Biotechnology Annual Review, Volume 10 (Biotechnology Annual Review)

v Preface What is Biotechnology Today? What is biotechnology today? More specifically, as one invites chapters for an...

Author: M. Raafat El-Gewely

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v

Preface

What is Biotechnology Today? What is biotechnology today? More specifically, as one invites chapters for an Annual Review of Biotechnology, what should be the range of topics? This is quite a complicated question. The world of biotechnology has changed profoundly with the advent of the Human Genome Project (HGP). The HGP was the first example of global discovery science – the attempt to take a complex biological object, the genome, and define all of its elements – the DNA sequences of the 24 different human chromosomes. For the first time, biologists had a genetics parts list for the human, namely an enumeration of all (most) of our genes (and by translation, our proteins). This raised the possibility for global studies in which all (most) of the elements of a system could be studied quantitatively or in terms of their interactions. Accordingly, genomic discovery science led quite naturally to two other applications of discovery science – the transcriptome, a quantitative measure of all (many) the mRNAs present in a particular cell, organ or organism and the proteome, a similar quantitative measure of all (many) the proteins present in a particular biological system. In a similar vein, others are studying metabolites, the metabolome, phenotypes, the phenome, etc. This ability to carry out global discovery science for the multiplicity of different types of biological information raised the possibility this information could be used to actually understand biology through an approach termed systems biology. What is systems biology? In its simplest terms, systems biology is the identification of the elements in a system and an analysis of their interactions while the system is functioning so as to understand the systems or emergent properties of the system. Systems biology probably started with the integrative physiologists of the early 20th century who were interested in homeostasis, and the effects of environmental perturbations such as hormones on homeostasis. The systems biology of today is quite different in that it can interrogate many different types of biological information in a global manner. Systems biology has just recently been employed in this manner and it has a number of interesting features. Systems biology is global, quantitative, hypothesis-driven, iterative, integrative (different types of biological information must be integrated together to understand systems), dynamic (systems must be studied as they execute their developmental or physiological functions), and multi-scale (systems extend from a few molecules carrying out a particular function, such as the metabolism of a sugar, to biological networks in cells to cells and organs and organisms; thus, the physical scale across which biological systems operate is enormous). It is important to point out that high-throughput global technologies must be invented and improved to capture the many different types of information. Likewise, it is essential to develop the computational, mathematical, and statistical tools for capturing, storing, analyzing, integrating, modeling, and finally dispersing biological information.

vi What is the context from which this global systems biology emerged? It has all happened in the last 5–10 years. Several factors have been important. (1) The completion of the HGP led to the genetics parts list, discovery science, and it initiated the development of high-throughput technical platforms such as automated DNA sequencing. These high-throughput platforms are, of course, essential to the capture of large and global data sets, which constitute the foundation of systems biology. (2) Biology has come to recognize the power of cross-disciplinary biology because of the technologies and computational tools that remain to be developed. Thus, computer scientists, mathematicians, and statisticians, as well as physicists, must develop the computational and modeling tools; the engineers, physicists, chemists, and biologists must develop the new technologies for high-throughput biology. (3) The internet has given us the capacity to store and dispense large amounts of information. (4) Finally, the idea that biology is an informational science has emerged – and this is the foundation for thinking about systems biology. Let us consider biological information in this context. Biological information falls into two categories – the digital information encoded in the genome, and the environmental signals that impinge of the digital genome to initiate developmental and physiological responses. The digital genome has two major types of information – the genes encode proteins, the molecular machines of life, and the cis-control elements that specify, in conjunction with their cognate transcription factors, the behavior of individual genes (when, where, and how much mRNA is expressed) and, the cis elements establish the linkages and architectures of the gene regulatory networks – those networks of transcription factor genes which control the peripheral batteries of genes executing developmental and physiological functions on the one hand, and which are triggered by the protein networks of signal transduction on the other hand. Thus, the major challenge of systems biology is defining and understanding the interactions of the protein and gene regulatory networks. Finally, biological information is hierarchal in its expression starting at the DNA and moving outward to ecological systems – that is, it goes from DNA to mRNA to protein to protein interactions and biomodules to protein networks to cells to organs to organisms to populations of organisms and finally to ecologies. Environmental signals modify the initial digital input of information at each of these successive informational levels – hence, global data sets must be gathered on as many of these informational levels as possible and, ultimately, these data sets must be integrated to begin understanding how the corresponding system works. Thus, systems biology is global and all encompassing with regard to its need to gather biological information. In this context, the contents of Volume 10 encompass one or more of the various types of biological information that constitute the foundations of systems biology. Indeed, one chapter is on a systems approach to human health. Several are on DNA mediated technologies and five are on varying aspects of proteomics. The remainders of the papers are on several of the enormous ranges of policy questions emerging from modern biology. We can begin to glimpse from these chapters the enormous challenges facing modern biology in general and fascinating promises (poised against a very challenging reality) that systems biology has made for providing an integrated picture of biological complexity. The fascinating question is how long will it take

vii systems biology to begin filling its enormous promise to understand biological complexity, for in time it surely will begin to comprehend and even reengineer this complexity.

Leroy Hood, M.D., Ph.D. President The Institute for Systems Biology 1441 North 34th Street, Seattle, WA 98103-8904 Phone: 1-(206) 732-1201; Fax: 1-(206) 732-1299 [email protected] http://www.systemsbiology.org/

ix

EDITORIAL BOARD FOR VOLUME 10 CHIEF EDITOR Dr. M. Raafat El-Gewely Department of Molecular Biotechnology Institute of Medical Biology University of Tromsø 9037 Tromsø, Norway Phone: þ 47-77 64 46 54 Fax: þ 47-77 64 53 50 E-mail: [email protected] EDITORS Dr. MaryAnn Foote Associate Director Medical Writing Department Amgen, Thousand Oaks, CA 91320-1879, USA Phone: þ 1-805-447-4925 Fax: þ 1-805-498-5593 E-mail: [email protected] Dr. Guido Krupp Director & Founder artus GmbH Koenigstr. 4a D-22767 Hamburg, Germany Phone: þ 49-40-41 364 783 Fax: þ 49-40-41 364 720 E-mail: [email protected] website: http://www.artus-biotech.com ASSOCIATE EDITORS Dr. Marin Berovic Department of Chemical and Biochemical Engineering University of Ljubljana Hajdrihova 19, Ljubljana Slovenia E-mail: [email protected] Dr. Thomas M.S. Chang Artiﬁcial Cells & Organs Research Centre McGill University

3655 Drummond St., Room 1005 Montreal, Quebec, Canada H3G 1Y6 Phone: þ 1-514-398-3512 Fax: þ 1-514-398-4983 E-mail: [email protected] Dr. Thomas T. Chen Department Molecular and Cellular Biology University of Connecticut 91 North Eagleville Rd, Unit 3125 Storrs, Connecticut 06269-3149, USA Phone: 1-860-486-5481 Fax: 1-860-486-5005 E-mail: [email protected] Dr. Frank Desiere Nestle´ Research Centre, P.O. Box 44, CH-1000 Lausanne 26 Switzerland E-mail: [email protected] Prof. Franco Felici Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari Universita` di Messina Salita Sperone, 31 98166 Messina, Italy Phone: þ 39 090 6765197 Fax: þ 39 090 392733 E-mail: [email protected] Dr. Leodevico L. Ilag Xerion Pharmaceuticals AG Fraunhoferstrasse 9 82152 Martinsried Germany Phone: þ 49 89 86307 201 Fax: þ 49 89 86307 222 E-mail: [email protected]

x Dr. Kuniyo Inouye Laboratory of Enzyme Chemistry Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Sakyo-ku, Kyoto 606-8502, Japan Phone: þ 81-75-753 6266 Fax: þ 81-75-753 6265 E-mail: [email protected] Dr. Alfons Lawen Senior Lecturer Monash University, Clayton Campus Department of Biochemistry and Molecular Biology Room 312, Building 13D Clayton, Victoria 3800 Phone: þ 61-3-9905 3711 Fax: þ 61-3-9905 4655 E-mail: [email protected] Dr. Jocelyn H. Ng Hirsch-Gereuth-strasse 56 81369 Munich Germany Phone: þ 49 89 78018945 E-mail: [email protected]

Dr. Eric Olson Program Executive, Strategic Development Vertex Pharmaceuticals, Inc 130 Waverly Street Cambridge, MA 02139, USA Phone: þ 1-617-444-6917 E-mail: [email protected] Dr. Steﬀen B. Petersen Biostructure and Protein Engineering Laboratory Department of Biotechnology University of Aalborg Sohngaardsholmsvej 57 DK-9000 Aalborg Denmark Phone: 45-9-635 8469 Fax: 45-9-814 2555 E-mail: steﬀ[email protected] Prof. Vincenzo Romano-Spica Professor of Hygiene University Institute of Motor Science, IUSM Piazza Lauro e Bosis 15, 00194 Rome, Italy Phone/Fax: þ 39-06-36733247 E-mail: [email protected]

xv

Contents Preface Editorial Board List of contributors

v ix xi

Rapid translation system: A novel cell-free way from gene to protein Michael Hoﬀmann, Cordula Nemetz, Kairat Madin and Bernd Buchberger

1

Protein expression and refolding – A practical guide to getting the most out of inclusion bodies Lisa D. Cabrita and Steve Bottomley Towards a systems biology understanding of human health: Interplay between genotype, environment and nutrition Frank Desiere Public health issues related with the consumption of food obtained from genetically modiﬁed organisms Andrea Paparini and Vincenzo Romano-Spica p75 Neurotrophin receptor signaling in the nervous system Yuiko Hasegawa, Satoru Yamagishi, Masashi Fujitani and Toshihide Yamashita

31

51

85

123

Phage display for epitope determination: A paradigm for identifying receptor–ligand interactions Merrill J. Rowley, Karen O’Connor and Lakshmi Wijeyewickrema 151 DNA vaccines and their application against parasites – promise, limitations and potential solutions Peter M. Smooker, Adam Rainczuk, Nicholas Kennedy and Terry W. Spithill 189 Drug-induced and antibody-mediated pure red cell aplasia: A review of literature and current knowledge Ralph Smalling, MaryAnn Foote, Graham Molineux, Steven J. Swanson and Steve Elliott

237

Using the biologic license application or new drug application as a basis for the common technical document MaryAnn Foote Guidelines and policies for medical writers in the biotech industry: An update on the controversy MaryAnn Foote

259

Radioimmunotherapy of non-Hodgkin’s lymphoma: Clinical development of the Zevalin regimen Charles P. Theuer, Bryan R. Leigh, Pratik S. Multani, Roberta S. Allen and Bertrand C. Liang

265

251

xvi Biosimulation software is changing research Richard L.X. Ho and Lenore Teresa Bartsell

297

Index of authors

303

Keyword index

305

1

Rapid translation system: A novel cell-free way from gene to protein Michael Hoffmann*, Cordula Nemetz, Kairat Madin, and Bernd Buchberger Roche Diagnostics GmbH, Nonnenwald 2, 82372 Penzberg, Germany Abstract. Proteome research has recently been stimulated by important technological advances in the ﬁeld of recombinant protein expression. One major breakthrough was the development of a new generation of cell-free transcription/translation systems. The open and ﬂexible character of these systems allows direct control over expression conditions via the addition of supplements to the expression reaction. The possibility of working with linear expression templates instead of cloned plasmids and the ease of downstream processing, circumventing the need for cell-lysis, makes them ideally suited for high-throughput screening applications. Among these novel cell-free systems, the Rapid Translation System (RTS) developed by Roche is the ﬁrst one that is scalable from micrograms to milligrams of protein. This review describes the basic principles of RTS which diﬀerentiate it from traditional in vitro expression technologies, starting from template generation to high-end applications like labeling for structural biology research. Recent results obtained by RTS users from diﬀerent institutions are presented to illustrate each step of a novel cellfree protein expression workﬂow and its beneﬁts compared to traditional cell-based expression. Keywords: in vitro translation, cell-free expression, template optimization, solubility, protein– protein interactions, high-throughput screening, scale-up, wheat germ, E. coli lysate.

1. Introduction to scalable cell-free protein expression Most of our knowledge about the molecular basis of protein synthesis including the genetic code, mRNA, ribosome functions, protein factors involved in translation, initiation and control of translation, co-translational protein folding, etc. was originally derived from in vitro studies carried out with cell-free expression systems. Now, in addition to their contributions to basic science, such systems oﬀer a broad range of new technological applications. Since the ﬁrst experiments in the early 1960s, cell-free protein biosynthesis has been improved continuously. An important step in this was the method of preparing S30 bacterial extracts and the introduction of exogenous messages into the system by Nirenberg and Matthaei [1]. Later, the coupled in vitro transcription–translation was developed by adding exogenous DNA as a template for the expression reaction by Zubay [2]. Owing to the simplicity of preparation, the Zubay bacterial extracts became very popular. However, a major shortcoming of most test-tube translation and transcription–translation systems remained their limited lifetime and, as a consequence, their low yield of expressed proteins. A major experimental breakthrough addressing this insuﬃciency was the invention of the continuous exchange cell-free (CECF) translation and coupled transcription–translation systems by Spirin in 1988 [3]. *Corresponding author: E-mail: michael.hoﬀ[email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10001-X

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

2 The principle of CECF relies on passive diﬀusion via a semipermeable membrane. Instead of incubating the reaction mixture in a ﬁxed volume of a test-tube, the reaction is performed under conditions where the reaction products (Pi, NMPs and polypeptides) are continuously removed from, and the consumable substrates (amino acids, NTPs and energy-regenerating compounds) are permanently supplied to the reaction chamber. This setup allows protein expression to continue for up to 24 h with maximized protein yields, because the accumulation of inhibitory reaction by-products or the exhaustion of substrates is avoided. This contrasts the situation in in vitro protein synthesis reactions performed in batch mode (without continuous removal and supply), where a plateau is reached after approximately 2 to 4 h. The CECF-principle can be implemented by using a device containing two chambers that are separated by a semi-permeable membrane, allowing continuous supply of substrates and removal of inhibitory by-products from the reaction chamber (Fig. 1). Only the enzymatic machinery required for coupled transcription and translation and the expressed protein remain in the reaction compartment permanently. Using the CECF format, protein yields of several milligrams per milliliter have been reached [4]. An optimization of the energy regeneration system and an enhanced bacterial lysate helped further to increase the productivity [5]. Roche Applied Science has introduced commercial versions (RTS 500 E. coli HY Kit, RTS 9000 E. coli HY Kit and RTS 500 ProteoMaster E. coli HY Kit) of the CECF format based on E. coli extracts. In their RTS 500 and RTS 9000 reaction devices, up to 5 mg of an individual protein can be synthesized per ml in 24 h. All RTS reactions can be conveniently carried out in

protein DNA template

membrane

supply

supply

reaction change inhibitory by-products

feeding chamber

feeding chamber

membrane

Fig. 1. Schematic illustration of the continuous-exchange cell-free (CECF) reaction principle.

3 the RTS ProteoMaster Instrument under reproducible conditions, ensuring that the CECF process is optimally supported by shaking and temperature control. The workﬂow leading from a gene of interest to its preparative expression in the Rapid Translation System is outlined in the next chapter and illustrated with examples. 2. Generation of optimized expression templates Similar to any other expression system, successful cell-free expression of a DNA encoding a speciﬁc protein of interest depends on a correct starting material, i.e., a high-quality cDNA library or an isolated cDNA clone, sequenced and characterized and thereby shown to be free of frameshifts or stop codons. This basic condition met, one has to additionally keep in mind that (1) it often makes sense to speciﬁcally adapt the coding sequence to the expression system in question, and (2) regulatory elements for transcription and translation initiation and termination have to be added up- and downstream of the coding sequence. 2.1 Rational cDNA design While it is obvious that the addition of promoter and terminator sequences as well as a ribosomal binding site are absolutely essential to start and stop an E. coli-based expression reaction, sequence optimization is often considered as a recommended ‘‘nice-to-do’’, but maybe a dispensable option. However, it is worth spending some eﬀort on sequence optimization, and conﬁrm that it works under the best conditions from the very beginning instead of going back to the start at a later stage of the expression project because suboptimal sequences were used. The Rapid Translation System uniquely oﬀers the possibility to use the tailor-made bioinformatics tool ProteoExpert to reliably perform this task of sequence optimization (see www.proteoexpert.com for further details). The ProteoExpert service runs on a Roche-independent server at Biomax Inc. in Martinsried/Germany and can be accessed via the internet using SSLconnections. The algorithm included in ProteoExpert does not calculate RNA secondary structures, although the changes it suggests ﬁnally result in a modiﬁed and therefore optimized RNA structure. Instead of trying to model the existing mRNA structure, any wild-type sequence submitted is compared to a set of data derived from several hundred genes whose expression yields have been very thoroughly determined experimentally and fed into a database. Since the correlation between the yields and certain biophysical parameters derived from the sequences of these previously expressed proteins is known, these same parameters can be optimized for each newly investigated target sequence as well, resulting in suggestions for yield-improved sequence variants. The mutations

4 A 1 2 3 4 5 6 7 8 9 10 wt

B 1 2 3 4 5 6 7 8 9 10 wt

Fig. 2. Examples of enhanced yields obtained after template optimization with ProteoExpert. 1–10: Variants suggested by ProteoExpert; wt: Wild-type sequence used as template. (A) Antibody fragment (55 kD, hybrid). (B) Human p58 (unknown function).

proposed by ProteoExpert can be incorporated into the coding region of the target gene very easily by PCR. The output of each ProteoExpert calculation contains a list of primer sequences that can be used directly in PCRs for template generation. Bioinformatic algorithms in general are not precise enough to determine exactly which sequence variant will ﬁnally give the highest yield in an actual expression reaction. Therefore, ProteoExpert suggests that 10 diﬀerent variants be derived from the wild-type sequence, each of them with a very high probability to have a higher expression yield than the wild-type sequence. Testing 5 of these 10 variants was suﬃcient for the identiﬁcation of the maximumyield sequence variant in about 75% of all optimization calculations carried out so far (see Fig. 2 for examples). Up to now, ProteoExpert has been able to ﬁnd sequence variants with signiﬁcantly increased yields in about 80% of all sequences that were initially expressed at very low levels or not expressed at all. When compared to ProteoExpert, other approaches like direct modeling of the mRNA secondary structure were shown to mostly fail because they still require an empiric decision about which of the several alternative structures should be chosen for sequence variant generation. Also, most other tools are not based on biochemical experiments and are not tailor-made for a speciﬁc expression system. All the base substitutions proposed by ProteoExpert are translationally silent. Following calculation of optimized DNA templates by ProteoExpert, these DNAs can be generated using appropriate primers and the RTS E. coli Linear Template Generation Set (LTGS). The resulting linear DNA can then be expressed directly using Roche’s RTS 100 E. coli HY system. Researchers at Roche now use ProteoExpert routinely at the beginning of each cell-free expression project. For example, in a case where the yield obtained for an RTS-expressed SH3 domain was too low and therefore a bottleneck to continue with an expression project ﬁnally aiming at X-ray or NMR studies, the use of ProteoExpert at an early stage in the whole project allowed to work with an optimized sequence that gave several milligrams of protein, as required for the subsequent experimental steps (cf. Section 6.2).

5 wt

m1 m2 m3 m4 m5 m6 m7 m8 m9 m10

Fig. 3. Analysis of sequence optimizations suggested by the ProteoExpert software. Shown is an anti-His6-tag Western blot of a wild-type SH3 domain (wt) and 10 mutants (m1–m10). His6-tagged expression templates were generated by PCR and expressed in 50 ml batch reactions.

2.2 Generation of templates for small-scale expression reactions and scale-up A range of diﬀerent T7-driven plasmids (called pIVEX, plasmids for in vitro expression) for RTS in vitro expression have been designed. They include vectors with His6-, HA-, Avi-tags, or vectors for the reversible fusion of the target protein to MBP or GST to increase its solubility and/or facilitate puriﬁcation. Standard cloning procedures via PCR and restriction enzymes can be used to insert target cDNAs into these vectors. One template generation method, much more convenient than restriction site cloning into expression plasmids, is used to linear templates generated by PCR (Fig. 3). The up- and downstream regions (T7 promoter/ribosomal binding site/T7 terminator) can be added to the product of a ﬁrst gene-speciﬁc PCR using the commercially available RTS Linear Template Generation Sets. DNA fragments included in these kits carry the necessary regulatory regions. The ends of these DNA fragments overlap with the ends of the ﬁrst gene-speciﬁc PCR product and can be linked to them in a second (so called ‘‘overlap-extension’’) PCR, using primers provided in the kit (Fig. 4). As for pIVEX vectors, diﬀerent options for protein tagging are provided (His6, Avi-tag, HA or MBP fusion). Linear templates generated using this method can be used directly in smallscale expression reactions producing up to 20 mg protein/50 ml. These reactions are carried out in 96-well-plates or PCR tubes without prior PCR product puriﬁcation or cloning. If a certain number of sequence variants (e.g., silent – for yield optimization, or mutational – to study the eﬀect of amino acid point mutations on protein function) are to be studied in parallel, this process can be easily automated (see Section 4.2). If plasmids instead of linear templates are preferred or ﬁnally required for large scale expressions, molecules generated during the two-step PCR as shown in Fig. 4 must be linked to a plasmid backbone. When individual molecules are cloned out of a pool of linear expression cassettes and inserted into PCR cloning vectors, one cannot exclude the risk of picking clones that carry mutations introduced by previous high-cycle-number PCRs. To circumvent this problem, the BD In-FusionTM PCR Cloning Technology (BD Biosciences Clontech, Palo Alto, CA), provides a powerful alternative to standard cloning: the product of the ﬁrst, less error-prone gene-speciﬁc PCR reaction can be taken instead of the complete linear expression cassette and inserted (‘‘in-fused’’) into

6 First PCR: Addition of overlap regions Gene-specific primers (customer designed)

Second PCR: Addition of regulatory elements and a tag

T7 Promoter Primer

Downstream regulatory element encoding DNA

Upstream regulatory element encoding DNA

T7 Terminator Primer tag T7 Promoter

T7 Terminator

Fig. 4. Application of the overlap extension PCR technique. Linear expression fragments are generated by two PCR steps. The overlap regions added to the gene of interest during the ﬁrst PCR hybridize to DNA sequences that carry the regulatory elements. In the early cycles of the second PCR, the 30 ends are extended. The full-length fragment is ﬁnally ampliﬁed via short external oligonucleotides.

a linearized and puriﬁed pIVEX vector which already contains the regulatory elements and tags. It has recently been demonstrated that this method, which does not depend on restriction digestion or T/A-overhangs but only on the activity of the BD In-FusionTM enzyme, also works in a dry-down, microtiter plate-based format, making it easy to adapt high-throughput applications. Figure 5 provides a summary of methods recommended for the generation of RTS expression templates. 3. Small-scale expression and optimization Cell-free synthesis of proteins has the advantage of allowing direct access to the reaction conditions, e.g., by oﬀering the possibility of adding co-factors, chaperones or other supplements, and to permit synthesis of cytotoxic proteins. Optimal conditions (e.g., reaction time, temperature) for best expression results can be quickly identiﬁed and easily reproduced. Some examples are discussed in the following sections. 3.1 Influence of temperature The inﬂuence of temperature on expression of the MIA (Melanoma Inhibitory Activity) protein, a secreted protein from human melanoma cell lines whose

7 RTS E. coli Workflow wt sequence

ProteoExpert E. coli cDNA primer suggestions for sequence optimization

LinTempGenSet E. coli

1st PCR *

PCR product 1 2nd PCR

pIVEX E. coli In-Fusion cloning

circular template E. coli

PCR product 2: linear template E. coli RTS 100 E. coli HY

µgs of protein

sequence optimization expression screening expression scale-up

RTS 500/9000 HY

mgs of protein * 1 to 10 reactions, depending on how many variables are compared.

Fig. 5. Methods for template generation in RTS and use of these templates for expression on diﬀerent scales.

expression levels are closely correlated with the capability of melanoma cells to form metastasis, was investigated [6]. As recombinant synthesis of functionally active MIA is a time-consuming and diﬃcult procedure and refolding is problematic due to the importance of disulﬁde formation, expression of mutant MIA proteins was performed via in vitro protein transcription/translation. Correct refolding of the molecule, known to be critical for function, had only been achieved by high eﬀort procedures before. For the screening of mutants, RTS proved to be able to produce the amounts of protein needed for initial testing. The highest amount of correctly folded MIA was achieved using the RTS 500 E. coli HY Kit at 30 C, whereas the highest percentage of correctly folded protein was obtained at 25 C. Functional testing proved the correct folding and activity of MIA. Downstream analysis of RTS-expressed mutant MIA allowed to derive information regarding the importance of deﬁned amino acids for protein structure and function, and to identify individual amino acid residues important for folding (Fig. 6). 3.2 Influence of ligand addition on solubility In another study on the solubility of recombinant proteins, the ligand binding domain of a human nuclear steroid hormone receptor was investigated. Due to being detached from the native protein structure, the single domain was insoluble when expressed both in vivo and in vitro. The addition to the RTS reaction of a receptor agonist known to bind to the single domain had a strong solubilizing eﬀect. Western blot analysis revealed a shift of the soluble protein

8

100 80 60 40

MIA_G61R MIA_Y69H

MIA_G56D

MIA_D29G MIA_D34A MIA_V48I MIA_L52Q

0

MIAdel82 MIAdel79 MIAdel73 MIAdel66

20 reaction buffer MIAwt

Invasion [per cent]

120

Fig. 6. Functional activity of MIA wild-type protein and various mutants tested in invasion assays. The RTS samples were used in a Boyden Chamber with approximately equal amounts of protein in each sample. An RTS sample of the empty vector was used as negative control (reaction buﬀer). Invasion of melanoma cells was set as 100%. Invasion is inhibited by MIAwt and by several MIA mutants, whereas MIAdel73, MIAdel66, MIA_D34A, MIA_V48I, MIA_L52Q and MIA_G61R lost functional activity. For experimental details see Ref. [7].

M

S

w/o P

1x S

10x P

S

P

S

50x P

Addition of agonist

S

supernatant

P

pellet

Fig. 7. Ligand-dependent solubility of a human nuclear receptor expressed in RTS 100. A ligand known to bind to the expressed receptor was added to the expression reaction in diﬀerent amounts. Reaction pellets and supernatants were analyzed by anti-His6-tag Western blotting.

fraction from 30% to more than 90% in the presence of the ligand (Fig. 7) (unpublished results). It is only in cell-free expression systems that the addition of a ligand for solubilization is practical. Consequently, RTS displays a wide range of possibilities for screening approaches if insoluble proteins are investigated. 3.3 Co-expression and subunit complementation Protein–protein interactions play a critical role in nearly all cellular processes. Thus, a practical strategy for studying the function of a particular protein

9 of interest is to identify other proteins that interact with it. This approach may lead to the isolation of new components participating in the same pathway, or the identiﬁcation of previously characterized factors that can help elucidate the function of a protein under study. Moreover, for hetero-oligomeric proteins which assemble as intermediates or metastable species during folding, co-expression of the diﬀerent subunits is often essential to build a functional assembly. The most widely used technique for identifying protein–protein interactions in genome-wide proteomics studies is the two-hybrid system. However, it has been noted that this system suﬀers from technical drawbacks inherent in the cellular approach: non-speciﬁc interactions, for example, can generate a high proportion of false positive results. It is therefore important to obtain biochemical evidence for speciﬁc protein–protein interactions using a second independent method. Combining the genetic technique with a simple in vitro assay to detect physical protein–protein interactions allows validation of twohybrid results. In vitro, proteins can be co-expressed easily and the inﬂuence of binding partners or ligands can be analyzed. RTS is an open system and is very well suited for co-expression experiments. Lorenz and Thiesen, for example, explored the possibilities of synthesizing Kox1, a member of the KRAB zinc ﬁnger protein family, and its interaction partner TIF1b in RTS [8]. They took advantage of the known interaction of these two proteins as an indicator of proper folding. cDNAs were subcloned in diﬀerent vectors suitable for T7 polymerasedriven transcription and their potential to give rise to the respective proteins was analyzed by Western blotting. Reconstitution experiments of Kox1/ TIF1b complexes followed by immunoprecipitation showed that complex formation only occurred if the two binding partners were co-translated (Fig. 8). The data led to the hypothesis that TIF1b protein helped the Kox1 proteins to adopt a proper conformation for association. In contrast to this, when working with E. coli, researchers had experienced inclusion body formation and consequently denatured protein, whereas expression of Kox1 in other systems like yeast or baculovirus appeared to be toxic for the host cells and was not possible at all. In another interaction study, two fragments of the E. coli protein MalE, an exported periplasmic receptor for high-aﬃnity transport of maltodextrins, were expressed separately under standard RTS 500 conditions and their reassembly was analyzed [9]. Both fragments were expressed at comparable steady state levels, whereas in vivo, the production of any MalE fragment smaller than 30 kDa had not been detectable previously because of excessive degradation. This suggested that proteolytic activity of RTS lysates was lower than in bacterial cells. A short N-terminal fragment was correctly produced, whereas a C-terminal fragment was mainly found in an aggregated and insoluble form. The complementation of both fragments was assessed by their ability to bind cross-linked amylose, providing a rapid and simple assay for probing native

10 A 1 2 3 4 5 6

TIF1β

kD

94 67

Kox 1 43 30 IP anti TIF1β + + − + − −

Fig. 8. Analysis of possible interactions between the Kox1 protein and TIF1b after individual vs. co-expression in RTS100. Immunoprecipitation with anti-TIF1b antibodies as a means to show association is indicated at the bottom of each lane. Positions of TIF1b and Kox1 as well as of molecular weight standards are indicated. Kox1 and TIF1b were both expressed from T7-driven pRSET vectors. Immunoprecipitation after 20 ml aliquots of His-Kox1 and His-TIF1b RTS reactions had been added together (lane 1); immunoprecipitation after 20 ml Kox1 RTS reaction had been added to a HeLa cell extract representing 500 mg total protein (lane 2); 10 ml aliquot of His-Kox1 RTS reaction (lane 3); immunoprecipitation of in-vitro cotranslated His-Kox1 and His-TIF1b (lane 4); 10 ml aliquot of in-vitro cotranslated His-Kox1 and His-TIF1b RTS reaction (lane 5); and 10 ml aliquot of His-TIF1b RTS reaction (lane 6). The upper part of the blot has been stained with monoclonal anti-TIF1b and anti-His tag antibodies, the lower part with rabbit anti-His tag antibodies. For details see Ref. [8]. Picture with courtesy of Springer and P. Lorenz, Universita¨t Rostock.

MalE conformation [10]. Independently produced MalE fragments were unable to bind amylose, as expected, presumably because residues involved in maltose binding are equally distributed between both fragments. In contrast, the simultaneous expression of both fragments in a single RTS reaction resulted in an active complex that could be puriﬁed with a yield of approximately 200 mg/ml. The N- and C-terminal fragments were detected after SDSpolyacrylamide gel electrophoresis and Coomassie staining in stoichiometric amounts, indicating that they had been present on the column as a stable and active complex (Fig. 9). Turbidity measurements were then used to determine the solubility of both fragments. Since precipitation occurred when the C-terminal fragment was expressed individually, misfolding in absence of the complementary N-terminal fragment was concluded. In contrast, the smaller N-terminal fragment remained soluble in the expression solution. Interestingly, when both fragments were simultaneously expressed in the RTS reaction, turbidity decreased to a value comparable to the sample containing only the N-terminal fragment. These results were conﬁrmed by analyzing the protein contents of supernatants and pellets after centrifugation of RTS extracts (Fig. 10). In summary, it could be concluded that the presence of the N-terminal fragment prevented aggregation of the C-terminal fragment and mediated the ﬁnal assembly of active MalE. Therefore, the RTS strategy for co-expressing protein fragments led to identiﬁcation of an intramolecular chaperone-like function for the N-terminal

11 A

whole extracts 1

2

3

4

B

eluates 5

6

7

8

1

kDa

2

−175 −83 −62 −48 −33 C-fragment

−25 −17

N-fragment

−7

Fig. 9. (A,B) RTS production of MalE fragments. (A) Production and puriﬁcation of MalE fragments were analyzed by SDS-polyacrylamide gel electrophoresis and stained by Coomassie blue. Lanes 1–4 whole RTS extracts after 18 h at 30 C; 1 empty vector pIVEX2.4a; 2 pIVME-N; 3 pIVME-C; 4 pIVME-N þ pIVME-C; lanes 5–7 maltose eluates from cross-linked amylose columns; 5 pIVME-N; 6 pIVME-C; 7 pIVME-N þ pIVME-C; lane 8 molecular weight standards. Arrows indicate the position of MalE fragments. (B) Native gel stained by Coomassie blue. Lane 1 wild-type MalE; lane 2 puriﬁed complex from coexpression of MalE fragments.

A

B soluble fraction insoluble fraction

1

Turbidity (Abs550nm)

2.0 1.5

2

3

4

5

6

7

kDa −175 −83 −62 −48

1.0

−33

0.5

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0

−17 1

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Fig. 10. (A,B) Solubility assays. (A) Turbidity of RTS extracts after 18 h at 30 C; 1 pIVME-N; 2 pIVME-C; 3 pIVME-N þ pIVME-C. (B) Soluble/insoluble production of MalE fragments was analyzed by SDS-polyacrylamide gel electrophoresis and stained by Coomassie blue. Lanes 1–3 RTS supernatants; 1 pIVME-N; 2 pIVME-C; 3 pIVME-N þ pIVME-C; lanes 4–6, RTS pellets; 4 pIVME-N; 5 pIVME-C; 6 pIVME-N þ pIVME-C; lane 7 molecular weight standards. Arrows indicate the position of MalE fragments. For details see Ref. [9]. Picture with courtesy of Springer and J-M Betton, Institut Pasteur, Paris.

fragment of MalE. Interestingly, such a critical role in protein folding for N-terminal fragments has been recently hypothesized for several other proteins as well [11]. The results of these studies validate co-expression of proteins or single domains as a successful application of the RTS system and demonstrate its

12 suitability to detect protein–protein interactions or to study the role of binding partners for proper folding. In contrast to in vivo expression systems, the in vitro environment of RTS oﬀers considerable ﬂexibility for co-expressing two or more proteins without the requirement of constructing plasmids carrying compatible origins of replication.

4. High-throughput protein expression and analysis 4.1 Introduction The analysis of all proteins encoded by the enormous sequence data collected from the various genome projects is a major challenge and requires the synthesis of many target proteins in parallel, often combined with PCR-based mutagenesis. Common approaches to evaluate protein function include the introduction of point mutations, deletions or insertions, and techniques for domain fusion. Since linear expression constructs can directly be expressed in vitro, it is easy to combine Expression-PCR (E-PCR) with other wellestablished PCR mutagenesis methods. In cases where research focuses on the rapid production of pharmaceutically relevant proteins and their functional and structural analysis for the development of inhibitory or activating drugs, in vitro expression displays considerable advantages. As the proteins are not produced in vivo, direct functional analysis, e.g., by Surface Plasmon Resonance (SPR), without the otherwise necessary puriﬁcation from a complex E. coli cell is possible. Using in vitro expression PCR, the time required for protein engineering is dramatically shortened. The whole process of two PCR steps followed by in vitro expression is feasible in less than 16 h. The proteins can subsequently be subjected to activity assays as demonstrated for example, by studies on the ﬂuorescence of green ﬂuorescent protein (Fig. 11). Random mutagenesis, PCR misincorporation procedures and recombination strategies may be combined with linear template generation by PCR in a similar way. After mutation or shuﬄing of the gene of interest, an expression fragment is constructed via overlap extension methods. In contrast to the introduction of single point mutations, the PCR products resulting from random mutagenesis are not homogeneous but represent a mixture of diﬀerent variants. The constructs can be ligated into RTS pIVEX vectors to divide the pool into single species that can be screened. Small-scale expression reactions in 50 ml volumes can be carried out in microtiter plate-based 96 well formats, and the whole process can be easily adapted to high-throughput screening (HTS) or automated liquid handling. Plasmid preparation is the only step in the whole workﬂow where transformation and growth of E. coli cells is required, while the steps of growth and lysis of large-volume cultures of more than 100 ml during downstream processing can be avoided.

13 A kD

B

GFP GFP T203Y

30

20

fluorescence activity

45

fluorescence activity

75

10

502 nm GFP T203Y

502 nm GFP

Fig. 11. GFP and mutated GFP (T203Y) were expressed in RTS100 E. coli HY from linear templates. (A) 0.5 ml of each reaction solution were separated by SDS-PAGE, blotted and detected with Anti-His6 Peroxidase conjugate. (B) Fluorescence activity was measured by exciting the protein at 395 nm and monitoring the emission at 430–580 nm. The reaction solutions were diluted 1:80.

