, Josien R, Lee SY, et al. TRANCE (Tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominanctly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 1997; 186: 2075. 2194. Wong BR, Rho J, Arron J, et al. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 1997; 272: 25190. 2195. Wong CK, Cheung PF, Ip WK and Lam CW. Interleukin-25induced chemokines and interleukin-6 release from eosinophils is mediated by p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor-κB. Am J Respir Cell Mol Biol 2005; 33: 186–194. 2196. Wood N, Bourque K, Donaldson DD, et al. IL-21 effects on human IgE production in response to IL-4 or IL-13. Cell Immunol 2004; 231: 135–145. 2197. Wood N, Whitters MJ, Jacobson BA, et al. Enhanced interleukin (IL)-13 responses in mice lacking IL-13 receptor α2. J Exp Med 2003; 197: 703–709. 2198. Woods JM, Katschke KJ, Tokuhira M, et al. Reduction of inflammatory cytokines and prostaglandin E2 by IL-13 gene therapy in rheumatoid arthritis synovium. J Immunol 2000; 165: 2755–2763. 2199. Worth LL, Jaffe N, Benjamin RS, et al. Phase II study of recombinant interleukin 1α and etoposide with relapsed osteosarcoma. Clin Cancer Res 1997; 3: 1721–1720. 2200. Wu C-Y, Ferrante J, Gately K, et al. Characterization of IL-12Rβ chain-deficient mice. J Immunol 1997; 159: 1658. 2201. Wu C-Y, Wang X, Gadina M, et al. IL-12 receptor β2 (IL-12Rβ2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding. J Immunol 2000; 165: 6221–6228. 2202. Wu C, Demeure C, Kiniwa M, et al. IL-12 induces production of IFN-γ by neonatal human CD4 T cells. J Immunol 1993; 151: 1938–1949. 2203. Wu DMH, Zhang Y, Parada NA, et al. Processing and release of IL-16 from CD4+ but not CD8+ T cells is activation dependent. J Immunol 1999; 162: 1287–1293. 2204. Wu K, Bi Y, Sun K, et al. IL-10-producing type 1 regulatory T cells and allergy. Cellular Molec Immunol 2007; 4: 269–275. 2205. Wu Q, Sun Y, Wang J, Lin X, et al. Signal via lymphotoxin-β on bone marrow stromal cells is required for an early checkpoint of NK cell development. J Immunol 2001; 166: 1684–1689. 2206. Wu Q, Wang Y, Wang J, et al. The requirement for membrane lymphotoxin for the presence of dendritic cells in lymphoid tissues. J Exp Med 1999; 190: 629. 2207. Wu S, Boyer CM, Whittaker RS, et al. Tumor necrosis factor as an autocrine and paracrine growth factor for ovarian cancer: Monokine induction of tumor cell proliferation and tumor necrosis factor α production. Cancer Res 1993; 53: 1939. 2208. Wurster AL, Rodgers VL, Satoskar AR, et al. Interleukin 21 is a T helper (Th) cytokine that specifically inhibits the differentiation of naive Th cells into interferon γ producing Th1 cells. J Exp Med 2002; 196: 967–977. 2209. Wuthrich RP, Jenkins TA, Snyder TL. Regulation of cytokine stimulated vascular cell adhesion molecule-1 expression in renal tubular epithelium cells. Transplantation 1993; 55: 172.

273 2210. Xia D, Li F, Xiang J. Engineered fusion hybrid vaccine of IL-18 gene-modified tumor cells and dendritic cells induces enhanced antitumor immunity. Cancer Biother Radiopharm 2004; 19: 322–330. 2211. Xiang J. Expression of costimulatory 4-1BB ligand induces significant tumor regression and protective immunity. Cancer Biother Radiopharm 1999; 14: 353–361. 2212. Xie MH, Aggarwal S, Ho WH, et al. Interleukin (IL)-22, a novel human cytokine that signals through the interferon related proteins CRF2 and IL-22R. J Biol Chem 2000; 275: 31335–31339. 2213. Xin X, Cai Y, Matsumoto K, Agui T. Endothelin-induced interleukin-6 production by rat aortic endothelial cells. Endocrinology 1995; 136: 132. 2214. Xu D, Chan WL, Leung BP, et al. Selective expression and functions of interleukin 18 receptor on T helper (Th) type I but not Th2 cells. J Exp Med 1998; 188: 1485. 2215. Xu DP, Sauter BV, Huang TG, et al. The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma. Gene Ther 2005; 12: 1526–1533. 2216. Xu H, Okamoto A, Ichikawa J, et al. TWEAK/Fn14 interaction stimulates human bronchial epithelial cells to produce IL-8 and GM-CSF. Biochem Biophys Res Commun 2004; 318: 422–427. 2217. Xu Z, Buckley MJ, Evans CH, Agarwal S. Cyclic tensile strain acts as an antagonist of IL-1β actions in chondrocytes. J Immunol 2000; 165: 453–460. 2218. Yacoub A, Mitchell C, Lebedeva IV. mda-7 inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling. Cancer Biol Ther 2003; 347–252. 2219. Yaden Y, Kuang WS, Yang-Feng T, et al. Human protooncogene c kit, a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J 1987; 6: 3341–3351. 2220. Yamada K, Takane N, Otabe S, et al. Pancreatic β-cell-selective production of tumor necrosis factor induced by interleukin-1. Diabetes 1993; 42: 1026–1031. 2221. Yamaguchi K, Kinosaki M, Goto M, et al. Characterization of structural domains of human osteoclastogenesis inhibitory factor. J Biol Chem 1998; 273: 5117. 2222. Yamagushi Y, Suda T, Suda J, et al. Purified Interleukin 5 supports the terminal differentiation and proliferation of purified eosinophil precursors. J Exp Med 1988; 167: 43. 2223. Yamamoto M, Yoshizaki K, Kishimoto T, Ito H. IL-6 is required for the development of Th1 cell-mediated murine colitis. J Immunol 2000, 164: 4878–4882. 2224. Yamanaka R, Honma J, Tsuchiya N, et al. Tumor lysate and IL-18 loaded dendritic cells elicits TH1 response, tumor-specific CD8+ cytotoxic T cells in patients with malignant glioma. J Neurooncol 2005; 72: 107–113. 2225. Yamasaki K, Taga T, Hirata Y, et al. Cloning and expression of the human interleukin 6 (BSF-2/IFNβ2) receptor. Science 1988; 241: 825–828. 2226. Yang FC, Agematsu K, Nakazawa T, et al. CD27/CD70 interaction directly induces natural killer cell killing activity. Immunology 1996; 88: 289–293. 2227. Yang Y-C, Ciarletta AB, Temple PA, et al. Human IL-3 (multiCSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell 1986; 47: 3–10. 2228. Yang Y, Mercep M, Ware CF, Ashwell JD. FAS and activationinduced ligand mediated apoptosis of T cells hybridomas: inhibition of Fas ligand expression by retinoic acid and glucocorticoids. J Exp Med1995; 181: 1673.

274 2229. Yang YC, Riccaciardi S, Ciarletta A, et al. Expression cloning of a cDNA encoding a novel human hematopoietic growth factor: Human homologue of murine T cell growth factor P40. Blood 1989; 74: 1880–1884. 2230. Yao L, Pike SE, Setsuda J, Parekh J, et al. Effective targeting of tumor vasculature by angiogenesis inhibitors vasostatin and interleukin-12. Blood 2000; 96: 1900–1905. 2231. Yao Z, Fanslow WC, Seldin MF, et al. Herpes virus saimiri encodes a new cytokine IL-17 which binds a novel cytokine receptor. Immunity 1995; 3: 811. 2232. Yao Z, Painter SL, Fanslow FC, et al. Interleukin-17: a novel cytokine derived from T cells. J Immunol 1995; 155: 5483. 2233. Yao Z, Spriggs MK, Derry JMJ, et al. Molecular characterization of the human interleukin (IL)-17 receptor. Cytokine 1997; 9: 794. 2234. Yasamura S, Lin WC, Weidemann E, et al. Expression of interleukin 2 receptors on human carcinoma cell lines and tumor growth inhibition by interleukin-2. Int J Cancer 1994; 59: 225–234. 2235. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesisinhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998; 95: 3597. 2236. Ye P. Rodriguez FH, Kanaly S, et al. Requirement of Interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment and host defense. J Exp Med 2001; 194: 519. 2237. Ye Q, Fraser CC, Gao W. Modulation of LIGHT-HVEM costimulation prolongs cardiac allograft survival. J Exp Med 2002; 195: 795–800. 2238. Yee NS, Langen H, Besmer P. Mechanism of kit ligand, phorbol ester and calcium-induced down-regulation of c-kit receptors in mast cells. J Biol Chem 1993; 268: 14189. 2239. Yeh K-Y, McAdam AJ, Pulaski BA, et al. IL-3 enhances both presentation of exogenous particulate antigen in association with Class I major histocompatibility antigen and the generation of primary tumor specific cytotolytic T lymphocytes. J Immunol 1998; 160: 5773–5780. 2240. Yellin MJ, Sippel K, Inghirami G, et al. CD40 molecules induce down-regulation and endocytosis of T cell surface T cell-B cell activating molecules/CD40L: potential role in regulating helper effector functions. J Immunol 1994; 152: 598. 2241. Yellin MJ, Winniloff S, Fortune SM, et al. Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) upregulation and IL-6 production and proliferation. J Leukocyte Biol 1995; 58: 209. 2242. Yen D, Cheung J, Scheerens H, et al. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest 2006; 116: 1310–1316. 2243. Yiling L, Granelli-Piperno A, Bjorndahl JM, Phillips CA, Trevillyan JM. CD28-induced T-cell activation. J Immunol 1992; 149: 24. 2244. Yin T, Schendel P, Yang Y-C. Enhancement of in vitro and in vivo antigen-specific antibody responses by IL-11. J Exp Med 1992; 175: 211. 2245. Yin T, Tsang ML, Yang YC. JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes. J Biol Chem 1994; 269: 26614–26617. 2246. Yokota T, Arai N, DeVries J, et al. Molecular biology of interleukin 4 and interleukin 5 genes and biology of their products that stimulate B cells, T cells and hematopoietic cells. Immunol Rev 1988; 102: 137–187.