4.2 Example: Screening of single-point muteins Researchers at Roche Pharmaceuticals developed a production platform which enables them to generate, express and purify recombinant proteins automatically in a small-scale format for initial construct evaluation [12]. All reaction steps were performed in microtiter plates allowing the automated and parallel processing of up to 96 protein variants through all steps including mutations, truncations and species variations. The expression cassettes used in this study comprised sequences for C-terminal aﬃnity puriﬁcation via an N-terminal hexahistidine-tag and for site-directed biotin labeling. Selective biotin-labeling of the fusion proteins was performed by using a biotin-accepting peptide (BAP) sequence called Avi-tag, which is enzymatically biotinylated by E. coli biotin ligase during the in vitro translation [13]. After a robotically performed Ni-NTA puriﬁcation procedure, a fraction of each puriﬁed protein was immobilized on streptavidin-coated microchips via the mono-biotinylated BAP sequence and analyzed by protein–protein interaction measurements using SPR technology. As a model protein, the authors chose the N-terminal binding domain of the human Insulin-like Growth Factor-Binding Protein-4 (IGFBP-4). IGFBP-4 is a 24 kDa protein that binds Insulin-like Growth Factor 1 (IGF-I) and 2 with high aﬃnity, presumably via its N-terminal region (mini-BP4). To characterize the interaction of mini-BP4 and IGF-I and to identify the amino acids that are involved, site directed mutagenesis was performed by PCR with the mini-BP4 gene. Altogether 30 diﬀerent single-point muteins were synthesized (Fig. 12).

A

m

miniBP4 bridged LEE wt V49L V49I V49M V49F V49Y V49W Y50R Y50C R53Y R53M R53F R53H Y61W Y61W Y61F K68Q L70Y L70W L70M L70I L70F L73I L73W L73M L73F M74Y M74W M74I M74F H75D #

14

B

1

2

3

4

5

A B C D E F G H

m

Western blot 1234 BCCP mini-BP4

Slot-Blot (native protein)

Fig. 12. (A) Ethidium bromide-stained, 2% agarose-gel showing mini-BP4 constructs generated by PCR. mini-BP4 Wild-type mini-BP4 after the PCR synthesis reaction. Bridged:mini-BP4 elongated with the bridging primers bridgeF1 and bridgeR1 to form overlaps with the DNA modules; LEE wt the His-mini BP4-Avi wild-type construct; V49L-H75D 31 diﬀerent mini-BP4 constructs. LEE Y61W was produced twice; þ positive control DNA; m: marker. (B) Nondenaturating slot-blotting as a fast and highly speciﬁc detection method. In the slot blot ( positions A1–G4) and in the Western blot (lanes 3, 4) His-mini-BP4-Avi muteins were detected via the biotinylated AviTag with SA-HRP conjugate. The positive control in position C5 and in lane 1 is monobiotinylated PEX2 protein. Positions H4 and A5 correspond to lane 2 and are negative controls of incubated RTS 100 HY lysate containing no Linear Expression Element. BCCP provides no background signal in non-denatured slot blotting (H4, A5) but was detected after denaturing Western blotting (lanes 2–4). The arrows indicate the same samples detected by slot and Western blotting. Reproduced from Ref. [12] with courtesy of Schra¨ml, Roche Pharmaceuticals, Penzberg, Germany and Springer.

The inﬂuence of the site directed mutations on the binding aﬃnity of the mini-BP4 protein to its 7.6 kDa IGF-I protein binding partner was determined and the eﬀect of each speciﬁc mutation on binding was demonstrated (Fig. 13). Thus, the sequence-speciﬁc, co-translational biotinylation of fusion proteins containing an Avi-tag allowed their speciﬁc detection, quantiﬁcation and site-directed immobilization on surfaces coated with streptavidin. The combination of fast template-generation, in vitro expression, robot-assisted protein refolding and puriﬁcation as well as the automated interaction analysis turned out to be a valuable system for the production and analysis of protein variants. 5. Expression scale-up and protein purification For studies on protein structure (e.g., NMR), the recombinant protein is often required in milligram amounts. Finding an appropriate expression system or optimizing the yield of a given one is often a question of trial-and-error methods. In RTS, a more rational approach exists: as a ﬁrst step, cDNA templates for optimized expression yields can be calculated via ProteoExpert.

15

RU

IGF-1

Response units

160

biot. mini-BP4

140 120

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100 80 60

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40

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IGFl inject 350

SA-biosensor

400

450

500

550

600

Time (s) wt-miniBP4

K68Q

RU

RU

Time (s)

Time (s)

V49I RU

Y61F RU

Time (s)

Time (s)

Fig. 13. Thirty-one mini-BP4 constructs were analyzed in SPR-interaction analysis (Biacore 3000) with IGF-I. The mini-BP4 muteins were site-directed immobilized on streptavidin-coated sensor chips via the biotin-labeled AviTag; 26 muteins were functionally active, 6 muteins were inactive and 11 muteins revealed a complex binding behavior (4 exemplary sensorgrams are shown). To determine the refolding eﬃcacy of the three diﬀerent buﬀers, equal amounts of Mini-BP4 mutein were immobilized (RU loaded) on the ﬂow cells of a BIAcore SA chip. The chip was saturated (RMAX) by an 800-nM IGF-I injection and the ligand-binding activities of the immobilized mutein were calculated by the ratio RMAX/RU loaded. Refolding of the constructs revealed highest ligand-binding activity (20%) with a redox/arginine buﬀer system (A). A moderate ligand-binding activity (3%) was reached with buﬀer I (B). As expected, no activity was observed with the reducing buﬀer system (C). The curve plot of the diﬀerently treated V49F mutein is given here, as example. Reproduced from Ref. [12] with courtesy of M. Schra¨ml, Roche Pharmaceuticals, Penzberg, Germany and Springer.

16 Using the modiﬁed primer sequences proposed by this program, a number of diﬀerent linear expression constructs can be produced rapidly in parallel PCRs and fused to the necessary regulatory regions via the RTS E. coli Linear Template Generation Sets in a second PCR step (see Section 2.1). Next, a smallscale expression of 2 h in RTS 100 E. coli HY reactions allows to identify the template with the highest productivity. This template can then be preserved e.g., by BD In-FusionTM cloning. After transformation and plasmid ampliﬁcation, the circular expression template is ready for expression scale-up in RTS 500 ProteoMaster or RTS 9000 E. coli HY reactions. The use of the RTS ProteoMaster Instrument thereby guarantees reproducible expression conditions for the scale-up. The complete RTS workﬂow starting from template optimization by ProteoExpert via expression PCR and cloning of the best DNA variant to high-level expression in RTS 500 or RTS 9000 has been carried out successfully with diﬀerent proteins. For structural analysis, the SH3 domain of a human kinase was investigated following this procedure, resulting in an expression level of 3 mg/ml in RTS 500 E. coli HY reactions [14]. This protein amount was enough for homogenous puriﬁcation via an His6-tag (Fig. 14). The RTS system proved also to be very eﬀective for synthesis and puriﬁcation of chloramphenicol acetyl transferase (CAT) for interaction studies [15]. Here, a ProteoExpert-optimized sequence coding for CAT was cloned into a pIVEX construct that contained a sequence for an N-terminal Avi-tag. By adding

M: Multicolor marker S: Starting material F1, F2: Flow through W: Wash E1, E2: Elution

M

S

F

W

E1

E2

Fig. 14. His6-tag puriﬁcation of an SH3 domain (9 KD) after sequence optimization and expression in RTS 500 E. coli HY.

17 RU 1400

Dissociation

Binding of Bio-CAT on SA-chip

1200 RU

pIVEX 2.8dWT CAT/pIVEX 2.8d CAT RTS 500 E. coli HY

1000 Association .DIM

1 2

800 600 400

CAT

200 0 −200 200 250 300 350 400 450 500 550 600 650 Time s

Fig. 15. Optimized expression of CAT (chloramphenicol acetyltransferase) in RTS500 E. coli HY followed by functional studies. Left: Concomitant biotinalytion and expression reactions of pIVEX 2.87d vT (wild-type) CAT (lane 1) and pIVEX 2.8d CAT (lane 2, codon optimized by ProteoExpert algorithm) analyzed by Coomassie staining. Right: SPR Analysis of Avi-Tag CATbio immobilized on Biacore SA chip from 1, 10 and 100 nM solutions, binding/dissociation of polyclonal anti CAT antibody is shown. Insert Immobilization of Avi-Tag CATbio onto Biacore SA Chip from 1-, 10- and 100-nM solution.

biotin, biotin protein ligase and ATP, speciﬁc monobiotinylation was accomplished during expression reactions. The successful scale-up from RTS 100 to RTS 500 E. coli HY was ﬁnally followed by an SPR analysis to measure the interaction with an anti-CAT antibody (Fig. 15). 6. Protein labeling In the last decade, it became clear that the set of 20 canonical amino acids prescribed by the universal genetic code needs to be enlarged to span all dimensions of chemical variability that could be potentially advantageous for functional diversiﬁcation of proteins. Canonical amino acids attached to cognate tRNAs can be chemically or enzymatically modiﬁed or even loaded onto desired tRNAs before they enter the ribosome. Speciﬁc protein labeling with modiﬁed amino acids is used in a wide variety of experimental setups to obtain information on conformational changes, protein folding, ligand and co-factor binding, or to monitor protein thermal stability [16], and to simplify spectra for structure elucidation [17]. Recent examples of artiﬁcial (i.e., tailor-made) proteins include novel classes of functionally designed protein pH-sensors, variants of green ﬂuorescent proteins or luminescent proteins with enhanced stability. 6.1 Labeling with fluorescence-enhanced amino acids Speciﬁc incorporation of spectrally enhanced Trps (e.g., 5-ﬂuorotryptophan, 5-FW) is a widely used approach to study protein–protein interactions.

18 Conformational changes in proteins, e.g., due to denaturation or ligand and substrate binding, can be analyzed by ﬂuorescence spectroscopy. Trp ﬂuorescence is sensitive to solvent eﬀects, and its emission spectrum and quantum yield strongly depend on the protein structure and local microenvironment. However, since the intrinsic ﬂuorescence of diﬀerent proteins resulting from naturally occurring Trp residues overlap, it is often impossible to assign and interpret ﬂuorescence changes that result from intermolecular associations. Replacement of natural Trp residues in proteins by appropriate analogues is therefore an important option. It is usually achieved by the tightly controlled overproduction of the protein in a Trp auxotrophic E. coli strain growing in minimal medium containing the desired Trp analogue [18]. High level cell-free expression techniques like RTS oﬀer an excellent alternative possibility for the eﬃcient label incorporation into recombinant proteins, allowing the convenient and uniform labeling of virtually any amino acid. In addition, common problems of standard in vivo labeling protocols associated with toxic eﬀects of the label precursors, reduced protein yields or low label incorporation into protein samples can be eliminated. In an example underlying this principle, Sengupta et al. studied the homoand heterodimer formation of the E. coli RcsB protein, a key regulator in enteric and plant pathogenicity [19]. RcsB is known to interact with the coactivator RcsA during transcriptional regulation of the expression of bacterial capsules. The researchers showed replacement of the natural Trp residue in RcsB by various Trp analogues as well as high level production of the modiﬁed RcsB derivatives using cell-free expression. The isolated RcsB alloproteins proved to be suitable for protein interaction studies by ﬂuorescence spectroscopy and evidence was obtained that RcsB also oligomerizes due to molecular association of the C-terminal eﬀector domains. No negative eﬀects due to the utilized amino acid analogues on the kinetics or eﬃciency of cell-free protein production were observed. As demonstrated by ﬂuorescence spectroscopy, cell-free production of RcsB alloproteins in RTS aﬀected neither protein–protein interactions (homo- or heterodimer formation) nor DNA binding activities. The incorporation of electron-rich selenium-containing noncanonical counterparts of Trp and Met into proteins, often used due to the rare occurrence of these amino acids in sequences, via in vitro translation system, represents a useful route for solving the phase problem in protein X-ray crystallography. In a study carried out by Budisa et al. [20], b-Selenolo[3,2-b]pyrrolyl-L-alanine ([3,2]Sep), a surrogate of tryptophan (Trp), and selenomethionine (SeMet), an analogue of methionine (Met) were incorporated into GFP (green ﬂuorescent protein). GFP was used as the model protein for the replacement experiments since it has a known three-dimensional structure and well-established biochemical, biophysical and genetic properties. The seleno-proteins were obtained at yields comparable to those of the wild-type protein and also crystallized under similar conditions (Fig. 16).

19 A

B C Se

S

H2N

H2N

O

HO Methionine (Met)

O HO Selenomethionine (SeMet) O

D d c

N H

Tryptophan (Trp) τM = 5580 M−1cm−1 (at 280 nm)

a 1

O OH

OH NH2

b

NH2 N H β-Selenolo[3,2-b]pyrrolyl-alanine; [3,2]Sep or Selenolo-Tryptophan τM = 12880 M−1cm−1 (at 267 nm)

2

Fig. 16. Green ﬂuorescent protein (GFP) engineering by in vitro substitution of its Trp and Met residues with indicated amino acid analogues. (A) Structures of GFP with marked chromophore and ﬁve Met residues (upper plot) and only one Trp residue (lower plot) based on its PDB coordinates and represented as a ribbon plot. (B) Structural representations of side chains of canonical amino acids Met and Trp and their analogues and surrogates selenomethionine (SeMet) and b-Selenolo[3,2-b]pyrrolyl-L-alanine ([3,2]Sep), respectively. (C) Analysis of the expression proﬁles in RTS HY lysates after overnight reaction: a wt-GFP; b SeMet-GFP; c [3,2]Sep-GFP; d control (i.e., reaction without plasmid). Arrows indicate the position of overexpressed native and substituted proteins. Lysates were separated by 12% SDS-polyacrylamide gel and stained with Coomassie blue. (D) (box): Photographs of crystals (1: [3,2]Sep-GFP; 2: SeMet-GFP). Reproduced from [20] with courtesy of N. Budisa, Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany and Springer.

6.2 Labeling with isotopes for NMR and X-ray NMR spectroscopy is uniquely capable of providing information on the structure, function, and dynamics of proteins and other biomolecules in solution. Recent advances in NMR spectroscopy promise to revolutionize the sensitivity of the technique as well as the molecular weight range amenable to investigation. However, milligram quantities of proteins are typically required for such studies. The assignment and structure determination of peptides and proteins with less than approximately 100 amino acids can usually be accomplished utilizing two-dimensional experiments such as the NOESY, COSY, and TOCSY that rely on 1H signals [21]. With increasing molecular weight and the concomitant increase in the number of 1H resonances, multidimensional (two, three, and four-dimensional) spectra resulting from labeling with other isotopes (usually 15N and 13C) must be used to solve the 1H overlap problem. Most of the work carried out nowadays therefore relies on uniform 15N, 13C and 2H labeling of the protein. Information about speciﬁc residues and speciﬁc sites can also be obtained by selective incorporation of only one or a few amino acids labeled with 15N, 13C, and possibly 2H. This selective labeling strategy can dramatically simplify the NMR spectrum. It is routinely possible to determine the structures of proteins up to 25 kDa using a combination of 15N and 13C labeling.

20 Recent experimental advances have increased the molecular weight range accessible to such studies to complexes as large as 800 kDa [22]. In vitro protein synthesis shows a number of important advances for the production of labeled NMR samples. Although diﬀerent cell-based methods exist for the production of uniformly 15N and 13C-labeled proteins [23], there are several problems that occur when expressing proteins in in vivo systems. The proteins must be puriﬁed from a range of other cellular proteins, a process that can be very tedious. Additional complications arise when trying to express selectively labeled proteins using a cell-based approach. One major problem results from metabolism of the amino acids once they are taken up by the cell. For example, aspartate can be converted into glutamate, asparagine, and lysine. Thus, addition of labeled aspartate can result in the inadvertent labeling of other amino acids as well. Similar metabolic interconversion pathways exist in E. coli for many amino acids. Adding a complement of unlabeled amino acids helps to suppress endogenous amino acid synthesis. However, even in this case, scrambling of the label has been shown to occur, albeit at a reduced level [24]. Another problem is that signiﬁcant amounts of the often expensive labeled amino acids are either thrown away with the medium after the cells have been harvested, or incorporated into other cellular proteins. Cell-free protein expression has the potential to overcome this limitation since the labeled amino acids can be just added by replacing the unlabeled ones, the expressed protein is labeled exclusively and label scrambling hardly occurs due to the absence of cellular metabolism. In the future, with continuing research into cell-free technology, in particular with regard to protein yields and the inhibition of amino acid metabolic pathways, it is expected that most speciﬁcally labeled proteins for NMR studies will be produced by in vitro methods. As an example, Fernholz et al. tested the labeling eﬃciency of the Rapid Translation System with an SH3 domain as a model protein [25]. The expression yield was substantially increased (more than 6-fold) by optimizing the sequence of the gene by silent mutations (cf. Section 2.1). Using the improved expression construct, the incorporation of 15N-labeled derivatives of each canonical amino acid was evaluated and analyzed via NMR (Fig. 17). Eighteen of the 20 amino acids (all except Glu and Gln) could be speciﬁcally incorporated. Scrambling was observed for only three amino acids (Ser, Asp and Asn). The yields in all expression reactions were comparable to the reaction using unlabeled amino acids ( 3 mg of soluble protein). Isotope eﬀects were not detected, even when deuterated amino acids had been used. These results demonstrate that cell-free protein expression has unique advantages over cellular expression regarding selective labeling of proteins. Due to a dramatic reduction of the time needed for cross-peak assignments of 15N-HSQC spectra, it can be considered as a valuable tool to solve protein structures in signiﬁcantly shorter time.

21 A

B

Pro Val Gly Ala Leu

Arg His Lys

C

D

Met Cys Thr Ser

Trp Phe Tyr

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5

104 106 108 110 112 114 116 118 120 122 124 126 128 130 104 106 108 110 112 114 116 118 120 122 124 126 128 130

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5

15

Fig. 17. N-HSQC spectra of diﬀerently labeled SH3 domain. (A) Aliphatic residues. (B) Basic residues. (C) Aromatic residues. (D) Heteroaliphatic residues.

7. Advanced applications and limitations 7.1 Expression of disulfide-bonded proteins Expression of recombinant proteins in E. coli cells often results in intracellular aggregation of the produced proteins. This is also observed in cell-free synthesis, especially for large, complex mammalian proteins. However, in cell-free synthesis, one can control and inﬂuence the reaction. In order to be biologically active, recombinant polypeptide chains have to fold into their native threedimensional structure, e.g., by interacting with GroE or DnaK chaperones. The issue of protein folding becomes even more complicated for mammalian proteins, especially extracellular molecules like antibodies or receptors harboring multiple disulﬁde bonds. Modiﬁcations of the conventional cell-extract made it possible to obtain such target proteins in a functional form. It has been demonstrated that E. coli cell-free protein synthesis reactions allow the formation of disulﬁde bonds when the free sulfhydryl groups of endogenous proteins contained in the lysate are blocked by sulfhydryl-speciﬁc alkylating agents [26]. Treating the cell-extract with iodoacetamide (IAM) completely eliminated the reducing activity without a severe reduction in protein synthesis capability. IAM was chosen as the thiol modifying agent since it eﬃciently blocks thiolmediated catalysis without changing the ionic charge on proteins. In addition, the use of chaperone-enriched extract further enhanced the solubility of expressed proteins. The use of IAM-treated extract along with the addition of DsbC disulﬁde isomerase and an oxidizing glutathione mixture enabled the expression of an enzymatically active murine urokinase, a serine protease,

22 1000

Active rUK (ug/ml)

800 600

400 200 0 0

10

20

30

Incubation time (hr)

Fig. 18. Expression of the protease domain of murine urokinase (rUK). Plasmid pIVEX2.3rUK was incubated in RTS500 HY systems with normal (open circles) or IAM-treated ( full circles) extract in the presence of glutathione mixture (4 mM GSSG and 1 mM GSH). 40-ml samples were taken at the indicated time points and assayed for enzymatic activity. Stn'd extract S

P

IAM - extract S

P rPA(9)

rUK(6)

sFcgR 1(4)

scFv a-Hag (2)

CD40Fc (10)

Fig. 19. Enhanced solubility of disulﬁde bonded proteins due to expression in chaperone-enriched, IAM-treated extract. Various proteins with diﬀerent numbers of disulﬁde bonds were expressed with standard (Stn’d) extract or GroE-enriched extract after IAM treatment. S Soluble fraction; P pellet. Numbers of disulﬁde bonds are indicated in parentheses.

demonstrating the feasibility of using cell-free protein synthesis for the rapid and general expression of bioactive eukaryotic proteins (Fig. 18). In terms of protein solubility, it was found that IAM-treatment alone is often not suﬃcient to allow production of protein in a soluble and functionally active form. The inclusion of chaperones like GroE, present as overexpressed components of the IAM-treated E. coli lysate, helped in many cases to overcome this problem (Fig. 19).

23 Another important example is the expression of functional single-chainantibody molecules and their derivatives in RTS. The relatively small number of disulﬁde bonds present in scFv molecules and their conserved position makes them ideally suited as targets for cell-free expression. Basically the same conditions and additives (e.g., chaperones) can be used for the cell-free expression of all molecules belonging to this class, irrespective of their speciﬁcities. In addition to this, the presence of highly conserved framework regions ﬂanking the variable parts on the cDNA level makes scFv-sequences good candidates for high-throughput template generation, because a very limited set of primers is suﬃcient to generate linear expression templates encoding many diﬀerent speciﬁcities. In an unpublished study by Ylera and LeGall, a diabody (bispeciﬁc molecule composed of two scFv fragments connected via a stretch of amino acids as linker) speciﬁc for CD3 and CD19 was found to speciﬁcally bind the antigen-expressing target cells as monitored by FACS analysis, provided that diabody was expressed in an IAM-treated lysate, but not when produced in a conventional, unmodiﬁed lysate. Interestingly, a side-product derived from the main target protein by proteolytic cleavage was present in high amounts in the conventional but not the IAM-treated lysate, presumably because the IAM treatment blocked the functional sites of the proteases. 7.2 Expression of toxic proteins Cell-free expression systems are especially useful for the expression of toxic proteins. As an example, D-amino acid oxidase (DAO) from T. variabilis was expressed for the ﬁrst time using RTS 500 [27]. DAO is the prototype of FAD-containing oxidases, and in vivo expression in E. coli is FAD-dependent. However, higher expression levels seem to interfere with growth of E. coli cells, presumably by disturbance of cell wall metabolism and/or toxic eﬀects of enzymatically produced H2O2. By addition of the essential cofactor FAD to the cell-free expression reaction the yield of soluble and functional active DAO was increased dramatically (Fig. 20). It can be assumed that by identifying and supplementing limiting cofactors such an approach can also be used for the expression of other critical proteins to increase their solubility and functionality. This shows again that cell-free expression systems are particularly advantageous as reaction conditions and concentrations of added components can be easily modiﬁed. 7.3 Expression of peptides Expression of short polypeptide chains in RTS is challenging because small molecules are more easily attacked by proteases than larger protein complexes. High concentrations are diﬃcult to obtain because they depend on the use of the CECF technology which in turn is based on the use of a semipermeable

24 1000

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800 1 2

34

600

400

200

0 0

500

1000

1500

2000

2500

FAD Conc. [mM]

Fig. 20. Dependency of DAO volume activity on the FAD supplement concentration. The inserts show the SDS-PAGE analysis of DAO expression mixture at 0 mM FAD (lane 1 supernatant; lane 2 pellet fraction) and at 2000 mM FAD (lane 3 supernatant; lane 4 pellet fraction). Reproduced from [27] with courtesy of Frank Wedekind, Roche Applied Science, Penzberg, Germany and Springer.

membrane of a certain cut-oﬀ (usually 10 kDa). Since smaller peptides are not retained in the reaction compartment of the CECF devices, only batch reactions excluding the dialysis eﬀect normally make sense for peptide synthesis, or reaction products must be isolated from both CECF reaction compartments. Researchers at Phylos GmbH (Zwingenberg, Germany) showed recently that expression of peptides in RTS 100 E. coli HY batch reactions is possible from a circular template (unpublished results). Since they failed with the detection of the small peptides when expressing them alone, they introduced an additional tag, thereby increasing the size of the peptides and allowing their detection by radioactive labeling and ﬂuorescence imaging. Product functionality, especially the possibility to immobilize the peptides on beads, will be the subject of further studies. 7.4 Expression of transmembrane proteins The synthesis of eukaryotic membrane proteins in a preparative scale in prokaryotic systems is another challenging task. Membrane proteins have been expressed in RTS 100 and 500 at high yields, however, in insoluble form [28]. Four types of membrane proteins were selected that biosynthetically become integrated into the membrane of the endoplasmic reticulum and are resident proteins of the endoplasmic reticulum and the plasma membrane, respectively: a type I membrane protein (Mtj1p), a type II membrane protein

25

+ CHAPS (8.5 mM) + additional salt

+ CHAPS (8.5 mM)

+ Deoxycholic acid (0.5 mM)

+ Mega10 (6 mM)

+ Mega8 (10 mM)

+ betain (500 mM)

silent mutations (PM22)

M

+additional salt

kDA

without addition

Coomassie-stained gel with pellets

200 120 80 50

Mtj1p

30 20

reaction

1

2

3

4

5

6

7

8

9

Fig. 21. Optimization of the synthesis of Mtj1p in RTS 500 E. coli HY (reaction time: 24 h). Protein synthesis is shown in the presence of higher salt concentrations, silent mutations, betain and diﬀerent detergents. The ﬁgure shows the sediments corresponding to 5 ml of the reaction mixtures on a Coomassie-stained gel.

(Sec62p), two multispanning membrane proteins (Trp4/TRPC4, Trp8/CaT-L/ TRPV6) and a tail-anchored membrane protein (Ubc6). Out of ﬁve model membrane proteins four were produced in milligram amounts in RTS 500. The non-native proteins could be used for immunization and as protein standards for quantiﬁcation of the respective proteins in biological material by semiquantitative Western-blotting. In addition to producing membrane proteins in high yields, it was observed that detergents can play a signiﬁcant role in the eﬃciency of translation: as shown for Mtj1p in Fig. 21, the presence of detergents in RTS 500 E. coli HY reactions aﬀected the yield of some of the expressed proteins but did not give rise to more soluble product. Based on these ﬁndings the investigators proposed that in the absence of detergent certain newly synthesized membrane proteins may accumulate on the surface of ribosomes via hydrophobic interactions and that this may lead to inactivation of ribosomes. 8. Switching between and direct comparison of cellular vs. cell-free expression Since constitutive T7-driven protein expression is characteristic not only for RTS but for a number of other vectors for cellular expression as well (e.g., pRSET vectors or the pDEST vector series used in the context of the Gateway platform), many of these vectors can be directly used for cell-free expression. If, for example, expression of a panel of eukaryotic proteins fails because of toxicity, low yield or low solubility, one can quickly get an idea about expression eﬃciency in RTS simply by running a small-scale pilot expression experiment using already existing vectors in batch mode. Control of gene expression by the

26 strong phage T7 promoter that yields constitutive expression induced by endogenous T7 RNA polymerase expression is advantageous for protein production in terms of yield, but since many heterologous proteins are toxic to E. coli cells, basal expression of the genes may lead to reduced cell growth, increased cell death, and an overall failure of protein synthesis. In a cell-free context, this toxicity eﬀect is much more unlikely as shown by the following examples. In a systematic study of optimization of protein expression the Rapid Translation System was used for proteins that had been expressed in T7-driven Gateway vectors before or in parallel [29]. When expressing a total of 21 ORFs from pDEST17 vectors either in the E. coli strain BL21(DE3) pLysS or directly in an RTS 100 E. coli HY batch reaction, about 50% of all tested proteins were expressed successfully in each case. When the software tool ProteoExpert was used in conjunction with the RTS E. coli Linear Template Generation Set (LTGS) to optimize a subset of low-yield sequences, it proved to be a very important part of the cell-free workﬂow not available in the cellular context, improving dramatically the success rate and yield of the cellfree reactions. In summary, a total of 21 ORFs were tested. Of these, 10 were successfully expressed in BL21pLysS E. coli (as detected by Coomassie stain), and 12 in the RTS 100 E. coli HY Kit. All 21 ORFs were submitted to ProteoExpert for optimization calculations, linear expression constructs were generated with RTS E. coli Linear Template Generation Sets and ﬁnally expressed in RTS 100 E. coli HY. Eighteen samples had higher yields of protein when expressed in vitro. Remarkably, removal of the att (attachment) sites included in the Gateway pDest vectors, and a change of the His6-tag position from N- to C-terminus led to a signiﬁcant increase in expressability and cell-free protein yield. In another case, the expression of chemokine-like factor 1 (CKLF1) from the T7-driven vector pRSET in RTS was compared with results obtained in E. coli BL21(DE3)pLysS [30]. SDS-PAGE and Western blotting showed that the recombinant protein was synthesized in the cell-free system but not in E. coli cells, showing the advantages of a cell free protein expression system over in vivo expression for recombinant CKLF1 (Fig. 22). The fact that cellular expression of this protein had also failed utilizing the Glutathione S-transferase (GST) Gene Fusion System from Amersham Pharmacia Biotech made these researchers conclude that CKLF1 was possibly toxic to E. coli, and therefore unable to be expressed in living cells. This again demonstrates that cellfree biosynthesis systems provide a good alternative method for expressing proteins such as CKLF1 that cannot be expressed using traditional methods. 9. RTS – the next generation: Eukaryotic cell-free expression in wheat germ lysates Taking all these examples together, it is clear that the expression of proteins has many advantages in a cell-free environment but also that – in spite of

La ne

2 La ne

2 La ne

La ne

1

La ne

1

3

27

A

B 14 kDa

Fig. 22. Expression of His-tagged CKLF1 in the Rapid Translation System. Left: Comparison between expression in vitro and in vivo using the same expression vector as template. Western blotting analysis probed with (A) anti-CKLF1 and (B) anti-His; lane 1 CKLF1 synthesized with RTS 500; lane 2 CKLF1 expressed in E. coli. Right: SDS-PAGE analysis of cell-free protein synthesis. One ml of the reaction solution diluted 10-fold with PBS was applied to denaturing SDSPAGE. Lane 1 Protein marker; lane 2 pRSET C-CKLF1 utilized as template. A notable band with apparent molecular mass of 14 kDa was observed; lane 3 pRSET C without gene of interest used as template (negative control).

sequence optimization and fast screening methods – eukaryotic target proteins remain challenging in a system based on E. coli lysates. CECF technology has therefore recently been transferred to a eukaryotic environment and adapted to a lysate based on wheat germ extracts [31,32]. Such lysates are highly advantageous in terms of success rate. Even non-optimized wild-type sequences can be expressed at levels detectable non-radioactively by Western blotting. This has been shown for many eukaryotic proteins that are not expressed at all in E. coli lysates, even when optimized mutants are generated. Higher stability and better compatibility of eukaryotic mRNAs with the eukaryotic translational machinery than with E. coli ribosomes may explain this ﬁnding. From those proteins successfully expressed in both systems, solubility (and presumably also function) have been found to be signiﬁcantly higher, possibly due to the presence of eukaryotic chaperones and lower protease activity of the wheat germ lysate compared to E. coli extracts. The RTS Wheat Germ system (visit www.proteinexpression.com to check for commercial availability) diﬀers from other available eukaryotic cell-based systems in two important aspects. Including CECF technology with running times of up to 24 h (instead of 2–4 h batch reactions) also, for small-scale reactions of 50 ml, it aims at the expression of proteins in a range that allows detection by Coomassie stain or Western blotting instead of radioactive labeling. With an also upcoming RTS 500 Wheat Germ platform, the Rapid

28 wt sequence

ProteoExpert E. coli

primer suggestions for sequence optimization LinTempGenSet E. coli

cDNA

sequence optimization expression screening expression scale-up

1st PCR

PCR product 1* 2nd PCR

LinTempGenSet Wheat Germ 2nd PCR

In-Fusion cloning PCR product 2: linear template wheat germ

PCR product 2: linear template E. coli

RTS 100 E. coli HY

pIVEX WG pIVEX E. coli e.g. Ncol/Smal RTS 500/9000 E. coli HY

RTS 100 WG CECF

RTS 100/500 WG CECF protein

protein protein

protein

*same product can be used for E. coli and WG 2nd PCR

Fig. 23. Overall summary of expression workﬂow for template generation and expression in RTS E. coli and wheat germ.

Translation System will become the ﬁrst scalable eukaryotic cell-free protein expression system, with amounts allowing to easily link up expression reactions to downstream applications like functional assay, immobilization on chips etc. A consequence of this focus on yield, however, is that posttranslational modiﬁcations, e.g., microsome-dependent glycosylations, cannot be carried out in RTS, since – unlike ribosomes – microsomes work stoichiometrically instead of catalytically and cannot carry out their function throughout the running time of a preparative-scale expression reaction. The approaches for expressing a given target protein in RTS E. coli vs. wheat germ are very similar, except for the fact that a template optimization for a eukaryotic protein expressed in the eukaryotic wheat germ lysate is not required. Template generation strategies like overlap extension PCR and BD In-FusionTM cloning work similarly in both systems based on the same genespeciﬁc primers. As restriction sites between expression plasmids are also compatible switching from E. coli to wheat germ or vice versa is easy and straightforward (Fig. 23). 10. Summary and conclusions Cell-free protein expression is a promising technology for keeping pace with the exponentially increasing amount of genetic sequence information and for empowering the exciting ﬁeld of protein and pathway evolution. The consensus in the scientiﬁc community is that the 21st Century will be the era of proteome

29 research, and cell-free systems are key technologies that will support this expanding research area. The Rapid Translation System (RTS) from Roche Applied Science combines a unique set of innovative technologies to a powerful new protein expression approach. It oﬀers unique possibilities such as rational template design, expression of toxic proteins, co-expression of multiple polypeptides, eﬃcient labeling for NMR and X-ray studies, and rapid production of engineered protein variants. RTS is a rapidly evolving platform overcoming the limitations of traditional cell-based methods in terms of speed, throughput and ﬂexibility. Based on an isolated translational machinery under optimized reaction conditions, it enables scientists to expand their research projects into multiplexed and automated formats.

References 1. Nirenberg MW and Matthaei JH. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polynucleotides. Proc Natl Acad Sci USA 1961;47: 1588–1602. 2. DeVries JK and Zubay G. DNA-directed peptide synthesis. II. The synthesis of the a-fragment of the enzyme beta-galactosidase. Proc Natl Acad Sci USA 1967;57:1010–1012. 3. Spirin AS, Baranov VI, Ryabova LA, Ovodov SY and Alakhov BY. A continuous cell-free translation system capable of producing polypeptides in high yield. Science 1988;242: 1162–1164. 4. Kigawa T, Yabuki T, Yoshida Y, Tsutsui M, Ito Y, Shibata T and Yokoyama S. Cell-free production and stable-isotope labelling of milligram quantities of proteins. FEBS Lett 1999; 442:15–19. 5. Kim D-M and Swartz JR. Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol Bioeng 1999;66:180–188. 6. Guba M, Bosserhoﬀ AK, Steinbauer M, Abels C, Anthuber M, Buettner R and Jauch KW. Overexpression of Melanoma Inhibitory Activity (MIA) enhances extravasation and metastasis of A-mel 3 melanoma cells in vivo. Br J Cancer 2000;83:1216–1222. 7. Bosserhoﬀ AK. Recombinant expression of functional active MIA (Melanoma Inhibitory Activity) protein for mutation analysis using the RTS system. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 173–179. 8. Lorenz P and Thiesen HJ. In-vitro translation of KRAB zinc ﬁnger transcriptional repressor proteins and their interaction with their TIF1b co-repressor. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 151–157. 9. Betton J-M. Using maltose-binding protein fragment complementation to probe protein– protein interactions by co-expression in the RTS system. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 143–149. 10. Betton JM and Hofnung M. In vivo assembly of active maltose binding protein from independently exported protein fragments. EMBO J 1994;13:1226–1234. 11. Ma B, Tsai CJ and Nussinov R. Binding and folding: in search of intramolecular chaperonelike building block fragments. Protein Eng 2000;13:617–627. 12. Nemetz C, Wessner S, Schweitzer R, Graentzdoerﬀer A and Buchberger B. Rapid protein engineering by expression-PCR. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003.

30 13. Schra¨ml et al. Rapid generation of protein variants and subsequent analysis by surface plasmon resonace. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 69–79. 14. Cronan JE. Biotination of proteins in vivo. J Biol Chem 1990;265,18(Issue of June 25): 10327–10333. 15. Fernholz, et al. Production of a speciﬁcally labeled protein in mg quantities for NMR analysis. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 55–60. 16. Schlo¨ssmann T, et al. In situ mono-biotinylation of cell-free expressed proteins using the AviTag technology. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 61–67. 17. Biekofsky RR, Martin SR, McCormick JE, Masino L, Fefeu S, Bayley PM and Fenney J. Thermal stability of calmodulin and mutants studied by 1H–15N HSQC NMR measurements of selectively labeled 15N-Ile proteins. Biochemistry 2002;28:6850–6859. 18. Kelly MJ, Ball LJ, Krieger C, Yu Y, Fischer M, Schiﬀmann S, Schmieder P, Kuhne R, Bermel W, Bacher A, Richter G and Oschkinat H. The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand binding studies reveal the location of the active site. Proc Natl Acad Sci USA 2001;6:13025–13030. 19. Ross JBA, Szabo AG and Hogue CWV. Enhancement of protein spectra with tryptophan analogs: ﬂuorescence spectroscopy of protein–protein and protein–nucleic acid interactions. Methods Enzymol 1997;278:151–190. 20. Sengupta K, et al. Incorporation of ﬂuorescence labels into cell-free produced proteins. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 81–88. 21. Budisa N, et al. Expression of ‘Tailor-Made’ proteins via incorporation of synthetic amino acids by using cell-free protein synthesis. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 89–98. 22. Skelton NJ and Chazin WJ. Solution structure determination of proteins by nuclear magnetic resonance spectroscopy. Drugs Pharm Sci 2000;101:683–726. 23. Riek R, Flaux J, Bertelsen EB, Horwich AL and Wuthrich K. Solution NMR techniques for large molecular and supramolecular structures. J Am Chem Soc 2002;124:12144–12153. 24. McIntosh LP and Dahlquist FW. Biosynthetic incorporation of 15N and 13C for assignment and interpretation of nuclear magnetic resonance spectra of proteins. Quart Rev Biophys 1990; 23:1–38. 25. Lian LY and Middleton DA. Labeling approaches for protein structural studies by solution state and solid-state NMR. Prog Nucl Mag Reson Spect 2001;39:171–190. 26. Kim D-Y, et al. Cell-free expression of proteins containing multiple disulﬁde bonds. In: CellFree Protein Expression, Swartz James R (ed), Springer, 2003, pp. 125–131. 27. Lehmann D and Wedekind F. Optimization of cell-free expression of FAD-dependent D-amino acid oxidase. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003. 28. Maurer P, et al. Cell-free synthesis of membrane proteins on a preparative scale. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003. 29. Langlais C, et al. The linear template generation set: Optimization of protein expression in the RTS 100 HY. BIOCHEMICA, 03/2003. 30. Liu P and Ma D. Expression of recombinant chemokine-like factor 1 with a cell-free protein biosynthesis system. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 165–171. 31. Madin K, Sawasaki T, Ogasawara T and Endo Y. A highly eﬃcient and robust cell-free protein synthesis system prepared from wheat embryos: Plants apparently contain a suicide system directed at ribosomes. Proc Natl Acad Sci USA 2000;97:559–564. 32. Akbergenov RZ, et al. Complementary interaction between the central domain of 18S rRNA and the 50 untranslated region of mRNA enhances translation eﬃciency in plants. In: Cell-Free Protein Expression, Swartz James R (ed), Springer, 2003, pp. 199–208.

31

Protein expression and refolding – A practical guide to getting the most out of inclusion bodies Lisa D. Cabrita and Stephen P. Bottomley* Monash University, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, P.O. Box 13D, Melbourne, Victoria 3800, Australia Abstract. The release of sequence data, particularly from a number of medically and biotechnologically important genomes, is increasing in an exponential fashion. In light of this, elucidating the structure and function of proteins, particularly in a ‘‘high throughput’’ manner, is an important quest. The production of recombinant proteins however is not always straightforward, with a number of proteins falling prey to low expression problems, a high susceptibility to proteolysis and the often despised production of inclusion bodies. Whilst expression as inclusion bodies can often be advantageous, their solubilization and renaturation is often a time consuming and empirical process. In this review, we aim to outline some of the more common approaches that have been applied to a variety of proteins and address issues associated with their handling. Keywords: inclusion body, protein folding, protein aggregation, protein engineering, protein expression, refolding, protein puriﬁcation

Introduction One of the areas of great importance in this post-genomic era is the ability to rapidly express and purify a protein of interest. With the vast amount of sequence data now available, numerous sites around the world are now attempting structural genomic projects in which a vast array of proteins are being expressed, puriﬁed and structurally deﬁned. In addition to these highthroughput approaches almost all biomedical labs, academic or industrial, are expressing proteins of interest for structural, functional and therapeutic investigations. The general scheme in this research involves rapid cloning of the genes of interest and then expression of the protein, usually in the host E. coli. At present, E. coli remains the ‘‘king’’ of expression hosts, due to its advantages such as ease of handling, cost eﬀectiveness and high success rates. However, it has some associated disadvantages such as the inability to perform many post-translational modiﬁcations and expression of the protein product often leads to its deposition as inclusion bodies. Inclusion body formation is often dreaded because there are a bewildering number of approaches which a researcher can utilize to obtain soluble proteins. However, in the past few years, there has been an increase in products and protocols which are generally applicable to the inclusion body problem. New strains of E. coli and fusion partners are now available, which oﬀer a range of options to minimize, or *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10002-1

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

32 maximize, inclusion body formation. In addition, a more rational approach to protein refolding has become established, which allows the user to establish very quickly, whether refolding is a viable option. In this review, we will initially outline the theoretical background to protein folding and then we will focus on ﬁrstly, the ways in which inclusion body formation can be avoided during the growth of E. coli and then techniques that can be used to refold any inclusion bodies that are formed. Mechanisms of protein folding and misfolding The classic work of Anﬁnsen demonstrated that it is possible to reversibly unfold and refold proteins, indicating that the amino acid sequence of a protein contains all the information required for successful folding [1]. Moreover, these experiments also revealed that a protein is able to form its native conformation whilst avoiding the vast array of other structures accessible to the polypeptide chain as it folds. These alternative structures are often stable, non-native conformations that self-associate and result in the formation of aggregated material. The process of folding/misfolding therefore, is of great biomedical importance, as protein aggregation forms the basis for an increasing number of diseases such as cystic ﬁbrosis, liver cirrhosis and many neurodegenerative diseases [2]. In the in vitro context, protein aggregation is a very common feature that occurs during recombinant protein expression in E. coli, which dramatically reduces the yield of soluble, useful protein. The folding pathway of a protein is extremely complex. Pioneering work by Alan Fersht and colleagues has shown that many small proteins, of less than 150 amino acid residues, fold via a two-state pathway in which only the native and unfolded ensembles are signiﬁcantly populated [3]. A combination of protein engineering and theoretical techniques have revealed that a folding protein follows multiple paths to a native-like transition state ensemble which collapses to form the native conformation. In contrast to small proteins, proteins consisting of more than 150 amino acid residues generally fold via multi-state pathways in which intermediate species are populated (Fig. 1). Some of these intermediates (Io) are obligatory and lie on the productive folding pathway, whereas others (IA) lie on non-productive pathways which culminate in the formation of aggregated material. There is a plethora of examples demonstrating that factors such as mutation, temperature and protein concentration can disrupt the energetics of the folding reaction, making misfolding and aggregation a more likely (though usually undesirable) endpoint. During recombinant protein expression where inclusion body formation can result, many of these factors as well as the strength of the promoter and cell type play a signiﬁcant role, as discussed below. Recent experimental data has lead to the hypothesis that all proteins have the potential to form aggregates [4]; in addition the converse also holds that under speciﬁc solution conditions, a denatured protein should be able to successfully refold to its native state.

33 U

Io

N

Factors that disrupt the productive folding

landscape:

IA + IA

temperature, pH, salt concentration, mutation

A Fig. 1. Possible routes for the folding of a protein. From the unfolded state (U), the protein can proceed through an obligatory intermediate (Io) which leads to successful adoption of the native state (N). Alternatively misfolding can occur through an oﬀ-pathway reaction in which an aggregation prone intermediate (IA) is formed, either directly from U or through structural changes in Io, which can self-associate and form aggregates (A).

The problem faced, however, is identiﬁcation of these speciﬁc conditions, which is a bottleneck in dealing with refolding of solubilized inclusion bodies. In the rest of this review we will discuss many of the options available to ﬁrstly avoid inclusion body formation and secondly refold proteins that are present within inclusion bodies. Recombinant protein expression in E. coli It has been well described in several excellent reviews, that the expression of recombinant proteins within E. coli is aﬀected by several factors. These include, but are not limited to: plasmid copy number, mRNA stability, upstream elements, temperature and codon usage and these are covered in detail elsewhere [5–7]. It appears therefore, that successful production of protein is a combination of these factors and to a degree, some might argue, a stroke of luck. So while the expression of an uncharacterized protein is at best, diﬃcult to predict, numerous advances have been made to improve both expression and solubility. This has been made possible through the development of novel tags, fusion partners, and vector systems, such that the choices for recombinant expression in E. coli are forever expanding. E. coli strains, promoters and fusion partners In regulating protein expression, the choice of promoter/vector system is important, as one system may be more suited to a target than another. There are several promoters that are available (Table 1), perhaps the most well known variety being T7-derived, as found in the pET vectors (Novagen). These IPTG inducible promoters have in the past been associated with ‘‘leaky’’ expression prior to induction, which is a pitfall for proteins that are toxic to cells. This however, has been addressed with the co-transformed pLysS and pLysE plasmids that are able to act as strong repressors. This issue aside, the

34 Table 1. Examples of promoters that are used in E. coli expression. Promoter

Example vector

Inducer

T7 T5 trc tac lac trp araB PL (l) phoA PLtetO-1

pET (Novagen) pQE (Qiagen) pTrcHis (Invitrogen) pMAL (New England Biolabs) pTriplEx2 (Clontech) pLEX (Invitrogen) PBAD (Invitrogen) pKC30 pBKIGF2B-A pLP-PROTet-6xHN (Clontech)

IPTG IPTG IPTG IPTG IPTG tryptophan L-arabinose temperature shift (42 C) phosphate tetracycline

T7 is a strong promoter which allows high level expression of target proteins. Unfortunately, it is overexpression which can also lead to the formation of intracellular inclusion bodies, sometimes in far greater yields than the soluble version. Again, this can sometimes be regulated, by altering the expression time, induction temperature and IPTG levels [5]. The T7-based vectors have previously included aﬃnity tags for protein detection and puriﬁcation, though they are also now designed with other features. Of interest are the ‘‘fusion partners,’’ that in general, ‘‘fuse’’ the protein of interest with another, to circumvent a solubility issue and simultaneously aid in puriﬁcation. The classic GST fusion system has been successful for a large number of proteins [8–11], however more recently, there has been the development of alternatives (Table 2 and [12]). One of these is the 42 kDa maltose binding protein (MBP), which has been successfully used to aid in increasing the solubility of a range targets [13–15] and has been shown recently to have little eﬀect during crystallization [16] – an advantage, as it is generally accepted that ‘‘tags’’ need to be removed to aid in crystallography. A similar tag is Nus A (Novagen), a 54 kDa protein, which was identiﬁed as being the most soluble protein out of a pool of almost 4000 E. coli proteins [17]. In addition to aiding in folding, some tags such as the S-tag (Novagen), for instance, which comprises of 15 amino acids, can be used to detect, quantitate and purify soluble protein [18]. Poor expression of a target protein can amongst many things, be associated with codon bias – that is, codons used infrequently in the prokaryotic system. The Rosetta (Novagen) and BL21-CodonPlus strains (Stratagene) of E. coli have the advantage of co-expressing plasmids that code for the rare tRNAs and have been successfully used recently for a number of proteins (cellobiose phosphorylase [19], phosphoenolpyruvate carboxylase [20], Dictyostelium 5NT [21]). Moreover, the development of E. coli that accommodates disulﬁde bond formation also can improve the yields of certain targets. The AD494(DE3) strain (Novagen) has a single mutation in the thioredoxin reductase gene,

35 Table 2. Examples of fusion partners and tags which aid solubility and tracking protein expression. Tag

Size

Location

Refs.

Glutathione S-transferase (GST) Maltose Binding Protein (MBP) S-Tag Chloramphenicol acetyltransferase NusA Ubiquitin Thioredoxin Z-domain (derived from Protein A)

26 40 15 24 54 76 11 58

N N, C N, C, internal N N N N N

[11] [15] [18] [90] [17] [91] [92] [93]

kDa kDa aa kDa kDa aa kDa aa

and has enabled the production of proteins including C1-inhibitor (containing two disulﬁde bonds) [22], chitinases (one disulﬁde) [23] and a superoxide dismutase (two disulﬁdes) [24]. The Origami strain (Novagen), however, combines a double mutation in both the thioredoxin reductase and the glutathione reductase gene, hence providing a more favorable oxidizing environment within the cytoplasm. In addition, the recent Rosetta-Gami strain (Novagen) is a powerful combination of the Rosetta and Origami strains, encompassing rare codon usage and disulﬁde bond formation. Such a strain may be particularly useful for eukaryotic or intracellular proteins, which typically display alternative codon usage and require disulﬁde bond formation, respectively. Cell-free expression Cell-free expression (‘‘in vitro transcription–translation’’) has been an invaluable tool for many years, exploiting the E. coli, wheat germ and rabbit reticulocyte systems to generate protein. In more recent times, the demand for cell-free expression has increased, particularly as it is seen as a viable alternative to other expression systems for recalcitrant proteins. This technology was developed some 30 years ago, with the production of rat growth hormone, using an S30 extract derived from mice [25]. It has since been brought to the forefront by some ground breaking work by Spirin and coworkers [26] and also by Kim and Choi [27]. Spirin developed a ‘‘continuous-ﬂow’’ system whereby amino acids and energy sources are supplied into a reaction chamber, while synthesized proteins and used substrates are removed using an ultraﬁltration membrane. In contrast, Kim and Choi probed a ‘‘semicontinuous’’ method, involving the use of two chambers separated by a dialysis membrane, allowing for the replenishment of substrates and removal of by-products. With this system they were able to produce 1.2 mg/ml of chloramphenicol o-acetyltransferase, in 14 h and since then, have increased its eﬃciency by over 70% to produce 0.3 mg of protein per hour [28].

36 Roche also now oﬀers the in vitro transcription–translation ‘‘Rapid Translation System,’’ which has been successful for a number of targets and whose capabilities are expanding, particularly with the incorporation of molecular chaperones, detergents and other additives to assist in folding. Although not seen to be as eﬃcient as the E. coli system at present, the cell free system based upon wheatgerm has also been used [29–31]. Being a eukaryotic system, it is being explored as an alternative, particularly for the production of some proteins that will not otherwise express in E. coli. The cell-free approach to protein expression has suﬀered in the past due to concerns relating to its low eﬃciency, membrane clogging and as an eﬀect, questionable reproducibility. At present however, many advancements are being made, such that it may encourage more ‘‘mainstream’’ use and with continued improvements, it may perhaps be a competitive rival to the traditional E. coli expression system in the future.

Preparation of inclusion bodies Whilst a researcher may attempt all of the tried and proven methods of obtaining soluble expression, it is also to be noted that the inclusion bodies persist and that rather than work against them, it is best to try and work with them (Fig. 2). Their formation can at times be advantageous, particularly if the protein is toxic to the cell, or is likely to be the subject of proteolytic attack. The inclusion body itself is a dense amorphous aggregate of misfolded protein and is generally formed if the protein has a high propensity to misfold and aggregate, or if the cellular protein production machinery itself is overwhelmed and hence unable to operate eﬃciently. It has been predicted that as much as 20–40% of human gene constructs will express as inclusion bodies in E. coli [32]. Being in the post-genome era, the view towards ‘‘high throughput’’ cloning/expression/puriﬁcation allows for the rapid isolation of numerous proteins. However ‘‘diﬃcult targets,’’ such as those that persist to express as inclusion bodies, may be left behind in such a pursuit. This is of course understandable, considering that the preparation, solubilization and renaturation of inclusion bodies is indeed laborious and varies greatly for any individual protein. Providing set ‘‘rules’’ therefore, is virtually impossible and ﬁnding conditions that enable a protein to refold from inclusion bodies can be likened to the factorial screens used in crystallography – it may require trial and error and many attempts. Inclusion bodies are easily identiﬁable, with a morphology similar to strings or clusters [33] and their isolation from other cellular components can be easily achieved. They are contained within the insoluble fraction that is obtained after cell lysis and subsequent centrifugation. While it is generally accepted that inclusion bodies can comprise 40–90% of the target protein, successful renaturation is dependent upon their level of purity. Studies with lysozyme have

37 Lyse cells (sonication, french press) Centrifuge, retain pellet (insoluble fraction)

Purify inclusion bodies Chromatography (denaturing conditions)

Centrifugation

Gel Filtration, Ni-NTA

Triton X-100, high salt, EDTA

Solubilization 4−6 M GdnHCl

Dialysis (step)

Activity

6−8 M Urea

Refolding On-column refolding (gradient, stepwise)

0.5-2% SDS

Dilution (step, dropwise)

Folded or Aggregated? Circular Dichroism

Non-denaturing PAGE

Size exclusion chromatography

Light scattering

Ultracentrifugation

Fig. 2. A schematic outlining of a procedure for the puriﬁcation from inclusion bodies.

shown that the presence of proteinaceous contaminants, during renaturation, decreases the eﬃciency of refolding, presumably as protein contaminants can promote co-aggregation [34]. Inclusion bodies are therefore washed several times with detergents such as Triton X-100 (0.1 ! 4% (v/v)), sodium deoxycholate (2% (w/v)), sarkosyl and even low molar concentrations (0.5 ! 1 M) of denaturants such as guanidine hydrochloride or urea. They essentially remove the cellular contaminants, that adsorb onto the hydrophobic inclusion bodies. After washing, the inclusion bodies are solubilized, usually with the use of 4–6 M GdnHCl or 8 M urea. GdnHCl is the stronger denaturant of the two, because during prolonged incubations at alkaline pH, urea can suﬀer from the formation of isocyanate ions, which can modify amino acid side chains [35,36]. Aside from denaturants, solubilizing alternatives also include detergents such as sarkosyl [37,38], SDS [39] and also alkaline pH [40,41]. It is important to note that during unfolding, there must be reduction of any disulﬁde bonds present, which is usually achieved using b-mercaptoethanol or DTT (5 mM ! 100 mM).

38 This is in light of a pivotal study which demonstrated that disulﬁdes persisted in high denaturant concentrations in the absence of reducing agents [42]. Normally, solubilization can be performed in any buﬀer that is compatible with the protein of interest (Tris/Hepes/Phosphate), generally a neutral pH [7–8] is suitable for solubilization. Upon solubilizing the inclusion bodies, ample incubation time should be incorporated to allow for complete unfolding. Incubation at either room temperature or 30 C for 1–4 h is an acceptable time frame, while some have opted for 16–24 h at 4 C. At times, inclusion bodies may be diﬃcult to solubilize, hence the use of some form of agitation and increase in temperature may be necessary. Some researchers also choose to purify the inclusion bodies further, by incorporating column chromatography (conducted under denaturing conditions) such as ion exchange, size exclusion or metal aﬃnity [43–45]. Again this has been seen to enrich the proportion of monodispersed protein which can increase the yield of the refolded target protein [46]. More recently, Gu and colleagues took the approach of purifying inclusion bodies exclusively by gel ﬁltration using a macroporous medium (e.g., 4% agarose). By coupling with the use of a French press to reduce cell debris, the column matrix excluded only the inclusion bodies and hence allowing their separation [47]. This was used as an alternative to the standard centrifugation steps mentioned earlier. Refolding solubilized inclusion bodies After solubilization, the renaturation process aims to eﬀectively remove the denaturant and thiol reagents and allow the protein to refold. Whilst this appears trivial, the refolding process is a competing reaction with misfolding and aggregation events and as such, numerous factors impact on its success. Studies on proteins, especially those larger than 150 amino acids have shown that for many, folding involves the formation of an intermediate species, often resembling a ‘‘molten globule.’’ Such a species represents a branch point within folding, where it may also lead to misfolding and subsequent protein aggregation. With the molten globule containing some secondary structure, but little tertiary structure, hydrophobic patches normally buried within the protein are exposed to solvent [48]. Under appropriate conditions, therefore, these regions can promote inappropriate interactions that may lead to aggregation. Several issues are therefore of importance in aiming to minimize the aggregation reaction: ﬁnal concentration of protein to be refolded, the components of the refolding buﬀer and method of refolding. Importance of protein concentration during refolding The amount of protein refolded has an impact on the yields that will be obtained. As folding competes with aggregation, it is generally acknowledged

39 that refolding at low protein concentrations (10–100 mg/ml) is the most successful approach. There have been instances where proteins have refolded into high concentrations of up to 5 mg/ml, prochymosin [49], lysozyme [50] carbonic anhydrase [51], albeit in low concentrations of denaturant. On the most part, however, the lower the ﬁnal protein concentration attained during refolding, the greater the eﬃciency. Components of the refolding buffer The components of a refolding buﬀer vary widely, depending on the protein of interest with pH, ionic strength, redox conditions and ligands all inﬂuencing the outcome (Tables 3 and 4). Most commonly Tris or Hepes based buﬀers at neutral pH with NaCl (between 50 mM and 500 mM) are used, however again this depends on the protein of interest. There are also numerous additives which can be included, that have had varying success with numerous targets. They include detergents, polar additives, weak chaotrophs, osmolytes and cations (Table 3). These additives to one degree or another, either act as stabilizers (stabilizing the native state/solubilizing intermediates) or promote correct folding by preventing aggregation. Another common approach is to include proteinase inhibitors (PMSF, aprotinin, leupeptin, etc.) within the refolding buﬀer, if the protein of interest is prone to proteolysis.

Table 3. Additives that have been successfully used for refolding. Additive

Concentration

Eﬀect

L-Arginine

0.4–0.5 M 10–50% 0.4 M

stabilizer stabilizer stabilizer

up to 1 M 0.1–2 M 0.1–1 M 0.1–1% 0.01% 0.3 mM 30 mM 0.1% up to 4 M up to 0.05% (w/v) 1–3 M

solubilizer chaotroph chaotroph detergent detergent detergent detergent detergent detergent osmolyte osmolyte salt salt cation chelator buﬀer

Glycerol Sucrose/glucose Non-detergent sulfobetaine (NDSB) 256/201 Urea Guanidine HCl Triton X-100 Tween-80 Lauryl maltoside CHAPS SDS Lauroylsarcosine PEG 3350 TMAO Sodium citrate/Sulfate NaCl/Ammonium sulfate MgCl2/CaCl2 EDTA Tris

0.2–0.5 2 mM–10 mM 20 mM 0.4–1 M

40

Table 4. Examples of proteins refolded from inclusion bodies. Protein

IB puriﬁcation

Solubilization

Refolding

Technique

Ref.

Human IL-15

Ni-NTA

[94]

2 M urea, 5 mM BME

Ni-NTA

[95]

Plasminogen activator-1 Resistin

0.05% Tween 80 Ni-NTA

8 M Urea 1 mM DTT, 10% glycerol 1% SDS 20 mM BME pH 9 4 M GdnHCl 6 M GdnHCl

Gradient

Clostridium diﬃcile

2 M Urea 2% Triton X-100 –

Dilution Step dialysis

[96] [56]

Alpha lytic protease

S sepharose

5 M Urea

Pulse dilution

[45]

ACC synthase Glutamyl-tRNA reductase

0.1% Triton X-100 2 M Urea 0.2% Triton X-100 0.5% NP-40/3 M urea/ Gel ﬁltration (superdex 200)

6 6 5 6

1 M NaCl, 0.01% Tween 80 1 mM DTT, 0.1% mannitol 2 M urea/1 M urea, pH 11 5–20% sucrose/glucose 200 mM methionine 1% glycerol, 33 mM Chaps 20% glycerol

Dilution Ni-NTA

[58] [64]

Gel ﬁltration

[65]

Transposase Tc1A

M urea, 10 mM DTT M GdnHCl mM DTT M GdnHCl

10% glycerol, 5 mM MgCl2, 1 mM DTT

41 Disulfide bond formation For proteins with native disulﬁde bonds a redox system during renaturation is required, for their correct formation. The combination of reduced and oxidized forms in molar ratios ranging from 1:1 up to 10:1 are generally used to form disulﬁdes correctly. Glutathione (GSH/GSSG) is a common reagent, however the combination of cysteine and cystine or DTT/oxidized glutathione are alternatives. While air/molecular oxygen is suitable to promote disulﬁde bond formation, a redox system accelerates the shuﬄing of disulﬁde bonds. It is generally catalyzed by trace amounts of metal ions and also by slightly alkaline pH (8–9) [52]. On the contrary, to minimize the eﬀects of oxygen oxidizing thiols, the presence of EDTA can be beneﬁcial [53]. The time taken for the ‘‘shuﬄing’’ to take place varies between proteins, anywhere from 2 h to 150 h, though 16–48 h is common place for eﬃcient disulﬁde shuﬄing, although this must be determined empirically. A recent report using controlled air oxidation demonstrated the successful production of prochymosin. Menzella and colleagues carried out oxidation by introducing air at a ﬂowrate of 0.1 L/min, in the presence of Cu2 þ as a catalyst. With this and the use of additives (L-arginine), they were able to recover up to 67% of active protein from inclusion bodies [49]. There are however, conﬂicting reports on the impact of inappropriate disulﬁde bonding, with suggestions that it may not necessarily lead to aggregation, considering a carboxymethylated version of lysozyme (cysteines blocked) was still found to be aggregation-prone [54]. Methods of refolding There are several methods to refold proteins including: dialysis, dilution and use of column chromatography techniques. The method which is used depends on the propensity of the protein to aggregate and the kinetics of refolding. The temperatures at which refolding takes place can vary, though to minimize aggregation, 4 C is best. Dialysis Here, the concentrated denatured protein is dialysed against a refolding buﬀer, such that the concentration of the denaturant decreases as it is buﬀerexchanged. It is this slow removal process which allows for the refolding to take place [15,55]. Unfortunately, the slow removal of the denaturant often results in the formation of the exposure of long lived intermediate species over a long period of time and hence there may be increased propensity for the protein to aggregate. A variation of a one-step dialysis as previously described, is the use of refolding buﬀers with decreasing denaturant concentrations [56]. By dialysing in

42 a step-wise fashion (usually 1 to 3 progressively lower denaturant concentrations), this allows for an equilibrium to be established. Again, whilst the refolding is more controlled in this environment, long-lived intermediate species can still present a challenge. Dilution The dilution method can be described as ‘‘rapid’’ or ‘‘slow.’’ In the rapid dilution method, the denatured protein is delivered to a refolding buﬀer, such that in a very short period of time, the concentrations of both the protein and denaturant decrease rapidly. For instance, if the protein were denatured in 8 M urea and diluted 10-fold into buﬀer, the ﬁnal concentration of the denaturant is 0.08 M. Like dialysis, the aim of dilution is to remove the denaturant, so that its ﬁnal concentration would be low. There are instances though, where refolding into low molar concentrations (1–2 M) aids in the solubility of the protein. The period of time used in ‘‘rapid’’ dilution may be detrimental to the proteins, particularly if they refold over a time period of minutes to hours. By forcing the protein to adopt its conformation in a limited time frame, it may increase its chances of misfolding and hence aggregating, however this approach has been successful for a range of proteins (monogalactosyldiacylglycerol [57], 1-aminocyclopropane-1-carboxylate synthase [58], antichymotrypsin [59], transglutaminase [60]). Slow dilution is an alternative, as it is a more gentle approach, by dramatically decreasing the eﬀective concentration of the refolding protein. The ‘‘dropwise’’ or ‘‘pulsed dilution’’ method, involves the solubilized inclusion bodies being delivered very slowly to the refolding buﬀer (using a pump) and has been used successfully for proteins such as a1-antitrypsin [61] and also a-lytic protease [45]. On-column refolding This approach has been used for several proteins with success and oﬀers an alternative where other methods may not be applicable. If a protein is tagged with a hexa-histadine, it can be immobilized on a Ni2 þ aﬃnity column. By applying buﬀers with a decreasing concentration of denaturant, either stepwise or by using a gradient, the protein can be refolded and then eluted [62,63]. Gel ﬁltration is an alternative, whereby the denatured protein is loaded onto the column and refolds as it is passed through with buﬀer [64,65]. A variation of this is to equilibrate the column with a linear gradient, with decreasing concentrations of denaturant, so the protein refolds gradually [66]. Flow rates required for successful on-column refolding appear to vary with slower ﬂow rates improving recovery in some cases [47] whilst in others, faster ﬂow rates were best [67].

43 Recently, the on-column refolding technique has been improved by immobilizing common ‘‘foldases’’ onto the column matrix and exploiting them as a folding ‘‘platform.’’ This has been used successfully with GroEL [68,69] and DsbA/DsbC [70]. One associated concern with on-column refolding is the clogging of ﬁlters by aggregated protein, however, if performed with samples that have been carefully ﬁltered/centrifuged and with a sensible column matrix, may minimize these problems. Other refolding techniques One technique which is a departure from the aforementioned ‘‘classic’’ techniques involves the use of reverse micelles, which has been explored recently. Vinogradov and colleagues trapped their enzyme in a water–sodium bis2-ethylhexyl sulfosuccinate-isooctane reverse micellar system and reported the recovery of monomeric protein, where other techniques had failed. By varying the size of the micelles, they were able to manipulate the degree of oligomerization of the protein [71]. Other folding aids There are several molecular chaperones that can be incorporated either in vivo or in vitro to aid in folding. The most well known E. coli chaperones include GroEL-GroES, DnaK-DnaJ-GrpE (Hsp70) and also ClpA/ClpB (Hsp100) which have been used successfully in renaturation studies [72–75]. While molecular chaperones can promote correct folding, foldases can accelerate the process. The three types of foldases include: peptidyl prolyl cis/ trans isomerases (PPI’s) (arranges prolines into correct conformation, an otherwise a lengthy process) [76], disulﬁde oxidoreductase (DsbA) and disulﬁde isomerase (DsbC) (which promote disulﬁde bonds, found in E. coli) [77,78] and protein disulﬁde isomerase (PDI) (a eukaryotic protein catalyzing oxidation and isomerization) [79]. Folding screens As trying to refold a protein can be a time-consuming process, several commercial screens are now available to focus on potential conditions that return the best yield of monomeric protein. One such kit is Hampton Research’s FoldIt screen, which uses a variety of additives, redox conditions and pH and similar kits are available through Novagen and Sigma-Aldrich. Such kits are the result of sparse screens that have been successful in the past [80–82] to refold a number of proteins. With the commercially prepared kits, it enables a number of refolding conditions to be sampled simultaneously on a small scale, the aim being to ﬁnd a suitable condition that can be up-scaled for puriﬁcation.

44 An elegant example of using factorial screens was with work done on procathepsin S and cathepsin S, which could not be previously refolded, in vitro. An initial screen was used to identify conditions for folding, targeting both L-arginine and pH as two important factors. While pH was important for both, arginine was more beneﬁcial for procathepsin only. Once these conditions were established, it was then followed up, using a second screen to improve yields and hence produce the proteins on a larger scale. It was concluded that procathepsin required detergent, arginine and a redox system, while cathepsin only required glycerol and the redox couple [83]. This in itself illustrated the powerful nature of the factorial screen and how seemingly similar proteins may have very diﬀerent requirements for refolding.

Correctly folded or aggregated material? Once refolding is complete, one must determine whether the procedure has yielded folded or aggregated protein and whether disulﬁde bonds (if any) have been formed correctly. Perhaps the simplest means of assessing the quality of the protein is by using a known activity assay. While this is possible for some, the need to determine the nature of the protein using other techniques is important, considering the high throughput production of proteins from diﬀerent genomes will undoubtedly yield targets with no known function or structure. The use of size-exclusion chromatography and non-denaturing PAGE can be employed, as is circular dichroism (CD) which reports on the secondary structure. There are distinct features in a typical CD scan that report the presence of a helical, b sheet or random coil structure (Fig. 3). While proteins will generally be a combination of a helical and b sheet content, for aggregated

α helix

20 15

β sheet

ellipticity

10

turn

5 0 −5

−10

random coil 180

200

220

240

260

wavelength (nm) Fig. 3. A typical far-UV CD spectrum of classical secondary structure elements.

45 material, a random coil may predominate. Fluorescence techniques such as dynamic light scattering [84], which determines the average diameter of particles in solution and lateral turbidimetry [13] can also report the presence of aggregates [85], as can ultracentrifugation [86] and also electron microscopy [87]. Assessing the nature of disulﬁde bonds can be deﬁned by a number of methods. Perhaps the most straightforward is the use of reducing and nonreducing gel electrophoresis. In the absence of reducing agent, there should be a deﬁned ‘‘band shift,’’ accompanied with any additional bands that were record of linked domains. For a single domain protein, a ‘‘laddering’’ eﬀect is a telltale sign of intermolecular disulﬁde bond formation. The use of thiol speciﬁc dyes (DTNB (Ellman’s reagent)) [88], mass spectrometry [89] and gel electrophoresis (‘‘cysteine counting’’) [52] are all common techniques that are also employed. Conclusion Handling inclusion bodies is time consuming and often a frustrating trial-anderror process. There are also no clear rules that apply to all proteins, as where an approach suitable to one may also be just as detrimental to another. Despite this, working with and conquering the inclusion body may be beneﬁcial, particularly when it is impossible to express soluble protein in reasonable amounts. As more proteins are successfully refolded the rules may become clearer. This will aid biotechnology and may also aid medicine as the tricks which are used to refold proteins may be useful in preventing the array of diseases in which misfolding plays a central role. Abbreviations IPTG GST MBP CD DTT

isoproyl-thiogalactoside, glutathione-S-transferase, maltose binding protein, circular dichroism, dithiothreitol

Acknowledgments The authors would like to thanks Michelle Chow for her help with the manuscript. This work was in part funded by both the Australian Research Council and the National Health and Medical Research Councils of Australia. SPB is a Monash University senior Logan research fellow and an RD Wright Fellow of the NHMRC.

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51

Towards a systems biology understanding of human health: Interplay between genotype, environment and nutrition Frank Desiere* Nestle´ Research Center, P.O. Box 44, 1000 Lausanne 26, Switzerland; Institute for Systems Biology, Seattle, Washington, USA Abstract. Sequencing of the human genome has opened the door to the most exciting new era for the holistic system description of human health. It is now possible to study the underlying mechanisms of human health in relation to diet and other environmental factors such as drugs and toxic pollutants. Technological advances make it feasible to envisage that in the future personalized drug treatment and dietary advice and possibly tailored food products can be used for promoting optimal health on an individual basis, in relation to genotype and lifestyle. Life-Science research has in the past very much focused on diseases and how to reestablish human health after illness. Today, the role of food and nutrition in human health and especially prevention of illness is gaining recognition. Diseases of modern civilization, such as diabetes, heart disease and cancer have been shown to be eﬀected by dietary patterns. The risk of disease is often associated with genetic polymorphisms, but the eﬀect is dependent on dietary intake and nutritional status. To understand the link between diet and health, nutritional-research must cover a broad range of areas, from the molecular level to whole body studies. Therefore it provides an excellent example of integrative biology requiring a systems biology approach. The current state and implications of systems biology in the understanding of human health are reviewed. It becomes clear that a complete mechanistic description of the human organism is not yet possible. However, recent advances in systems biology provide a trajectory for future research in order to improve health of individuals and populations. Disease prevention through personalized nutrition will become more important as the obvious avenue of research in life sciences and more focus will need to be put upon those natural ways of disease prevention. In particular, the new discipline of nutrigenomics, which investigates how nutrients interact with humans, taking predetermined genetic factors into account, will mediate new insights into human health that will ﬁnally have signiﬁcant positive impact on our quality of life. Keywords: systems biology, genomics, transcriptomics, proteomics, metabolomics, nutrigenomics, pharmacogenetics, pharmacogenomics, health, nutrition, diet, disease, prevention, diagnostics, bioinformatics, molecular databases, metabolism, networks, cells, polymorphisms, SNPs, epigenetics.