Cytokines 2247. Yokota T, Coffman RL, Hagiwara H, et al. Isolation and characterization of lymphokine cDNA clones encoding mouse and human IgA-enhancing factor eosinophil colony-stimulating activities: relationship to interleukin 5. Proc Natl Sci (USA) 1987; 84: 7388–7392. 2248. Yokota T, Lee F, Rennick D, et al. Isolation and characterization of a mouse cDNA clone that expresses mast cell growth factor activity in monkey cells. Proc Natl Acad Sci USA 1984; 81: 1070–1074. 2249. Yokota T, Otsuka T, Mosmann T, et al. Isolation and characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expressed B cell and T cell stimulating activities. Proc Natl Acad Sci USA 1986; 83: 5894–5898. 2250. Yoneda J, Kuniyasu H, Crispens MA, et al. Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice. J Natl Cancer Inst 1998; 90: 447. 2251. Yoshimoto T, Bendelac A, Watson C, Hu-Li J, Paul WE. Role of NK1.1+ T cells in a Th2 response and in immunoglobulin E production. Science 1995; 270: 1845. 2252. Yoshimoto T, Paul WE. CD4 pos NK1.1 pos T cells promptly produce interleukin-4 in response to in vivo challenge with antiCD3. J Exp Med 1994; 179: 1285. 2253. Yoshimoto T, Takeda K, Takara T, et al. IL-12 upregulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-γ production. J Immunol 1998; 161: 3400. 2254. Yoshimoto T, Tsutsui H, Tominaga K, et al. IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc Natl Acad Sci USA 1999; 96: 1362. 2255. Yoshimoto T, Wang W-R, Yoneto T, et al. Role of IL-16 in delayed type hypersensitivity reaction. Blood 2000; 95: 2869–2874. 2256. Yoshimura A, Ohkubo T, Niguchi T, et al. A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phoshorylated interleukin-3 and erythropoietin receptors. EMBO J 1995; 14: 2816. 2257. Yoshimura T, Matsushima K, Tanaka S, et al. Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines. Proc Natl Acad Sci USA 1987; 84: 9233–9237. 2258. Yoshimura T, Takeda A, Hamano S, et al. Two-sided roles of IL-27: Induction of Th1 differentiation on Naïve CD4+ T cells versus suppression of proinflammatory cytokine production including IL-23-induced IL-17 on activated CD4+ T cells partially through STAT3-dependent mechanism. J Immunol 2006; 177: 5377–5385. 2259. Younes A, Consoli U, Snell V, et al. CD30 ligand is expressed in lymphoma patients with CD30+ tumors. J Clin Oncol 1997; 15: 3355–3362. 2260. Younes A, Consoli U, Zhao S, et al. CD30 ligand is expressed in normal and malignant human B lymphocytes. Brit J Haematol 1996; 93: 569–571. 2261. Yousefi S, Hemmann S, Weber M, et al. IL-8 is expressed by human peripheral blood eosinophils. Evidence for increased secretion in asthma. J Immunol 1995; 154: 5481–5490. 2262. Yssel H, de Waal Malfyt R, Roncarolo MG, et al. IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J Immunol 1992; 149: 2378. 2263. Yssel H, Schneider PV, Lanier LL. Interleukin 7 specifically induces the B7/BB1 antigen on human cord blood and peripheral blood T cells and T cell clones. Int Immunol 1993; 5: 753–759.

Walter M. Lewko and Robert K. Oldham 2264. Yu JS, Wei MX, Chjiocca A, et al. Treatment of glioma with engineered interleukin 4 secreting cells. Cancer Res 1993; 53: 3125–3128. 2265. Yu KY, Kwon B, Ni J, et al. A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHTmediated apoptosis. J Biol Chem 1999; 274: 13733–13746. 2266. Yuan Q, Gurish MF, Friend DS, Austen KF, Boyce JA. Generation of a novel stem cell factor-dependent mast cell progenitor. J Immunol 1998; 161: 5143–5146. 2267. Yuan X, Hu J, Belladona ML, et al. Interleukin-23-expressing bone marrow-derived neural stem cell-like cells exhibit antitumor activity against intracranial glioma. Cancer Res 2006; 66: 2630–2638. 2268. Yumoto K, Ishijima M, Rittling SR, et al. Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice. Proc Natl Acad Sci USA 2002; 99: 4556–4561. 2269. Yun TJ, Chaudary PM, Shu GL, et al. OPG/FDCR-1, a TNF family member, is expressed in lymphoid cells and is upregulated by ligating CD40. J Immunol 1998; 161: 6113. 2270. Yun TJ, Tallquist MD, Aicher A, et al. Osteoprotegerin, a crucial regulator of bone metabolism, also regulates B cell development and function. J Immunol 2001; 166: 1482–1491. 2271. Zachariae C, Ternowitz T, Larsen CG, Nielsen V, ThestrupPedersen K. Epidermal lymphocye chemotactic factor specifically attracts OKT4-positive lymphocytes. Arch Dermatol Res 1988; 280: 354–357. 2272. Zamir O, Hasselgren PO, O’Brien W. Thompson RC Fischer JE. Muscle protein breakdown during endotoxemia in rats and after treatment with interleukin-1 receptor antagonist (IL-1Ra). Ann Surg 1992; 216: 381. 2273. Zang XG, Gu JJ, Lu ZY, Yasukawa GD, et al. Ciliary neurotrohpic factor, interleukin 11, leukemia inhibitory factor and oncostatin M (OSM) are growth factors for human myeloma cell lines using the interleukin 6 signal transducer gp130. J Exp Med 1994; 179: 1337. 2274. Zarling JM, Shoyab H, Marquardt H, et al. Oncostatin M: a growth regulator produced by differentiated histiocyte lymphoma cells. Proc Natl Acad Sci USA 1986; 83: 9739. 2275. Zeng R, Spolski R, Finkelstein SE, et al. Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function. J Exp Med 2005; 201: 139–148. 2276. Zerafa N, Westwood JA, Cretney E, et al. TRAIL deficiency accelerates hematological malignancies. J Immunol 2005; 175: 5586–5590. 2277. Zetterstrom M, Sundgren-Anderson AK, Ostlund P, et al. Delineation of the proinflammatory cytokine cascade in fever induction. Ann NY Acad Sci 1998; 856; 48–52. 2278. Zhai Y, Hsu TL, Yu GL, Kwon BS, et al. LIGHT, a novel ligand for lymphotoxin β receptor and TR2/HVEM induces apoptosis and suppresses tumor formation via gene transfer. J Clin Invest 1998; 102: 1142–1151. 2279. Zhang GX, Gran B, Yu S, et al. Induction of experimental autoimmune encephalomyelitis in IL-12Rβ2-deficient mice: IL-12 is not required in the pathogenesis of inflammatory demyelination in the central nervous system. J Immunol 2003; 170: 2153–2160. 2280. Zhang H, Merchant MS, Chua KS, et al. Tumor expression of 4-1BB ligand sustains tumor lytic T cells. Cancer Biol Ther 2003; 2: 576–586. 2281. Zhang JZ, Maruyama K, Iwatsuki K, Kaneko F. Regulatory effects of 1,25-dihydroxyvitamin D3 and a novel Vitamin D3 analog MC903 on secretion of interleukin 1α (IL-1α) and IL-8

275

2282.

2283.

2284.

2285.

2286.

2287.

2288. 2289.

2290.

2291.

2292.

2293. 2294.

2295.

2296.

2297.

2298.

2299.

2300.

by normal human keratinocytes and a human squamous cell carcinoma cell line (HSC-1). J Dermatological Sci 1994; 7: 24–31. Zhang S, Kaplan MH. The p38 mitogen-activated protein kinase is required for IL-12-induced IFN-γ expression. J Immunol 2000; 165: 1374–1380. Zhang X, Franco A, Myers K, et al. Relation of TRAIL receptor and FLIP expression to TRAIL-induced apoptosis of melanoma. Cancer Res 1999; 59: 2747. Zhang X, Sun S, Hwang I, Tough D, Sprent J. Potent and selective stimulation of memory phenotype CD8+ T cells in vivo by IL-15. Immunity 1998; 8: 591. Zhang XG, Gu JJ, Yasukawa K, et al. Ciliary neurotrophic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are growth factors for human myeloma cell lines using the interleukin 6 transducer gp130. J Exp Med 1994; 179: 1337–1342. Zhang Y, Center DM, Wu DMH, et al. Processing and activation of pro-interleukin-16 by caspase-3. J Biol Chem 1998; 273: 1144. Zhang Y, Kornfeld H, Cruickshank WW, et al. Nuclear translocation of the N-terminal prodomain of interleukin-16. J Biol Chem 2001; 276: 1299–1303. Zheng L, Fisher G, Miller RE, et al. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature 1995; 377: 348. Zheng M, Bocangel D, Doneske B, et al. Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10. Cancer Immunol Immunother 2007; 56: 205–215. Zheng T, Nathanson M, Elias JA. Histamine augments cytokine stimulated interleukin-11 production by human lung fibroblasts. J Immunol 1994; 153: 4742. Zheng Y, Danilenko DM, Valdez P, et al. Interleukin-22, a TH17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 2007; 445: 648–651. Zheng Y, Ouaaz F, Bruzzo P, et al. NF-κB RelA (p65) is essential for TNF-α-induced Fas expression but dispensable for both TCR-induced expression and activation-induced cell death. J Immunol 2001; 166: 4949–4857. Zhou L-J, Tedder TF. A distinct pattern of cytokine expression by human CD83+ blood dendritic cells. Blood 1995; 86: 3295. Zhou P, Devadas K, Tewari D, Jegorow A, Notkins AL. Processing, secretion and anti-HIV-1 activity of IL-16 with or without a signal peptide in CD4+ T cells. J Immunol 1999; 163: 906–912. Zhou P, Goldstein S, Devadas K, Tewari D, Notkins AL. Human CD4+ cells transfected with IL-16 cDNA are resistant to HIV-1 infection: inhibition of mRNA expression. Nat Med 1997; 3: 659–664. Zhu M, Oishi K, Lee SC, Patterson PH. Studies using leukemia inhibitory factor (LIF) knockout mice and a LIF adenovirus vector demonstrate a key anti-inflammatory role for this cytokine in cutaneous inflammation. J Immunol 2001; 166: 2049–2054. Zilocchi C, Stopociaro A, Chiodoni C, et al. Interferon-γ rejection of interleukin 12-transduced carcinoma cells required CD+ T cells and granulocyte-macrophage colony stimulating factor. J Exp Med 1998; 188: 133. Zimmermann N, Hogan SP, Mishra A, et al. Murine eotaxin-2: A constitutive eosinophil chemokine induced by allergen challenge and IL-4 overexpression. J Immunol 2000; 165: 5839–5846. Zitrogel L, Tahara H, Robbins PD, et al. Cancer immunotherapy of established tumors with IL-12. Effective delivery by genetically engineered fibroblasts. J Immunol 1995; 155: 1393–1403. Zitzmann K, Band S, Bachs S, et al. Novel interferon-lambdas induce proliferative effects in neuroendocrine tumor cells. Biochem Biophys Res Commun 2006; 344: 1334–1341.