Introduction Systems biology is gaining importance in today’s life-science research. Interestingly, the ﬁrst attempts to systems biology, go back to the 1960s. At the time such attempts were called modeling of cellular processes by study means of ‘‘systems theory and biology.’’ Many mathematicians and engineers tried to develop approaches that allow analyzing biological systems in a physical way. It was realized at the time, that when they tried to interact with an organism as a physical system, they found themselves interacting with it in many *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10003-3

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

52 more ways than they had instrumentations for. It then became clear that for a complex system as cells or organisms there were many more capabilities, and several more modes of interaction than just a limited set of canonical rules [1]. Up until that point, many scientists and biologists alike have focused on reducing life to its constituent parts, ﬁrst focusing on the cell, then working their way down to the molecular level. Today, two apparently opposing opinions are in discussion. The ﬁrst claims that a cellular system can readily be described in all its parts and even be simulated, maybe using the tools of systems biology. The other opinion cast serious doubts that this can be achieved due to fundamental reasons and limitations. This opinion is certainly well documented by the work of Robert Rosen (1934–1998), a theoretical biologist who strived to answer the question the Nobel physicist Erwin Schro¨dinger posed in 1943: ‘‘What is Life?’’ To this day, what it is that makes an organism alive has remained unanswered by conventional biology, chemistry and physics. Schro¨dinger’s works on complexity and biological systems claim that these cannot be decomposed or predicted because of their anticipatory nature and that a biological system is not a just a complex machine [2]. But let us now focus on the achievements that have catalyzed the massive advances in understanding of biological systems through the ﬁeld of biotechnology and later genomics, leading ﬁnally to the more holistic (and mysterious) term of ‘‘systems biology.’’ With the development of highthroughput technologies for molecular biology in the 1980s and 1990s, that amongst other achievements have resulted in the completion of the human genome [3,4], quantitative data on the transcriptome [5], proteome [6] and metabolome level [7], an increasing interest in formal mathematical models of cellular activities as gene expression and regulation has been triggered. It was realized that the vast amount of data available would require new concepts for the understanding and new tools for the description of life as a whole. Systems biology and systems theory, which study the organization and behavior of living systems, seemed indeed a natural conceptual framework for such a task. Systems biology attempts to reconstruct living systems as a series of overlapping models. It exploits all the theoretical and experimental advances of the various genome projects, allying them to computational, mathematical and engineering disciplines. This is done in an attempt to create predictive models of cells, organs, biochemical processes, and complete organisms. Consequently, systems biology today has the potential to advance our knowledge and understanding of complex biological systems, from simple cells to complete organisms and potentially to whole ecosystems. The understanding of biological systems is not an altruistic matter for the beneﬁt of advancing philosophy and theoretical sciences. No, there are real problems to be solved. The world’s demography has pushed medical treatment to higher levels over the last decades, mainly due to aging populations and increased life-expectancy [8]. With individuals realizing that they will enjoy longer lives, the issue of disease-prevention has become an important concern.

53 The quest for new treatments and prevention of illness has let pharmaceutical companies become powerful, big corporations which drive many areas of modern life science and a big proportion of the biotechnology industry to this date. The discovery of new pharmaceutical treatments, especially those which will bring large amounts of cash back to the industry, the so called ‘‘block-buster drugs,’’ seem to be the ultimate goal for research and development in academic and public institutes, biotechnology companies and the private medical research centers alike. In parallel, researchers have promoted disease prevention also through adequate nutrition and it was realized that scientiﬁc breakthroughs in both areas would require a massive investment into modern nutrition research through ‘‘systems biology.’’ To accelerate the mission, research institutes and scientiﬁc groups dealing with systems biology have been created in recent years. Founded in the year 2000, the ‘‘The Institute for Systems Biology’’ (www.systemsbiology.org) is for many people the pioneer in the new ﬁeld, and has managed to inﬂuence the pace and direction of modern biology. That trend has now gained broad acceptance as a new scientiﬁc ﬁeld, prompting the National Institutes of Health (NIH) to identify in 2003 systems biology and multidisciplinary research as key components in a new set of agency initiatives for the NIH Roadmap for Medical Research of the next decade. New projects under the theme of New Pathways to Discovery would include Bioinformatics and Computational Biology, Structural Biology, Building Blocks and Pathways, Molecular Libraries and Molecular Imaging, and Nanomedicine. Starting from the year 2004, the NIH will fund these topics which will at the same time require an improved computational infrastructure for biomedical research, libraries of chemical molecules, new molecular and cellular imaging tools, and nanoscale technology devices for viewing and interacting with basic life processes. This policy describes clearly the challenges ahead of us to investigate biological systems, particularly in the context of human health, treatment of disease and prevention of illness. Technologies of systems biology Systems biology focuses on complex biological systems that are composed of molecular components. Understanding systems biology requires the integration of experimental and computational research data [9]. Systems biology is the attempt to systematically study all the concurrent physiological processes in a cell or tissue by global measurement of diﬀerentially perturbed states. The ultimate goal of systems biology is the integration of data from these observations into models that might, eventually, represent and make possible the simulation of the physiology of the cell [10,11]. Although biological systems are made-up of their components, the essence of a system lies in dynamics and it cannot be described merely by enumerating components of the system. At the same time, it is inappropriate to believe that only system structures, such as network topologies, are important without

54 Table 1. Web resources and databases for systems biology.

Institute for Systems Biology (www.systemsbiology.org) MIT Computational and Systems Biology Initiative (CSBI) (csbi.mit.edu/) Bauer Center for Genomics Research (CGR) at Harvard University (www.cgr.harvard.edu/) Bio-X at Stanford University (biox.stanford.edu/) Cell Systems Initiative at the University of Washington (csi.washington.edu/) Genomes to Life program at the US Department of Energy (DOE) (doegenomestolife.org/) Biomolecular Systems website at the Paciﬁc Northwest National Laboratory (PNNL) (biomolecular.org) Institute for Computational Biomedicine at the Weill Medical College of Cornell University (icb.med.cornell.edu/)

paying suﬃcient attention to diversities and functionalities of the components (Table 1). Both structure of the system and its components play indispensable roles forming a holistic view of the state of the system. The goals of systems biology are: (1) (2)

(3) (4)

Understanding of the components of a biological system, such as genes, proteins and metabolites, as well as their physical structures, Understanding of dynamics of the system, both quantitative and qualitative analysis as well as construction of theories/models with powerful prediction capability, Understanding of control methods of the system, and Understanding of design methods of the system.

The following sections will give a more detailed overview of the sub-disciplines of systems biology, which characterize the cellular components. Finally these components will have to be put into context, which will be the focus towards the end of this review. Genomics The availability of completely sequenced genomes catalyzed the emergence of systems biology and has truly revolutionized biology. For the ﬁrst time since the advent of molecular biology, biological questions are now addressed by studying the complete set of a system in contrast to the previous investigation of function(s) of individual genes and gene products one or a few at a time. Before, high-throughput analytical instruments like the DNA sequencer or mass spectrometers for protein determination had been invented, this reductionist approach proved to be extremely fruitful, leading to the discovery of an impressive number of biological principles. However, it was quickly realized that in nature, cellular components function together with other components. As Henri Poincare´ already pointed out in 1952 [12] ‘‘the aim of science is not things in themselves, but the relations between them; outside these relations there is no reality knowable.’’ Indeed, biological processes should be considered as a

55

Number of completed genomes

complex network of interconnected components. In other words, for any biological process, one might consider a ‘‘modular approach’’ in which the behavior and function of the corresponding network are studied as a whole. In addition to studying some of its components individually, the ﬁrst step to reach that goal was the determination of complete genomes of organisms. The signiﬁcance of the ﬁnished human genome sequence [3,4] and other genomes of model organisms for the ﬁeld of systems biology cannot be overstated. Without these genomes, holistic studies would simply not be possible. Still, our knowledge is steadily increasing, which is underlined by the latest detailed analysis of human chromosome 6 [13]. A great abundance of biological information was revealed that was previously unrecognized within the draft of the human genome. Comparative genomics using the genomes of the mouse, rat, puﬀer- and zebra-ﬁsh allowed reﬁned predictions of which stretches of DNA are actually genes, and a more sophisticated interpretation of the underlying genomic data. The power of comparative genomics is quickly growing as the genome sequences of other nematodes are sequenced [14], as well as chicken, chimpanzee, frog, and cow that are already in the production queue, become available. Currently there are about 203 complete genomes of living organisms in the public domain (www.ebi.ac.uk/genomes/, Fig. 1), with more than 800 on their way of being ﬁnished. These numbers underline the growing importance of comparative genomics. However, it must be stated that gene-prediction remains to be a signiﬁcant challenge and it can be anticipated that our current data about location and number of genes will constantly have to be updated [15]. The genome of an organism represents an ideal coordinate system for systems biology, a precisely deﬁnable digital core of information for an organism [16]. Genes are the ‘‘genetic parts list’’ to which all other biological information can be linked. Transcripts are directly related to genes. Proteins are related to transcripts and then to genes. All the information is hierarchical in

160 140 120 100 80 60 40 20 0 1995 1996 1997 1998 1999 2000 2001 2002 2003

Fig. 1. Number of completed genomes (http://www.ebi.ac.uk/genomes).

56

Fig. 2. Regulatory gene network for endomesoderm speciﬁcation: the view from the genome. The architecture of the network is based on perturbation and expression data, on data from cisregulatory analyses for several genes, and on other experiments (reproduced with permission from Hamid Bolouri and Eric Davidson, http://sugp.caltech.edu/endomes/) [17].

nature: DNA, mRNA, protein, protein interactions, informational pathways, informational networks, cells, tissues or networks of cells, an organism, populations and whole ecologies. It is therefore tempting to construct a geneindex in which every gene of organisms are listed and numbered and to use it as a central core for linking any kind of biological information to it. This concept has partially been applied to publicly accessible genome resources e.g., Ensembl (www.ensembl.org) and RefSeq (www.ncbi.nlm.nih.gov/RefSeq). Genomic sequences also provide access to regulatory sequences in genomes, which are a vital component to solving the regulatory code [17]. Also, genomic sequences open access to polymorphism studies; some of these variations are responsible for diﬀerences in physiology and disease predisposition. These components combined make-up the elements in the ‘‘periodic table of life.’’ With these components in hand, the immediate challenge is to place them in the context of their informational pathways and networks.

57 The logical extension to studying the genome is the determination of interindividual diﬀerences within the genome of people. Only a small number of common polymorphisms explain the bulk of heterozygosity [18]. Human genetic diversity appears on the level of individual polymorphisms, known as single nucleotide polymorphisms (SNPs), as well as in the speciﬁc combinations of alleles (haplotypes) as observed at closely linked sites. The goal of the International HapMap Project for example is to develop a haplotype map of the human genome, to describe the common patterns of human DNA sequence variation. The HapMap is expected to be a key resource for researchers in ﬁnding genes aﬀecting health, disease, responses to diet and other environmental factors. SNPs, single-nucleotide polymorphisms, are small genetic variations between people that can signiﬁcantly alter the function of proteins. Most importantly, the altered function may have signiﬁcant eﬀects on how the individual reacts to treatment of drugs, allergies to environmental substances and digestion of foods [19]. The latest release of dbSNP (118) at the NCBI contains an impressive amount of 5,798,183 SNPs for human of which 2,359,534 are validated. These polymorphisms now have to be investigated for their signiﬁcance in altering biological function of proteins and pathways. Knowledge about SNPs is most important for treatment using drugs. Altered protein function might not carry a drug to its target cells or tissues cripple the enzymes that activate a drug or aid its removal from the body, or alter the structure of the receptor to which a drug is supposed to bind. Variation in immune-system genes can also inﬂuence how particular drugs are tolerated. Together, these subtle genetic variations mean that the dose at which a drug will work may vary hugely from person to person. The so widely utilized ‘‘one-size-ﬁts-all’’ prescription leads to life-threatening adverse reactions and to drugs completely failing to do their job. Well-documented examples of active SNPs are available from the P450 protein family, enzymes in the liver that oxidize foreign chemicals. Three of these P450 genes that are particularly important for drug metabolism of commonly prescribed drugs, have been shown to be highly polymorphic and some have already been linked to failure in certain patients [20]. Other examples show that the eﬃciency of the painkiller codeine depends on a particular polymorphism [21] and that the anticoagulation drug warfarin can cause serious adverse drug reactions depending on the genotype of the patient [22]. Another example shows that the base excision repair enzyme MED1 is associated with nonpolyposis colorectal tumors, a very common form of hereditary cancer. The gene’s protein product, MED1, is an enzyme that normally helps cells repair potentially cancer-causing damage to genes. However, a defective MED1 enzyme did not only prevent repairs in normal cells and permitted a cancer to start, but in particular, the enzyme also interfered with the eﬀectiveness of some types of chemotherapy [23]. Genomic polymorphisms will only be able to be investigated with many complete human genomes available, an achievement that can be envisaged by the

58 end of the ﬁrst decade of the 21st century. It is anticipated that within about 10 years, advances in nanotechnology and other methods will allow the fast and cheap sequencing of individuals’ genomes, which in turn will lead to advances in predictive medicine. As scientists are able to look at 30,000 or more genes for each patient, doctors could use such genome sequences to predict what health problems the individual patient is likely to face. Genome shotgun sequencing and microarrays have given us the tools to identify people with SNPs [24]. Individuals can now be proﬁled with increasing eﬃciency, and used to highlight polymorphic genes that inﬂuence our response to speciﬁc drugs or foods. These developments have resulted in a completely new discipline called ‘‘Pharmacogenetics’’ – the study of the inﬂuence of genetic variation on drug responses [25]. Similarly, the science of nutrigenomics seeks to provide a molecular understanding for how common dietary chemicals (i.e., nutrition) aﬀect health by altering the expression and/or structure of an individual’s genetic makeup. Thus, the new ﬁeld of nutrigenomics opens the way for ‘‘personalized nutrition.’’ In other words, by understanding our nutritional needs, our nutritional status, and our genotype, nutrigenomics should enable individuals to manage better their health and well-being by precisely matching their diets with their unique genetic makeup. The success of these methods will largely depend on the large-scale discovery of SNPs, their validation and the discovery of diet-related genes. To achieve this task more research into nutritional sciences using systems biology will have to be initiated thus identifying nutritionally relevant genes in order to study their response to nutrients systematically. The understanding of the human genome is not completed with the genome sequence established and the polymorphisms determined. New discoveries further complicate the understanding of genomes. Inheritable changes in gene function can occur without a change in the DNA sequence. Epigenetic mechanisms such as DNA methylation, histone acetylation, and RNA interference, and their eﬀects in gene activation or inactivation might be involved in imprinting and parental imprinting in which a gene’s activity depends on whether it is inherited from the mother or the father [26]. There is evidence to suggest that factors such as lifestyle and diet leave a trail of epigenetic footprints across our genome, which is then inherited [27]. In a striking example, Duke University researchers have demonstrated recently in mice how extra vitamin doses during pregnancy in the mother’s diet changes the color of pups [28]. This study is the ﬁrst one to ﬁnd a clear mechanism of the eﬀect of maternal nutrition on disease and phenotype. The nutrients used in the study, B12, folic acid, choline and betaine, had silenced the gene that rendered mice fat and yellow, but had not altered its sequence. The gene was in fact methylated, and thus switched oﬀ, linking prenatal diet to diseases like diabetes, obesity and cancer. Thus, knowledge about the genomic make-up of individuals will be crucial in health research (Table 2).

59 Table 2. Web resources and databases for genomics.

The National Human Genome Research Institute (www.nhgri.nih.gov) Nature Genome Getaway (www.nature.com/genomics/human/) Ensembl Human Genome browser (www.ensembl.org) European Bioinformatics Institute EBI (www.ebi.ac.uk/) National Center for Biotechnology Information NCBI (www.ncbi.nlm.nih.gov/genome/guide/ human/) International HapMap Project (www.hapmap.org) The Human Epigenome Project HEP (www.epigenome.org) Database of single nucleotide polymorphisms dbSNP (www.ncbi.nlm.nih.gov/SNP)

Transcriptomics The genome describes the ultimate potential of an organism, and the transcriptome, all complementary DNA sequences, describes the utilization/ expression of that potential. Transcripts can readily be identiﬁed by expressed sequence tags (ESTs). EST sequencing eﬀorts still represent an economic and fast way to characterize expressed genes. EST sequencing still remains an essential resource for genome exploitation and annotation. This is particularly important with the increasing availability of draft genome sequences from diﬀerent organisms and the mounting emphasis on gene function and regulation [29]. Simultaneous analysis of gene-expression can be performed using the technology that allows synthesis or immobilization of known complementary DNA sequences on microscopic arrays and later hybridizing RNA obtained from living cells onto the array. Microarrays exploit the preferential binding of complementary single-stranded nucleic-acid sequences and the underlying principle is the same for all microarrays. An unknown sample is hybridized to the array of immobilized DNA molecules whose sequence is known. Each array features thousands of diﬀerent DNA probe sequences arranged in a deﬁned matrix and thus can identify thousands of genes simultaneously, which means that genetic analysis can be done on a huge scale. Transcriptome proﬁling, using microarrays [30,31] or serial analysis of gene expression (SAGE) [32], can measure the relative abundance of transcripts simultaneously for thousands of genes under various experimental conditions. This technology has revolutionized the way in which researchers analyze gene expression in cells and tissues. It allows researchers to determine which genes are being expressed in a given cell type at a particular time and under particular conditions. They can be used to compare the status of gene expression in two diﬀerent cell types or tissue samples, for example, healthy versus diseased tissue, and to examine changes in gene expression-proﬁle at diﬀerent stages in the cell cycle or during embryonic development. Other uses of microarrays include comparative genomic hybridization studies [33], genotyping individuals for genetic diﬀerences that might be associated with disease [34], assignment of probable functions to newly discovered genes by comparison with the expression patterns of known genes,

60 to identify key players in signaling pathways and to uncover new categories of genes [35] (Fig. 3). Other areas of application engulf today the identiﬁcation of new targets for therapeutic drugs, in disease diagnosis, and in toxicogenomics [36], the study of the genetic basis of an individual’s response to environmental factors such as drugs and pollutants. Transcription proﬁling is today applied in all major areas of biology. One of the most remarkable studies to date and a great example for a systems biology approach is the description of a geneco-expression network for global discovery of conserved genetic modules [5].

Experimental Design

Experiment

Data analysis

Data storage

RT-PCR Labeling Pooling of samples Hybridization to array Scanning

Image analysis Statistics Normalization Clustering Annotation

MAGE-ML, MIAME Relational database ArrayExpress GEO Stanford microar. DB

Protein extraction Sample fractionation Separation (2D-GE, LC) Digestion ESI/MALDI/FT-MS

Analysis of spectra Statistical evaluation Identification: Database search, Quantification Annotation

mzXML, PEDRO Relational database No repository yet! GenBank, EMBL, BIND, DIP, etc.

Analysis of spectra Database search Statistical evaluation Annotation, Clustering Network Modeling

SBML Relational database No repository yet! KEGG, E-cell, EMP etc.

Transcriptome Determination of genome-wide transcript levels via DNA array: Treated vs. non-treated Normal vs. abnormal tissue

Proteome Determination of all proteins in a cell or body fluid via (quantitative) mass spectrometry: Treated vs. non-treated Normal vs. abnormal tissue

500

Intensity, counts

400

Metabolome Determination of all metabolites in a cell or body fluid: Treated vs. non-treated Normal vs. abnormal tissue

300 200 100 0 600

1000

1400 1800 m/z , au

2200

2600

Metabolite extraction Sample fractionation Separation (LC, GC) Identification: MS, GC, NMR

y

x z

Fig. 3. Comparison of data analysis strategies for transcriptome, proteome and metabolome studies. Abbreviations: RT-PCR, reverse transcriptase polymerase chain reaction; MAGE-ML, MicroArray Gene Expression Markup Language; MIAME, Minimum Information About a Microarray Experiment; GEO, Gene Expression Omnibus; 2D-GE, 2 Dimensional Gel Electrophoresis; ESI, Electro Spray Ionization; MALDI, Matrix Assisted Laser Desorption/ Ionization; FT, Fourier Transform; MS, Mass Spectrometry; mzXML, mass spectrometry eXtensible Markup Language [37]; PEDRO, software, to support the capture, storage and dissemination of proteomics experimental data [37,38]; EMBL, European Molecular Biology Laboratory; BIND, Biomolecular Interaction Network Database; DIP, Database of Interacting Proteins; LC, Liquid Chromatography; GC, Gas Chromatography; NMR, Nuclear Magnetic Resonance spectrometry; SBML, Systems Biology Markup Language; KEGG, Kyoto Encyclopedia of Genes and Genomes; EMP, database of Enzymes and Molecular Pathways.

61 In a truly massive approach co-expressed pairs of genes were identiﬁed over 3182 DNA microarrays from humans, ﬂies, worms, and yeast. An estimated number of 22,163 conserved co-expression relationships were identiﬁed using statistical clustering algorithms providing new evidence for the involvement of genes in core biological functions. The relative ease of producing such a large number of data is obscured by the diﬃculty of dealing with the results due to a lack of simple and accepted approaches to analyzing large-scale gene expression data. Visualizing and presenting such large gene expression data is not trivial [30]. Despite these diﬃculties, the ﬁeld of gene expression analysis is helping devise strategies that also allow distinguishing between the expressions of alternatively spliced transcripts. It has been estimated that 30%–60% of all human genes encode for more than one transcript. The impact of these alternative gene-products on function and regulation has become a major focus for research and has led to the establishment of various databases harboring information on alternatively spliced transcripts [39,40]. Further investigation is required to determine the cause and eﬀect of alternative splicing in a genome, transcriptome and proteome context. The impact of transcriptome studies in human health research has been shown for many ﬁelds in recent times. Their applications include assessing the safety of food, drugs, vaccines, medical devices and other products of consumer interest [41–46]. DNA arrays for the identiﬁcation of food-borne bacterial pathogens and viruses [47] can be used to reduce the incidence of food poisoning, illness and death associated with bacterial or viral contamination of meat, seafood, dairy products and other foods. Also in clinical settings, the identiﬁcation of organisms in patients admitted to hospitals with systemic bacterial infections can be envisaged. The capacity to type unambiguously all the common bacteria on a single chip within a few hours of sampling will allow high-speed testing in agricultural, manufacturing and clinical settings. It might be possible that gene-expression patterns will be used to simplify widely used diagnostic descriptions of cancers. When currently as many as 7000 disease-concepts with 42,000 names (and synonyms) are used worldwide to describe diﬀerent cancers and the number of validated gene-expression proﬁles for cancers grows, these proﬁles may oﬀer a useful way to streamline this list and standardize cancer classiﬁcation on a rational basis [48]. Another possible application will be the test of eﬃcacy and safety of pharmaceuticals, both in clinical trials and treatment. Genotyping by DNA arrays could be used to stratify patients participating in clinical trials into populations of responders and non-responders to enhance the accuracy of drugtesting results, and allowing drugs to be tailored to speciﬁc subsets of the population according to clearly identiﬁable markers in the patient population [30]. DNA arrays could also be used to examine the physiological eﬀects of a speciﬁc diet, allowing the analysis of pathways and the identiﬁcation of reactions in which food and its components are involved in Ref. [49].

62 Table 3. Web resources and databases for transcriptomics. Microarray Informatics at the EBI (www.ebi.ac.uk/microarray) Pat Brown’s lab at Stanford University (cmgm.stanford.edu/pbrown/) Stanford Microarray Database (genome-www5.stanford.edu/) Microarray Gene Expression Data (MGED) Society (www.mged.org/) Database of alternatively spliced proteins ASP at UCLA (www.bioinformatics.ucla.edu/ HASDB/)

This technology will be used as a valuable tool to identify mechanisms by which nutrients interact with the body and how individuals respond to food intake in a speciﬁc diet (Table 3). DNA microarrays are currently becoming useful analytical tools for disease proﬁling. However, there is a pressing need for other proﬁling technologies that go beyond measuring RNA levels, particularly for disease-related investigations. DNA microarrays have limited utility for the analysis of biological ﬂuids and for the discovery of markers directly in the ﬂuid. To reach that goal, there is a need to assay protein levels and activity. Numerous alterations may occur in proteins that are not reﬂected in changes at the RNA level, providing a compelling rationale for additional, direct analysis of gene expression at the protein level. The next challenge is to integrate RNA data with protein data [50]. Proteomics Proteomics technologies attempt the large-scale determination of gene and cellular function directly at the protein level. Mass spectrometry (MS) has increasingly become the method of choice for analysis of complex protein samples. MS-based proteomics is a discipline made possible by the availability of gene and genome sequence databases and technical and conceptual advances in instrumentation technology [51]. Proteomics has also established itself as an indispensable technology to interpret the information encoded in genomes. Protein analysis by MS so far, has been most successful when applied to small sets of proteins isolated in speciﬁc functional contexts. The systematic analysis of the much larger number of proteins expressed in a cell is now also rapidly advancing, mainly due to the development of new experimental approaches. A single bacterial cell may produce 4000 proteins whose abundances and activities may vary throughout an experiment, while the number of proteins expressed in higher eukaryotes is likely to be at least 10-fold greater. Attempts to catalogue, visualize, and analyze proteomics experiments have therefore become a major challenge (Table 4). Further to the identiﬁcation of proteins, their quantiﬁcation can now be addressed. However, no single method or instrument exists that is capable of identifying and quantifying the components of a complex protein sample. Two methods are popular: 2-dimensional electrophoresis (2DE) followed by MS or

63 Table 4. Steps involved in a typical proteomics experiment. Protein isolation from a biological sample (e.g., a cell extract) following some experimental treatment. Fractionation of the resulting proteins (or peptides, the products of proteome digestion) by methods such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) or liquid chromatography (LC). Protein or peptide detection by MS. Protein identiﬁcation through manual interpretation or database correlation of mass spectra.

limited protein puriﬁcation with automated peptide MS/MS. When accurate quantiﬁcation is desired, stable-isotope tagging of proteins or peptides is made. While 2DE clearly has its shortcomings [52–54], the use of liquidchromatography combined with tandem MS (LC MS/MS) experiments appears to be an extremely promising technology. However, mass spectrometers are inherently poor quantitative devices. The data collected by this method is comprehensive requiring more sophisticated tools for its analysis than what is presently available. Current challenges for the analysis of MS based data are the development of tools for their high-throughput analysis [55–57]. First steps into that direction do however exist. For example, statistical models to estimate the accuracy of the peptide assignments called ‘‘PeptideProphet’’ [58]. For computing probabilities that proteins are present in a particular sample can be done by ‘‘ProteinProphet’’ [59]. In order to tackle the quantitative analysis of peptide LC-MS/MS experiments, stable-isotope tagging has been developed. Diﬀerent stable-isotopes can be readily diﬀerentiated in a mass spectrometer owing to their mass diﬀerence leading to an accurate indication of the abundance ratio for the two samples. This new technique has been applied successfully in several experiments [6,60–62]. An interesting aspect for studying the inner workings of a cellular system is to study their protein machines. Most proteins exert their function by way of protein–protein interactions. Enzymes are often held in tightly controlled regions of the cell by such protein–protein interactions. Thus, protein–protein interactions provide a wealth of information on the fundamental aspects of cellular life. The ﬁrst of such screens is the yeast-two-hybrid (Y2H) technology [63]. Recently, two studies of biochemical puriﬁcations combined with mass spectrometry (MS) were conducted: one uses ‘‘high-throughput MS protein complex identiﬁcation’’ (HMS-PCI) [64], the other employing ‘‘Tandem aﬃnity puriﬁcation (TAP) followed by MS identiﬁcation’’ [65]. Some of the most biologically informative results have come from the analysis of large protein complexes, like the analyses of the spliceosome, followed by the yeast nuclear pore complex [66,67]. The complexity of the biological system on the protein level is further rendered more diﬃcult through protein post-translational modiﬁcation. These posttranslational modiﬁcations modulate the activity of most eukaryote proteins.

64 Their analysis is now pursued using mass spectrometric peptide sequencing and analysis technologies. Furthermore, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modiﬁcations [68]. Proteomics is an essential component of systems biology research because proteins are responsible for many crucial processes in the cell. This technology became extremely valuable for the description of biological processes such as protein abundance, linkage maps to other proteins or to other types of biomolecules including DNA and lipids. Proteomics can also address for example protein expression proﬁling, activities, modiﬁcation states, and subcellular location. Unfortunately, with the exception of quantitative protein expression proﬁles and protein–protein interactions none of these properties can currently be measured systematically, quantitatively and with high throughput. But rapid advances in technology suggest that these limitations may be only momentary. The few studies where the same biological system was subjected to diﬀerent types of systematic measurements already oﬀer insights into the power of the method. For instance, mRNA expression proﬁles and protein expression proﬁles seem to be largely complementary and therefore contribute to a more reﬁned description of the system that each observation by itself is unable to provide [10]. Combining diﬀerent genomic and proteomic results obtained from the same biological system will substantially increase our understanding of complex biological processes. More speciﬁcally, the systems biology studies based on diverse and high-quality proteomic data are already deﬁning functional biological modules and reveal previously unrecognized connections between biochemical processes and modules. The new hypotheses that are generated by this approach can be tested either by traditional methods or by the targeted generation of more genomic and proteomic data [10,69–71]. A promising quantitative proteomic proﬁling method (MS/MS) has recently been reported for glycoproteins using isotope protein tagging as well as automated tandem mass spectrometry [72]. The method is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the speciﬁc release of formerly N-linked glycosylated peptides via peptide-N-glycosidase F. The application of this approach to the analysis of plasma membrane proteins and human blood serum proteins promises great potential for the functional analysis of biological systems and for clinical diagnostics or prognostics. The result could be that an individual global-health proﬁle based on protein identiﬁcations will become feasible, revolutionizing the ﬁeld of disease diagnosis and health monitoring. It may be possible in the future that a small sample of blood can reveal an image of the physiological and pathological states of every tissue in the body [73]. In conclusion, the ever-advancing proteomics research represents one of the most promising technologies for the investigation of human health. Only two years ago, scientists reported a simple blood test based on proteomics a technology that successfully detects ovarian cancer even in its early stages [74].

65 Table 5. Web resources and databases for proteomics. ExPASy Proteomics tools (expasy.org/tools/) A Research Pointer to the Applied Proteomics and Proteomics Technologies (http:// proteomicssurf.com) spectroscopyNOW (http://www.spectroscopynow.com) Human Proteome Organization HUPO (http://www.hupo.org) Institute for Systems Biology (http://www.systemsbiology.org/)

Now, clinical laboratories are ready to employ the test. The emerging ﬁeld of clinical proteomics will provide early diagnostic methods leading the way for potentially curing such diseases (Table 5). Metabolomics Our metabolism is an expression of a transient steady state in the dynamics of cellular biosynthesis. Proteins function either as enzymes, receptors, transporters, channels, hormones and other signaling molecules or provide structural elements for cells, organs or the skeleton. Metabolites, in contrast, serve in an extensive broad range of functions within the cell. Metabolites are usually rapidly ‘‘converted’’ in enzymatic and chemical reactions, serve as building blocks for macromolecules or may serve as transient energy-storage. Therefore, the identiﬁcation, quantiﬁcation and the reactions of metabolites are important in the context of systems biology. Metabolomics is considered to be the study of the entire set of metabolites in a cell, tissue or organ sample [75–78]. In many respects, metabolites are the ﬁnal stage of biological cellular activity along the line from gene to mRNA to protein to function to phenotype (Fig. 4). Analytical approaches that take the chemical complexity and dynamic range of the metabolome into account employ usually an extraction of metabolites from a cell by diﬀerent techniques followed by parallel analyses of those subfractions. This strategy is required to segregate the metabolome into more manageable subclasses with similar chemical properties that also helps minimizing chemical side-reactions between them. The subclasses are subjected to parallel analytical techniques to record metabolite proﬁle information. Segregation of the subclasses while parallel analyses helps visualize a greater portion of the metabolome. In most cases the methods use classical chromatographic separation techniques that may comprise Fourier-transform infrared spectroscopy (FTIR), electrospray mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. A promising route to the metabolome is the comprehensive metabolic analysis coupled with statistical methods of cluster and phenotype analysis alike. An individual’s health status is rapidly reﬂected at the metabolic state. Thus, it might be possible for health-care and nutrition practitioners to make recommendations for a speciﬁc treatment or food for their condition. To reach this goal, a suitable database based on a large

66

Fig. 4. A network of metabolic pathways illustrating the complexity of metabolism as it is known today (excerpt reproduced with permission from Roche Applied Science’s Biochemical Pathways Michal: Biochemical Pathways, 1998 ß Spektrum Akademischer Verlag, Heidelberg, Berlin).

number of measurements of accurate metabolite concentrations from healthy people is required. Consequently, the development of a public metabolite atlas might be necessary. Speciﬁc quantiﬁcation of metabolites has been used to characterize metabolic processes in a multitude of focused metabolic pathways studies. The developed methods have been optimized to produce high-quality data that describe the compounds of interest. Today, these data constitute of the metabolic states of individuals. However, this type of analysis is poorly suited to simultaneously gathering information on the multitude of metabolites that characterize an organism’s nutritional processes. Another technique, metabolic proﬁling, has been devised to monitor, in parallel, hundreds or even thousands of metabolites, using high-throughput techniques. This is done to enable screening for relative changes rather than absolute concentrations of compounds. Most analytical techniques for proﬁling small molecules consists of HPLC or gas chromatograph (GC) coupled to mass spectrometry. Mass spectrometers are generally more sensitive and more selective than any other types of detectors. When coupled with the appropriate sample-introduction and ionization techniques, mass spectrometers can selectively analyze both organic and inorganic compounds. Nevertheless, the metabolites have to be separated prior to detection, by chromatographic techniques that are coupled online to the mass detector. Gas chromatography is used to separate compounds on the basis of their relative vapor pressures and aﬃnities for the material in the chromatography column, but is restricted to compounds that are volatile and heat stable. HPLC separations are better suited for the analysis of labile and high-molecular-weight compounds and for the analysis of non-volatile polar compounds in their natural form. The vast information gathered using high-throughput screening with GC-and HPLC-MS techniques require advanced informatics technologies for analysis. Yet proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy is dealing with metabolite proﬁling and allowing information to be gathered on the

67 ﬂow of metabolites through biological processes and the control of the pathways. High-resolution 1H-NMR spectroscopy, with the advantage of detection of any proton-containing metabolite, appears to become more important in the future in metabolite proﬁling. NMR-techniques have been used in the past mainly to analyze metabolite changes in mammalian body ﬂuids and tissues and this method may be extended by detecting other nuclei, for example 31P or naturalabundance isotopes such as 13C. When metabolomics is applied in studies where substrates enriched in 13C, metabolite analysis can even be taken onto a dynamic level by allowing the ﬂuxes to be determined quantitatively. Such automated biochemical proﬁling techniques will become an important component of multi-disciplinary integrated approaches in metabolic and functional genomics studies. The previously described technologies of genomics, transcriptomics and metabolomics, have produced a complete ‘‘parts-catalog’’ of the molecular components in many organisms. The next challenge would be to reconstruct and simulate the overall cellular functions. Recently, advances have been made in the area of ﬂux balance analysis and mathematical modeling [79]. Fundamental physicochemical laws and principles are used to systematically describe the living cell. However, serious limitations to this goal are the inability to rationally and exhaustively analyze biochemical networks and to accurately take all parameters into account, e.g., conservation of mass, energy and redox potential as well as mass transfer. An attempt to derive a global model of metabolisms of a cell is presented in the E-Cell software for cell simulation. Given a set of reaction rules and initial values, users can run simulations and observe dynamic changes in quantities and concentrations of intra- and extracellular metabolites and substances through graphical user interfaces. Activities of biochemical reactions can be monitored, as well as amounts of substances can be subject of change (increased/ decreased) by the users at any time during the simulation. e-Cell system makes it possible to conduct in silico metabolic experiments [80]. Furthermore, the availability of many annotated genomes paves the way for a systematic application of ﬂux-balance methods to a large variety of organisms. However, such a high-throughput goal crucially depends on the capacity to build metabolic ﬂux models in an automated fashion [81] (Table 6). Pulling it together The availability of genome sequences, expressed protein repertoires and identiﬁed metabolites for several organisms, including humans have allowed the transition from classic analytical biology to ‘‘systems biology.’’ In this new approach, biological processes of interest, mostly systems, are studied as complex networks of functionally interacting macromolecules and reactions. These functional genomics approaches can be helpful to accelerate the identiﬁcation of the genes and gene products involved in particular modules,

68 Table 6. Web resources and databases for metabolomics.

Metabolomics at University of Wales Aberystwyth (http://dbk.ch.umist.ac.uk/metabol.htm) Biochemical pathways (ExPASy) (http://us.expasy.org/tools/pathways/) Biopathways consortium (http://www.biopathways.org/) BRENDA, the Comprehensive Enzyme Information System (http://www.brenda.uni-koeln.de) EcoCyc and MetaCyc (http://www.ecocyc.org/) GeneCards (http://bioinformatics.weizmann.ac.il/cards/) KEGG – Kyoto encyclopedia of genes and genomes (http://www.genome.ad.jp/kegg/) E-cell project (http://www.e-cell.org/) Main metabolic pathways on Internet (http://home.wxs.nl/ pvsanten/mmp/main.htm) Metabolic Control Analysis (MCA) (http://dbk.ch.umist.ac.uk/mca_home.htm) MPI for Molecular Plant Physiology (http://www.mpimp-golm.mpg.de/ﬁehn/index-e.html) PathDB Biochemical Pathways (http://www.ncgr.org/pathdb/) Compugen’s Biocarta (http://www.biocarta.com/) Interactive metabolic reconstruction on the web WIT (http://wit.mcs.anl.gov/WIT2/) EMP Database of Enzymes and Metabolic pathways (http://emp.mcs.anl.gov)

Table 7. Useful databases for protein interaction

Database of Interacting Proteins DIP [86] (dip.doe-mbi.ucla.edu/) BIND [87] (http://bind.ca) PathCalling Yeast Interaction Database [63] (portal.curagen.com/) Mammalian protein–protein interaction database (PPI) [88] (fantom21.gsc.riken.go.jp/PPI/) Molecular Interaction database MINT [89] (160.80.34.4/mint/) General Repository Interaction Datasets GRID [90] (biodata.mshri.on.ca/grid/servlet/Index)

and to describe the functional relationships between them. However, the data emerging from individual ‘‘omic’’ approaches should be viewed with caution because of the occurrence of false-negative and false-positive results [82]. One of the problems biologists face is that the data set too large to comprehend in full. Novel and useful databases are being developed in recent times reﬂecting progress in diﬀerent aspects of genomics [83], prompting the saying that we live in ‘‘the age of databases.’’ In the new age of computational biology, it is not enough to publish scientiﬁc results in the literature, but the data has to be stored in a structured way both for retrieval and to connect to other resources on the web. Computer databases ﬁrst rose to prominence in life science as central repositories for nucleic acid and protein sequences. Their interrogation via e.g., the BLAST sequence search tool [84] is now performed frequently by biologists. After the establishment of GenBank in 1982 [85], many other databases have been developed that will be important for systems biology (Table 7). Some of these databases for example contain searchable indices of known protein-protein interactions. The current limiting factor in these databases however is the quality of information. High-quality information of validated protein–protein interactions

69 is so far only available for yeast [91] and the fruit-ﬂy [92]. Very few largescale high quality data sets for mammalian systems are available in the public domain. TRANSFAC [93] and SCPD [94] catalog interactions between proteins and DNA (i.e., transcription factor interactions), and databases of metabolic pathways have also recently been established e.g., EcoCyc [95], KEGG [96], and WIT [97]. A growing number of databases are under development for storing gene-expression data sets, as for example ArrayExpress [98], Gene Expression Omnibus [99] and the Stanford Microarray Database [100]. This recent explosion, in both the variety and volume of information of interest poses two challenges to database users and developers alike. First, the information must be maintained systematically in a format that is compatible with both single queries and global searches. Often, the desired information is present in the database but is not annotated consistently for all entries. We therefore need systems that integrate data globally [11]. Apart from computer-generated databases, high-quality databases require very often manual work of curators. This time intensive approach is well exempliﬁed in the Human Protein Reference Database (HPRD) [101] (www.hprd.org/). Information relevant to the function of human proteins in health and disease is collected including protein–protein interactions, post-translational modiﬁcations, enzyme/ substrate relationships, disease associations, tissue expression, and sub-cellular localization. The data is collected from more than >300,000 published articles for a non-redundant set of 2750 human proteins. The HPRD database as well as others of its kind put existing information in computer-readable format. They represent bioinformatics platforms that are useful in cataloging and mining the large number of proteomic interactions and alterations that are about to be discovered with systems biology approaches. Storing existing knowledge in structured ways is the key challenge and the cornerstone for the new biology (Fig. 5).