276 2301. Zoltnick A, Ransom J, Frank G, et al. Interleukin 4 is a growth factor for activated thymocytes: possible role in T cell ontogeny. Proc Nat Acad Sci USA 1987; 84: 3856. 2302. Zoltnik A, Fisher M, Roehm N, and Zipori D. Evidence for effects of (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow derived macrophages. J Immunol 1987; 138: 4275. 2303. Zou J, Presky DW, Wu CY, Grubler U. Differential associations between the cytoplasmic regions of the interleukin-12 receptor subunits β1 and β2 and the Jak kinases. J Biol Chem 1997; 272: 6073–6077. 2304. Zsebo KM, Williams DA, Geissler EN, et al. Stem cell factor is encoded at the S1 locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 1990; 63: 312–224. 2305. Zsebo KM, Wypych J, McNiece IK, et al. Identification characterization and biological characterization of hematopoietic stem cell factor form Buffalo rat liver conditioned medium. Cell 1990; 63: 195–210. 2306. Zsebo KM, Yuschenoff VN, Schiffer S, et al. Vascular endothelial cells and granulopoiesis: interleukin-1 stimulates release of G-CSF and GM-CSF. Blood 1988; 71: 99. 2307. Zuany-Amorim C, Ruffie C, Haile S, et al. Requirement for γ/δ T cells in allergic airway inflammation. Science 1998; 280: 1265. 2308. Zubiaga AM, Munoz E, Huber BT. IL-2 and IL-4 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis. J Immunol 1992; 149: 107–112. 2309. Zucali JR, Dinarello CA, Oblon DJ, et al. Interleukin 1 stimulates fibroblasts to produce granulocyte-macrophage colony stimulating activity and prostaglandin E2. J Clin Invest 1986; 77: 1857. 2310. Zurawski G, de Vries JE. Interleukin 13, an interleukin 4 like cytokine that acts on monocytes and B cells but not T cells. Immunol Today 1994; 15: 19–29.

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

D. Goldstein et al.

289

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]

D. Goldstein et al.

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].

References 1. Adams, F., J. R. Quesada, et al. (1984). “Neuropsychiatric manifestations of human leukocyte interferon therapy in patients with cancer.” J Am Med Assoc(252): 938–941. 2. Aderka, D., Y. Levo, et al. (1987). “Reduced production of tumor necrosis factor (TNF) by mononuclear cells of hairy cell leukemia patients and improvement following leukemia therapy.” Cancer(60): 2208–2212. 3. Aderka, D., R. Michalevicz, et al. (1988). “Recombinant interferon alpha-C for advanced hairy cell leukemia.” Cancer(61): 2207–2213. 4. Ahre, A., M. Bjorkholm, et al. (1984). “Human leukocyte interferon and intermittent high dose melphalan-prednisone administration in the treatment of multiple myeloma. A randomized clinical trial from the myeloma group of central Sweden.” Cancer Treat Rep(68): 1331–1338. 5. Ajani, J., A. H. Rios, et al. (1989). “Phase I and II Studies of the combination of recombinant human interferon-alpha and 5-fluorouracil in patients with advanced colorectal carcinoma.” J of Biol Resp Modif(8): 14–46. 6. Allan, N. C., S. M. Richards, et al. (1995). “On behalf of the UK Medical Research Council’s Working Parties for Therapeutic Trials in Adult Leukaemia – UK Medical Research Council randomized multicentre trial of interferon alpha N1 for chronic myeloid leukaemia:

294

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17. 18.

19.

20.

21.

22.

23.

24.

Improved survival irrespective of cytogenetic response.” Lancet 1392–1397. Allen, M. J., M. Vaughan, et al. (2000). “Protracted venous infusion 5-fluorouracil in combination with subcutaneous interleukin-2 and alpha-interferon in patients with metastatic renal cell cancer: a phase II study.” Br J Cancer(83): 980–985. Andersen, J. and R. V. Smalley (1993). “Interferon alfa plus chemotherapy for non-Hodgkin’s lymphoma: Five-year follow up.” New England J Med(329): 1821–1822. Armitage, J. O. and B. Coiffier (2000). “Activity of interferon alpha in relapsed patients with diffuse large B-cell and peripheral T-cell Non-Hodgkin’s Lymphom.” Ann Oncology(11): 359–361. Aviles, A., S. Cleto, et al. (2001). “Interferon alfa 2b as maintenance therapy in poor risk diffuse large B-cell lymphoma in complete remission after intensive CHOP-BLEO regimens.” Eur J Haematol(66): 94–99. Basham, T. Y., M. Kaminski, S., et al. (1986). “Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma.” J Immunol(137): 3019. Bergsagel, D. E., R. H. Haas, et al. (1987). “Interferon alfa-2b in the treatment of chronic granulocytic leukemia.” Invest New Drugs(5): 9–17. Billard, C., F. Sigaux, et al. (1986). “Treatment of hairy cell leukemia with recombinant alpha interferon: II. In vivo down-regulation of alpha interferon receptors on tumor cells.” Blood(67): 821–826. Billard, C., R. A. Diez, et al. (1989). “Lack of in vivo downregulation of interferon alpha receptors in non-Hodgkin lymphoma cells from patients unresponsive to interferon-alpha therapy.” J Interf Res(9(Suppl 2) ): S202. Bjorkstrand, B., H. Svensson, et al. (2001). “Alpha-interferon maintenance treatment is associated with improved survival after high-dose treatment and autologous stem cell transplantation in patients with myeloma: a retrospective registry study from the European group for blood and marrow transplantation.” Bone Marrow Transplant (EBMT)(27): 511–515. Blade, J., J. F. San Miguel, et al. (1998). “Maintenance treatment with interferon alpha-2b in multiple myeloma; a prospective randomised study from PETHEMA.” Leukemia(12): 1144–1148. Borden, E. (1979). “Interferons, rationale for clinical trials in neoplastic disease.” Ann Intern Med(91): 472–479. Borden, E. C., J. F. Holland, et al. (1982). “Leukocyte-derived interferon (alpha) in human breast carcinoma. The American Cancer Society Phase II trial.” Ann Intern Med(97): 1–6. Borden, E. C., G. C. Sen, et al. (2007). “Interferons at age 50: past, current and future impact on biomedicine.” Nat Rev Drug Discov(6(12) ): 975–990. Browman, G. P., D. Bergsagel, et al. (1995). “Randomized trial of interferon maintenance in multiple myeloma:A study of the National Cancer Institute of Canada Clinical Trials Group.” J Clin Oncol(13): 2354–2360. Bunn, P. A., K. A. Foon, et al. (1984). “Recombinant leukocyte A interferon: An active agent in advanced cutaneous T-cell lymphomas.” Ann Intern Med(101): 484–487. Bunn, P. A. J., D. C. Ihde, et al. (1989). “The role of recombinant interferon alfa-2a in the therapy of cutaneous T-cell lymphoma.” Cancer(57): 1689–1695. Carrel, S., A. Schmidt-Kessen, et al. (1985). “Recombinant interferon-gamma can induce the expression of HLA-DR and DC-or DR-negative melanoma cells and enhance the expression of HLAABC and tumor-associated antigen.” Eur J Immunol(15): 118–123. Cascinelli, N. (1995). “Evaluation of efficacy of adjuvant rIFNa 2A in melanoma patients with regional node metastases.” Proc Am Soc Clin Oncol(14): 1296.

Interferons: therapy for cancer 25. Case, D. C. J., H. L. Sonneborn, et al. (1986). “Phase II study of rDNA alpha-2 interferon (Intron A) in patients with multiple myeloma utilizing an escalating induction phase.” Cancer Treat Report(70): 1251–1254. 26. Cheson, J. M., D. A. Sorensen, et al. (1998). “Treatment of hairy cell leukemia with 2-chlorodeoxyadenosine via the Group C protocol mechanism of the National Cancer Institute: a report of 979 patients BD.” J Clin Oncol(16): 3007–3015. 27. Childs, R., A. Chernoff, et al. (2000). “Regression of metastatic renal cell carcinoma after non-myeloablative allogeneic peripheral blood stem cell transplantation.” N Engl J Med(343): 750–758. 28. Chisesi, T., G. Capnist, et al. (1987). “Interferon alfa-2b and chlorambucil in the treatment of non-Hodgkin’s lymphoma.” Invest New Drugs(5): 535–540. 29. Clark, R. H., N. V. Dimitrov, et al. (1984). “Leukocyte interferon as a possible biological response modifier in lymphoproliferative disorders resistant to standard therapy.” J Biol Resp Mod(3): 613–619. 30. Clark, R. H., N. V. Dimitrov, et al. (1989). “A phase II trial of intermittent leukocyte interferon and high dose chlorambucil in the treatment of non-Hodgkin’s lymphoma resistant to conventional therapy.” Am J Clin Oncol(12): 75–77. 31. Clemens, M. (1985). “Interferons and oncogenes.” Nature(313): 531–532. 32. Cole, B. F., P. Solal-Celigny, et al. (1988). “Quality-of-life-adjusted survival analysis of interferon alfa-2b treatment for advanced follicular lymphoma: an aid to clinical decision making.” J Clin Oncol(16): 2339–2344. 33. Cole, B. F., R. D. Gelber, et al. (1996). “Quality-of-life-adjusted survival analysis of interferon alpha-2b adjuvant treatment of high risk resected cutaneous melanoma: An Eastern Cooperative Oncology Group study.” J Clin Oncol(14): 2666. 34. Collaborators, M. R. C. R. C. (1999). “Interferon-alpha and survival in metastatic renal carcinoma: Early results of a randomized controlled trial.” Lancet(353): 14–17. 35. Cooper, M. R. (1988). “Interferons in the management of multiple myeloma.” Sem Oncol(15): 21–25. 36. Cooper, M. R., A. Fefer, et al. (1986). “Alpha-2-interferon/melphalan/prednisolone in previously untreated patients with multiple myeloma: A Phase I-II trial.” Cancer Treat Report(70): 473–476. 37. Costanzi, J. J. and R. B. Pollard (1987). “The use of interferon in the treatment of multiple myeloma.” Sem Oncol(14): 24–28. 38. Costanzi, J., J, M. R. Cooper, et al. (1985). “Phase II study of recombinant alpha 2 interferon in resistant multiple myeloma.” J Clin Oncol(3): 654–659. 39. Creagan, E. T., D. L. Ahmann, et al. (1984). “Phase II study of recombinant leukocyte A interferon (RIFN-Alpha-A) in disseminated malignant melanoma.” Cancer 1(54): 2844–2849. 40.Cunningham, D., R. Powles, et al. (1998). “A randomised trial of maintenance interferon following high-dose chemotherapy in multiple myeloma; long term follow-up results.” Br J Haematol(102): 495–502. 41. Davis, T. A., G. Maloney, et al. (2000). “Combination Immunotherapy of Relapsed or Refractory Low-Grade or Follicular Non-Hodgkin’s Lymphoma with Rituximab and Interferon – 2a.” Clin Cancer Res (6): 2644–2652. 42. DeMaeyer-Guignard,J.andE.DeMaeyer(1985).“Immunomodulation by interferons: Recent developments.” Academic Press(6): 69–86. 43. Dewit, R., C. A. B. Boucher, et al. (1988). “Clinical and virological effects of high-dose recombinant interferon alpha in disseminated AIDS-related Kaposi’s sarcoma.” Lancet(2): 1214–1217. 44. Dinney, C. P. N., D. R. Bielenberg, et al. (1998). “Inhibition of basic fibrobroblast growth factor expression, angiogenesis and growth of human bladder carcinoma in mice by systemic interferon alpha administration.” Cancer Res(58): 808.