PEX14 SEC35 VMA22 TIP20

YPR105C YLR315W YMR181C YOR164C YOR331C

Fig. 5. Visualization of protein interaction using the PathCalling resource. The TIP20 protein, a transport protein that interacts with Sec20p, required for protein transport from the endoplasmic reticulum to the golgi apparatus, shows interaction with protein YPR105C, which itself interacts with many other proteins. This information allows for a rapid evaluation of the functionality of a protein within the context of whole proteome.

70

YBR093C

YAL038W YCR012W

YOL127W YIL0697

YIL13 YER074W YDR171W

YHR174W YGR254W YOL086C

YPL075W YLR127W

YOL120C YML024W

YDR050C

YNL301C YNL216W YIL0697 YER179W

YNL199C YPR048W

YPR048W

YPR048W

Fig. 6. Visualization of a selection of the 331 genes containing network described in Ref. [11] using Cytoscape version 1.1.1. Proteins were selected from the full yeast genome based on their having signiﬁcant expression change at least 1 of 20 conditions: The wild type (wt) strain and nine genetically altered yeast strains, perturbed environmentally by growth in the presence (+gal) or absence ( gal) of 2% galactose sugar. Each altered strain has a complete deletion of one of GAL genes, which encode proteins needed for the metabolism of galactose. Cytoscape is used to display all information regarding nodes (proteins) and edges (interactions). Here, nodes are represented by grey circles, and interactions/edges are represented by colored lines.

The most complex adventure we are facing now is to achieve a description of cellular biology. Current theories are able to capture and model only a small portion of the data at a time. General approaches to integrate, visualize and model information about cells that will help broaden biological understanding are necessary. To increase the reliability of gene function annotation, multiple independent datasets need to be integrated. Such integration will be crucial for systems biology to achieve its promise (Fig. 6). In order for databases to interact, and researchers to exchange information about their biological observations of a system, a common representationlanguage for storing biochemical models is required. The Systems Biology Markup Language (SBML) was created for that purpose [102]. It is a machinereadable format for the representation of computational models in systems biology. It is expressed in XML (www.w3.org/XML/), and contains structures for representing compartments, species and reactions, as well as optional unit deﬁnitions, parameters and rules. SBML will be crucial for the storage and exchange of data between databases. The rapidly expanding biological datasets of physical, genetic and functional interactions present a daunting task for data visualization and evaluation [103].

71 Completely new concepts are required in order to help scientists understand complex data. The Cytoscape software, for example, attempts to integrate biomolecular interaction networks with high-throughput expression data and other molecular states into a uniﬁed conceptual framework. It is applicable to any system of molecular components and interactions, and most powerful when used in conjunction with large databases of protein–protein, protein– DNA, and genetic interactions that are increasingly available for humans and model organisms. The tools provide functionality to layout and query interaction networks; visually integrate the network with expression proﬁles, phenotypes, and other molecular states and linking to databases for functional annotations. An important facet of the tool is that it is extensible through plug-ins, allowing rapid development of additional computational analyses [104]. Another approach is presented in the Osprey software [90] that represents interactions in a ﬂexible and expandable graphical format and provides options for functional comparisons between datasets. Systems biology involves interaction between experiment and simulation, attempting to create ever more accurate models of processes, such as the functioning of an organ over a period of time. Initially, a rough working model is created and used to design experiments that will verify or refute the predictions of that model. The model is modiﬁed to incorporate results and new simulations that in turn require further experiments. In this way, both the model and experiments evolve together until a satisfactory simulation can be achieved (Fig. 7). The above-mentioned databases are only covering the cellular level. However, the ﬁnal goal to capture information about individuals will require databanks TCTTGTCGCACGCAACTT TTGAGGATTTTTAAAGGG TGTCTATACCAAACGGA GAGGAGTAATGATGAGT GGTTAAGAATCCATACTT CAAGCAGAATTCGGGGC GGTTACCAAGCGAC

Biological question

DNA Cells

RNA

Vmax .[S]

In-silico experiment, simulation

[S]+ K m Models Networks

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v=

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500 400

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300

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Metabolites

200 100 0 600 1000 1400 1800 2200 2600 m/z, au

Biological System Databanks

Result: New insight

Fig. 7. Systems biology iterative research. Data about the living cell will reside in structured databases that are used to test-out new hypothesis and for proof of new models describing the system. In an iterative process, going back and forth between in-vitro, in-vivo, and in-silico experiments, new insight is created.

72 with information about people, biopsies or body ﬂuid samples, stored to be analyzed for genetic and biochemical assessment. The UK Biobank project (www.ukbiobank.ac.uk/) will pursue exactly this goal. Up to half a million participants aged between 45 and 69 years will be involved in the study. They will be asked to contribute a blood sample, lifestyle details and their medical histories to create a national database of unprecedented size. With such databases, fears and uncertainties dealing with ethical issues have surfaced and are under continuous debate. The concerns associated with single-gene disorders, such as privacy, conﬁdentiality, potential employment, or insurance discrimination and the rights of family members, are relevant. Additional factors include the nuanced meaning of genetic risk in complex diseases that result from genetic, environmental, and lifestyle interactions. The blurred boundary between medicine and genetic enhancement and the social implications of predicting diseases among a large fraction of the population, not to mention the gulf between identifying susceptibility and providing preventive treatment are subject of discussion. There is clearly a need to foster a public debate about the customization of diets or medical treatment to match the genetic proﬁles of consumers in the interest of preventing or managing chronic health conditions. This discussion needs to be initiated as fast as possible. To ease those fears, the latest decision of the US senate passed a bill, which would bar employers from using genetic information in making employment decisions, and prohibit health insurers from using genetic information to deny coverage or set rates [105] (Table 8). Health: the focus of systems biology ‘‘Let food be your medicine and medicine be your food.’’ Hippocrates, the father of modern medicine, c. 400 BC Biological research using molecular information on all cellular levels is addressing human health in a completely new ways. Disease prevention through Table 8. Web resources and databases for data-integration.

Physiome Project (http://www.physiome.org/) Systems Biology Software at the Keck Graduate Institute (http://www.cds.caltech.edu/ hsauro/) Virtual Cell Project of The National Resource for Cell Analysis and Modeling (http:// www.nrcam.uchc.edu/) E-Cell Project (http://www.e-cell.org/) Microbial Cell Project (http://microbialcellproject.org/) World Wide Web Instructional Committee Virtual Cell (http://www.ndsu.nodak.edu/instruct/ mcclean/vc/) Cytoscape (www.cytoscape.org/) GoMiner (http://discover.nci.nih.gov/gominer/) Database of functional networks at EMBL (http://www.ebi.ac.uk/research/pfmp/)

73 nutritional intervention and/or intelligent medical treatment is realized to be crucial for increased human quality of life. The combination of individual assessment of health status and the resulting personalized interventions can be envisaged in this decade. It can be estimated that by the year 2010 predictive genetic test will be available for as many as a dozen common disorders [106]. Individuals who choose to learn about their susceptibility to these diseases will be able to use this information to take preventive measures. For example, a woman at increased risk for developing breast cancer may want to have more frequent mammograms. A man susceptible to coronary heart disease may take medication to lower his cholesterol. Other people may reduce their risk for disease by changing their diet, getting more exercise and avoiding environmental agents that trigger disease. Genes are being identiﬁed that inﬂuence how a person responds to a given drug. Increasingly, doctors will prescribe drugs based on the genetic proﬁles of their patients. This individualized treatment will allow using the drug most likely to treat disease symptoms and also to minimize adverse drug reactions. Such an approach will usher in an era of personalized medicine. The tools of systems biology, by virtue of measuring all constituents of an organism, will have large implications for disease prevention via diet or other environmental factors such as lifestyle. Understanding human health will depend of a holistic view of our body’s biology and the numerous environmental cues to which we are constantly exposed. These include pollutants, toxins, pathogens, commensals and also radiation. Our gastrointestinal system, for example, is the organ with greatest contact to our environment; it is inhabited with a large number of bacteria, termed the microbiome [107]. Exploring the human microbiome during the diﬀerent states of health, using molecular techniques have partly been initiated. These studies will lead to crucial insights about the relationship of the micro cosmos in our gut and us. These surveys are important for a number of reasons [108]. As adults, our total microbial population that is residing mainly in the intestine is composed of 500 to 1000 species. Their total number is at least one order of magnitude bigger than our somatic and germ cells altogether, with their total number of genes exceeding our own genes by a factor of 100. The microbiota residing in our body functions as a multifunctional organ with multiple implications for our health. In addition to the numerous but poorly characterized beneﬁcial eﬀects of the endogenous microﬂora on human health, a proper understanding of abundance and variations therein will be critical for recognizing potential patterns that are predictive of health or disease. We have virtually no information on the levels of microbial diversity and abundance that are optimal for maintenance of human health, or of those that are associated with disease. With only few gut microorganisms sequenced [109–112] we are just starting to learn about microbial partitioning within human micro-environments. We still understand little about inter-individual variability or variability as a function of time. Gut bacteria have also been

74 Duodenum and Jejunum: 102-105 cfu ml−1 Lactobacillus Streptococcus Bifidobacterium Enterobactericeae Staphylococcus Yeast

Ileum and Caecum: 103-109 cfu ml−1 Bifidobacterium Bacteroides Lactobacillus Streptococcus Enterobactericeae Staphylococcus Clostridium Yeast

Stomach: 100-103 cfu ml−1 Lactobacillus Streptococcus Staphylococcus Enterobactericeae Yeast

Colon: 1010-1012 cfu g−1 Bacteroides Eubacterium Clostridium Peptostreptococcus Streptococcus Bifidobacterium Fusobacterium Lactobacillus Enterobactericeae Staphylococcus Yeast

Fig. 8. Composition of the human gastro-intestinal micro-biota. The overall number of microorganisms in our body is estimated to be bigger than the number of all our somatic and germ cells [108,114,115].

implicated in colon cancer development, but their role in tumor invasion, which is modulated by environmental factors, has been unclear. One report states that a metalloprotease from Listeria monocytogenes, in combination with a host protease, produces a peptide that stimulates motility and invasion of colon cancer cells [113]. The pro-invasive factor was identiﬁed as peptide derived from bovine b-casein. This peptide could be generated in vitro by the combined actions of the L. monocytogenes metalloprotease Mpl and a trypsin-like serine protease present in the collagen used in the cell invasion assay. That data shows convincingly that the combined action of diet, bacteria and host elements can produce health impairments. Thus, detailed knowledge about the somatic and germ cells which make up our corpus has to be extended with knowledge about the microbiome and its interaction with our body (Fig. 8). Intensive research has focused in the past on protection of individuals from various stresses using food ingredients such as anti-oxidants [116]. However, a recent report underlines the signiﬁcance of natural products for human health apposed to puriﬁed ingredients and especially their importance for prevention of disease. Lycopene, a carotenoid found in tomato products, was long known for its anti-oxidant eﬀects [117]. It is used frequently as a puriﬁed additive to foods. In this new study [118] tomato powder was shown to inhibit the development of prostate cancer compared with a control diet, whereas a diet containing a pure synthetic lycopene supplement did not. The authors also found that the equally measured restriction on energy intake due to experimental conditions during the experiment produced a reduction in prostate cancer mortality that was independent of the eﬀect of tomato powder. This new

75 Soul

Food Drugs Pollu tants

Body composition

Genome

Health

Exogenous bacteria

Age Disease

Stress Lifestyle Other factors

Microflora

Physiology

Fig. 9. Interaction of the environment with the human body. Environmental factors will inﬂuence healthy state of an organism taking individual genetic factors into account. Genetic predisposition may lead to body-dysfunction later in life that is modulated by nutrition and other environmental factors.

study is important on several levels. Perhaps most important, it weighs heavily in the debate about whether cancer prevention is best achieved via whole foods versus via single compounds. Another striking aspect is that caloric restriction can readily lead to disease prevention [119]. It has to be realized from this study that the ultimate biologic activity of a given food or nutrient depends on a large number of variables, including food processing and preparation method, gastrointestinal tract physiology, interactions between compounds in the food, and interactions between foods eaten together at the same meal. Clearly, we have barely begun to scratch the surface of understanding how the nutrient compounds within natural food interact within our biologic systems. The promise of systems biology is to grasp the potential complexity of the relevant eﬀects in humans, untangling these interactions in the laboratory (Fig. 9, Table 9).

Conclusions and outlook What has systems biology achieved for the complete description of human biology? With all the advances we have to realize that getting from a gene to a human being is not as straightforward as some had hoped. Although a starting point of a genomic approach to health research is identifying the mechanisms how components interact in a healthy state and also which genes

76 Table 9. Web resources for health genomics.

European Nutrigenomics Organisation NuGO (www.nugo.org) Nutrigenomics.UCDavis.edu (nutrigenomics.ucdavis.edu) The Centre for Human NutriGenomics (http://www.nutrigenomics.nl/) The IFR Food and Health Network (http://www.foodandhealthnetwork.com/) Nutrition, Metabolism and Genomics Group http://nutrigene.4t.com Center for Nutrigenomics TU Munich (http://www.nutriogenomics.com/) Human Genome Project Information from the DOE (link) NCBI Science primer Pharmacogenomics (http://www.ncbi.nlm.nih.gov/About/primer/ pharm.html) International Society of Pharmacogenomics (www.pharmacogenomics.org.uk/) PharmGKB (http://www.pharmgkb.org)

are associated with disease, the sheer complexity of our biology is projecting this goal far out into the future. Increasing evidence suggests that the genetic makeup may partially explain why people of diﬀerent ancestry experience disease or metabolize nutrients diﬀerently. Yet, these genetic clues have to become ﬁrm enough to guide medical practice. Genomics, proteomics, and bioinformatics are just beginning to inﬂuence the practice of medicine, most notably in diagnosis of disease and development of drugs and recommendations for nutritious foods. To accelerate this inﬂuence, physicians must be better prepared. They need to understand the nature of the tests and the kinds of information from which they will make clinical inferences and assist patients in making clinical decisions, always taking cultural and ethical considerations into account [120]. Translating genomic information into successful clinical trials will require advances on several fronts. Despite extensive preclinical studies, the vast majority of clinical trials fail because the drugs do not work as anticipated in patients or lead to intolerable side eﬀects, mainly due to the lack of basic information about physiology and the diﬃculty to predicting which treatments is likely to succeed. Current medical practice treats illnesses after they appear. However, with the extended human lifespan, averting one illness enables a person to live long enough to contract another. Therefore, disease prevention and how to reach a global healthy state of our body must be the new focus of research. Nutrition and life-style of individuals can play a primary role in that battle. At some point in the future, genomic information and individual susceptibility data will be part of our healthcare system in which we will try to intervene or prevent at the earliest possible time, rather than what we are doing now, that is treating after an event occurred. Crucial factors for our new health-care consciousness of the public will be genome, proteomic, metabolic and informatics technologies, moving away from the reactive ‘‘ﬁx-it’’ medical treatment towards a proactive, prospective and preventive medicine. This new health concept will start with a personalized assessment of individual nutritional status, environmental factors and life-style factors like sport and risk to disease and ﬁnalize in an individual lifestyle and healthcare plan.

77 Therefore, we need to obtain detailed knowledge about how proteins participate in the physiological processes in our bodies. We must know what the normal healthy state of our body is and how we might intervene to prevent any inconvenient condition of health. As for predictive medicine, we will require extensive information to analyze the genetic contribution to disease and, at least for the common aﬄictions, we will need to know the environmental components as well. Nutrition can certainly help in dealing with the growing problem of obesity. But how can we change the eating habits of our children? Should we exercise greater control over what people eat? But do we know which diet is adapted for us? How can we judge if there is too few scientiﬁc data. Clearly, the completion of our genome sequence is not the end of our quest, neither is it the beginning; maybe, it is only the end of the beginning. References 1. Wolkenhauer O. Systems Biology: The Reincarnation of Systems Theory Applied in Biology?. New York, Columbia University Press, 2001, pp. 258–270. 2. Rosen R. Essays on life itself. New York, Columbia University Press, 1999. 3. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, StangeThomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, DoucetteStamm L, Rubenﬁeld M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Raymond C, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la BM, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP,

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85

Public health issues related with the consumption of food obtained from genetically modiﬁed organisms Andrea Paparini1 and Vincenzo Romano-Spica1,2,* 1

University of Rome ‘‘Foro Italico’’ (IUSM), Rome, Italy Catholic University Medical School, Rome, Italy

2

Abstract. Genetically Modiﬁed Organisms (GMOs) are a fact of modern agriculture and a major ﬁeld of discussion in biotechnology. As science incessantly achieves innovative and unexpected breakthroughs, new medical, political, ethical and religious debates arise over the production and consumption of transgenic organisms. Despite no described medical condition being directly associated with a diet including approved GM crops in large exposed populations such as 300,000,000 Americans and a billion Chinese, public opinion seems to look at this new technology with either growing concern or even disapproval. It is generally recognized that a high level of vigilance is necessary and highly desirable, but it should also be considered that GMOs are a promising new challenge for the III Millennium societies, with remarkable impact on many disciplines and ﬁelds related to biotechnology. To acquire a basic knowledge on GMO production, GM-food consumption, GMO interaction with humans and environment is of primary importance for risk assessment. It requires availability of clear data and results from rigorous experiments. This review will focus on public health risks related with a GMO-containing diet. The objective is to summarize state of the art research, provide fundamental technical information, point out problems and perspectives, and make available essential tools for further research. Are GMO based industries and GMO-derived foods safe to human health? Can we consider both social, ethical and public health issues by means of a constant and eﬀective monitoring of the food chain and by a clear, informative labeling of the products? Which are the so far characterized or alleged hazards of GMOs? And, most importantly, are these hazards actual, potential or merely contrived? Several questions remain open; answers and solutions belong to science, to politics and to the personal opinion of each social subject. Keywords: biotechnology, food safety, genetically modiﬁed organisms, genetically engineered organisms, genetically manipulated organisms, transgenic, animals, plants, horizontal transfer, DNA uptake, DNA intake, genetic modiﬁcation, genetic manipulation, novel food, GMO, GE, crops, recombinant, agriculture, food allergies, diet, regulations, labeling, food intolerance, antinutrients, herbicide tolerance, insect-resistant, EPSPS, BT, BAR, cry, nptII, public health, antibiotic resistance.

Introduction For thousands of years, thoroughly unaware of even the existence of nucleic acids, farmers and plant breeders have been performing a rudimental yet eﬀective form of gene transferring and selection, a process called genetic ‘‘manipulation’’ or ‘‘modiﬁcation.’’ Breeding animals is an example, but most commonly crossing and saving seeds of the strongest or most fruitful plants enabled to slowly maintain or improve the most desired characteristics for farming. As all crop and domesticated animal species have undergone human selection since the dawn *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10004-5

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

86 of time, they can all be considered ‘‘genetically modiﬁed’’ with respect to the wild types, even if old farmers did not know they were selecting genes. Today, humans have knowledge of the underlying mechanisms and, above all, can manipulate genetic information. The general principle is that the genetic code is universal as also the mechanisms involved in transcription and translation. Among the several advantageous traits, that drove natural or genetically engineered (GE) selection, there was an improved growth and yield, an enhanced parasite or herbicide resistance, a better nutritional value or quality. Employing novel and more powerful instruments and knowledge, modern agronomists and zootechnologists are performing the same old process, somehow, trying to reach the same old goals. The ability to modify a speciﬁc organism at a molecular level represents the newest instrument of the contemporary form of genetic selection. New promising perspectives arise, but also new questions and concerns. The recombinant DNA revolution discloses key developments for the societies of the ‘‘biotech’’ Millennium. By means of the recombinant DNA (rec-DNA) technology, it is now possible to remove or introduce a new trait, in a plant or animal, in a much faster and more precise fashion than ever. Furthermore, novel biomolecular strategies allow scientists to selectively control, or prevent the transgene escape from genetically modiﬁed plants (Table 1) [1,2]. About three decades ago, the recombinant DNA technology broke into the scientiﬁc scene with its impressive impact. In 1973, Stanley Cohen and

Table 1. List of useful websites. Each listed website address is intended to begin with ‘‘http://www.’’. FDA U.S. Food and Drug Administration fda.gov/ cfsan.fda.gov/ lrd/biotechm.html cfsan.fda.gov/ lrd/biotechm.html#inf cfsan.fda.gov/ lrd/biotechm.html#prod cfsan.fda.gov/ lrd/biotechm.html#label cfsan.fda.gov/ lrd/biotechm.html#reg fda.gov/cvm/biotechnology/bioengineered.html

FDA Home Page Biotechnology Information for Consumers Completed Consultations on GM foods Food labelling Regulations and Guidance on Safety Assessments Biotechnology in Animals and Feeds

EPA U.S. Environmental Protection Agency epa.gov/ epa.gov/ebtpages/pesticides.html epa.gov/pesticides/biopesticides epa.gov/ebtpages/pestpesticherbicides.html epa.gov/epahome/lawregs.htm epa.gov/ebtpages/treatechnobiotechnology.html epa.gov/ebtpages/humafoodsafety.html

EPA Home Page Pesticides Biopesticides Herbicides Laws and Regulations Biotechnology Food Safety

(Continued)

87 Table 1. (Continued) USDA U.S. Department of Agriculture usda.gov/ usda.gov/agencies/biotech/index.html

USDA Home Page Agricultural biotechnlogy

APHIS – USDA animal and Plant Health Inspection Service (USDA) aphis.usda.gov/ aphis.usda.gov/programs/programs.html aphis.usda.gov/programs/biotechregsvcs.html

APHIS Home Page APHIS Programs Biotechnology Regulatory Services

GIPSA – USDA Grain Inspection, Packers and Stockyards Administration (USDA) usda.gov/gipsa/biotech/biotech.htm GIPSA Home Page WHO World Health Organization who.int/en/ who.int/health_topics/food_safety/en/ FAO Food and Agriculture Organization of the United Nations fao.org/ fao.org/ag/ fao.org/ag/guides/subject/b.htm fao.org/biodiversity/index.asp?lang ¼ en fao.org/biotech/index.asp?lang ¼ en fao.org/ethics/index_en.htm fao.org/ag/AGA/AGAP/FRG/Feedsafety/ feedsafety.htm http://apps.fao.org/ EU European Union europa.eu.int/index_en.htm europa.eu.int/pol/food/index_en.htm europa.eu.int/comm/biotechnology/ introduction_en.htm europa.eu.int/comm/index_en.htm europa.eu.int/pol/agr/index_en.htm europa.eu.int/pol/food/index_en.htm europa.eu.int/comm/food/food/ biotechnology/index_en.htm europa.eu.int/comm/food/food/foodlaw/ principles/index_en.htm europa.eu.int/comm/environment/index_en.htm europa.eu.int/comm/food/food/biotechnology/ novelfood/index_en.htm europa.eu.int/comm/food/plant/ gmplants/index_en.htm europa.eu.int/comm/food/food/biotechnology/ strategy/index_en.htm europa.eu.int/comm/food/food/biotechnology/ gmfood/legisl_en.htm europa.eu.int/comm/food/food/biotechnology/ authorisation/list_author_gmo_en.pdf europa.eu.int/comm/food/food/ labellingnutrition/foodlabelling/index_en.htm

(Continued)

WHO Home Page Food safety

FAO Home Page Agriculture Biotechnology Biological Diversity in Food and Agriculture Biotechnology in Food and Agriculture Ethics in Food and Agriculture FAO Feed and Food Safety Gateway FAO Statistical Databases

EU Home Page Food Safety Biotechnology European Commission European Commission European Commission European Commission feed safety European Commission General Food Law European Commission European Commission

– Agriculture – Food Safety – Food and – Principles of – Environment – Novel Food

European Commission – GM plants and seeds European Commission – Strategy for Europe on Life Sciences and Biotechnology European Commission – Legislation of GM Food & Feed Genetically modiﬁed (GM) foods authorised in the European Union European Commission – Food Labelling

88 Table 1. (Continued) Colorado State Univesity colostate.edu/programs/lifesciences/ TransgenicCrops/index.html colostate.edu/programs/lifesciences/ TransgenicCrops/terminator.html colostate.edu/programs/lifesciences/ TransgenicCrops/hotlabel.html colostate.edu/programs/lifesciences/ TransgenicCrops/hotrice.html colostate.edu/programs/lifesciences/ TransgenicCrops/current.html colostate.edu/programs/lifesciences/ TransgenicCrops/future.html colostate.edu/programs/lifesciences/ TransgenicCrops/risks.html colostate.edu/programs/lifesciences/ TransgenicCrops/defunct.html

Transgenic crops Terminator Technology Food Labelling Golden Rice Transgenic Crops Currently on the Market Future Transgenic Products Risks and Concerns Discontinued Transgenic Products

ISAA International Service for the Acquisition of Agri-biotech Applications isaaa.org/ ISAA Home Page isaaa.org/Publications/pubs.htm ISAAA Publications isaaa.org/Publications/Downloads/ James, C. 2000. Global Status of Briefs%2021.pdf Commercialized Transgenic Crops isaaa.org/Publications/Downloads/ James, C. 2001. Global Status of Briefs%2024.pdf Commercialized Transgenic Crops isaaa.org/Publications/Downloads/ Brookes G and Barfoot P. GM Rice: Briefs%2028.pdf Will This Lead the Way for Global Acceptance of GM Crop Technology? Union of Concerned Scientists ucsusa.org/ Union of Concerned Scientists Home Page ucsusa.org/food_and_environment/ Biotechnology biotechnology ucsusa.org/food_and_environment/ Engineered foods allowed on the market biotechnology/page.cfm?pageID ¼ 337 GMO-Watch Internet site of the Biosafety Assessment, Technology and Sustainability (BATS) Institute gmo-watch.org GMO-Watch Home Page gmo-watch.org/GVO-report140703.pdf Bruderer S and Leitner KE. Modiﬁed (GM) Crops: molecular and regulatory details. PBS pbs.org/wgbh/harvest/

University of Sussex http://www.biols.susx.ac.uk/Home/ Neil_Crickmore/Bt/index.html boils.susx.ac.uk/home/Neil_Crickmore/ Bt/toxins2.html (Continued)

‘‘Harvest of fear’’ – Exploring the intensifying debate over genetically-modiﬁed (gm) food crops. Bacillus thuringiensis toxin nomenclature Bacillus thuringiensis delta-endotoxin list

89 Table 1. (Continued) FURTHER DOCUMENTS AND LINKS General topics ncbi.nlm.nih.gov ncgr.org cast-science.org iﬁc.org croplifeamerica.org usinfo.state.gov/gi/global_issues/ biotechnology.html biome.ac.uk betterfoods.org fb.org bioigene.it/biotech iss.it Commercial websites agbios.com agbios.com/dbase.php aventis.com monsanto.com mycogen.com Bayer.com bejo.com/ pioneer.com Syngenta.com seminis.com dupont.com

National Center for Biotechnology Information National Center for Genome Resources Council for Agricultural Science and Technology International Food Information Council CropLife America U.S. State International Information Programs BIOME The Alliance for Better Foods American Farm Bureau IUSM Biotechnology Home Page Istituto Superiore di Sanita` AgBios Home Page AgBios – GM Crop Database Aventis Home Page Monsanto Home Page Mycogen Home Page Bayer Bejo Zaden Pioneer Syngenta Seminis Vegetable Seed DuPont

Herbert Boyer, developed the technique of DNA cloning, which allowed genes to be manipulated and transferred between diﬀerent biological species [3,4]. Their discovery marked the birth of genetic engineering together with the discovery by Temin and Baltimore of the reverse transcriptase [5,6]. Almost 10 years later, in 1982, the ﬁrst transgenic mice were obtained by microinjection, into fertilized mouse eggs, of a DNA fragment containing the promoter of the mouse metallothionein-I gene fused to the structural gene of rat growth hormone [7]. Successively, in 1988 the ﬁrst tobacco plant was successfully transfected. Thereafter, it only took about two years for genetically engineered crops to enter food production, in the early 1990s and only another decade for the total area, cultivated with GM crops in the world, to reach 44.2 million hectares in 2000 [8]. Between the 1970s and the early 1980s, molecular biology and genetic engineering have been continuously, rapidly and eﬀectively contributing to biology, medicine and biotechnologies. This evolution represents an example of the rapid reduction in the gap between basic research advancements and know-how applications, almost reciprocally overlapping in the biotech ﬁelds, showing potentialities, concerns and promises for the next millennium.

90 Recombinant DNA technology enabled scientists to change an organism’s genetic endowment by direct manipulation of DNA sequences. This procedure, also known as genetic engineering, involves (i) elimination (e.g., knock out animals) or (ii) introduction of speciﬁc foreign genes, even belonging to unrelated species. In particular, the latter strategy (ii) produces a ‘‘transgenic’’ organism, in which a foreign DNA (a transgene) is incorporated into the genome during an early stage of development. The transgene is present in both somatic and germ cells, is expressed in one or more tissues and is inherited by the oﬀspring in a Mendelian fashion. A gene, to be transcribed by the cell, requires regulatory sequences: a promoter and a terminator. These genetic elements determine the activity of a gene and the time and modalities of its expression. The product of a speciﬁc coding sequence can be modulated or ‘‘switched on’’ or ‘‘of’’ by the presence or absence of such an element. Therefore, a broader deﬁnition of ‘‘transgenesis’’ includes the introduction of foreign regulatory sequences in the hosting organisms, and not only the speciﬁc coding sequences. Transgenic plants Creation of the ﬁrst transgenic plants dates back to the early 1980s, when four groups working independently at Washington University in St. Louis, Missouri, the Rijksuniversiteit in Ghent, Belgium, Monsanto Company in St. Louis, Missouri, and the University of Wisconsin successfully inserted foreign genes in plant cells. Their scientiﬁc achievements were then published in three diﬀerent journals, in 1983 [9–12]. Today, transgenic plants are currently produced by introduction of genes conferring several properties such as: resistance to insects, viruses or herbicides, improved nutritional value and ﬂavor, resistance to environmental stresses (such as drought, salinity, pollution, extreme temperatures), capacity to produce heterologous substances with pharmacological properties, prolonged organoleptic stability, extended conservation and improved value of ﬂowers (ﬂoriculture). Noteworthy, not all of the above traits are already present in commercially available plants or employed in edible crops. Indeed, some of them are just successful applications of recombinant DNA technology, may have an exclusive scientiﬁc value or be yet only at an experimental phase. Furthermore, several transgenic food products that received approval for marketing have been discontinued for a variety of reasons, even after being available on the market for years. A few examples of such abandoned products can be found on the University of Colorado website (Table 1). Examples of discontinued foods include transgenic tomatoes that soften more slowly than conventional, a tomato paste made of another line of transgenic tomatoes with the same trait, some insectresistant potato lines, a herbicide-tolerant ﬂax, some insect-resistant corn lines. Interestingly, beside various clear failures based on either medical or environmental issues, other products that showed satisfactory results during trials,

91 Identification of the desired trait

Identification of the source of the gene (donor organism)

Isolation of the gene from that source

Adjustment of the gene to confer the desired trait

Transfection of the plant

Test for the presence of the desired trait

Field trials, to make sure that :

Initiation of product safety trials

1) There are no detrimental effects of the gene 2) The gene works the way it was conceived

Transmission to regulatory agencies as required Fig. 1. Development of a transgenic plant.

were discontinued only due to the reluctance of buyers and/or the adverse public opinion about GM-food. Transgenic plants, currently available on the market, include corn, tomato, potato, rape soybeans, maize, canola, potatoes and papayas (Table 1) [8,13]. A new transgenic crop can be developed through a complex serial procedure (Fig. 1). As shown in Fig. 2, once a speciﬁc gene of interest has been chosen and isolated, the transfection may be carried out by several diﬀerent strategies: (1)

Agrobacterium tumefaciens. This method involves the use of a plant parasite, A. tumefaciens that is a well-known bacterium, causing large tumors in some dicotyledons. Its infectious capacity is associated with the

92 Isolation of the desired gene a) A. tumefaciens method

b) Gene gun method

c) Other strategies Gene inserted intoTi plasmid Particles coated with DNA and transformation of A. tumefaciens

Cells shot with Gene gun and Bacterium mixed with plant cells. DNA incorporated into Ti plasmid moves into cells and inserts plant chromosome DNA into plant chromosome Selection and screening of transformed cells. Regeneration of the plant from a single transformed cell Fig. 2. Alternative methods of plant transformation.

(2)

(3)

presence of a plasmid called Ti (Tumor inducing), which is eventually transferred to the infected plant cell, and that carries the tumor-associated genes. A coding sequence of the plasmid is then integrated into the host chromosome and hence inherited by all the cells [14]. This phenomenon results in permanent cell transformation and unregulated massive growth (tumor). By substituting the naturally integrating region of the Ti plasmid with the transgene of interest, it is possible to insert foreign genes in plant cells infected by A. tumefaciens and to obtain a transgenic organism. A disadvantage of this system is that the bacterium does not infect all plant species. Gene gun. The gene gun method circumvents the host-restriction limitation, typical of the A. tumefaciens method. Tiny gold or tungsten particles are preventively coated with the DNA fragment to be inserted, and successively shot into the cell [15]. Once DNA enters the cell, it becomes free to integrate into the host chromosome. When the integration occurs, permanent transfection of the host plant cell is achieved. Further approaches, commonly used for plant transformation, include inﬁltration, electroporation of cells and tissues, direct protoplast transformation, electrophoresis of embryos, microinjection, pollen-tube pathway, silicon carbide- and liposome-mediated transformation [15]. In particular, electroporation was ﬁrst employed in 1985, to transfect tobacco and maize protoplasts [16], whereas transformation of tobacco protoplasts by direct DNA microinjection was carried out about a year later [17].

93 The nucleic acid fragment that is used to transfect the host plant cell is formed by one or more units, each containing three genetic elements: the promoter, the transgene and the terminator [13]. The whole unit is called ‘‘gene cassette’’ and the promoter and the terminator represent its regulatory sequences. The promoter of the transgene is one of the most critical choices to make, when a genetically modiﬁed organism is to be constructed. It locates at the 50 end of the gene cassette and its sequence can aﬀect the expression level of the transgene, the histological fate of the product and the time of the synthesis. Most of the manipulated crops, approved and commercialized today, utilize the 35s constitutive promoter (P-35s) of the Cauliﬂower Mosaic Virus (CaMV), although its sequences, available from several sources (e.g., patents, gene bank or petitions), show some diﬀerences when compared. Table 2 and Fig. 3 include a list of promoters used in transgenic crops and their occurrences.

Table 2. The frequency of occurrence of introduced promoters into approved GM crops. Used promoters

Donor organisms (origin)

An anther speciﬁc promoter Bacterial dP-35s E-OCS nda P-35s P-4AS1 P-5126del P-ALS P-Als P-CDPK P-E35s P-E8 P-FMV P-HelSsu P-Kti3 P-mac P-mas P-napin P-nos and 2 P-nos P-OCS,35s P-PCA55 P-PEPC P-Ptac P-ract P-Ssu P-TA29

/ / Cauliﬂower Mosaic Virus Agrobacterium tumefaciens / Cauliﬂower Mosaic Virus Cauliﬂower Mosaic Virus Zea mays Nicotiana tabacum Arabidopsis thaliana Zea mays Cauliﬂower Mosaic Virus Lycoperiscon esculentum (tomato) Figworth Mosaic Virus Heliantus annus Glycine max (soybean) A. tumefaciens and Cauliﬂower Mosaic Virus Agrobacterium tumefaciens Brassica rapa Agrobacterium tumefaciens Cauliﬂower Mosaic Virus and A. tumefaciens Zea mays Zea mays Bacterial Oryza sativa (rice) Arabidopsis thaliana Nicotiana tabacum

(Continued)

Number of occurences of each promoter 2 22 1 1 1 42 1 1 1 1 1 12 1 8 1 1 1 1 1 10 1 1 1 1 2 9 6

94 Table 2. (Continued) Used promoters

Donor organisms (origin)

P-ubiZM1(2) P-b-Conglycinin

Zea mays Glycine max (soybean)

Number of occurences of each promoter 1 1

The donor organisms of promoters are indicated. Some promoters may be present in more than one copy in a single product, since a regulatory sequence may have been used for more than one transgene and since several copies of a transgene may be present in the same product. This frequency of appearance is not taken into account in the table. dP-35s: double 35s promoter, promoter region from Cauliﬂower Mosaic Virus. The double (d) represents a duplicated region in the promoter. E-OCS: octopine synthase enhancer from A. tumefaciens Ti plasmid, pTiACH5. nda: No Data Available. P-35s: 35s Cauliﬂower Mosaic Virus promoter. P-4AS1: promoter containing four tandem copies of AS1 (activating sequence 1) and a single portion of 35s Cauliﬂower Mosaic Virus promoter synthetic polylinker sequence. P-5126del: a modiﬁed Z. mays anther speciﬁc promoter. P-ALS: tobacco ALS1 promoter. P-PCDK: promoter derived from a corn calcium dependent protein kinase (CDPK) gene. P-E35s: 35s promoter from the Cauliﬂower Mosaic Virus with the duplicated enhancer region. P-E8: ethylene responsive gene promoter. P-FMV: a promoter derived from Figworth Mosaic Virus (FMV). P-HelSsu: RuBisCo SSU (ribulose-1,5-bisphosphate carboxylase small subunits 1A) promoter, from Helianthus annuus. P-Kti3: Kunitz trypsin inhibitor 3 (Kti 3) promoter. P-mac: P-mas and P-35s hybrid. P-mas: promoter region of mannopine synthase gene of pTiA6. P-napin: the promoter of the nopamin gene from Brassica rapa which functions in developing seeds. P-nos: promoter region of the nopaline synthase gene. P-ocs: promoter of the octopine synthase gene. P-PCA55: the promoter region of the anther speciﬁc gene CA55 from Zea mays. P-PEPC: green tissue-speciﬁc phosphoenolpyruvate carboxylase (PEPC) promoter from corn. P-Ptac: bacterial Ptac promoter. P-ract: 50 region of the rice actin 1 gene containing the promoter and ﬁrst intron. P-Ssu: (also called P-SsuAra) the A. thaliana ribulose-1,5-bisphosphate carboxylase small subunits1A promoter. P-TA29: the promoter region of anther-speciﬁc gene TA29 from Nicotiana tabacum. P-ubiZM1(2): the ubiquitin promoter plus ubiquitin intron and a 50 untranslated region from Z. mays. P-b-Conglycinin: seed-speciﬁc promoter derived from the a0 -subunit of the Glycine max b-Conglycinin gene (modiﬁed from Bruderer S and Leitner KE, 2003).

More than 39 genes have already been used in currently approved GM crops. Among the most common are the bacterial neomycin-phosphotransferase II gene (nptII), the phosphinothrycin acetyl transferase from S. hygroscopicus (BAR) the cry gene and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, which will be described later. Table 3 includes a list of transgenes and the donor organisms, whereas their frequency of occurrence is depicted in the Fig. 4. The third element of the gene cassette is the terminator. It functions as a regulatory sequence controlling the halt of the transcription by RNA polymerase and the poly-adenylation signal. It situates at the 30 end of the transgene. Table 4 and Fig. 5 include the terminators most commonly found in GMOs and their frequency of occurrence. Improved resistance to parasites The cry gene encodes a bacterial insecticidal crystal protein (Cry protein or ICP) ﬁrst described in the gram-positive soil bacterium Bacillus thuringiensis (Bt) [18,19]. To be toxic, the Cry protein must be ingested by the insect larva.

95 Bacterial (16.5%)

nda (0.8%) P-FMV (6.0%)

P-35s (41.4%)

P-nos and 2xP-nos (7.5%) P-Ssu (6.8%) P-TA29 (4.5%) Others (16.5%) Fig. 3. Frequency of occurrence of the most often used promoters in the currently approved genetically engineered crop plants. P-35s: 35s Cauliﬂower Mosaic Virus promoter. P-35s includes P-35s, P-E35s and dP-35s. nda: No Data Available. P-FMV: Figworth Mosaic Virus Promoter. P-nos and 2 P-nos are, respectively, the promoter region of the nopaline synthase gene, from A. tumefaciens, and the tandem duplicate promoter region of the nopaline synthase gene, from A. tumefaciens. P-Ssu: A. thaliana ribulose-1,5-bisphosphate carboxylase small subunits 1A promoter. P-TA29: promoter region of anther-speciﬁc gene TA29 from Nicotiana tabacum (modiﬁed from Bruderer S and Leitner KE, 2003).

The mechanism of action involves solubilization of the crystal in the insect midgut, proteolytic processing of the protoxin by midgut proteases, binding of the Cry toxin to midgut receptors, and insertion of the toxin into the apical membrane to create ion channels or pores [20]. Lethality is believed to be due to destruction of the transmembrane potential, with the subsequent osmotic lysis of cells lining the midgut [21]. Each B. thuringiensis strain synthesizes up to ﬁve diﬀerent Cry proteins. Besides them, cytolysins (Cyt toxins), further toxins that act by a diﬀerent mechanism, are also found within the crystal. Both these two classes of toxins are referred as delta-endotoxins (d-endotoxins). Other than d-endotoxins, Bt produces also various further virulence factors, including secreted insecticidal protein toxins, alfa-exotoxins, beta-exotoxins, hemolysins, enterotoxins, chitinases and phospholipases. The Cry proteins have diﬀerent speciﬁcity and their combination within a given strain, deﬁnes the activity spectrum of that strain [22]. The ecology of Bt is not fully understood yet. Discording hypothesis have been postulated about the evolutionary advantage for the bacterium, associated with the production of the toxin, but according to Aronson et al., the bacterium is likely to have a subtle symbiotic interaction, perhaps with plants, to account for the extensive production of the highly speciﬁc and eﬃcacious toxins [21]. Several subfamilies of the cry gene have been discovered, named and classiﬁed and in 1993, a B. thuringiensis d-endotoxin nomenclature committee was created

96 Table 3. Frequency of occurrence of introduced genes in approved GM crop plants with the corresponding donor organisms. Multiple insertions of a gene into a genome were counted as one event. Introduced genes

Donor organisms (origin)

aad accd AccS ALS bar barnase barstar Bay TE bla Chimeric S4-HrA CMV cp CMV/PRV cp

E. coli Pseudomonas chlororaphis Lycoperiscon esculentum (tomato) Arabidopsis thaliana Streptomyces hygroscopicus Bacillus amyloliquefaciens Bacillus amyloliquefaciens Umbrellaria californica (California bay) E. coli Nicotiana tabacum Cucumber Mosaic Virus strain C Papaya Ringspot Virus and Cucumber Mosaic Virus Watermelon Mosaic Virus 2 strain FL and Cucumber Mosaic Virus Zucchini Yellow Mosaic Virus strain FL and Cucumber Mosaic Virus Agrobacterium tumefaciens sp. strain CP4 B. thuringiensis subsp. Kurstaki B. thuringiensis subsp. Kurstaki HD-73 B. thuringiensis var. aizawai B. thuringiensis subsp. kurstaki B. thuringiensis subsp. Tenebrionis B. thuringiensis subsp. kumamotoensis B. thuringiensis subsp. Tolworthi E. coli Corynebacterium E. coli Glycine max (soybean) Achromobacter sp. Strain LBAA E. coli Zea mays Klebsiella ozaenae Agrobacterium tumefaciens E. coli Streptomyces viridochromogenes Lycoperiscon esculentum (tomato) Potato Potato Leaf Roll Virus (PLRV) Potato Virus Y (PVY) strain O E. coli bacteriophage T3 E. coli

CMV/WMV2 cp CMV/ZYMV cp CP4EPSPS cry1Ab cry1Ac cry1F cry2Ab cry3A cry3Bb1 cry9C dam dapA gentR GmFAD2-1 gox GUS mEPSPS nitrilase nos nptII pat PG pinII PLRVrep PVYcp sam-K tetR

Number of occurrences of each gene 7 1 1 1 14 8 6 1 6 ( þ 7 part.*) 1 1 1 2 2 12 6 5 1 1 6 1 1 1 1 1 1 7 5 1 5 1 28 ( þ 1 part.*) 11 2 1 2 1 1 1

(*) denotes the number of GM crops containing only partial copies of the corresponding genes. It should be noted that plants containing only partial genes were not counted towards the total. aad (from E. coli): 300 (9)-O-aminoglycoside adenylyltransferase. accd: 1-amino-cyclopropane-1-carboxylic acid deaminase, an

97 [23,24]. A full and up-to-date list of the known d-endotoxins, along with the respective NCBI (National Center for Biotechnology Information) accession number, authors, publication year, source strain and further comments is available online on the University of Sussex website (Table 1) [25]. Numerous subspecies of Bt have been described so far. Together, all these subspecies can kill a large variety of host insects and even nematodes, but each strain does so with a high degree of speciﬁcity [26]. In a sprayed form, Bt and its puriﬁed toxins, have been used around the world for about 40 years as highly selective and inexpensive insecticides, for their recognized safety to humans, animals and environment [20]. However, despite its advantages, when exposed to physical factors (e.g., UV light), the toxins rapidly degrade into nontoxic/ environmental friendly compounds, with no further eﬃcacy on insect larvae. To circumvent this rapid inactivation, frequent applications of suspensions of spores and inclusions are required to maintain a constant and eﬀective level of pesticide in the ﬁeld [21]. A novel approach to such limitation is represented by the application of biotechnology. Transgenic plants, modiﬁed to express Bt toxins, also known as Bt-protected plants, were ﬁrst created during the early 1990s by cloning the cry genes into diﬀerent crops varieties [27]. Today, the most frequently found genes are cry1Ab and cry3A, present in about 29% of the insect-protected plants

essential precursor for the biosynthesis of the plant hormone ethylene. AccS: 1-amino-cyclopropane-1carboxylic acid synthase, an essential precursor for the biosynthesis of the plant hormone ethylene. bar (from S. hygroscopicus): phosphinothricin acetyl transferase. barnase: ribonuclease enzyme (RNAse). barstar: the coding region of the barstar gene from B. amyloliquefaciens. The barstar gene encodes for a ribonuclease inhibitor (barstar enzyme Bay TE: the 12:0 acyl carrier protein (ACP) thioesterase gene which codes for an enzyme in the fatty acid biosynthetic pathway found in developing seeds. bla: beta-lactamase. Chimeric S4-HrA encodes an acetolactate synthase (ALS) enzyme from Nicotiana tabacum. CMV cp: Cucumber Mosaic Virus coat protein gene. CMV/PRV cp: coat protein gene of Papaya Ringspot Virus (PRV) HA 5-1. CMV/WMV2 cp: coding region of the WMV2 cp gene fused to the 48 nucleotides from the 50 terminus of the CMV cp gene. CMV/ZYMV cp: ZYMV cp coding region fused to the CMV translation initiation codon. CP4EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase, isolated from Agrobacterium sp. (strain CP4). cry1Ab, cry1Ac, cry3A, cry9C, cry1F, cry3Bb1, cry2Ab cry: insecticidal bacterial crystal protein (Cry protein) found in the bacterium Bacillus thuringensis. Dam: DNA adenine methylase from E. coli. DapA: enzyme dihydrodipicolinic acid synthase. GentR: gentamycin resistance gene. GmF AD2-1: delta-12 desaturase. gox: glyphosate oxidase (GOX) from the bacterium Ochrobactrum anthropi. GUS: beta glucuronidase. mEPSPS: a modiﬁed form of wild type 5-enolpyruvyl-3-phosphoshikimate synthase gene from Zea mays which encodes an insensitive enzyme to inactivation by glyphosate nitrilase (from K. pneumoniae subspecies ozaenae): nitrilase. nos: nopaline synthase. nptII: neomycin phosphotransferase aminoglycoside (30 ) phosphotransferase type II gene, from E. coli transposon Tn5 (or Kanamycin resistance gene). pat: phosphinothricin acetyltransferase from Streptomyces viridochromogenes. PG: polygalacturonase. PinII: potato a potato DNA containing 18 bp untranslated leader, pinII protein coding region with intron and about 920 bp of 30 sequence (30 untranslated region of the RNA and putative transcription termination region), which encodes for a protease inhibitor. PLRVrep: full-length ORF1 and ORF2 from Potato Leaf Roll Virus (PLRV), which encode a fusion protein having both helicase and RNA-dependent RNA polymerase activity. PVYcp: coat protein from potato virus Y strain 0. sam-K: modiﬁed Sadenosylmethionine hydrolase gene derived from E. coli bacteriophage T3 that encodes an enzyme, Sadenosylmethionine hydrolase (SAMase). Tet-R: tetracycline resistance gene (modiﬁed from Bruderer S and Leitner KE, 2003).

98 cry family (13.5%)

aad (4.5%) bar (9.0%)

barnase (5.2%) bla (3.9%) Others (23.2%)

CP4EPSPS (7.7%)

gox (4.5%) nitrilase (3.2%) pat (7.1%) nptII (18.1%) Fig. 4. Frequency of occurrence of the most often used genes in the currently approved genetically engineered crop plants. aad (from E. coli): 300 (9)-O-aminoglycoside adenylyltransferase. bar (from S. hygroscopicus): phosphinothricin acetyl transferase. barnase: ribonuclease enzyme (RNAse). bla: beta-lactamase. CP4EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase, isolated from Agrobacterium sp. (strain CP4). gox: glyphosate oxidase (GOX) from the bacterium Ochrobactrum anthropi. nitrilase (from K. pneumoniae subsp. ozaenae): nitrilase. nptII: neomycin phosphotransferase aminoglycoside (30 ) phosphotransferase type II gene, from E. coli transposon Tn5 (or Kanamycin resistance gene). pat: phosphinothricin acetyltransferase from Streptomyces viridochromogenes. cry: insecticidal bacterial crystal protein (Cry protein) found in the bacterium Bacillus thuringiensis. The cry gene family was grouped as a whole and includes: cry1Ab, cry1Ac, cry3A, cry9C, cry1F, cry3Bb1, cry2Ab (modiﬁed from Bruderer S and Leitner KE, 2003).

carrying cry genes (Table 3). These latter transgenes are mostly modiﬁed or truncated forms of the native sequence [13]. Many synthetic variants have been obtained to modulate the expression of the endotoxin and to modify its features in general. Physical inclusion of the toxin within the transgenic plant cell: (i) provides a protected environment, that prolongs the activity of the insecticide by delaying its degradation, (ii) reduces costs and needs of higher dosages, and (iii) enables the toxin to reach even insects presents within the stalk, and not only on the plant surface. Moreover, by cloning the cry transgene under control of diﬀerent promoters it may be possible to selectively express the Bt toxin in certain tissues of the plant or in speciﬁc time lags. Thus, Bt-protected plants, by providing highly eﬀective control of major insect pests, such as the European corn borer, southwestern corn borer, tobacco budworm, cotton bollworm, pink bollworm, and Colorado potato beetle, ensure better yields, lower costs and reduced reliance on conventional chemical pesticides with wider insecticidal spectra and lower speciﬁcity [28]. Especially this

99 Table 4. A lists of terminators, the organism from which they originated, and how often they are found in current GM crops. Used terminators

Donor organisms (origin)

Bacterial nda T-35s T-7s T-ALS T-Als T-E9 T-g7 T-Kti3 T-mas T-napin T-nos T-ocs T-ORF25 T-phaseolin T-pinII T-SSU T-tahsp 17 T-tml T-Tr7

/ / Cauliﬂower Mosaic Virus Glycine max (soybean) Nicotiana tabacum Arabidopsis thaliana Pea Agrobacterium tumefaciens Glycine max (soybean) Agrobacterium tumefaciens Brassica rapa Agrobacterium tumefaciens Agrobacterium tumefaciens Agrobacterium tumefaciens Phaseolus vulgaris (green bean) Selanum tuberosum Glycine max (soybean) Triticum aestivum (Wheat) Agrobacterium tumefaciens Agrobacterium tumefaciens

Number of occurrences of each terminator 22 3 17 2 1 1 12 3 1 2 1 35 5 1 1 2 1 1 4 2

Some terminators may be present in more than one copy in a single product, since a regulatory sequence may have been used for more than one transgene and several copies of a transgene may be present in the same product. This frequency of appearance is not taken into account in the table. T-7s: the 30 untranslated region of the soybean alpha subunit of the beta-Conglycinin gene. T-ALS: ALS: tobacco ALS1 terminator. T-Als: Arabidopsis thaliana ALS1 terminator. T-E9: 30 untranslated region of the pea ribulose-1,5-bisphosphate carboxylase small subunit E9 gene. T-g7: 30 untranslated end of the TL-DNA gene 7. T-Kti3: Kunitz trypsin inhibitor 3 (Kti 3) terminator. T-mas: polyadenylation region from mannopine synthase gene of pTiA6. T-napin: napin gene terminator. T-nos: 30 untranslated region of the nopaline synthase gene. T-ocs: terminator of the octopine synthase gene. T-ORF25: terminator from A. tumefaciens. T-phaseolin: 30 fragment of the phaseolin gene of green bean. T-PinII: terminator sequence from Solanum tuberosum proteinase inhibitor II gene. T-SSU: the 30 untranslated region from the G. max ribulose-1,5-bisphosphate carboxylase small subunit gene. T-tahsp 17: 30 untranslated region of the coding sequence for the heat shock protein 17.3. T-tml: polyadenylation region of tml gene from pTiA6. nda: no data available. T-35s: 30 nontranslated region of the Cauliﬂower Mosaic Virus 35s gene. T-Tr7: the 30 region from A. tumefaciens t-DNA transcript 7 (modiﬁed from Bruderer S and Leitner KE, 2003).

latter advantage is considered highly desirable for what concerns public, animal and environmental safety. A few examples of the speciﬁcity of some used recombinant Cry proteins are listed in Table 5. Cry protein expression, in transgenic bacteria that naturally colonize tomato plants, has been reported as well. This approach involves the use of GM microorganisms with insecticidal activity, capable of surviving on leaf surfaces for several weeks. Also such a novel strategy should allow for a reduction in pesticide application [29].

100 others (16.2%)

Bacterial (18.8%)

T-tml (3.4%) nda (2.6%) T-ocs (4.3%)

T-35s (14.5%)

T-nos (29.9%)

T-E9 (10.3%)

Fig. 5. The frequency of occurrence of the most often used terminators introduced into the currently approved genetically engineered crop plants. T-nos: 30 untranslated region of the nopaline synthase gene. T-ocs: terminator of the octopine synthase gene. T-tml: polyadenylation region of tml gene from pTiA6. nda: no data available. T-35s: 30 untranslated region of the Cauliﬂower Mosaic Virus 35s gene. T-E9: 30 untranslated region of the pea ribulose-1,5-bisphosphate carboxylase small subunit E9 gene (modiﬁed from Bruderer S and Leitner KE, 2003).

Table 5. Speciﬁcity of some recombinant, insecticidal d-endotoxins, currently employed in transgenic plants. Susceptible insect orders (and species in same cases) are indicated. d-endotoxin

Toxic to these insect orders/species

Cry1Aa Cry1Ab Cry1Ac

Lepidoptera Lepidoptera Lepidoptera (cotton bollworm, tobacco budworm and pink bollworm) Lepidoptera Lepidoptera, Diptera Coleoptera (Colorado potato beetle, elm leaf beetle and yellow mealworm) Coleoptera (corn rootworm species) Diptera Lepidoptera, Coleoptera

Cry1B, Cry1C, Cry1D, Cry1F Cry2 Cry3A Cry3Ab, Cry3Bb1 Cry4 Cry5

Examples of Bt insect-resistant crops (approved but not necessarily currently commercialized) include corn (primarily for control of European corn borer, but also corn earworm and Southwestern corn borer), cotton (for control of tobacco budworm and cotton bollworm), potato (for control of Colorado potato beetle), tomato (for control of lepidopteran pests including, but not limited to, cotton bollworm, pink bollworm, tobacco budworm) (Table 1) [8,13]. Despite their approval, extensive use of Bt-protected plants is raising serious concerns among scientists, politicians and the public. There are worries regarding the possible spread of Bt-resistant insects. This eventuality may present

101 a threat to the environment as well as for the durability of this novel insect control technology. As in the presence of any constant selective pressure, the possible spread of individuals carrying mutant genes, conferring advantages against the challenging factor, is favored. Since the mid-1980s many cases of resistance to B. thuringiensis, mostly induced experimentally under laboratory conditions, have been discovered. Insects have also demonstrated their enormous genetic plasticity with hundreds of species found resistant to various insecticides [30]. To prevent the onset of undesired Bt-resistant pests, strict directives were given by regulatory agencies, such as the American Environmental Protection Agency (EPA) (Table 1). One of the adopted strategies is called ‘‘Hi-dose/ Refuge’’ [20]. Such an approach involves the coexistence of both transgenic ﬁelds expressing high levels of crystal protein (i.e., 25 times the protein concentration necessary to kill susceptible larvae or more) and conventional crop ﬁelds. The latter are called structured refugia and consist of restricted areas, devoid of GM plants that represent the selective pressure. The principle is to express Cry toxins at such a high dose that nearly all heterozygotic carriers of resistance alleles will be killed. Assuming an extensive random mating between rare resistant individuals and the numerous sensitive insects, harbored in the nearby refuge, a population of homozygous resistant insects would be unlikely to emerge. Resistance to B. thuringiensis toxins seems to be inherited in a recessive fashion at least in some species [31,32]. Studies on the feeding behavior of bollworm and tobacco budworm larvae, in mixed stands of traditional and transgenic insect-resistant cotton suggest that larvae of both species frequently moved among plants, feeding indiscriminately on BTK and non-BTK plants [33]. A further ardent debate, associated with the use of insect protected plants was sparked in 1999, by a short paper published by the entomologist John Losey and colleagues [34]. This study pointed out unpredicted toxic properties of the pollen from Bt corn, on larvae of the monarch butterﬂy, Danaus plexippus. The caterpillars of this species feed exclusively on milkweed leaves and, thus, are not a target pest of Bt corn. Nonetheless, when insects were reared on milkweed leaves, dusted with pollen from Bt corn, higher mortality, with respect to controls, could be observed. Based on these ﬁndings, several controversial studies have been conducted [35]. According to the results of, at least, three of them, the commercial large-scale cultivation of current Bt-maize hybrids did not seem to indicate a signiﬁcant risk for the monarch population [36–38]. Herbicide tolerance Besides insects, another serious problem in agriculture is the weed infestation of the cultivation. ‘‘Weeds’’ is a generic word to describe wild plants growing where they are not wanted, as in gardens or crops ﬁelds. These highly aggressive and fast-growing plants represent a plague for the cultivation for their (i) ability

102 to compete for water, light and nutrients, (ii) possible contamination of the crop seeds with undesired seeds and toxins and (iii) interference with the crop harvest. In the worst cases, weeds infestation can kill crops, with a dramatic loss in terms of yield percentage [39]. A speciﬁc biochemical pathway involving the 5-enolpyruvylshikimate-3phosphate synthase (EPSPS) gene, to produce aromatic amino acids, has been described in plants, fungi and bacteria. This enzyme is the target of the glyphosate (N-phosphonomethyl glycine), one of the most popular herbicides whose reaction kinetics was described in 1983 [40–42]. In 1985, Stalker et al. showed that the glyphosate-resistant phenotype was associated with a mutation, resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein [43]. Bacteria with the mutant form of the gene were viable and able to make aromatic amino acids in presence of glyphosate. A few years later, Klee et al. cloned an Arabidopsis thaliana EPSPS gene and then fused it with a Cauliﬂower Mosaic Virus 35s Promoter, before reintroducing the recombinant gene into Arabidopsis. The resultant overproduction of EPSPS led to glyphosate tolerance in transformed callus and plants [44]. Thus, unlike such transgenic plants, weeds infesting herbicide tolerant (HT) crops, are selectively susceptible to glyphosate treatments and can be selectively killed. Since Klee’s experiment, employment of approved HT plants has grown remarkably, world-wide. Between 1996 and 2001, herbicide tolerance has constantly been, by far, the most prevalent trait among GM plants (with insect protection being second) and in 2001, about 77% of the global area grown with transgenic crops (52.6 million hectares) was occupied by herbicide tolerant plants (largely soybean) [45]. Examples such transgenic crops (not exclusively for food or feed purposes) include sugar beet, Argentine canola, Polish canola, chicory, carnation, soybean, cotton, ﬂax, linseed, tobacco, rice and maize (Table 1). Regarding the safety aspects associated with the adoption and consumption of this transgene, several studies have been conducted. In a comparative analysis, in 1996, Padgette et al. demonstrated the nutritional equivalence of seeds and selected processing fractions, from glyphosate-tolerant soybean lines and their parental, conventional cultivar [46]. A similar comparative approach was also conducted to evaluate the composition of a glyphosate-tolerant line of corn, with respect to that of conventional corn, grown in the United States in 1998 and in the European Union in 1999. Also this study suggested that the GM corn was compositionally equivalent to, and as safe and nutritious as, conventional corn hybrids [47]. The safety of EPSPS derived from Agrobacterium sp. strain CP4 (CP4 EPSPS) was assessed [48]. CP4 EPSPS is introduced and expressed in glyphosatetolerant soybeans (Table 3). An in vitro digestion model was employed to infer and demonstrate the digestibility of the transgenic product, whereas acute administration of high dosages of CP4 EPSPS, showed no toxic eﬀects of the heterologous protein, in mice. In that paper, potential allergen concerns were excluded as well.

103 Additional studies suggested that glyphosate-tolerant soybeans are as safe as traditional soybeans, with respect to food and feed safety, even after treatment with commercial levels of glyphosate [49–52]. Recently, Chang et al. demonstrated that the EPSPS protein in GM soybean, cloned, expressed and puriﬁed from an E. coli strain, showed no signiﬁcant allergenicity in the Sprague Dawley rats [53]. Golden rice For underdeveloped and low-income countries including highly populated areas extending throughout Asia, Africa and Latin America, reliance on rice, as a primary food staple, contributes to vitamin A deﬁciency (VAD), a serious illness causing juvenile blindness, pregnancy-related mortality and death in million people per year. Indeed, upon milling, provitamin A content in rice endosperm, which ultimately represents the edible part of the rice grain, is insuﬃcient to meet the required daily allowance of several hundreds micrograms. Golden rice is the name given to a transgenic, nutritionally-enhanced plant, created in 2000 to obtain a functioning provitamin A (beta carotene) biosynthetic pathway in rice endosperm [54]. The transgenic plant accumulates beta carotene in the endosperm and may provide a supplementary dietary source of provitamin A for people feeding mainly on rice [55]. With regard to this, however, medical criticism seems to be mainly focused on the eﬀective adequacy, of the provided dietary supplement, as an eﬀective solution for VAD [56]. Thus, current experimental eﬀorts on Golden rice, are aimed to increase the provitamin A accumulation in the endosperm, by the identiﬁcation of the metabolic, rate-limiting bottlenecks that caused a limited beta carotene synthesis in the prototype lines. To enhance or improve nutritional properties is another task achievable by development of genetically modiﬁed foods. Golden rice represents an immediate example of the complex debate involving not only scientiﬁc or technical aspects, but also ethical, social, economical and political decisions related to its commercialization and diﬀusion. Modiﬁed and widely spread species, such as rice or wheat, that have provided important and precious foods in the history of human population survival may represent a successful and promising advancement, but open up reasonable concerns related mostly to the novelty of the GM procedures [57,58]. World population growth rate and globalization processes are changing the traditional view of food and agriculture, pushing toward new solutions for a sustainable development. Transgenic animals and animal technology One of the scientiﬁc aims of animal transgenesis in livestock, is to understand the various regulatory mechanisms of genes and the physiological role of the diﬀerent proteins in vivo. Downstream applications of such acquired knowledge

104 include maintenance, improvement or attainment of proﬁtable or desirable traits in farm animals. After preliminary studies, conducted on embryos of diﬀerent organisms such as sea urchin, Candida elegans, Xenopus, Drosophila and mice, successive experiments involved swine, rabbits and sheep that were modiﬁed in the attempt to obtain animals, with higher levels of circulating hormones [59]. Among the ﬁrst employed transgenes, were the human growth hormone (hGH) and the bovine growth hormone (bGH), which are normally secreted by the pituitary gland [60]. A mouse metallothionein-I promoter was used to control the synthesis in the ﬁrst manipulated animals. In these studies, the monitored growth rate was not enhanced in any of the transgenic animals, however deﬁnite biological eﬀects were observed, in comparison to the littermate controls. Although the complexity of the ﬁeld promptly revealed to the scientiﬁc world, these pioneer studies showed the enormous potential of animal transgenesis and thoroughly fulﬁlled the initial ambitious expectations. Today, though in a more developmental phase with respect to GM crops, research in animal technology is a very important and active sector of biotechnology. Advances in transgenic biology, gene therapy, ‘‘knock-out’’ gene technologies, and cloning may lead to other novel products/strategies that enhance productive eﬃciency. Nevertheless, passionate and ardent debates constantly arise worldwide. For instance, there are common fears of a reduced genetic diversity caused by the intensiﬁcation of livestock production associated to the adoption of genetically uniform varieties of animals. To avoid this unlikely event, several strategies (such as cryopreservation of semen and embryos, coupled with artiﬁcial insemination and embryo transfer, as well as somatic cloning) have been devised. Today, it seems unlikely that in a near future genetically modiﬁed livestock may play a major role, in developing countries, as a major food source. Among food animals, only engineered ﬁsh (salmon in particular) are presently under active consideration by US regulators. Current research and development in zootechnology, are mainly focused on (a) the quality of livestock feeds, through nutrient content improvement of forages, and the monitoring of food chain safety, (b) the digestibility of low quality feeds and auxological aspects (c) the eﬀective control of several animal diseases, (d) the possible secretion of heterologous substances (e.g., antibodies, vaccines, pharmaceuticals, dietary supplements) in milk of transgenic, dairy animals. Such research lines are likely to represent nearest applications of genetic engineering in animal technology. The recent observation of the Bovine Spongiform Encephalopathy (BSE), showed the need to respect the natural animal physiology and consider adequate preventive actions. The main public health aspects related to zootechnology developments will particularly need to deal not only with the deﬁnition of health safety aspects for human beings, food chain, environment, but also with the development of eﬀective screening tools and monitoring procedures for surveillance and traceability.

105 Social, ethical and legal issues The so-called ‘‘consumer sovereignty’’ requires that information be made available, so that people may make food choices based on their own ethical, social, cultural or religious values. Proper policy provisions and clear regulations to inform people about the degree, to which food has been genetically engineered, is highly desirable and appropriate. Regulation of genetic engineering in the US is overseen by three agencies: the Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA), the United States Food and Drug Administration (FDA), and the aforementioned EPA. The regulatory paradigm, used by the FDA to approve novel foods, is based on the concept of ‘‘substantial equivalence’’ [61,62]. According to this guideline, the allergen, nutrient and toxin content of the new GM food must fall within the normal range of the equivalent, conventional food. The concept of substantial equivalence provides the framework for a comparative approach to identify the similarities and diﬀerences, between GMOs and their traditional counterparts that have a known history of safe use. Using this method for the evaluation of several GM crops already approved world-wide, Cockburn concluded that foods and feeds derived from genetically modiﬁed crops are as safe and nutritious as those derived from traditional crops [63]. On the other hand, Kuiper et al. argue that the concept is not a safety assessment in itself because it identiﬁes hazards, but does not assess them. Moreover, application of the concept of substantial equivalence encounter several diﬃculties in its application [64]. The European Union plans to establish a ‘‘farm to fork’’ tracking system that would regulate the traceability for transgenic foods, and several proposals are in progress. It is noteworthy that public health would favor these directives not only for GMOs per se, but also for those foods derived from GMOs (e.g., oils or processed materials), that may not contain heterologous DNA or protein, but consider rec-DNA technology in their production processes. Food labeling represents a complex issue with several concerns. A hypothetical ‘‘zero tolerance’’ approach is not favorably seen or applicable (Table 1: ‘‘Harvest of fear’’ PBS website). Some argue that warning labels on approved GM foods may imply harmful eﬀects on health and create false alarm, even though those eﬀects have been excluded, by the pre-market safety trials. It seems also plausible that distinctive food labeling, segregation of GM and non-GM foodstuﬀ (during storage, shipping and processing) and transgenic organism traceability would be diﬃcult to achieve and may have a signiﬁcant impact on the costs of the goods. Thus, for the coming years, food labeling may still represent a major ﬁeld of discussion, for novel as well as for conventional foods [65]. Indications, laws and regulations are frequently modiﬁed due also to the rapid changes in the biotechnology ﬁeld and the relatively short juridical experience of society in this new area.