D. Goldstein et al. 45. Druker, B. J., M. Talpaz, et al. (2001). “Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.” N Eng J Med(344): 1031–1037. 46. Dutcher, J. P., T. Logan, et al. (2000). “Phase II trial of interleukin 2, interferon alpha and 5-fluorouracil in metastatic renal cell cancer: a cytokine working group study.” Clin Cancer Res September 2000 (6(9) ): 3442–3450. 47. Edwards, B. S., M. J. Hawkins, et al. (1984). “Comparative in vivo and in vitro activation of human NK cells by two recombinant interferons alpha differing in anti-viral activity.” Cancer Res(44): 3135–3139. 48. Edwards, B. S., J. A. Merritt, et al. (1985). “Low doses of interferon alpha result in more effective clinical natural killer cell activation.” J Clin Invest(75): 1908–1913. 49a. Eggermont, A. M., S. Suciu, et al. (2005). “Post-surgery adjuvant therapy with intermediate doses of interferon alfa 2b versus observation in patients with stage IIb/III melanoma (EORTC 18952): randomised controlled trial.” Lancet(366(9492) ): 1189–1196. 49b. Eggermont, A. M., S. Suciu, M. Santinami, et al. (2008). “Adjuvant therapy with pegylated interferon-2b versus observation alone in resected stage III melanoma: final results of EORTC 18991, a randomised phase III trial.” Lancet(12): 372 (9633): 117–126. 50. Einhorn, N., K. Cantel, et al. (1982). “Human leukocyte interferon.” Am J Clin Oncol(5): 167–172. 51. Einhorn, S., A. Ahre, et al. (1982). “Interferon and natural killer activity in multiple myeloma. Lack of correlation between interferon-induced enhancement of natural killer activity and clinical response to human interferon-alpha.” Int J Cancer 30(I): 167–172. 52. Einhorn, S., H. Blomgren, et al. (1978). “Interferon and spontaneous cytotoxicity in man.” Acta Med Scand(204): 477–483. 53. Elhilali, M. M., M. Gleave, et al. (2000). “Placebo-associated remissions in a multicentre, randomized, double-blind trial of interferon gamma-1b for the treatment of metastatic renal cell carcinoma.” BJU Int 2000 (86(6) ): 613–618. 54. Escudier, B., A. Pluzanska, et al. (2007). “Bevacizumab plus interferon alfa-2a for treatment of metastatic renal cell carcinoma: a randomised, double-blind phase III trial.” Lancet(370(9605) ): 2103–2111. 55. Eton, O., S. Legha, et al. (2000). “Phase III randomized trial of cisplatin vinblastine and dacarbazine (CVD) plus interleukin-2 (IL2) and interferon-alpha-2b (INF) versus CVD in patients with metastatic melanoma. 2000.” Proc Am Soc Clin Onco(l 19): 2174. 56. Evans, L. M., L. M. Itri, et al. (1989). “Alfa interferon 2a in the treatment of AIDS related Kaposi’s sarcoma.” J Interf Res(9): S207. 57. Ezekowitz, R. A. B., J. B. Mulliken, et al. (1992). “Interferon alfa2a therapy for life-threatening hemangiomas of infancy.” N Eng J Med(326): 1456. 58. Falkson, C. I., J. Ibrahim, et al. (1998). “Phase III trial of dacarbazine with tamoxifen in patirnts with metastatic malignant melanoma: an Eastern Cooperative Oncology Group study.” J Clin Oncol(16): 1743–1751. 59. Faltynek, C. R., G. L. Princler, et al. (1986). “Relationship of the clinical response and binding of recombinant interferon alpha in patients with lymphoproliferative diseases.” Blood(67): 1077–1082. 60. Farkilla, M., M. Ilvanainen, et al. (1984). “Neurotoxic and other side effects of high-dose interferon in amyotrophic lateral sclerosis.” Acta Neurol Scand(69): 42–46. 61. Figlin, R. A., J. B. deKernion, et al. (1985). “Treatment of renal cell carcinoma with alpha (human leukocyte) interferon and vinblastine in combination: A phase I-II trial.” Cancer Treat Report(69): 263–267. 62. Fisher, R. I., B. W. Dana, et al. (2000). “Interferon Alfa Consolidation After Intensive Chemotherapy Does Not Prolong the ProgressionFree Survival of Patients With Low-Grade Non-Hodgkin’s Lymphoma: Results of the Southwest Oncology Group. Randomized Phase III Study 8809.” J Clin Oncol (18): 2010–2020.

295 63. Flanigan, C., B. A. Blumnetsein, et al. (2000). “Cytoreduction nephrectomy in metastatic renal cancer: The results of South west Oncology group trial 8949.” Proc Am Soc Clin Oncol(19): 2a. 64. Foon, K. A., S. A. Sherwin, et al. (1984). “Treatment of advanced non-Hodgkin’s lymphoma with recombinant leukocyte A interferon.” N Engl J Med(311): 1148–1152. 65. Ford, R. J., D. Kwok, et al. (1986). “Production of B cell growth factor(s) by neoplastic B cells from hairy cell leukemia patients.” Blood(67): 573–577. 66. Fosså, S., G. Martinelli, et al. (1992). “Recombinant interferon-alpha-2a with or without vinblastine in metastatic renal cell carcinoma: Results of a European multi-centre phase III study.” Ann Oncol(3): 301–305. 67. Frank, M., K. J. Klose, et al. (1999). ”Combination therapy with octreotride and alpha-interferon: effect on tumour growth in metastatic endocrine gastropancreatic tumours.” Am J Gastroenerol(94): 1381–1387. 68. Gams, R., D. Gordon, et al. (1984). “Phase II trial of human polyclonal lymphoblastoid interferon in the management of malignant lymphomas.” Proc Am Soc Clin Oncology(3): 65. 69. Gastl, G., H. Denz, et al. (1985). “Treatment with low dose human recombinant interferon-alpha-2 ARG induces complete remission in patients with hairy cell leukemia.” Onkologie(8): 143–144. 70. Gastl, G., W. Aulitzky, et al. (1986). “Alpha interferon induces remission in hairy cell leukemia without enhancement of natural killing.” Blut(52): 273–279. 71. Gastl, G., W. Aulitzky, et al. (1986). “A biological approach to optimize interferon treatment in hairy cell leukemia.” Immunobiology(172): 262–268. 72. Gastl, G., M. Werter, et al. (1989). “Comparison of clinical efficacy and toxicity of conventional and optimum biological response modifying doses of interferon alpha-2C in the treatment of hairy cell leukemia: A retrospective analysis of 39 patients.” Leukemia(3): 453–460. 73. Gelmann, E. P., O. T. Preble, et al. (1985). “Human lymphoblastoid interferon treatment of Kaposi’s sarcoma in the acquired immune deficiency syndrome.” Am J Med(78): 737–741. 74. Giacomini, P., A. Aguzzi, et al. (1984). “Modulation by recombinant DNA leukocyte (a) and fibroblast (b) interferons of the expression and shedding of HLA- and tumor-associated antigens by human melanoma cells.” J Immunol(133): 1649–1655. 75. Giacomini, P., L. Imberti, et al. (1985). “Immunochemical analysis of the modulation of human melanoma-associated antigens by DNA recombinant immune interferon.” J Immunol(135): 2887–2894. 76. Gibson, J., K. Cameron, et al. (1988). “Clinical response of hairy cell leukaemia to interferon-α: results of an Australian study.” Med J of Aust(149): 293–296. 77. Gibson, J., K. Gallagher, et al. (1988). “Peripheral blood lymphocyte subsets and natural killer cell number and function during α-interferon treatment for hairy cell leukemia.” Aust New Zeal J Med(18): 897–898. 78. Gill, P. S., W. J. Harrington, et al. (1995). “Treatment of adult T-cell leukemia-lymphoma with a combination of interferon alfa and zidovudine.” N Engl J Med(332): 1744–1748. 79. Gisslinger, H., H. Ludwig, et al. (1989). “Long-term interferon therapy for thrombocytosis in myeloproliferative diseases.” Lancet(1): 634–637. 80. Gleave, M. E., M. Elhilali, et al. (1998). “Interferon gamma-1b compared with placebo in metastatic renal cell carcinoma.” New Engl J Med(338): 1265–1271. 81. Goldstein, D., F. Guadagni, et al. (1989). “Clinical and biological effects of intraperitoneal interferon gamma in a phase 1A/b trial in ovarian carcinoma.” J Interf Res(9(2) ): S115. 82. Golomb, H. M., A. Jacobs, et al. (1986). “Alpha-2 interferon therapy of hairy cell leukemia: A multi-center study of 64 patients.” J Clin Oncol(4): 900–905.