106 Labeling enables consumers to get a clear and comprehensive information on the contents and the composition of food and helps buyers to make an informed choice while purchasing their products. A recent survey was set up to determine whether perceiving obvious beneﬁts from eating genetically modiﬁed soybeans, would have altered, among consumers, personal risk assessment and desire for labeling in general [66]. The results of this study showed that consumers reading about the GM soybean with obvious consumer beneﬁts were signiﬁcantly more comfortable eating it, than those reading about the GM soybean with no obvious consumer beneﬁts. This interesting relationship, observed between perceived risks and beneﬁts, had also been shown in two previous studies [67,68]. Thus, activities or technologies that are judged high in risk tend to be judged low in beneﬁt, and vice versa. Such an observation has suggested the hypothesis that creation and commercialization of new transgenic cultivars with more direct beneﬁts for consumers, would increase public acceptance [66]. Indeed, as one can tell from the current most used transgenes, to date most modiﬁcations to crop plants have mainly beneﬁt producers. Consumers stand to beneﬁt by development of food crops with increased nutritional value, medicinal properties, enhanced taste and esthetic appeal [69]. As national regulations testify (Table 1) extremely diﬀerent public and governmental sensibilities concerning biotechnology, may be observed throughout the world [70–73]. This extremely heterogeneous condition should be addressed with eﬀective strategies such as new labeling systems and consensus conferences, to stimulate public information and public debate and to defeat the spread of misinformation, easily possible in a new and fast growing issue [74,75]. It is generally recognized that each adopted approach, aimed at the reconciliation between science, or technology, and the community, should be adapted to the cultural context of each country and society. Health risks: Present knowledge and potential hazards Health risks associated with GMOs can be classiﬁed as risks for the person and risks for the environment (Table 6). Present data do not provide evidences of speciﬁc hazards for populations exposed to a diet containing approved GMOs [76]. Epidemiological and experimental studies are in progress in diﬀerent countries and are supported by diﬀerent institutions including universities, research centers, companies, or supranational organizations. Prudential criteria are required due to the novelty of the procedures and of the possible derivable hazard, and the wide and quickly growing exposure levels that the world population encountered in a relatively short period of time. Risks for health comprehends: food poisoning, food intolerance, auto-intoxication, anti-nutrients. In principle, upon manipulation, including a genetic modiﬁcation, a food organism may become toxic. Food poisoning is an acute disturbance occurring

107 Table 6. Classiﬁcation of risks related to production and consumption of GM foods. Risks for the person #

Risks for the environment #

Toxic food Food poisoning

Extinction of existing species or varieties Of animals Of plants Interference with environmental balance Uncontrolled spread of transgenic species Gene transfer Unpredictable risks

Non toxic food Non immune-mediated (a) food intolerance (b) auto-intoxication (c) anti-nutrients Immune-mediated (food allergy) Gene Transfer Unpredictable risks

after consumption of food that is contaminated with toxic agents that are inherently unsuitable for human consumption. Apparently this may appear as a fearful and worrying risk. Instead, this possibility is easily detectable and controllable, as it is for any natural or synthetic novel food humans have met before. Major concerns are related to eﬀects over long periods or overreactions on susceptible population subgroups. Food intolerance and auto-intoxication are food-induced morbid states that do not involve a speciﬁc immune system reaction. Individuals with food intolerances may lack an enzyme that is needed to digest a certain food and show a pathological sensitivity to it. Autointoxications are caused by an accumulation of harmful metabolic intermediates or substances, through an endogenous origin. Genetic diﬀerences in the population may determine or enhance these eﬀects. The susceptibility to such reactions can be characterized by planning extended and accurate pilot studies. Anti-nutrients, although not necessarily toxic per se, can be plant compounds that decrease the nutritional value of that plant. Anti-nutrients usually make an essential nutrient unavailable or indigestible when consumed. For example, phytate, a common component of most seeds and cereals, forms a complex with many important minerals, making less of the minerals available [77]. Trypsin inhibitor, lectins, isoﬂavones, stachyose and raﬃnose are further examples of anti-nutrients that may be studied during nutritional assessments of novel foods [46]. The presence of these compounds in a plant is of essential importance not only for human nutrition but also for animal feeding. Further research will likely provide eﬀective protocols to focus on whether genetic engineering has accidentally changed the nutritional components associated with conventional cultivars of a crop. As antibiotic resistance markers are routinely used for the selection of transformed plant cells, concerns have been raised about whether the enzyme product of the DNA might be produced in transgenic plant cells. Although

108 various processing procedures would inactivate the enzyme in processed foods, ingestion of fresh or raw transgenic organisms may result in intake of active enzymes. Such an occurrence may cause the inactivation of orally ingested antibiotics but preliminary pilot studies showed that antibiotics would remain eﬀective. Moreover, antibiotic resistance is widely diﬀused in nature and is part of ancient phylogenetic processes involving both intrinsic and transferable resistance. It should be noted that all approved GMOs are currently created by genetic engineering of GRAS (Generally Recognized As Safe) organisms that have already been used or eaten without risks by humans and animals, since the dawn of time. Presently, current food safety regulations for traditionally bred food crops are, in practice, less stringent compared to those applied to GM foods [62]. Food allergies Food allergy is a speciﬁc immunomediated reaction to one or more food components; it is characterized by altered bodily reactivity (hypersensitivity) to an antigen, in response to a ﬁrst exposure. Atopic individuals may overreact to an allergen with diﬀerent clinical manifestations, both localized or systemic. This event can show diﬀerent degrees of severity and can also be fatal as in the anaphylactic shock. Agricultural biotechnology implies the introduction of novel proteins into the modiﬁed foods, and proteins can be allergens. There are two situations that may occur: ﬁrst, a known allergen may be transferred from a donor crop into a nonallergenic target crop. The second scenario is the creation of a novel allergen with a possible de novo sensitization of the population [78]. The case of the Brazil nut is a well known example, even if its conclusions underwent opposite interpretations: GMOs represent an ‘‘allergic hazard’’ or the allergic risk ‘‘can be prevented.’’ Methionine in relative low concentration in the protein fraction of soybean (Glycine max) seeds, compromises the nutritional value of such crops. Research was conducted in order to improve the quality of soybean meal as an animal feed and a transgene, coding for a storage protein from a Brazil nut (Betholletia excelsa), was successfully introduced and expressed into soybean. While it was known at that time that Brazil nuts were allergenic to some consumers, no one had ever identiﬁed which gene product from Brazil nuts was the responsible allergen, thus, several laboratory tests had to be conducted in turn, to determine whether potential allergens had been transferred to the GM plant. Investigations by Nordlee et al., on the transgenic soybeans, identiﬁed the methionine-rich 2S albumin as a major Brazil nut-derived allergen and demonstrated the possibility of transfer of allergens from a food, known to be allergenic, into another food, through genetic engineering. These data were the conclusion of a safety assessment study, begun in 1993, in collaboration with the University of Nebraska [79]. Following the 1993 preliminary ﬁndings,

109 all the ﬁeld trials were discontinued and all plant material and seeds, not held for laboratory study, destroyed. Despite this case, it should be noted that consumers’ exposure to approved and commercialized transgenic crops, is reaching signiﬁcant levels, especially in some countries such as the US or China. However, no reports exist regarding allergic reactions to the crops that have been approved for human consumption, and available data seem to conﬁrm the safety of the approved GM foods [80–82]. Biotechnology also oﬀers many promising perspectives, as concerns food allergies. In the future, biotechnology may be employed to characterize, eliminate or attenuate the potential of food allergens. Even if at an experimental phase, this represents a novel, yet encouraging approach. For instance, Herman et al. employed transgene-induced gene silencing to prevent the accumulation in soybean seeds of Gly m Bd 30 K protein, a major (i.e., immunodominant) soybean allergen [83]. Further, a reduced content of a known allergen was shown in GM rice. In this study, antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds showed that allergen content of seeds, from several transgenic rice plants, was markedly lower than that of the seeds from parental wild type rice [84]. Several approaches are currently being employed to assess the possible allergenic potential of the transgenic proteins introduced in GMOs [85,86,51,78]. The safety evaluation of transgenic foods is relatively easy when the allergenicity of the gene source is known. New and powerful tools may be represented by bioinformatics and modern databases. A typical use of bioinformatics, involves the search for possible homologies between the newly introduced protein and known allergens present in up-to-date databases [87]. Sequence analysis plays an important role in assessing the potential allergenicity of proteins used in transgenic foods, particularly for proteins that have not previously been part of the food supply. Sequence comparisons are used to indicate potential unexpected cross reactivity to existing allergens and to assess the potential for developing new sensitivities [88]. Bioinformatics plays an important role in this process and new laboratory protocols and surveillance procedures are being performed. Immunological and biomolecular assays involving the reactivity assessment of a novel food are available, including analysis of IgE antibodies from the serum of individuals with known allergies to the source of the transferred DNA or to materials that are broadly related to that protein or protein domains. The immunoreactivity can be tested also in appropriate animal models. Biochemical tests, focusing on the evaluation of the chemical stability of the foreign products after food processing, storage, cooking and digestion are usually performed, as well. Since food allergens may share physico-chemical properties that distinguish them from nonallergens, characterizing those properties may serve as an eﬀective

110 preventive tool to predict the inherent allergenicity of transgenic proteins introduced into novel foods. A candidate property is the stability to digestion. In a study involving an in vitro model of gastric digestion, Astwood JD et al. conclude that the stability to digestion is a signiﬁcant and valid parameter that distinguishes food allergens from non-allergens [89]. Additional factors, such as expression level of the novel protein in the edible portion of the food, may also bear signiﬁcant information. Further, the stability of potential allergens from GM plants, is also tested by heat stability analysis, to simulate cooking. As transgenic foods commonly eaten may contain transgenes (in variable quantities depending on the food and processing), concerns regarding the exposures to transgenic DNA sequences and the onset of speciﬁc allergies, aroused. Transgenic DNA degradation was tested in vitro, by means of a simulated digestion model, that showed how heterologous DNA fragments from GM foods, may survive passage through the small intestine [90]. However, in a comprehensive review Jonas et al. conclude that DNA from GMOs is equivalent to DNA from conventional, non-transgenic organisms and, consequently, any risks associated with the consumption of DNA will remain, irrespective of its origin [91]. Especially for infants, dietary nucleotides have been reportedly beneﬁcial, since they positively inﬂuence lipid metabolism, immunity, and tissue growth, development and repair [91–93]. Gene transfer and foreign DNA intake As previously mentioned, fragments of transgenic nucleic acids may be present in GM food or derivates. Transgenic sequences include the promoter, the transgene and the terminator [13]. The intake of such sequences, the ‘‘gene cassette,’’ is causing increasing worries in case a transfer event would occur upon ingestion. Hereafter, with ‘‘horizontal gene transfer,’’ we will refer to the passage, from engineered foods to gut epithelium, or cells from other tissues, or gut microﬂora. But, is this horizontal transfer from ‘‘diet’’ to human cells possible? Can a DNA sequence of hundreds of nucleotides pass the intestinal barrier? Can it integrate into the host genome or even be inherited by the oﬀspring? Can this event be possible in animal feeding and represent a risk for the food chain? Can gut microﬂora acquire antibiotic resistance through transgenic DNA? Researchers or public health are currently considering these and other related issues. Preliminary data are available to open up a discussion more than to clearly deﬁne the problem. Insertion of a DNA sequence in the genome of a human cell represents a mutation event and is feared to cause neoplastic transformation or other genetic modiﬁcations. However, to address this issue, it should be immediately noted that in principle the transgenic DNA is considered biochemically equivalent to the DNA from any other source and that almost every food contains DNA, either of animal or plant origin. Since the dawn of time, human gut, and that of other methazoans, has been

111 Table 7. ILSI Europe Workshop on Safety Considerations of DNA in Food, 26–28 June 2000 (modiﬁed from Jonas DA et al., 2001). rec-DNA: recombinant DNA. GMOs: genetically modiﬁed organisms. Statements of the ILSI Europe Workshop on Safety Considerations of DNA in Food, 26–28 June 2000 1. All DNA, including rec-DNA is composed of the same four nucleotides. 2. In view of the variability of dietary intake of DNA, consumption of foods derived from GMOs does not measurably change the overall amount of DNA ingested through the diet. 3. Taking into account the natural variations of DNA sequences, the present use of recombinant techniques in the food chain does not introduce changes in the chemical characteristics of the DNA. 4. There is no diﬀerence in the susceptibility of rec-DNA and other DNA to degradation by chemical or enzymatic hydrolysis. 5. The metabolic fate of DNA digestion products is not inﬂuenced by the origin of the DNA. 6. DNA is not toxic at levels usually ingested. Where there is potential for adverse eﬀects, e.g., in gout, this is due to excessive intake, not the origin of DNA. 7. Ingested DNA showed no indication of allergenic or immunogenic properties, that would be of relevance for consumption of GMOs-derived foods. 8. Uptake, integration and expression of any residual extracellular DNA fragments from foods by microorganisms of the gastro-intestinal tract cannot be excluded. However, each of these circumstances is a rare event and would have to happen sequentially. 9. In vivo uptake of DNA fragments by mammalian cells after oral administration has been observed. However, there are eﬀective mechanisms to avoid genomic insertion of foreign DNA. There is no evidence that DNA from dietary sources has ever been incorporated into the mammalian genome.

exposed to a constant ﬂow of dietary nucleic acids. This also implies that if some risks were associated with such an event, representing a selective pressure, counteracting evolutionary mechanisms would likely have arisen and spread. Nevertheless, even if the probability of gene transfer from GMOs to mammalian cells seems extremely low and statistically insigniﬁcant, it is highly important considering hypothetical health risks on the long period scale [91]. Evidence is available to address the issue of the bioequivalence and horizontal transfer, as stated by the ILSI Europe Workshop on Safety Considerations of DNA in Food (Table 7) [94]. Concerns regarding possible adverse eﬀects associated with gene transfer, should take into account the metabolic fate of DNA and RNA, and the content of rec-DNA with respect to the total amount ingested. Apart from diﬀerences in the sequence, recombinant DNA is composed of the same four nucleotides as nonrecombinant DNA. Chemical properties of such DNA molecules are not altered by rec-DNA techniques that are currently employed in the production of the approved GMOs. It seems reasonable, therefore, to expect, for transgenic sequences, the same degradation pathways of any other DNA fragment. Degradation is a generic term including several kinds of reactions either catalyzed by enzymes or caused by chemical

112 as well as physical agents [95,96]. Virtually all these events, can occur during food processing, storage, cooking and digestion, thus allowing only smaller nucleic acid fragments to reach the intestinal lumen [90]. During digestion, the acid environment of the stomach, along with pancreatic nucleases, and those secreted by intestinal epithelial cells, cause extensive hydrolysis of dietary DNA. Such breakage releases nucleotides that are subsequently processed through the gastro-intestinal (GI) tract to sugars, purine and pyrimidine bases. Proteolytic enzymes are responsible for nucleoprotein cleavage in the gut. As mentioned, nucleic acids content in raw foods can diﬀer signiﬁcantly, depending on the type of cells present in the foodstuﬀ, and can be considerably aﬀected by food processing. DNA and RNA in raw plant storage tissues, is lower than in animal muscle tissues and edible oﬀals, however, signiﬁcant amounts of dietary DNA and RNA are assumed daily [91]. The organisms with the highest content of nucleic acids are fungi, bacteria and yeast. In humans, with a certain variability depending on the diet and on the eﬀects of processing, this intake may range from 0.1 to 1.0 g/person per day. Processed foods have a lower nucleic acids content which can sometimes drop, to almost undetectable quantities, in case of highly reﬁned products. As regards rec-DNA content in GM crops, it should be noted that in some homozygous insect-protected and herbicide-tolerant cultivars, it may range from 0.00018 to 0.0011% of the plant genome, allowing an estimate of total per-capita intake of rec-DNA (mg/d) from GM maize, soya and potatoes as 0.38 mg/d, assuming that only GM crops would be consumed. This is about 0.00006% of a typical daily DNA intake of 0.6 g [91]. Some approved and commercialized plants, however, are heterozygous, and the nucleic acids intake is lower when this crop is consumed. Under physiological dietary exposure to GM crops, several individual events, most of them considered somewhat rare, would have to occur sequentially to cause gene transfer to gut microﬂora or mammalian cells (Fig. 6). In microorganisms, this event can occur by one of three diﬀerent mechanisms: conjugation, transduction, transformation. In particular, bacterial transformation involves the uptake of free extracellular DNA with an inheritable incorporation [95]. The host cell may either undergo a (i) transformation without expression of foreign proteins, or (ii) transformation with expression of heterologous products. In order to be able to actively take up DNA from the environment, bacteria have to achieve a state of competence, during which DNA molecules may be admitted to the cell. Stable incorporation relies on rare events of recombination, which ultimately depend on the extent of the genetic homology between the recipient cell and the fragment, or by formation of independent replicons. However, foreign DNA acquisition is not suﬃcient for the production of a heterologous protein in a transformed host cell. It always requires the presence of a coding sequence, proper regulatory elements, proper frequency of codon usage and, more generally, of a favorable cell environment which allows processing and expression of the heterologous protein.

113 The complete TE would have to be released into the GI tract, from the ingested GM food.

The TE would have to survive degradation by host nucleases or food associated nucleases.

The TE would have to compete for uptake with other DNA present in the GI.

Host mammalian or bacterial cells would have to be competent for transformation.

Uptake of the TE from a host cell would have to occur.

The TE would have to survive further degradation within the cell.

The TE would have to be inserted, into the host DNA, by repair or recombination events.

The TE would have to be expressed. Fig. 6. Individual events required for the transfer, in the gastro-intestinal (GI) tract, of ingested transgenic elements (TE), from a genetically modiﬁed plant, to microbial or mammalian cells, under physiological dietary exposure.

A foreign DNA fragment, that after cellular internalization is not integrated into the resident chromosome, might be expressed only transiently. Noteworthy, in the absence of a speciﬁc selective pressure that confers an advantage to the transformed cell with respect to the others, the spread of the trait may not occur. The importance of the gene transfer issue in gut microﬂora, may be better perceived considering how common this event is in nature. Unlike eukaryotes, bacteria have obtained a signiﬁcant proportion of their genetic diversity through the acquisition of sequences from distantly related organisms. Horizontal gene transfer produces extremely dynamic genomes and has, in turn, a great impact on bacterial population dynamics as well as on bacterial evolution and speciation [97,98]. Microenvironment inﬂuences bacterial competence acquisition.

114 Brautigam et al. assessed the eﬀects of food matrices on the natural transformation of B. subtilis and showed how this species may develop natural competence when grown in milk products. Frequency of transformation varied with the content of fat in the foodstuﬀ [99]. This general phenomenon may play a role in milk products obtained from GM dairy animals, but also in gut or soil microﬂora. On the other hand, over a million years of evolution, bacteria have also adopted an eﬀective mechanism to protect themselves from foreign DNA infection. The modiﬁcation/restriction system, for instance, is used by E. coli to distinguish between its own genome and foreign DNA introduced by bacteriophage infection, plasmid transfer or transfection [100]. DNA with familiar patterns of methylation are immune to attack, whereas those not recognized by the specialized cellular machinery are earmarked for degradation and promptly degraded by endogenous restriction enzymes [95]. An area of concern focuses on the possibility that antibiotic resistance genes used as markers in transgenic crops may be transferred to pathogenic bacteria. The most frequently used transgene is nptII, originating from the E. coli transposon 5 (Table 3 and Fig. 4). This gene confers resistance to selected aminoglycoside antibiotics. In 1997, nptII was found to be present in 61% of the surveyed GM crops. However, six years later, its employment in GM plants was found to be reduced to about 44% of the surveyed transgenic crops [13]. Antibiotic-resistance markers that are presently employed in GM crops for selection, belong to a class of the limited clinical importance of the antibiotic they inactivate, and their frequent occurrence in nature. It is also generally recognized that the increased number of resistant bacterial strains is more likely due to the widespread use, abuse and misuse of antibiotics, in human and zootechnical applications, rather than to the recent adoption of GM crops [101]. In 1993, Fuchs et al. showed that ingestion of genetically engineered plants, expressing the NPTII protein, did not determine particular safety concerns for human health. Puriﬁed NPTII protein produced in E. coli, was shown to be chemically and functionally equivalent to the NPTII protein produced in genetically engineered cotton seed, potato tubers and tomato fruit and to degrade rapidly under simulated mammalian digestive conditions [102,103]. As for other transgenic sequences, the chain of events that would transfer an antibiotic resistance marker, from a GM plant to a pathogenic bacterium is quite unlikely. A negative impact of GMOs on antibiotic eﬃcacy has been assessed and considered improbable by scientists and regulatory agencies [101]. However, in response to concerns about this remote possibility scientists are starting to use, in transgenic plants, other marker genes, such as the GFP gene from Aequorea Victoria, and alternative strategies [104]. Further approaches to the problem include removal of the selection marker after successful gene transfer.

115 Besides bacteria, it is also relevant for public health to assess the likelihood of uptake of free undigested transgenic fragments by mammalian cells. In recent studies, Jennings JC et al. failed to detect fragments of the cp4 epsps transgene in a variety of tissue samples from pigs, fed glyphosate-tolerant soybeans. Immunoreactive fragments of the transgenic protein were not detectable either. In a similar study, broiler chickens were fed a diet containing insectprotected plants. In accordance with the previous experiments, fragments of transgenic and endogenous plant DNA, as well as transgenic protein, were not detected in the chicken breast muscle samples [105,106]. These evidences are in contrast with those previously published by Schubbert et al. (1994), who observed that high doses of orally administered naked M13 phage DNA survived, transiently, in the GI tract and entered the bloodstream of mice [107]. Further experiments suggested a possible transport mechanism of foreign DNA, through the intestinal wall and Peyer’s patches, to peripheral blood leukocytes and into several organs [108]. Food-ingested M13 DNA fed to pregnant mice, could also be detected in several cells of various organs of the foetuses and newborn animals, suggesting a possible transfer through the transplacental route, but not via the germ line [109]. Thus, to challenge these preliminary observations with a more natural scenario, mice were fed soybean leaves, and the fate of the small subunit of the ribulose-1,5bisphosphate carboxylase (Rubisco) gene was followed in the mouse organism. A short fragment of the authentic Rubisco gene, was observed to be transferred to spleen and liver, of the mice [110]. In the same paper, authors failed to detect any GFP expression, in gut, spleen or liver, upon oral administration of GFPencoding constructs. Conversely, green ﬂuorescence was observed in mouse skeletal muscle tissues, after injection of the same constructs. Therefore, it seems plausible that small amounts of ingested DNA are not broken down under physiological digestive processes. This DNA may either enter the bloodstream or be excreted. It is generally accepted, however, that body’s normal defence systems eventually would destroy this DNA thus preventing potential adverse eﬀects. The signiﬁcance of the observations of Schubbert and Doerﬂer have been questioned by other recent studies that failed to detect transgenic proteins or transgenes in tissues from animals fed on GMOs [111–113]. It is generally accepted that the available data are not suﬃcient to demonstrate that plant DNA can be transferred, stably maintained and expressed in mammalian cells. Further, it should be considered that many genes used for the genetic modiﬁcation of food organisms come from the so called GRAS organisms, with a long and safe history of human coexistence. However the novelty of the problem requires further accurate research, experimental reproduction and prudence in result interpretation. Carcinogenesis, mutagenesis, reactivation of dormant viruses and even generation of new viruses have been recently postulated, for example in the case of horizontal transfers involving the promoter 35s (P-35s) from the

116 Cauliﬂower Mosaic Virus (CaMV). This regulatory sequence is used in most of the currently approved GM plants to give constitutive overexpression of transgenes (Fig. 3). Critics maintain that the naked recombinant promoter may have harmful eﬀects, for instance due to genetic instability. Rebuttals to these hypothesis are based on several evidences [114]. CaMV P-35s is a nucleic acid sequence and, as previously considered (Fig. 6), a multi-step chain of events would have to occur to escape the normal digestive breakdown process, penetrate a cell, insert itself into a human chromosome, and take on the control of expression of the resident genes. Moreover, a signiﬁcant percentage of the cauliﬂowers (10%) and cabbages normally sold in our markets and consumed, are found to be naturally infected by CaMV [115]. Therefore, it has been estimated that, historically, humans have been ingesting CaMV and its 35s promoter at levels that are over 10,000 times greater than those present in uninfected transgenic plants. It is actually this line of argument that led the USDA to endorse the use of CaMV P-35s as a safe promoter in GM crops. An integrated, multidisciplinary approach is required to address these and other issues related to gene transfer events and foreign DNA intake consequences. Accurate analysis of gene cassette sequence properties by molecular biology and bioinformatics tools is needed together with an open and rigorous interpretation of the results. Detection of possible health eﬀects by classical epidemiology studies is an important but limited strategy. Appropriate risk assessment requires a deep comprehension of the basic mechanisms involved in gene transfer and uptake, considering their potential role on extended exposed populations along a long-term scale. Conclusions Years of debate and intensive scientiﬁc work seem to exclude the presence of evident health hazards associated with the consumption of the authorized GMOs and of GMO-derived ingredients. When approved and marketed, such food was conﬁrmed to be devoid of additional risks with respect to its conventional, non-transgenic counterpart [76,86,100,116]. Risk assessment procedures and authorization protocols are available and consider the costbeneﬁt ratio of the introduction of GMOs in the food chain. However, it is not appropriate to generalize and the assessment of risks associated with the consumption of novel foods per se, would be meaningless if not conversely focused on a speciﬁc product. It is not presently possible to state that every GMO is potentially safe, but neither that a food is unsafe if ‘‘GM.’’ Availability of a rigorous and deep knowledge on the basic mechanisms involved in genetic modiﬁcation is the major limiting point for an eﬀective risk assessment. Intensive scientiﬁc eﬀort is in progress to thoroughly understand and foresee possible consequences on humans, animals and environment. Special care is required due to the particular novelty of these foods and their rapid diﬀusion in the absence of a co-evolution process. Social aspects and economical implications

117 strongly inﬂuence the commercialization of GMO products, in both the directions. In diﬀerent countries, several regulations are available and frequently updated. GMO foods represent a challenging issue for public health science, involving safe nutrition, health risks, monitoring tools, policy making. In the new Millennium, prevention tasks include also the survaillance on novel foods and the management of the health safety of Genetically Modiﬁed products.

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p75 Neurotrophin receptor signaling in the nervous system Yuiko Hasegawa1,2, Satoru Yamagishi1, Masashi Fujitani1,2, and Toshihide Yamashita1,* 1

Department of Neurobiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan 2 Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Abstract. The neurotrophin receptor p75NTR has long been known as a receptor for neurotrophins that promote survival and diﬀerentiation. Consistent with the role of neurotrophins, p75NTR is expressed during the developmental stages of the nervous system. However, p75NTR is re-expressed in various pathological conditions in the adult. We now know that p75NTR has the ability to elicit bi-directional signals, that result in the inhibition as well as the promotion of the neurite outgrowth. p75NTR is a key receptor for myelin-derived inhibitory cues that contribute to the lack of regeneration of the central nervous system. Keywords: neurotrophin, growth factor, myelin, peptide, neuron, oligodendrocyte, glia, receptor, p75, signal, G protein, rho, axon, neurite, regeneration, central nervous system, apoptosis, cell survival, synapse, migration.

Discovery of neurotrophins Pictures of isolated embryo-derived nerve cells in culture were presented by Rita Levi-Montalcini [1]. She explained that she was planning to investigate their growth under diﬀerent experimental conditions. This was a momentous achievement at that time and has lead to many advances in current biotechnology. She presented evidence that an ‘‘agent’’ released by sarcoma fragments could stimulate outgrowth of nerve ﬁbers from sensory and autonomic ganglia in culture [1]. The nature of the growth-stimulating agent led to the discovery of nerve growth factor (NGF). Remarkable events that followed were the use of snake venom to inactivate the agent. It resulted in the recognition that the venom itself had growth-stimulating properties. Since the venom had been derived from salivary glands, it was possible that salivary glands from other animals might also contain a similar factor. This lead to the discovery that adult male mouse salivary glands are an abundant source of the factor [2]. Thanks to the success in developing antibodies to the factor, she eventually identiﬁed NGF. However, the factor did not immediately command interest, as it stimulated neurite growth to a pathological degree but had no eﬀect on motor neurons or some neurons in the central nervous system (CNS). The careful crafting of her experiments *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10005-7

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

124 contributed to growing recognition of the signiﬁcance of her work, and in 1986 lead to her being awarded the Nobel Prize. One of her important strategies was to examine the growth of neurites from the neurons in culture to test the eﬀects produced by extracts made from the target tissue. It became a powerful tool in the search for other trophic factors [3]. It is also important for developing strategies to overcome the inability of CNS neurons to regenerate, which is described in this chapter. These observations set the stage for a molecular analysis of the mechanism of action of NGF by a number of groups, including those of Levi-Montalcini, Shooter, Thoenen and Barde. The second neurotrophin to be identiﬁed, brainderived neurotrophic factor (BDNF), was isolated in 1982 from pooled extracts of porcine brain [4]. The isolation of BDNF helped establish the concept that the fate and the shape of most vertebrate neurons can be regulated by diﬀusible growth factors, as only a small number of neurons are NGF responsive in the CNS. Neurotrophins and their receptors The neurotrophins are a family of structurally related, secreted proteins that have a profound inﬂuence on the development and functioning of the nervous system [5]. Four members of this family have been identiﬁed in birds and mammals: NGF, BDNF, neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). All consist of dimers of a small basic peptide, held together by disulﬁde linkages between the conserved cystein residues. The dimer appears as a symmetrical protein with variable, basic regions that determine receptor speciﬁcity. Neurotrophins aﬀect essentially all biological aspects of vertebrate neurons, including the survival, diﬀerentiation, growth and apoptosis of neurons by using a two-receptor system, which consists of the Trk tyrosine kinases and the p75 neurotrophin receptor (p75NTR) [6,7] (Fig. 1). Three kinds of Trk receptors: Trks A, B and C mediate the biological activity of neurotrophins with the following speciﬁcity (Fig. 2). TrkA interacts with NGF, TrkB with BDNF and NT4, while TrkC is the preferred receptor of NT-3, although this molecule could interact with TrkA and B. Trk receptor tyrosine kinases undergo rapid transphosphorylation following ligand binding, leading to a cascade of protein phosphorylations in the cell. The distinctive neuronal deﬁciencies in mice with null mutations in the trkA, trkB and trkC genes are similar to those observed in mice with null mutations in the NGF, BDNF and NT3 genes, respectively. These ﬁndings suggest that the Trks mediate the survival-promoting actions of neurotrophins on developing neurons. p75NTR was the ﬁrst member of a large family of receptors, which includes both TNF receptors, Fas (Apo-1/CD95), RANK, CD40, and approximately 25 other members to be molecularly cloned [8,9]. The deﬁning motifs of this receptor superfamily are cystein repeats in the extracellular domain, which form the ligand-binding domain.

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Fig. 1. The two receptor classes of the neurotrophins. p75NTR is a transmembrane glycoprotein receptor of approximately 75 kDa. There are four cystein rich repeats in the extracellular domain. Signaling of p75NTR occurs through cytoplasmic interactors which bind with the juxtamembrane linker region or the helical domain. The Trk receptors are transmembrane glycoproteins of approximately 140 kDa. They are tyrosine kinases with an extracellular ligand binding domain containing multiple repeats of leucine-rich motifs, two cystein clusters, two immunoglobulin-like domains.

Fig. 2. Speciﬁcity and cross-talk in the interactions of neurotrophins with their receptors.

All neurotrophins bind to p75NTR with an aﬃnity of 10 9 M [10]. This is a lower aﬃnity than that required for neurotrophin binding on neurons (typically 10 11 M). However, high aﬃnity binding of neurotrophins to neurons cannot simply be explained by the presence of Trk receptors, as the majority of the neurotrophin-binding sites formed by Trk receptors are of low aﬃnity. In addition, NT-3 binds p75NTR with high aﬃnity in embryonic chick sympathetic neurons, whereas this binding does not promote their survival [11]. Thus, it appears that the formation of high aﬃnity binding and speciﬁc sites for neurotrophins on neurons are most likely, but not all, a result of the association of p75NTR with Trk receptors [12].