296 83. Golomb, H. M., A. Fefer, et al. (1988). “Report of a multiinstitutional study of 193 patients with hairy cell leukemia treated with interferon-alfa 2b.” Sem in Oncol(15): 7–9. 84. Golomb, H. M., M. J. Ratain, et al. (1988). “Randomized study of the duration of treatment with interferon alfa-2B in patients with hairy cell leukemia.” J Natl Cancer Inst(80): 369–373. 85. Gore, M. E., M. Mohammed, et al. (2001). “Clinical resistance to STI-571 cancer therapy by BCR-ABL gene mutation or amplification.” Science(293): 876–880. 86. Greiner, J. W., P. H. Hand, et al. (1984). “Enhanced expression of surface tumor associated antigen on human breast and colon tumor cells after recombinant human leukocyte a-interferon treatment.” Cancer Res(44): 3208–3211. 87. Gresser, I. (1985). “How does interferon inhibit tumor growth?” Interferon(6): 93–126. 88. Grillo-Lopez, A. J., C. A. White, et al. (2000). “Rituximab: the first monoclonal antibody approved for the treatment of lymphoma.” Curr Pharm Biotechnol(1(1) ): 1–9. 89. Grob, J. J., B. Dreno, et al. (1998). “Randomised trial of interferon alpha-2a as adjuvant treatment in resected primary melanoma thicker than 1.5 mm without clinically detectable node metastases. French Co-operative Group on Melanoma.” Lancet(351): 1905–1910. 90. Groopman, J., M. Gottlieb, et al. (1984). “Recombinant alpha-2 interferon therapy for Kaposi’s sarcoma associated with the acquired immunodeficiency syndrome.” Ann Intern Med(100): 671–676. 91. Group, N. M. S. (1996). “Interferon-α 2b added to melphalanprednisone for initial and maintenance therapy in multiple myeloma.” Ann Intern Med(124): 212–222. 92. Group, T. M. T. C. (2001). “Interferon as therapy for multiple myeloma. An individual patient data overview of 24 randomized trials and 4012 patients.” Br J Haematol(113): 1020–1034. 93. Guadagni, F., J. Schlom, et al. (1989). “Selective InterferonInduced Enhancement of Tumor-Associated Antigens on a Spectrum of Freshly Isolated Human Adenocarcinoma Cells.” J Natl Cancer Inst(81): 502–512. 94. Gugliotta, L., G. P. Bagnara, et al. (1989). “In vivo and in vitro inhibitory effect of alpha interferon on megakaryocyte colony growth in essential thrombocythaemia.” Br J Hematol(71): 177–181. 95. Gutterman, J. U., A. R. Blumenschein, et al. (1980). “Leukocyte interferon-induced tumor regression in human metastatic breast cancer, multiple myeloma and malignant lymphoma.” Ann Intern Med(93): 399–406. 96. Hagenbeek, A., P. Carde, et al. (1998). “Maintenance of remission with human recombinant interferon alfa-2a in patients with stages III and IV low-grade malignant non-Hodgkin’s lymphoma. European Organization for Research and Treatment of Cancer Lymphoma Cooperative Group.” J Clin Oncol(16): 148. 97. Hancock, B. W., K. Wheatley, et al. (2004). “Adjuvant interferon in high risk melanoma; the AIM high study – UKCCR randomized study of adjuvant low-does extended duration interferon Alfa-2a in high risk resected malignant melanoma.” J Clin Oncol(22): 53–61. 98. Hehlmann, R., B. Anger, et al. (1988). “Randomized study on the treatment of chronic myeloid leukemia (CML) in chronic phase with busulfan versus hydroxyurea versus interferon-alpha.” Blut(56): 87–91. 99. Hehlmann, R., H. Heimpel, et al. (1994). “Randomized comparison of interferon alpha with Busulfan and hydroxyurea in chronic myelogenous leukemia.” Blood(84): 4064–4077. 100. Herberman, R. B. and J. R. Ortaldo (1981). “Natural killer cells: Their role in defenses against diseas.” Science(214): 24–30. 101. Heyns, A. P., Eldor. A., et al. (1985). “The antiproliferative effect of interferon and the mitogenic activity of growth factors are independent cell cycle events.” Exp Cell Res(161): 297.

Interferons: therapy for cancer 102. Hill, M., A. Norman, et al. (1995). “Royal Marsden phase III trial of fluorouracil with or without interferon alfa 2b in advanced colorectal cancer.” J Clin Onc(13): 1297–1302. 103. Holtl, L., C. Reiser, et al. (1999). “Cellular and humoral immune responses in patients with metastatic renal cell carcinoma after vaccination with antigen pulsed dendritic cells.” J Urol (161(3)): 777–782. 104. Horning, S. J., T. L. Merigan, et al. (1985). “Human interferon alpha in malignant lymphoma and Hodgkin’s disease.” Cancer Res(56): 1305–1310. 105. Huber, C., Batchelor. J. R., et al. (1984). “Immune responseassociated production of neopterin.” (160): 310–316. 106. Hudes, G., M. Carducci, et al. (2007). “Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma.” N Engl J Med(356(22) ): 2271–2278. 107. Huncharek, M., J. F. Caubet, et al. (2001). “Single agent DTIC versus combination therapy with or without immunotherapy in metastatic melanoma: a meta-analysis of 3273 patients from 20 randomized trials.” Melanoma Research(11): 75–81. 108. Isaacs, H. and J. Lindemann (1957). “Virus interference 1. The interferon.” Proc R Soc Oncol Biol(147): 257–262. 109. Itri, L. M., M. Campion, et al. (1987). “Incidence and clinical significance of neutralizing antibodies in patients receiving recombinant interferon alfa 2a by intramuscular injection.” Cancer(59): 668–674. 110. Ives, N. J., R. Stowe, L., et al. (2007). “Chemotherapy compared with biochemotherapy for the treatment of metastatic melanoma meta-analysis of 18 trials involving 2621 patients.” J Cin Onco(1): 5426–5434. 111. Jabbour, E., H. Kantarjian, et al. (2007). “PEG-IFN-alpha-2b therapy in BCR-ABL-negative myeloproliferative disorders: final result of a phase 2 study.” Cancer Res(110(9) ): 2012–2018. 112. Johnston, J. B., E. Eisenhauer, et al. (2000). “Long-term outcome following treatment of hairy cell leukemia with pentostatin; a National Cancer Institute of Canada study.” Semin Onco(27 (Suppl. 5) ): 32–36. 113. Jonasch, E. and F. G. Haluska (2001). “Interferon in Oncological Practice; Review of Interferon biology, Clinical applications, and toxicities.” The Oncologist(6): 34–55. 114. Joshua, D. E., R. Penny, et al. (1997). “Australian leukemia study group myeloma II: A randomised study of intensive combination chemotherapy with or without interferon in patients with myeloma.” Br J Haematol(97): 38–45. 115. Jumbou, O., J. M. N’Guyen, et al. (1999). “Long-term follow-up in 51 patients with mycosis fungoides and Sezary syndrome treated by interferon-alfa.” Br J Dermatol(140(3) ): 427–431. 116. Kaban, L. B., J. B. Mulliken, et al. (1999). “Antiangiogenic therapy of a recurrent giant cell tumour of the mandible with interferon alpha-2a.” Pediatrics(103): 1145. 117. Kantarajan, H. M., S. O’Brien, et al. (1999). “Treatment of Philadelphia chromosome-positive early chronic phase myelogenous leukemias with daily doses of interferon alpha and low-dose cytarabine.” J Clin Oncol(17): 284–293. 118. Kantarjian, H. M., T. L. Smith, et al. (1995). “Prolonged survival in chronic myelogenous leukemia after cytogenetic response to interferon alpha therapy.” Ann Int Med(122): 254–261. 119. Keiholz, U., S. H. Goey, et al. (1997). “Interferon alpha-2a and interleukin-2 with or without cisplatin in metastatic melanoma: a randomized trial of the European Organisation for Research and Treatment of Cancer Melanoma Cooperative Group.” J Clin Oncol(15): 2579–2588. 120. Kimby, E., J. Jurlander, et al. (2008). “Nordic Lymphoma Group. Long-term molecular remissions in patients with indolent lymphoma treated with rituximab as a single agent or in combination

D. Goldstein et al.

121. 122.

123.

124.

125.

126.

127.

128.

129.

130. 131.

132.

133.

134.

135.

136.

137.

138.

with interferon alpha-2a: a randomized phase II study from the Nordic Lymphoma Group.” Leuk Lymphoma(49(1) ): 102–112. Kirchner, H. (1984). “Interferons, a group of multiple lymphokines.” Semin Immunopathol(7): 347–374. Kirkwood, J. M., M. S. Ernstoff, et al. (1985). “Comparison of intramuscular and intravenous recombinant a-2 interferon in melanoma and other cancers.” Ann Intern Med(103): 32–36. Kirkwood, J. M., J. G. Ibrahim, et al. (2000). “High-and low-dose interferon alpha-2b in high-risk melanoma: first analysis of intergroup trial E1690/S9111/C9190.” J Clin Oncol(18): 2444–2458. Kirkwood, J. M., J. G. Ibrahim, et al. (2001). “High dose interferon alpha-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(19): 2370–2380. Kirkwood, J. M., M. H. Strawderman, et al. (1996). “Interferon alpha-2b adjuvant therapy of high-risk resected cutaneous lymphoma: the Eastern Cooperative Oncology Group Trial EST 1684.” J Clin Oncol (14): 7–17. Kleberg, U. R., S. Suciu, et al. (2004). “Final results of the EORTC 18871/DKG 80–1 randomised phase III trial. rIFN-alpha2b versus rIFN-gamma versus ISCADOR M versus observation after surgery in melanoma patients with either high risk primary or regional lymph node metastasi.” Eur J Cancer(40): 390–402. Kluin-Nelemans, H. C., G. Buck, et al. (2004). “Randomised comparison of low dose versus high dose interferon-alpha in chronic myeloid leukemia: prospective collaboration of 3 joint trials by the MRC and hOVON groups.” Blood(103): 4408–4415. Kreitman, R. J., W. H. Wilson, et al. (2001). “Efficacy of the antiCD22 recombinant immunotoxin BL22 in chemotherapy resistant hairy cell leukemia.” New Engl J Med(345): 241–247. Krigel, R. L., C. M. Slywotzky, et al. (1988). “Treatment of epidemic Kaposi’s sarcoma with a combination of interferon-alpha-2b and etoposide.” J Biol Resp Modif(7): 359–364. Krown, S. E. (1987). “The role for interferon in the therapy of epidemic Kaposi’s sarcoma.” Semin Oncol(2): 27–33. Kugler, A., G. Stuhler, et al. (2000). “Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids.” Nat Med(6(3) ): 332–336. Kuzel, T. M., K. Gilyon, et al. (1990). “Interferon alfa-2a combined with phototherapy in the treatment of cutaneous T-cell lymphoma.” J Natl Cancer Inst(82): 203–207. Kuzel, T. M., H. H. Roenigk, et al. (1995). “Effectiveness of interferon alpha 2a combined with phototherapy for mycosis fungoides and the Sézary syndrome.” J Clin Onc(13): 257. Laszlo, J., A. T. Huang, et al. (1983). “Phase I study of pharmacological and immunological effects of human lymphoblastoid interferon given to patients with cancer.” Cancer Res(43): 4458–4466. Lazzarino, M., A. Vitalle, et al. (1989). “Interferon alpha-2b as treatment for Philadelphia-negative chronic myeloproliferative disorders with excessive thrombocytosis.” Br J Haematol(72): 173–177. Leavitt, R. D., V. Ratanatharathorn, et al. (1987). “Alfa-2b interferon in the treatment of Hodgkin’s Disease and Non-Hodgkin’s lymphoma.” Sem Oncol(14(Suppl 2) ): 18–23. Lenhoff, S., M. Hjorth, et al. (2000). “Impact on survival of high dose therapy with autologous stem cell support in patients younger than 60 years with newly diagnosed multiple myeloma: a population based study.” Blood(95): 7–11. Liao, M. J., N. Lee, et al. (1994). “Distribution of interferon alpha 2 genes in humans.” J Interferon Res(14): 183–185.

297 139. Liekens, S., E. De Clercq, et al. (2001). “Angiogenesis: regulators and clinical applications. Biochemical Pharmacology.” Biochemical Pharmacology(61): 253–270. 140. Lindsay, K. L., C. Trepo, et al. (2001). “A randomized, double-blind trial comparing pegylated interferon alfa-2b to interferon alfa-2b as initial treatment for chronic hepatitis C.” Hepatology(34(2) ): 395–403. 141. Lingen, M. W., P. J. Polverini, et al. (1998). “Retinoic acid and interferon alpha act synergistically as antiangiogenic antitumour agents against human head and neck squamous cell carcinoma.” Cancer Res(58): 5551–5558. 142. Lippman, S. M., D. R. Parkinson, et al. (1992). “13-cis-retinoic acid plus interferon alfa 2a:Effective combination therapy for advanced squamous cell carcinoma of the cervix.” J Nat Ca Inst(84): 235–240. 143. Lotzova, E., C. A. Savary, et al. (1983). “Regulation of natural killer cell cytotoxicity by recombinant leukocyte interferon clone A.” J Biol Resp Modif(2): 482–498. 144. Louie, A. C., J. G. Gallagher, et al. (1981). “Followup observations on the effect of human leukocyte interferon in non-Hodgkin’s lymphoma.” Blood(58): 712–718. 145. Lucero, M. A., H. Magdalenat, et al. (1982). “Comparison of effects of leukocyte and fibroblast interferon on immunological parameters in cancer patients.” Eur J Cancer Clin Oncol(18): 243–251. 146. Ludwig, H. and E. Fritz (2000). “Interferon in multiple myelomasummary of treatment results and clinical implications.” Acta Oncol(39): 815–821. 147. Ludwig, H., A. Cortelezzi, et al. (1986). “Recombinant interferon alfa-2C versus polychemotherapy (VMCP) for treatment of multiple myeloma: a prospective randomized trial.” Eur J Ca Clin Onc(22): 1111–1116. 148. Maluish, A. E., J. R. Ortaldo, et al. (1983a). “Depression of natural killer cytotoxicity after in vivo administration of recombinant leukocyte interferon.” J Immunol(131): 503–507. 149. Maluish, A. E., J. R. Ortaldo, et al. (1983b). “Changes in immune function in patients receiving natural leukocyte interferon.” J Biol Resp Modif(2): 418–422. 150. Maluish, A. E., R. Leavitt, et al. (1984). “Effects of recombinant interferon-alpha on immune function in cancer patients.” J Biol Resp Modif(2): 470–481. 151. Mandelli, F., G. Avvisati, et al. (1990). “Maintenance treatment with recombinant interferon alpha 2b recombinant in multiple myeloma patients responding to conventional induction chemotherapy.” N Engl J Med(322): 1430–1434. 152. Mangia, A., G. L. Ricci, et al. (2005). “A randomized controlled trial of pegylated interferon alpha-2a (40 KD) or interferon alpha2a plus ribavirin and amantadine vs interferon alpha-2a and bavirin in treatment-naïve patients with chronic hepatitis C.” J Viral Hepat(12(3) ): 292–299. 153. Marotta, G., A. Frassoldati, et al. (2006). “Furthermore role of interferon-alpha administration after 2-deoxycoformycin in the treatment of hairy cell leukemia patient.” Eur J Haematol(77(2) ): 109–113. 154. Marumo, K., M. Murai, et al. (1984). “Human lymphoblastoid interferon therapy for advanced renal cell carcinoma.” Urology(24): 567–571. 155. Mauss, S. and H. Jablonowski (1995). “Efficacy, safety, and tolerance of low-dose, long-term interferon-alpha 2b and zidovudine in early-stage AIDS-associated Kaposi’s sarcoma.” J Acquir Immune Defic Syndr Hum Retrovirol(10(2) ): 157–162. 156. McClay, E., M. Mastrangelo, et al. (1992). “Effective combination chemo/hormonal therapy for malignant melanoma:

298

157.

158.

159. 160. 161.

162.

163.

164.

165.

166.

167.

168. 169.

170.

171.

172.

173.

174.

175.

Interferons: therapy for cancer Experience with three consecutive trials.” Int J Cancer(50): 553–556. McLaughlin, P., F. Cabanillan, et al. (1993). “CHOP-Bleo plus interferon for stage IV low-grade lymphoma.” Ann Onc(4): 205–211. Meeker, T., Lowder. J., et al. (1985). “Emergence of idiotype variants during treatment of B-cell lymphomas with anti-idiotype antibodies.” N Eng J Med(312): 1658. Meeker, T. C., J. Lowder, et al. (1985). “A clinical trial of antiidiotype therapy for B cell malignancy.” Blood(65): 1349. Mellstedt, H., A. Ahre, et al. (1979). “Interferon therapy in myelomatosis.” Lancet(1): 245–247. Merigan, T. L., K. Sikorak, et al. (1978). “Preliminary observations on the effect of human leukocyte interferon in non-Hodgkin’s lymphoma.” N Engl J Med(299): 1449–1453. Michalevicz, R. and M. Revel (1987). “Interferons regulate the in vitro differentiation of multilineage lympho-myeloid stem cells in hairy cell leukemia.” Proc Natl Acad Sci(84): 2307–2311. Moertel, C. G., J. Rubin, et al. (1989). “Therapy of metastatic carcinoid tumor and the malignant carcinoid syndrome with recombinant leukocyte alpha interferon.” J Clin Onc(7): 865. Motzer, R. J., B. A. Murphy, et al. (2000). “Phase III Trial of Interferon Alfa-2a With or Without 13-cis-Retinoic Acid for Patients With Advanced Renal Cell Carcinoma.” J Clin Oncol(18): 2972–2980. Motzer, R. J., t. A. Rakhi, et al. (2001). “ Phase I trial of 40-kd branched pegylated interferon alfa-2a for patients with advanced renal cell carcinoma.” J Clin Oncol(19): 1312–1319. Motzer, R. J., A. Rakhit, et al. (2001). “Randomized multicenter phase II trial of subcutaneous recombinant human interleukin-12 versus interferon-alpha 2a for patients with advanced renal cell carcinoma.” J Interferon Cytokine Res(21(4) ): 257–263. Motzer, R. J., T. E. Hutson, et al. (2007). “Sunitinib versus interferon alfa in metastatic renal-cell carcinoma.” N Engl J Med(115–24): 115–124. Muss, H. B., C. Welander, et al. (1985). “Interferon and doxorubicin in renal cell carcinoma.” Cancer Treat Report(69): 721–722. Musselman, D., D. H. Lawson, et al. (2001). “Paroxetine for the prevention of depression induced by high-Dose interferon Alfa.” New Engl J Med (344): 961–966. Neefe, J. R., J. E. Sullivan, et al. (1985). “Augmented immunity in cancer patients treated with alpha-interferon.” Cancer Res(45): 874–878. Negrier, S., B. Escudier, et al. (1998). “Recombinant human interleukin-2, recombinant human interferon alfa-2a, or both in metastatic renal-cell carcinoma.” N Engl J Med(338): 1273–1278. Negrier, S., D. Perol, et al. (2007). “For The French Immunotherapy Intergroup. Medroxyprogesterone, interferon alfa-2a, interleukin 2, or combination of both cytokines in patients with metastatic renal carcinoma of intermediate prognosis: results of a randomized controlled trial.” Cancer Res(110(11) ): 2468–2477. Neidhart, J. A., S. A. Anderson, et al. (1991). “Vinblastine Fails to Improve Response of Renal Cancer to Interferon Alfa-n1 (Wellferon); High Response Rate in Patients with Pulmonary Metastases.” J Clin Onc(9): 832–837. Neuman, H. A. and A. A. Fauser(1982). “Effect of interferon on pluripotent hemopoietic progenitors(CFU-GEMM) derived from human bone marrow.” Exp Hematol(10): 587. Nielsen, B., M. Hokland, et al. (1989). “Immunological recovery and dose evaluation in IFN-a treatment of hairy cell leukemia: Analysis of leukocyte differentiation antigens, NK and 2′,5′-oligoadenylate synthetase activity.” Eur J Haematol(42): 50–59.

176. O’Brien, S. G., F. Guilhot, et al. (2003). “Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronicphase chronic myeloid leukemia.” N Engl J Med (348(11) ): 994–1004. 177. O’Connell, M. J., J. P. Colgan, et al. (1986). “Clinical trials of recombinant leukocyte A interferon as initial therapy for favorable histology Non-Hodgkin’s lymphomas and chronic lymphocytic leukemia. An Eastern Cooperative Oncology Group Pilot Study.” J Clin Oncol(4): 128–136. 178. Oberg, K., G. Alm, et al. (1989). “Treatment of malignant carcinoid tumors with recombinant interferon alfa-2b:Development of neutralizing IFN antibodies and possible loss of antitumor activity.” J Nat Ca Inst(81(7)): 531-535. 179. Ohnishi, K., R. Ohno, et al. (1995). “A randomized trial comparing interferon-α with busulfan for newly diagnosed chronic myelogenous leukemia in chronic phase.” Blood(86): 906–916. 180. Ohno, R. and K. Kimura (1986). “Treatment of multiple myeloma with recombinant interferon alfa-2a.” Cancer(57): 1685–1688. 181. Oken, M. M., R. A. Kyle, et al. (1985). “A Phase II trial of interferon alpha-2 (rIFN-a2) in the treatment of resistant multiple myeloma (MM).” Proc Am Soc Clin Oncol(4): 215. 182. Oken, M. M., T. Leong, et al. (1999). “The addition of interferon or high dose cyclophosphamide to standard chemotherapy in the treatment of patients with multiple myeloma: phase III ECOG EST 9486.” Cancer(15): 957–963. 183. Oladipupo-Williams, C. K., I. Svet-Moldavskaya, et al. (1981). “Inhibitory effects of human leukocyte and fibroblast interferons on normal and chronic myelogenous leukemic granulocytic progenitor cells.” Oncology(38): 356–360. 184. Oldham, R. K. (1984). “ Biologicals and biologic response modifiers. The fourth modality of cancer therapy.” Cancer Treat Report(68): 221–232. 185. Olsen, E. A., S. T. Rosen, et al. (1989). “Interferon alfa-2a in the treatment of cutaneous T cell lymphoma.” J Am Acad Dermatol(20): 395–407. 186. Opravil, M., B. Hirschel, et al. (1999). “Related Articles. A randomized trial of interferon-alpha2a and zidovudine versus bleomycin and zidovudine for AIDS-related Kaposi’s sarcoma. Swiss HIV Cohort Study.” Int J STD AIDS(10(6) ): 369–375. 187. Osserman, E. T., W. F. Sherman, et al. (1980). “Preliminary results of the American Cancer Society-sponsored trial of human leukocyte interferon in multiple myeloma.” Proc Am Assoc Cancer Res(21): 161. 188. Österborg, A., M. Björkholm, et al. (1993). “Natural interferonalpha in combination with melphalan/prednisone versus melphalan/prednisone in the treatment of multiple myeloma stag.es II and III. A randomized study from the Myeloma Group of Central Sweden.” Blood(81): 1428–1434. 189. Ozer, H., J. R. Anderson, et al. (1987). “Combination trial of subcutaneous interferon alfa-2b and oral cyclophosphamide in favorable histology, non-Hodgkin’s lymphoma.” Invest New Drugs(5): 527–523. 190. Ozer, H., H. M. Golomb, et al. (1989). “Cost benefit analysis of interferon alfa 2-b in the treatment of hairy cell leukemia.” J Natl Cancer Inst(8): 594–602. 191. Pagannelli, K. A., S. S. Evans, et al. (1986). “B cell growth factorinduced proliferation of hairy cell lymphocytes and inhibition by Type I interferon in vitro.” Blood(67): 937–942. 192. Panem, S. (1985). “The Interferon Crusade.” The Brookings Institute. 193. Pangalis, G. A. and E. Griva (1988). “Recombinant alfa-2b-interferon therapy in untreated, stages A and B chronic lymphocytic leukemia.” Cancer(61): 869–872.

D. Goldstein et al. 194. Paulnock, D. M. and E. C. Borden (1985). “ Modulation of immune functions by interferons. In: Immunity to Cancer” 545–559. 195. Peest, D., H. Deicher, et al. (1995). “A comparison of polychemotherapy and melphalan/prednisone for primary remission induction, and interferon-alpha for maintenance treatment, in multiple myeloma. A prospective trial of the German Myeloma Treatment Grou.” Eur J Cancer Clin Oncol(31A): 146–151. 196. Pehamberger, H., H. P. Soyer, et al. (1998). “Adjuvant interferon alpha-2a in resected primary stage II cutaneous melanoma.” J Clin Oncol(16): 1425–1429. 197. Peterson, B. A., G. Petroni, et al. (1997). “Cyclophosphamide vs. Cyclophosphamide plus interferon alfa-2b in follicular low grade lymphomas.” Proc Am Soc Clin Oncol(16): 41–47. 198. Pfeffer, L. M., A. Dinarello. C, et al. (1998). “Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons.” Cancer Res(15(58) ): 2489–2499. 199. Pitha, P. (1993). “Nomenclature of the human interferon genes. For the Nomenclature Committee of the International Society for Interferon Research.” J Int Res(13): 443–444. 200. Pizzocaro, G., L. Piva, et al. (2001). “Interferon adjuvant to radical nephrectomy in Robson stages II and III renal cell carcinoma: a multicentric randomized study.” J Clin Oncol(19(2) ): 425–431. 201. Poiesz, B. J., F. W. Ruscetti, et al. (1980). “Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma.” Proc Natl Acad Sci USA(77): 7415–7419. 202. Pope, G. R., M. R. Hadam, et al. (1981). “Kinetics of natural cytotoxicity in patients treated with human fibroblast interferon.” Cancer Immunol Immunother(11): 1–6. 203. Porzsolt, F. (1992). “Adjuvant therapy of renal cell cancer with interferon alfa-2A.” Proc Amer Soc Clin Oncol(11): 202. 204. Pyrhonen, S., E. Salminen, et al. (1999). “Prospective randomized trial of interferon alfa-2a plus vinblastine versus vinblastine alone in patients,with advanced renal cell cancer.” J Clin Oncol(17(9) ): 2859–2867. 205. Quesada, J. R., E. M. Hersh, et al. (1984). “Alpha interfereon for induction of remission in hariy cell leukemia.” N Engl J Med(310): 15–18. 206. Quesada, J. R., R. Alexananian, et al. (1986). “Treatment ot multiple myeloma with trecombinanat alpha interferon.” Blood(67): 275–278. 207. Quesada, J. R., E. M. Hersh, et al. (1986). “Treatment of hairy cell leukemia with recombinant alpha-interferon.” Blood(68): 493–497. 208. Quesada, J. R., R. Alexananian, et al. (1987). “Alpha interferons in hairy cell leukemia (HCL). A five year follow up in 100 patients.” J Interf Res (7): 678. 209. Quesada, J. R., R. Alexananian, et al. (1988). “Recombinant interferon gamma in hairy cell leukemia, multiple myeloma, Waldenstrom’s macroglobulinemia.” Am J Hematol (29): 1–4. 210. Raefsky, E. L., L. C. Platanias, et al. (1985). “Studies of interferon as a regulator of hematopoietic cell proliferation.” J Immunol (135): 2507. 211. Trump D.L., Ravdin, P., M., E. C. Borden, et al. (1990). “Interferon alpha-N1 and continuous infusion vinblastine for treatment of advanced renal cell carcinoma.” J Biol Resp Mod. (9(1)): 108-111 212. Real, F. X., H. F. Oettgen, et al. (1986). “Kaposi’s sarcoma and the acquired immunodeficiency syndrome. Treatment with high and low doses of recombinant leukocyte A interferon.” J Clin Oncol(4): 544–551. 213. Revel, M., Kimch, A., et al. (1980). “Role of interferon-induced enzymes in the antiviral and antimitogenic effects of interferon.” Ann NY Acad Sc(350): 459–473.

299 214. Rohatiner, A. Z. S., P. F. Prior, et al. (1983). “Central nervous system toxicity of interferon.” Br J Cancer(47): 419–422. 215. Rohatiner, A. Z. S., et al. (1998). Proc AM Soc Clin Oncol(17): 11. 216. Rohatiner, A., C. G. A. Price, et al. (2001). “A randomized controlled trial to evaluate the role of interferon as initial and maintenance therapy in patients with follicular lymphoma.” Br J Cancer(85(1)): 29–35. 217. Rosenberg, S. A., J. C. Yang, et al. (1999). “Prospective randomized trial of the treatment of patients with metastatic melanoma using chemotherapy with cisplatin, dacarbazine, and tamoxifen alone or in combination with interleukin-2 and interferon alpha-2b.” J Clin Oncol(17): 968–975. 218. Rosenblum, M. G., B. L. Maxwell, et al. (1986). “In vivo sensitivity and resistance of chronic myelogenous leukemia cells to alpha interferon: Correlation with receptor binding and induction of 2′,5′- oligoadenylate synthetase.” Cancer Res(46): 4848–4852. 219. Rossi, G. (1985). “Interferon and cell differentiation. In: Interferon.” Academic Press(6): 31–68. 220. Rosti, G., F. Bonifazi, et al. (1999). “Cytarabine increases karyotypic response in alpha interferon treated chronic myeloid leukemia patients: results of a national prospective randomized trial of the Italian Cooperative Study Group on CML.” Blood(94): 600A. 221. Rozman, C., E. Montserrat, et al. (1988). “Recombinant alpha2 interferon in the treatment of B chronic lymphocytic leukemia in early stages.” Blood(71): 1295–1298. 222. Rupoli, S., S. Barulli, et al. (1999). “Low dose interferon-alpha2b combined with PUVA is an effective treatment of early stage mycosis fungoides: results of a multicenter study. Cutaneous-T Cell Lymphoma Multicenter Study Group.” Haematologica(84(9) ): 809–813. 223. Salmon, S. E., J. J. Crowley, et al. (1994). “Combination chemotherapy, glucocorticosteroids, and interferon alfa in the treatment of multiple myeloma. A Southwest Oncology Group Study.” J Clin Oncol(12): 2405–2414. 224. Samuelsson, J., H. Hasselbalch, et al. (2006). “A phase II trial of pegylated interferon alpha-2b therapy for polycythemia vera and essential thrombocythemia: feasibility, clinical and biologic effects, and impact on quality of life.” Cancer Res(106(11) ): 2397–2405. 225. Schaar, C. G., H. C. Kluin-Nelemans, et al. (2005). “Interferon alpha as maintenance therapy in patients with multiple myleoma.” Ann Oncol(3(16) ): 634–639. 226. Scheinfeld, N. (2007). “A brief primer on treatments of cutaneous T cell lymphoma, newly approved or late in development.” J Drugs Dermatol(6(7) ): 757–760. 227. Scheithauer, W., A. Cortelezzi, et al. (1989). “Combined alpha2C-interferon/VMCP polychemotherapy versus VMCP as induction therapy in multiple myeloma: A Prospective Randomized Trial.” J Biol Resp Modif(8): 109–115. 228. Schornagel, J. H., J. Verweij, et al. (1989). “Phase II Study of recombinant interferon alpha-A and vinblastine in advanced renal cell carcinoma.” J Urol(142): 253–254. 229. Schulof, R. S., M. J. Lloyd, et al. (1985). “Recombinant leukocyte A interferon in B-cell chronic lymphocytic leukemia: In vivo effects on autologous antitumor immunity.” J Biol Resp Modif(4): 310–323. 230. Senn, C. C. (1984). “Biochemical pathways in interferon action.” Pharmacol Ther(24): 235–257. 231. Sertoli, M. R., I. Brunette, et al. (1989). “Recombinant alpha-20 interferon plus vinblastine in the treatment of metastatic renal cell carcinoma.” Am J Clin Oncol(12): 43–45. 232. Shepherd, F. A., R. Beaulieu, et al. (1998). “Prospective randomised trial of two dose levels of interferon alfa with zidovudine for the treatment of Kaposi’s sarcoma associated with human immunodeficiency virus infection: a Canadian HIV clinical trials network study. et a.” J Clin Oncol(16): 1736–1742.

300 233. Shin, D. M., F. R. Khuri, et al. (2001). “Combined interferon-alpha, 13-cis-retinoic acid, and alpha-tocopherol in locally advanced head and neck squamous cell carcinoma: novel bioadjuvant phase II trial.” J Clin Oncol(19): 3010–3017. 234. Sidky, Y. A. and E. C. Borden (1987). “Inhibition of angiogenesis by interferons: Effects on tumor- and lymphocyte-induced vascular responses.” Cancer Res 1987(47): 5155. 235. Sigaux, F., S. Castaigne, et al. (1987). “Alpha-interferon in hairy cell leukaemia: direct effects on hairy cells or indirect cytotoxicity.” Int J Cancer Suppl(1):2–8. 236. Silver, R. T. (1993). “Interferon-alpha 2b:A new treatment for polycythemia vera.” Ann Intern Med: 1091–1092. 237. Silver, H. K. B., J. M. Connors, et al. (1985). “Prospectively randomized toxicity study of high-dose versus low-dose treatment strategies for lymphoblastoid interferon.” Cancer Treat Rep(69): 743–750. 238. Silver, R. T., M. Coleman, et al. (1987). “Interferon (rIFN) alfa 2b has activity in treating chronic myeloid leukemia (CML) in rIFN gamma failures.” Proc Amer Soc Clin Onc(6): 149. 239. Singh, R. K., M. Gutman, et al. (1995). “Interferons alpha and beta downregulate the expression of basic fibroblast growth factor in human carcinoma.” Proc Natl Acad Sci USA(92): 4562. 240. Sirohi, B., R. Powles, et al. (2007). “An open, randomized, controlled, phase II, single centre, two-period cross-over study to compare the quality of life and toxicity experienced on PEG interferon with interferon-alpha2b in patients with multiple myeloma maintained on a steady dose of interferon-alpha2.” Ann Oncol 3(18(8) ): 1388–1394. 241. Smalley, R. V., J. Vogel, et al. (1977). “Chronic granulocytic leukemia:Cytogenetic conversion of the bone marrow with cycle specific chemotherapy.” Blood(50): 107–113. 242. Smalley, R. V., S. S. Anderson, et al. (1991). “A randomized comparison of a low dose and a standard dose of alpha interferon (WellferonR) in Hairy Cell Leukemia.” Blood(78): 3133–3141. 243. Smalley, R. V., J. W. Andersen, et al. (1992). “Interferon Alfa Combined with Cytotoxic Chemotherapy for Patients with NonHodgkin’s Lymphoma – an ECOG Study.” New Eng J Med(327): 1336–1341. 244. Smalley, R. V., J. Connors, et al. (1992). “Splenectomy vs. Alpha Interferon: A Randomized Study in Patients with Previously Untreated Hairy Cell Leukemia.” Amer J Hematol(41): 13–18. 245. Smedley, H. M. and T. Wheeler (1983). “Toxicity of IFN. In: Interferon and cancer.” 203–210. 246. Solal-Celigy, P., E. Lepage, et al. (1998). “Doxorubicincontaining regimen with or without interferon alfa-2b for advanced follicular lymphomas: final analysis of survival and toxicity in the Groupe d’Etude des Lymphomes Folliculaires.” J Clin Oncol(16): 2332–2338. 247. Spinna, C. A., J. L. Dahey, et al. (1983). “Suppression of NK cell cytotoxicity in the peripheral blood of patients receiving interferon therapy.” J Biol Resp Modif(2): 458–459. 248. Stadler, W. M., T. M. Kuzel, et al. (1997). “Metastatic bladder cancer: advances in treatment.” Eur J Cancer(33): 23. 249. Stadler, R., H. G. Otte, et al. (1998). “Prospective randomized multicenter clinical trial on the use of interferon-2a plus acitretin versus interferon-2a plus PUVA in patients with cutaneous T-cell lymphoma stages I and II.” Blood(92(10) ): 3578–3581. 250. Steis, R. G., J. W. Smith, et al. (1988). “Resistance to recombinant interferon alfa 2a in hairy cell leukemia associated with neutralizing anti-interferon antibodies.” N Engl J Med(318): 1409–1413. 251. Steis, R. G., W. J. Urba, et al. (1988). “Immunogenicity of different types of interferons in the treatment of hairy-cell leukemia.” N Engl J Med(319): 1227. 252. Stewart, W. E. and D. K. Blanchard (1985). “Interferons, cytostatic and immunomodulatory effects.” Immunity to Cancer Eds Reif AE, Mitchell MS (Academic Press, Orlando, Fl, USA) 295–307.

Interferons: therapy for cancer 253. Strander, H., U. Abramson, et al. (1978). “Adjuvant interferon treatment of human osteosarcoma.” Rec Results Ca Res(68): 40. 254. Strander, H., K. Cantell, et al. (1980). “Interferon treatment of osteogenic sarcoma: A clinical trial. In: Conference on Modulation of Host Immune Resistance in the Prevention and Treatment of Induced Neoplasms.” (28): 377–381. 255. Talpaz, M., K. B. McCredie, et al. (1983). “Leukocyte interferoninduced myeloid cytoreduction in chronic myelogenous leukemia.” Blood(62): 689–692. 256. Talpaz, M., J. M. Trujillo, et al. (1985). “Suppression of clonal evolution in two chronic myelogenous leukemia patients treated with leukocyte interferon.” Br J Haematol(60): 619–624. 257. Talpaz, M., H. M. Kantarjian, et al. (1987). “Clinical investigation of human alpha interferon in chronic myelogenous leukemia.” Blood(69): 1280–1288. 258. Talpaz, M., R. Kurzrock, et al. (1989). “Recombinant interferonalpha therapy of Philadelphia chromosome-negative myeloproliferative disorders with thrombocytosis.” Am J Med(86): 554–558. 259. Taniguchi, T., S. Ohno, et al. (1980). “The nucleotide sequence of human fibroblast interferon cDNA.” Gene(20): 11–15. 260. Taylor-Papadimitriou, J., N. Ebsworth, et al. (1985). “Possible mechanisms of interferon-induced growth inhibition. In: Mediators in Cell Growth and Differentiation.” 283–298. 261. Tichelli, A., A. Gratwohl, et al. (1989). “Treatment of thrombocytosis in myeloproliferative disorders with interferon alpha-2a.” Blut(58): 15–19. 262. Toy, J. (1983). “The interferons.” Clin Exp Immunol(54): 1–13. 263. Trinchieri, G. and B. Perussia (1985). “Immune interferon: A pleiotropic lymphokine with multiple effects.” Immunol Today 1(6): 131–136. 264. Trump, D. I., P. Elson, et al. (1996). “Randomised controlled trial of adjuvant therapy with lymphoblastoid interferon in resected high risk renal cell carcinoma.” Proc Am Soc Clin Oncol(15): 253. 265. Tura, S. (1994). “Interferon alfa 2a as compared with conventional chemotherapy for the treatment of chronic myeloid leukemia.” New Engl J Med(330): 820–825. 266. Uze, G., G. Lutfalla, et al. (1990). “Genetic transfer of a functional human interferon-α receptor into mouse cells: Cloning and expression of its cDNA.” Cell(60): 225–234. 267. Varesio, L., E. Blasi, et al. (1985). “Recombinant IFN-gamma activates macrophages to become tumoricidal.” 268. Verma, S., I. Quirt, et al. (2006). “Systematic review of systemic adjuvant therapy for patients at high risk for recurrent melanoma.” Cancer Res(106 (7) ): 1431–1442. 269. Vilcek, J., H. C. Kelke, et al. (1985). “Structure and function of human interferon gamma. In: Mediators in Cell Growth and Differentiatinon.” 299–313. 270. Volberding, P., R. Valero, et al. (1984). “Alpha interferon therapy of Kaposi’s sarcoma in AIDS.” Ann NY Acad Sci(437): 439–447. 271. von Wussow, P., M. Frund, et al. (1988). “Immunogenicity of different types of interferons in the treatment of hairy cell leukemia.” N Engl J Med(319): 1226. 272. von Wussow, P., R. Hehlmann, et al. (1994). “Roferon (rIFNα2a) is more immunogenic than Intron A (rIFN-α2b) in patients with chronic myelogenous leukemia.” J Interferon Res(14): 217–219. 273. Vugrin, D., L. Hood, et al. (1985). “A Phase II trial of high-dose human lymphoblastoid alpha interferon in patients with advanced renal carcinoma.” Cancer Treat Rep(69): 817–820. 274. Wadler, S., E. L. Schwartz, et al. (1989). “5-fluorouracil and recombinant alpha-2a-interferon: An active regimen against advanced colorectal carcinoma.” J Clin Oncol(7): 1769–1775. 275. Wadler, S. and P. H. Wiernik (1990). “Clinical update on the role of fluorouracil and recombinant interferon alfa-2a in the treatment of colorectal carcinoma.” Sem Onc(17(Suppl 1) ): 16–21.

D. Goldstein et al. 276. Wagstaff, J., P. Loynds, et al. (1985). “A phase II study of rDNA human alpha 2 interferon in multiple myeloma.” Cancer Treat Rep(69): 495–498. 277. Weiner, L. M., P. J. Moldofsky, et al. (1988). “Antibody delivery and effector cell activation in a Phase II trial of recombinant gamma interferon and the murine monoclonal antibody CO-17–1-A in advanced colorectal carcinoma.” Cancer Res(49): 1609–1621. 278. Weiss, G. R., P. Y. Liu, et al. (1998). “13-cis-retinoic acid or all-trans-retinoic acid plus interferon-alpha in recurrent cervical cancer: a Southwest Oncology Group phase II randomized trial 1998.” Gynecol Oncol(71): 386–390. 279. Westin, J., S. Rödjer, et al. (1995). “Interferon alfa-2b versus no maintenance therapy during the plateau phase in multiple myeloma. A randomized study.” Br J Haematol(89): 561–568. 280. Wiernik, P. H., B. Schwartz, et al. (1990). “Successful treatment of hairy cell leukemia with beta ser interferon.” Am J Hematol. 281. Wirth, M. P. (1993). “Immunotherapy for metastatic renal cell carcinoma Urol.” Clin North Am(20): 283–295. 282. Wisloff, F. and N. Gulbrandsen (2000). “Health related quality of life and patient’s perceptions in interferon-treated multiple myeloma patients.” Nordic Myeloma Study Group Acta Onco(39): 809–813. 283. Wollina, U., A. Looks, et al. (2001). “Treatment of stage II cutaneous T-cell lymphoma with interferon alfa-2a and extracorporeal

301

284.

285.

286.

287.

288.

289.

290.

photochemotherapy: a prospective controlled trial.” J Am Acad Dermatol(44(2) ): 253–260. Worman, C. P., D. Catovsky, et al. (1987). “The UK experience withhuman lymphoblastoid interferon in HCL: A report of the first 50 cases.” Leukemia(1): 320–322. Worman, C. P., A. B. W. Nethersell, et al. (1987). “Natural IFN-a therapy in hairy-cell leukaemia (Namalva-Type IFN-Wellferon).” Klin Wochenschr(65): 685–687. Xu, D., S. Erickson, et al. (2000). “Interferon alpha down-regulates telomerase reverse transcriptase and telomerase activity in human malignant and nonmalignant hematopoietic cells.” Blood(96): 4313–4318. Yang, J. L., X. J. Qu, et al. (2007). “Erlotinib (OSI-774)-induced inhibition of transitional cell carcinoma of bladder cell line growth is enhanced by interferon-alpha.” BJU Int 2000(99(6) ): 1539–1545. Yoffe, G., M. Blick, et al. (1987). “Molecular analysis of interferon-induced suppression of Philadelphia chromosome in patients with chronic myeloid leukemia.” Blood(69): 961–963. Zinzani, P. L., Magagnoli. M., et al. (2000). “Long term-term follow-up of hairy cell leukemia patients treated with 2-chlorodeoxyadenosine.” Haematologica 2000 (85(9) ): 922–925. Zoon, K. C., R. Hu, et al. (1986). “Further Studies on the Purification and Partial Characterization of Human Lymphoblastoid Interferon Alphas. In: The Biology of the Interferon System.” 55–58.

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.

303

304

Monoclonal antibody therapy

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

325

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.

Robert O. Dillman

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

328

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%

Robert O. Dillman

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

Robert O. Dillman

331

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

332

Monoclonal antibody therapy

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

Robert O. Dillman

333

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

334

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

Robert O. Dillman

335

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.

336

Monoclonal antibody therapy

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

Robert O. Dillman

337

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.

338

Monoclonal antibody therapy

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].

Robert O. Dillman

339

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

Robert O. Dillman

341

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.

342

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

344

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

Robert O. Dillman

345

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

Monoclonal antibody therapy

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

Robert O. Dillman

369

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.

370

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