126 Although p75NTR was the ﬁrst neurotrophin receptor to be identiﬁed, its characterization had been in the shade for some years. This is, at least partly, due to the nonenzymatic activities of p75NTR, in sharp contrast to the inherent tyrosine kinase and the potent biological activities of Trk receptors. None of the receptor family members exhibit any intrinsic catalytic activity, and they pass their signals by associating with, or dissociating from, cytoplasmic interactors. In addition, studies on p75NTR have been complicated by the fact that it can interact with Trk receptors [13], and by the ﬁnding that its signaling capacity is modiﬁed by the coincident activation of Trk receptors. Nontheless, recent years have seen the emergence of a consensus regarding the signaling pathways activated by p75NTR and of potential biological function, and have lead to the elucidation of a number of p75NTR-interacting proteins [14]. We review here p75NTR signaling in the nervous system, especially focusing on the recent discovery that it transduces the signal from several myelin-derived inhibitors of neurite outgrowth, which are involved with the inability of CNS neurons to regenerate after injury. Interactions between p75NTR and the Trk receptors The present data suggest that p75NTR has two main physiological functions: modulating Trk receptor signaling and initiating autonomous signaling cascades. The precise molecular mechanisms that allow p75NTR to enhance NGF binding to TrkA and increase TrkA responsiveness to NGF remain uncertain, but two hypotheses have been put forward. First, p75NTR acts as a co-receptor that binds NGF and either concentrates it locally or presents it to TrkA in a favorable binding conformation. A number of studies have shown that disrupting NGF binding to p75NTR inhibits NGF-induced TrkA activation [15–18]. Complementary studies have shown that a mutant form of NGF, which binds TrkA but does not bind p75NTR, is less eﬀective than the wild type NGF in activating TrkA in cells where the two receptors are co-expressed [15,19]. These ﬁndings support the notion that NGF binding to p75NTR is necessary to facilitate TrkA activation in response to low levels of NGF. Second, p75NTR has an allosteric eﬀect on TrkA that confers high-aﬃnity NGF binding to the TrkA receptor irrespective of NGF binding to p75NTR. In support of this hypothesis, high aﬃnity NGF binding sites can be generated when TrkA is co-expressed with either a p75NTR mutant deﬁcient in neurotrophin binding, or using a chimaeric receptor consisting of the extracellular domain of EGF receptor and the cytoplasmic domain of p75NTR [20]. Further work will be required to reconcile these two models. Diverse function of p75NTR One of the most prominent biological functions of p75NTR (Fig. 3) may be that it induces cell death, as it contains a death domain sequence distantly related to the

127

Fig. 3. p75NTR is involved in several diﬀerent biological activities. p75NTR regulates both cell death and cell survival. Pro NGF seems to be the most eﬀective activator of cell death. Several interactors have been found to associate with these actions presumably depending on the cellular context. Some of the proteins that interact with p75NTR block cell cycle progression. p75NTR also regulates axon elongation by regulating the activity of RhoA both during development and after lesion.

intracellular domains of the Fas and TNF receptors [6,7]. This domain consists of a bundle of six short a-helices spanning 90 amino acids that form a novel type of fold [21]. Direct evidence of p75NTR-mediated apoptosis was ﬁrst described in 1993 by Bredesen and his colleagues who reported that p75NTR overexpression facilitates apoptosis, which is inhibited by NGF [22]. The cell death may be caused by the spontaneous signaling that occurs when the receptor multimerizes. In contrast, many in vitro studies have subsequently demonstrated that neurotrophins binding to p75NTR induce apoptosis. For example, NGF binding to p75NTR elicits apoptosis of diﬀerentiated rat oligodendrocytes [23–25], Schwann cells [26], hepatic stellate cells [27], sympathetic neuron precursor (MAH) cells [28], mesodermal cells [29], chick isthmo-optic nucleus neurons [30], trigeminal mesencephalic sensory neurons [31], and retinal ganglion

128 cells [32]. Therefore, the precise ligand dependency of this phenomenon is still in controversy and there are some reports that indicate p75NTR-promoted survival of the neurons. These in vitro studies may represent the extreme supraphysiological limits of p75NTR action, and the physiological role of the receptor should be determined at least in vivo. Basal forebrain cholinergic neurons express p75NTR at high levels, and recent evidence shows that there are indeed signiﬁcantly more cholinergic neurons in the complete p75NTR mutant mice [33]. Cell death in the avian retina in the developmental stages is reduced following the addition of the antibodies against NGF or the extracellular domain of p75NTR, which is expected to act as an inhibitor of endogenous p75NTR [34,35]. Mice that transgenically overexpress the intracellular domain of p75NTR show reductions in cortical, sympathetic and sensory neurons [36]. These ﬁndings show that p75NTR activates the cell death pathway in vivo. Neurotrophins are synthesized as precursors or pro-proteins, and proteolytic cleavage is necessary for the production of the mature neurotrophins. Recent evidence shows that uncleaved pro-NGF binds to p75NTR with high aﬃnity and causes cell death at signiﬁcantly lower concentrations than does mature NGF [37]. The binding aﬃnity of pro-NGF to TrkA is not as strong as that for mature NGF, suggesting that proteolytic processing is crucial in determining the signaling elicited by these two kinds of receptors. This aspect might be relevant, as increased levels of pro-NGF are found in the brains of patients of Alzheimer’s disease [38]. p75NTR also binds non-neurotrophin ligands, which include the neurotoxic prion protein fragment PrP (26–106) and the Ab-peptide of the amyloid precursor protein (APP) [39–42]. These peptides induce the cell death via p75NTR in culture. p75NTR is also a receptor for a glycoprotein of the rabies virus envelope, allowing the virus to enter the nervous system [43], and for the invertebrate ligand cystein-rich neurotrophic factor [44]. Other p75NTR-mediated activities have been proposed, including enhancing axonal outgrowth [45–47], inﬂuencing Schwann cell migration [48], promoting myelin formation [49], modulating synaptic transmission [50] and regulating the function of sensory neurons [51] and calcium currents [52]. Most of these biological activities can be attributed to neurotrophins binding to p75NTR. Recent work also implicates p75NTR in the regulation of axonal elongation that is elicited by several myelin-derived proteins that may contribute to the lack of regeneration of the injured adult CNS. Finally, it is noted that a short isoform of p75NTR has also been found. The transmembrane and intracellular domains of the short isoform are identical to that of the full-length p75NTR, but the short isoform lacks three of the four cystein rich repeats in the extracellular ligand binding domain [53]. Thus, this short isoform does not bind neurotrophins. Mice carrying a mutation in the p75NTR gene still express this short isoform, although the full-length of p75NTR is completely deleted [54]. The mice generated were the initial p75NTR-targeted

129 ones and have proven very useful for determining p75NTR action. However, the phenotype of the mice is diﬀerent from that of the complete p75NTR knockout mice [53], which shows up to 40% lethality, presumably as a result of blood vessel defects. The involvement of this short isoform in the formation of the blood vessels is not clear. This numerous amount of work uncovers surprisingly diverse function of p75NTR. p75NTR is involved in the pathogenesis of neurological diseases Although the expression of p75NTR is developmentally regulated in the nervous system, marked increases in p75NTR levels are observed under certain pathological conditions. In rats subjected to pilocarpine-induced seizure, expression of p75NTR is induced in entorhinal, piriform and hippocampal cortices, and its expression is associated with the cell death [55]. In the dorsal root ganglia, reduction of the p75NTR levels by antisense oligonucleotides prevents the loss of axotomized neurons [56]. Similarly, motor neuron loss occurring after transection of the neonatal facial nerve is reduced in mutant mice that carry a mutation in the p75NTR gene [57,58]. Conversely, administration of NGF into transected neonatal facial nerve of animals produces increased cell death [59]. p75NTR is re-expressed in neurons, at levels comparable to those seen during the developmental stages. Upregulation of p75NTR is also observed in the cerebral cortex in Alzheimer’s disease [60]. In a mouse model of amyotrophic lateral sclerosis, in which a mutant form of superoxide dismutase is overexpressed, there is re-expression of p75NTR in lumbar motor neurons, which are destined to die 4 months after birth [61]. In fact, expression of p75NTR is observed in motoneurons in the cervical spinal cords of patients with amyotrophic lateral sclerosis. After axotomy, cortical spinal neurons strongly reexpress p75NTR three days after lesion, when the neuronal death occurs. Activation of p75NTR by NT3 causes the death of these neurons, as shown by experiments employing antibodies against p75NTR or to NT3 [62]. These ﬁndings establish a link between p75NTR and neuronal death in neurological disorders. Signal transduction through p75NTR The ﬁrst indication of the signaling function of p75NTR was the observation that p75NTR mediates sphingomyelin hydrolysis and production of ceramide following neurotrophin binding [63]. Ceramide production is known to follow TNF binding to its receptor and to lead to NF-kB activation [64]. Likewise, in Schwann cells expressing p75NTR but not catalytic Trk receptors, NF-kB activation was observed following the addition of NGF [65]. As is the case with TNF receptor 1 signaling, it appears that activation of NF-kB prevents cell death [66,67].

130 In contrast to NF-kB activation that seems to mediate cell survival, there should be signals that elicit programmed cell death. These signals involve caspase activation, as well as Bax/Bad, Bcl-2 and Bcl-xL [68]. Inhibition of Jun kinase (JNK) activity blocks apoptosis through p75NTR, suggesting that JNK plays a signiﬁcant role in p75NTR-mediated apoptosis [69]. However, activation of JNK does not always explain p75NTR-mediated apoptosis, and there is evidence demonstrating that p53 and the p53-related protein p73 play a role [70]. As p75NTR has no intrinsic catalytic activity, cytoplasmic interactors should be recruited to produce the signals. A number of interactors have been identiﬁed mainly by yeast two-hybrid screening, trying to explain multiple intracellular signals (Fig. 4). The interactors, presumably involved in the cell death, include a ubiquitously expressed zinc ﬁnger protein designated as NRIF (neurotrophin receptor-interacting factor) [71]. The retinas of the NRIF / mice show reduced cell death, and this reduction is quantitatively similar to that seen in mice carrying a mutation in the p75NTR gene. As it localizes in the nucleus as well as in the cytoplasm, it is suggested that the neurotrophin-binding to p75NTR facilitates release from the intracellular domain of p75NTR, resulting in the translocation to the nucleus. A protein named NADE, the p75NTR-associated cell death executor, is found to be associated with p75NTR when activated by NGF, but not by BDNF,

Fig. 4. p75NTR recruits cytoplasmic interactors to signal. Several interactors of p75NTR intracellular domain have been identiﬁed that mediate diﬀerent biological functions. NADE, NRIF and NRAGE associate with cell death. NRIF, NRAGE and SC-1 are involved in cell cycle arrest. The GTPase RhoA is a regulator of axon elongation. TRAFs associate with p75NTR to activate NF-kB. The physiological relevance of some interactors is not clear.

131 NT3 or NT4/5 [72]. It also seems to contribute to the cell death inducing activity of p75NTR. The NRAGE (neurotrophin receptor-interacting MAGE) homolog was also identiﬁed as an interactor of p75NTR, and was shown to mediate NGF-dependent apoptosis in sympathetic neuron precursor cells [28]. When NRAGE is overexpressed in the transfected cells, it causes cell cycle arrest, suggesting that p75NTR may play a role in the control of growth. Nestin-positive neural stem cells proliferate at a higher rate than the wild type cells in the absence of the fulllength p75NTR, and the activation of p75NTR by BDNF promotes diﬀerentiation into neurons [73]. Therefore, the diﬀerentiation promoting eﬀect of p75NTR might be mediated by NRAGE, which should be the subject of future studies. Although NRIF is a zinc ﬁnger protein, another zinc ﬁnger protein, the Schwann cell factor-1 (SC-1), is involved in cell cycle arrest [74]. In transfected COS cells, the localization of SC-1 changes from the cytoplasm to the nucleus following NGF stimulation, but not BDNF stimulation. Expression of SC-1 in the nucleus results in a loss of BrdU incorporation. Several kinases have been shown to interact with the intracellular domain of p75NTR. A p75-associated kinase [75], as well as ERK1 and ERK2 [76], are also the interactors of p75NTR, although the functional signiﬁcance of the interactions is not clear. A variant of the b catalytic subunit of cAMP-dependent protein kinase (PKACb) is shown to be a p75NTR interacting protein, which phosphorylates p75NTR at serine 304 [77]. Intracellular cAMP in cerebellar neurons is transiently accumulated by ligand binding to p75NTR. Activation of cAMP-PKA is required for translocation of p75NTR to lipid rafts, and for biochemical and biological activities of p75NTR, such as inactivation of Rho and neurite outgrowth. Therefore, PKACb may be necessary for the proper recruitment of the activated p75NTR to lipid rafts, structures that represent specialized signaling organelles.

Axon elongation In good correlation with the function of neurotrophins, p75NTR is expressed abundantly in neurons during developmental stages. Motor neurons in the spinal cord, most sympathetic and sensory neurons in the peripheral nervous system, as well as cerebellar Purkinje cells and retinal ganglion cells all express p75NTR at high levels during the outgrowth of axons [78–82]. In dendrite-bearing neurons, p75NTR is also expressed during the time of dendritic arborization. Some neurons markedly up-regulate p75NTR after lesion or seizure [55,83]. Mice carrying a mutation in the p75NTR gene show deﬁcits in the outgrowth of thoracic intercostal nerves and forelimb motor axons [45] as well as retarded axonal arborization of the opthalamic branch [84]. As adults, these mice have deﬁcits in sensory and sympathetic target innervation [85]. Notably, these mice show a marked reduction of visual cortex innervation by thalamic axons, which are thought to use the pathway pioneered by the subplate axons as a scaﬀold,

132 and the growth cones are smaller and have a markedly reduced number of ﬁlopodia [86]. Although these in vivo biological eﬀects are likely to be due to reduced Trk activation, there are indications, at least in vitro, that the ligands binding to p75NTR promotes axon outgrowth. NGF stimulates neurite outgrowth from embryonic rat hippocampal neurons and chick ciliary neurons [45,87], which express p75NTR but not TrkA. These ﬁndings suggest that p75NTR plays some primary roles in the developmental stages as well as during pathological states. The key molecule that regulates these eﬀects may be small GTPase Rho. Rho GTPases are a family of highly related proteins that are best known for their eﬀects on the actin cytoskeleton. The representatives of the Rho family are Rho, Rac, and Cdc42. Several isoforms of Rho have been reported, and in neurons, RhoA is expressed at higher levels than RhoB and RhoC [88]. RhoA was shown to interact with the intracellular domain of p75NTR [45]. Interestingly, overexpression of p75NTR in 293 cells results in the activation of RhoA, whereas ligand binding to p75NTR abolishes the activation (Fig. 5). Inactivation of RhoA is suggested to be implicated in the neurite outgrowth of chick ciliary neurons, as incorporation of the active mutant of RhoA into the cells attenuates the eﬀect of NGF. This suggestion is substantiated by the fact that blocking Rho activity

Fig. 5. p75NTR is a bi-directional regulator of RhoA. RhoA was shown to interact with the intracellular domain of p75NTR [45]. Overexpression of p75NTR in 293 cells results in the activation of RhoA, presumably by the clustering of the receptor. Ligand binding to p75NTR, however, abolishes the activation of RhoA.

133 with the botulinus toxin C3 mimicks the eﬀects of NGF. It should be noted that neurite outgrowth by the inactivation of Rho is not speciﬁc to ciliary neurons [89,90]. The missing link between p75NTR and inactivation of RhoA was shown recently, where ligand binding to p75NTR was demonstrated to increase intracellular cAMP (Fig. 6) [91]. NGF induces inactivation of RhoA in cerebellar neurons and 293T cells, and this eﬀect is PKA dependent. PKA phosphorylates many target proteins, and one such target identiﬁed is RhoA. When serine 188 is phosphorylated, RhoA becomes inactive [92]. Taken together, it is possible

Fig. 6. Mechanisms of the axon elongation by p75NTR and neurotrophins. The ligand binding to p75NTR increases intracellular cAMP [77]. Inactivation of RhoA by neurotrophins binding to p75NTR in cerebellar neurons and 293T cells is PKA dependent. PKA phosphorylates serine 188 of RhoA, leading to inactivation of RhoA [92].

134 that inactivation of RhoA is the downstream component of cAMP-PKA. However, another interpretation of the data is that the inhibition of the PKA signal blocks the translocation of the receptor to lipid rafts and might result in the failure of transduction of the downstream signal. Inhibition of axon elongation by p75NTR Recent reports indicate that p75NTR is involved in the inhibition of axonal elongation by myelin, in sharp contrast with the notion that p75NTR contributes to promotion of axon elongation. Transgenic mice were generated in which NGF was expressed by astrocytes in the CNS under the control of the GFAP promoter. Sympathetic axonal sprouting into the brains was observed in these mice, however much more axonal sprouting occured if a mutation was inserted into the p75NTR gene. Interestingly, abberant axonal elongation is observed in myelin-rich areas where these axons would normally not grow [93]. The hippocampus of mice carrying a mutation in the p75NTR gene is hyperinnervated by cholinergic aﬀerents [94]. These seemingly contradictory ﬁndings suggest that p75NTR transduces bi-directional signals that elicit inhibition of neurite growth as well as axonal outgrowth. Recent surprising reports uncover the molecular mechanism of these biological eﬀects, providing molecular targets for the development of the therapies against injuries to the CNS. Inability of the adult CNS to regenerate Injury to the adult CNS is devastating because of the inability of central neurons to regenerate correct axonal and dendritic connections. It is now well established that axons of the adult CNS are capable of only a limited amount of regrowth after injury, and that an unfavorable growth environment plays a major role in the lack of regeneration. In 1911, F. Tello described the ﬁrst successful transplantation of a peripheral nerve into the adult mammalian CNS [95]. Previously denervated sciatic nerve pieces were implanted into the cortex of rabbits, and he observed fascicles and individual nerve ﬁbers that invaded into these peripheral nerves 2 to 4 weeks after surgery. He and Ramon y Cajal concluded that peripheral nerve Schwann cells reacted to the loss of their axons by the synthesis of attractive and neuritepromoting cues [96]. They suggested further that CNS glia would be devoid of such a reaction. The morphological features of axonal injury and degeneration in vivo were elegantly described by Ramon y Cajal. Aguayo’s group in the early 1980s showed that many neurons can regenerate over long distances if oﬀered a peripheral nerve as a substrate [97–99]. That CNS myelin is involved in the prevention of axonal regeneration in adult mammals was ﬁrst suggested by Berry [100]. He pointed out that nonmyelinated axons in the CNS would regenerate after chemical axotomy if damage did not occur to nearby myelinated ﬁbers, but not after mechanical axotomy, which

135 damages myelinated ﬁbers. As damage to the myelinated ﬁbers leads to the release of degeneration products of CNS myelin, it was proposed that this damage would be inhibitory to axonal growth. Subsequently, Schwab’s group tested this hypothesis by exposing perinatal DRG or sympathetic neurons to optic and sciatic nerve explants of adult rats in the presence of NGF. However, they observed few or no axons in the optic nerves during 2 weeks in culture, whereas abundant nerve ﬁbers invaded into the sciatic nerves [101]. As repeated freezing and thawing of the explants prior to culture gave the same results, the absence of neurite outgrowth in the adult optic nerve explants results from an intrinsic property of the adult CNS tissue rather than to reactions to the lesion or the culture condition. They postulated that myelin from the adult CNS is an inhibitory substrate for neurite outgrowth.

Three distinct myelin proteins inhibit axon growth Nogo Initially, biochemical analysis of rat brain myelin showed two protein constituents of MW 35 kDa and 250 kDa which were potent inhibitors of neurite outgrowth [102]. One monoclonal antibody called inhibitor-neutralizing antibody (mAB IN-1) was obtained and used extensively for subsequent in vitro and in vivo experiments. The inhibitory activity of a crude myelin extract was decreased to approximately 50% by this antibody, and that of puriﬁed bovine NI-220 (the homolog of rat NI-250) was decreased to 0–20% of initial levels by the antibody [103]. Starting with large amounts of bovine spinal cord, Schwab’s group succeeded in purifying the bovine homolog bNI-220. The corresponding cloned cDNA has the characteristics of a type 2 membrane protein and is derived from a gene which gives rise to three mRNAs [104–106]. This gene is designated Nogo. The three splice variants of Nogo are called NogoA, NogoB and NogoC, the latter two of which are widely expressed outside the CNS. NogoA possesses a unique amino-terminal region not shared by NogoB and NogoC. The two most strongly predicted transmembrane domains are separated by the 66-residue extracellular or lumenal loop, called Nogo-66. Nogo-66 causes growth cone collapse [107]. The Nogo-A speciﬁc aminoterminal region is also inhibitory for neurite outgrowth, and prevents the spreading of ﬁbroblasts. Immnunohistochemical studies have shown that Nogo proteins are present in neuronal cell bodies and axons as well as oligodendrocytes. Speciﬁcally, Nogo-A is most strongly expressed in oligodendrocytes in the white matter, although it was also detected in neuronal perikarya including those in the cerebral cortex, spinal motor neurons and DRG neurons, as well as in axons [108]. Whether neuronal Nogo-A plays a role in axonal growth or guidance in the developing nervous system should be determined in the future.

136 MAG Myelin-associated glycoprotein (MAG) is a transmembrane protein of the immunoglobulin superfamily, found in both peripheral and CNS myelin, where it plays a role in the formation and maintenance of myelin sheath. MAG was identiﬁed as the ﬁrst myelin-derived growth inhibitory protein by two groups. McKerracher et al. detected MAG inhibitory activity in myelin after extraction with octylglucoside, fractionation by ion exchange chromatography, and screening for inhibitory activity [109]. Filbin’s group demonstrated that MAG that was ectopically expressed in CHO cells inhibits neurite outgrowth [110]. Interestingly, MAG is a bifunctional regulator of axon growth. MAG can stimulate neurite outgrowth of young neurons, where endogenous levels of cAMP may be critical for this eﬀect of MAG [111]. A soluble form of MAG, capable of inhibiting neurite outgrowth from P6 DRG neurons, is released from damaged CNS myelin [112]. Soluble MAG constitutes the great majority of the neurite outgrowth inhibiting factors released from damaged myelin. OMgp Oligodendrocyte-myelin glycoprotein (OMgp) is the most recently identiﬁed protein that is an inhibitory component of myelin. In the course of the isolation of MAG as an inhibitory protein, Braun’s group observed two peaks of inhibitory activity, with MAG present in the ﬁrst peak. The group led by He separated the inhibitory protein in the second peak, and identiﬁed OMgp [113]. They identiﬁed OMgp as an inhibitor with the hypothesis that any GPI-anchored myelin proteins act as regeneration inhibitors [114]. OMgp, which is abundant in myelin, has potent growth cone collapsing and neurite outgrowth inhibitory activities. The available evidence suggests that OMgp is principally a neuronal protein with a limited amount of OMgp being expressed by oligodendrocytes [115], whereas the functions of neuronal OMgp have not been explored. The three inhibitors, Nogo, MAG and OMgp, have similar inhibitory activity and distribution in the myelin sheath, suggesting that all of them probably contribute to growth inhibition in the adult CNS. The Nogo receptor A protein that binds Nogo-66 was identiﬁed with high aﬃnity by an alkalinphosphatase-fusion protein expression screening strategy [116]. Transfection of the cDNA encoding this putative receptor into retinal ganglion cells at a developmental stage when they otherwise are unresponsive to Nogo-66 promotes growth cone collapse by GST-Nogo-66. Mutated forms of the receptor eliminates growth inhibition by Nogo-66. Therefore, this protein is a receptor for Nogo-66 (NgR). NgR is a glycosylphosphatidylinositol (GPI) anchor protein

137 that attaches to the outer leaﬂet of the plasma membrane, and is expressed in the CNS neurons as well as their axons [117,118]. As release of GPI-anchored proteins by phosphatidylinositol-speciﬁc phospholipase C from embryonic DRG results in the abolishment of growth cone collapse in response to Nogo-66, NgR mediates the signal from Nogo-66 in at least these neurons. Surprisingly, two other inhibitory components, MAG and OMgp, also bind to NgR. In an expression screening for NgR-interecting proteins, Strittmatter’s group isolated MAG as a binding partner for NgR [119]. Filbin’s team identiﬁed it by direct binding studies based on the similarity in molecular weight to candidates revealed in a previous characterization of MAG binding proteins [120]. NgR was also obtained by screening for proteins that bind to OMgp [114]. Therefore, NgR is necessary for inhibition of axon growth by MAG, Nogo-66 and OMgp in vitro, and ectopic expression of NgR leads insensitive neurons to become sensitive to these myelin-derived proteins. These ﬁndings bring these various molecules to an intersection at the level of NgR. Surprisingly, NgR expression is not very altered by axotomy [121], which suggests that it has a physiological role in the intact CNS, unrelated to injury and regeneration. Moreover, Niederost et al. claim that phospholipase treatment, to remove NgR and other GPI-linked cell surface molecules, does not block all of the inhibitory eﬀects of MAG on neurite outgrowth from cerebellar granule cells grown on polylysine [122]. Their results contrast markedly with the ﬁndings of others [119,120]. From the perspective of trying to develop a therapeutic approach, it is important to note that a fragment of Nogo-66 binds to NgR as a high aﬃnity antagonist [123]. The antagonist peptide, NEP1-40, reduces endogenous inhibitory activity, to promote sprouting of corticospinal tract axons, long distance growth and functional recovery. p75NTR transduces the signal from MAG, Nogo and OMgp Although NgR is a binding partner for MAG, Nogo-66 and OMgp, the GPIlinked nature of NgR suggests that there may be a second receptor subunit that spans the plasma membrane and mediates signal transduction. Identiﬁcation of the signal transducer of these proteins came from the experiments by Filbin’s team showing that nerve cells pretreated with neurotrophins overcome MAG’s power to squelch growth [124]. The ﬁnding hinted at a connection between p75NTR and MAG. Perhaps MAG cannot signal when neurotrophins occupy p75NTR, we reasoned. To learn whether the receptor might be playing both sides – as a growth stimulant and suppressor – we tested whether MAG requires p75NTR to relay its message. MAG’s eﬀect on nerve elongation in normal mice and in animals lacking the receptor was examined. Without p75NTR, MAG’s clout in blocking nerve extension withered [125]. Colocalization of p75NTR and MAG binding is seen in neurons. These results show that p75NTR may be a signal

138

Fig. 7. Signal transduction by the complex of the Nogo-66 receptor (NgR) and p75NTR. MAG, Nogo-66 and OMgp are ligands for the Nogo-66 receptor (NgR), and are all expressed by oligodendrocytes. These ligands do not share any recognized protein domains. p75NTR interacts with NgR as well as ganglioside GT1b, and mediates the inhibitory signaling of these myelinderived proteins by activating RhoA.

transducer of MAG (Fig. 7). Next, we looked for conspirators in the molecules’ ability to suppress nerve cell extension. p75NTR’s talent for eliciting nerve growth relies on Rho. As Rho shuts down and nerves branch out when p75NTR binds neurotrophins, we reasoned that p75NTR’s nerve-constraining alter ego also relies on Rho. To test the idea, Rho’s function in cells was crippled and then, MAG susceptibility vanished. Then we exposed normal and p75NTR-deﬁcient cells to MAG and measured the amount of active Rho by aﬃnity precipitation. MAG activates Rho only in the presence of the receptor, verifying p75NTR’s part in the eﬀects. Extrapolating from the observations that MAG is a ligand for NgR, the possibility that p75NTR associates with NgR to form a receptor complex for MAG, Nogo and OMgp was tested [126,127]. He’s group as well as Poo’s group demonstrated that at least a fraction of p75NTR binds with NgR using co-immunoprecipitation experiments. Postnatal cerebellar neurons from mice carrying a mutation in the p75NTR gene are insensitive to GST-Nogo-66 and OMgp-AP [126]. The inhibitory activity of these proteins in cerebellar granule neurons is decreased by the ectopic expression of a dominant negative form of p75NTR that lacks a cytoplasmic domain. Soluble p75NTR-Fc fusion protein also attenuates the eﬀects. These observations not only suggest that p75NTR is required for the inhibitory activity of these myelin-derived proteins, but also

139 provide a potent molecular target for developing therapeutic agent against injuries to the CNS. The p75NTR knockout mouse has become a prime target for regeneration experiments. However, it should be noted that p75NTR has diverse functions, including bi-directional signals regulating axon growth, thus alarming that silencing all the functions of p75NTR would be a less attractive treatment strategy. Axon growth inhibition signals from p75NTR Downstream from the receptor complex of p75NTR and NgR, Rho appears to be a key intracellular eﬀector for growth inhibitory signaling by myelin. In neurons, myelin and MAG inhibit growth, that is abolished by the botulinus toxin C3 which inactivates Rho [88]. More speciﬁcally, it is directly shown that Rho is activated by MAG-Fc in the cerebellar granule neurons shown by the aﬃnity precipitation of GTP-bound form of RhoA [125]. The precise mechanism of action of p75NTR is suggested by the ﬁnding that p75NTR releases Rho from Rho guanine nucleotide dissociation inhibitor (RhoGDI) (Fig. 8) [128], thus eliciting activation of Rho. Rho-GDI is an essential part of the signaling mechanism that suppresses the activity of Rho. Rho proteins are regulated either by enzymes that enhance GTP binding and activity (guanine nucleotide exchange factors) or by proteins that increase the hydrolysis of GTP (GTPase activating proteins) and thus decrease activity. Rho is kept in an inactive state in cells by Rho-GDI [129]. Rho-GDI inhibits the activity of Rho by binding to and sequestering Rho in the cytoplasm, by inhibiting the formation of active RhoGTP, and by blocking the binding of Rho to its eﬀectors. As mentioned above, RhoA was previously identiﬁed as an interactor of p75NTR by the yeast two-hybrid screening method [45]. As only the wild type of RhoA, which is predominantly in a GDP-bound form, but not the constitutive active form of RhoA, interacts with p75NTR, as shown by the co-immunoprecipitation assay, it is suggested that activation of RhoA is dependent on a direct interaction of RhoA and p75NTR. Interestingly, overexpression of the intracellualr domain of p75NTR as well as the full-length p75NTR activates RhoA ligand independently, suggesting that p75NTR may be a constitutive activator of Rho. However, the intracellular domain of p75NTR shows no similarity with the Dbl homology domain, which is shared by conventional guanine nucleotide exchange factors, demonstrating that p75NTR does not mediate the process of exchange reaction, in which GDP is replaced by GTP. Instead, direct interaction of the Rho-GDI with p75NTR initiates the activation of RhoA, by promoting the release of prenylated RhoA from RhoGDI, enabling RhoA to be activated by guanine nucleotide exchange factors. These ﬁndings establish Rho as a key player in inhibiting the regeneration of the CNS, and launched a new wave of studies that aimed to promote regeneration of injured axons by modulating this inhibitory pathway. For example, an inhibitor of Rho kinase, a downstream eﬀector of Rho, called Y-27632 has

140

Fig. 8. Mechanisms of axon growth inhibition by p75NTR. In the absence of MAG or Nogo, growth and regeneration occur as a result of Rho-GDI-induced suppression of Rho activity. Rho-GDI maintains Rho in an inactive state by binding, and prevents Rho from interacting with its eﬀectors. Activation of p75NTR promotes dissociation of Rho-GDI from RhoA, allowing RhoA to become activated through the exchange of GDP for GTP. The activated RhoA then interacts with its signaling molecules to elicit axon growth inhibition in some neurons.

been used to probe the role of Rho in growth inhibiting signaling [130,131]. Treating neurons with C3 transferase, a bacterial endotoxin that inactivates Rho, or with Y-27632, promotes growth on inhibitory substrates. Intriguingly, a peptide that blocks the pathway elicited by MAG, Nogo and OMgp was found [128]. The binding region of Rho-GDI on p75NTR was identiﬁed as the ﬁfth alpha helix in the p75NTR intracellular domain. The short sequence of the ﬁfth helix is similar to mastoparan, a 14-residue peptide of wasp venom that is capable of activating Rho [132]. A peptide ligand bonded to this region was previously reported by Ilag’s group [133] by screening a combinatorial library using a variation of the selectively-infective phage method. This peptide, designated Pep5, inhibits the interaction of p75NTR with Rho-GDI in vitro and in vivo. The inhibitory peptide completely abolishes the eﬀects

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Fig. 9. Hypothesis of p75NTR function. p75NTR positively and negatively regulates axon elongation. Balancing mechanisms of the opposite cues through p75NTR might be necessary for plasticity and axon-glial interaction as well as neural development and regeneration.

mediated by MAG or Nogo-66 in adult DRG neurons and postnatal cerebellar granule neurons [128], establishing the Rho-GDI-p75NTR association as an important mecahnism of p75NTR-induced suppression of axon growth by myelin proteins. An especially notable aspect is that the peptide does not inhibit other functions of p75NTR, such as axon elongation or cell death by neurotrophins.

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151

Phage display for epitope determination: A paradigm for identifying receptor–ligand interactions Merrill J. Rowley*, Karen O’Connor, and Lakshmi Wijeyewickrema Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia Abstract. Antibodies that react with many diﬀerent molecular species of protein and non-protein nature are widely studied in biology and have particular utilities, but the precise epitopes recognized are seldom well deﬁned. The deﬁnition of epitopes by X-ray crystallography of the antigen–antibody complex, the gold standard procedure, has shown that most antibody epitopes are conformational and speciﬁed by interactions with topographic determinants on the surface of the antigenic molecule. Techniques available for the deﬁnition of such epitopes are limited. Phage display using either gene-speciﬁc libraries, or random peptide libraries, provides a powerful technique for an approach to epitope identiﬁcation. The technique can identify amino acids on protein antigens that are critical for antibody binding and, further, the isolation of peptide motifs that are both structural and functional mimotopes of both protein and non-protein antigens. This review discusses techniques used to isolate such mimotopes, to conﬁrm their speciﬁcity, and to characterize peptide epitopes. Moreover there are direct practical applications to deriving epitopes or mimotopes by sequence, notably the development of new diagnostic reagents, or therapeutic agonist or antagonist molecules. The techniques developed for mapping of antibody epitopes are applicable to probing the origins of autoimmune diseases and certain cancers by identifying ‘‘immunofootprints’’ of unknown initiating agents, as we discuss herein, and are directly applicable to examination of a wider range of receptor–ligand interactions. Keywords: antibody, epitope, conformational epitope, discontinuous epitope, critical contact residues, phage displayed libraries, random peptide libraries, gene speciﬁc library, mimotopes, carbohydrate antigens, DNA antigens, ﬁlamentous bacteriophage, pIII, pVIII, pVI, receptor–ligand interactions, cysteine-constrained libraries, unconstrained libraries, immunofootprinting, autoimmunity, tumor-speciﬁc antigens, X-ray crystallography, homology modeling, vaccines.

Introduction Antibodies can bind with high aﬃnity and speciﬁcity to molecules of virtually any shape, and to antigens ranging from small organic compounds to large proteins. These characteristics have led to the widespread use of antibodies as laboratory reagents, in diagnostic tests, and for therapeutic purposes. The immune system, upon stimulation, produces antibodies of increasing aﬃnity by a process of natural selection. This antigen-driven selection that governs the production of antibodies has three key features: the generation of millions of diﬀerent antibody genes through the rearrangement of a limited number of germ-line gene segments; the expression of this repertoire of rearranged genes on the surface of B lymphocytes where the antibody functions as an *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10006-9

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

152 antigen receptor; and the further antigen-driven selection of clones of B lymphocytes for proliferation and diﬀerentiation into antibody-producing plasma cells [1]. Antibodies are raised by the immune system against regions on the surface of a protein known as epitopes. Epitopes or antigenic determinants are often classiﬁed as either continuous or discontinuous: a continuous or linear epitope typically comprises three or four adjacent amino acids over a short segment in the primary sequence, whereas discontinuous or conformational epitopes comprise residues that are distant in primary sequence but brought together in the folded native conformation. Whilst an epitope is often speciﬁed as a linear sequence of an antigenic molecule, antibodies in fact react optimally with structures formed by protein folding by which disparate residues for the antibody paratope come into contiguity on the surface of the antigenic molecule; the actual aﬃnity of antigen–antibody binding, and the conferring of speciﬁcity, can depend on the interaction of just a few contiguous albeit discontinuous amino acid residues [2]. Accordingly the distinction between the two types of epitopes to a degree is artiﬁcial. Thus, a linear peptide itself can adopt particular conformations and, within the tertiary structure of a protein, an epitope deﬁned as linear according to reactivity with a short peptide sequence may well represent just one part of a larger conformational epitope [2,3].

Techniques of epitope mapping The epitope–paratope interface involves a surface area of about 700 A˚2. The use of X-ray crystallography to examine structures of antibody–protein complexes has indicated that most epitopes contain 15–22 protein residues in contact with the combining site of the antibody [4–6]. Molecular modeling and site-directed mutagenesis studies on antibodies and protein antigens revealed that within a structural or conformational epitope, there is a subset of residues that contributed most of the free binding energy [7,8]. These contact residues constitute the functional epitope and can be scattered over two or three discontinuous polypeptide segments. On average at least three noncontiguous amino acids dominate a functional epitope, and about eight amino acid residues contribute to binding [8]. Complete deﬁnition of the structure of epitopes requires X-ray crystallography, but this requires a puriﬁed and homogenous source of antibody and of antigen, and is not readily applicable to most antigens. Initially, epitopes were most readily deﬁned by comparing the cross-reactivity of antibodies against deﬁned proteins with naturally occurring variants [9]. This technique was used extensively in early studies of antigen–antibody interactions, for antigens that included hen egg-white lysozyme in which lysozymes could be compared from diﬀerent species of birds [9,10]. However, such studies were limited by the availability of naturally occurring and sequenced variants.

153 With the development of molecular biology, epitope mapping with both monoclonal and polyclonal antibodies has been performed by dissecting the antigen into overlapping polypeptides in the form of recombinantly expressed fusion proteins or truncation mutants [11–13]. However the assembly of appropriate panels of truncation mutants is time-consuming and the procedure usually resolves the epitope region only to a sequence of some 100–200 amino acids. With other approaches, attempts are made to maintain the conformation of the original antigen. Hybrid molecules have been prepared, exempliﬁed by hybrids of the two isoforms of glutamic acid decarboxylase, GAD65 and GAD67 [14,15], or antigens are altered by site-directed mutagenesis to identify amino acids that contribute particularly to antibody binding [15,16]; these procedures likewise provide limited insight into the conformational structure of the true epitope. Alternatively, linear synthetic peptides can be used to identify epitopes. Geysen introduced the technique of synthesizing overlapping oligopeptides and probing for reactivity with the antibody under investigation [17,18], and showed the applicability of this technique to the mapping of ‘‘discontinuous’’ epitopes [18]. Since then many peptide-based technologies have been developed for mapping both B and T cell epitopes [19]. It was these studies that lead to the designation of ‘‘mimotopes,’’ as mimics of an epitope, with the term applied to peptides that bound to antibodies reactive with conformational epitopes, and hence acted as mimics of the epitopes without necessarily any linear sequence homology with the original antigen. Although peptide epitope mapping has been widely applied [20–22] and the peptides identiﬁed may react directly by ELISA with antibodies, or absorb reactivity, this reactivity is usually weak relative to that of the parent molecule; hence these sequences are likely fragments of a more complex antigenic structure [3]. For this reason, scanning for antibodies with overlapping sets of synthetic or recombinant peptides covering the antigen sequence has had only limited success. Screening large libraries of random peptides would allow the selection of any peptides that could fulﬁl the 3-dimensional requirements for recognition, whether or not the peptide occurred in the primary antigenic sequence, but studies that utilize such random peptides have been restricted by the lack of techniques to synthesize and screen appropriate libraries. Accordingly, the development of phage display has provided a powerful new methodology for epitope mapping that allows the ready identiﬁcation of noncontiguous ‘‘critical contact residues’’ that contribute to binding.

Phage display Phage display technology is the product of two elementary concepts. First, any insertion mutation at an appropriate location within a structural gene of a virus if it does not disrupt an essential function conferred by the gene product,

154 will lead to the display of the mutation-encoded peptide on the surface of the viral particle. Second, if multiple inserts are all random oligonucleotides, the resulting phage particles will comprise a vast library of peptides, each one displayed on the virus and linked to the DNA that encodes it in the mutated coat protein surrounding the enclosed mutant DNA [23]. The physical association between phenotype (the displayed peptide) and genotype (the encoding DNA) in the same phage particle is the unique and highly advantageous feature of phage displayed peptide libraries. Phage display technology was ﬁrst developed by Smith [24] who cloned a restriction enzyme digest of plasmid DNA into the gene III insertion site of the ﬁlamentous phage f1, thus creating a library of fusion proteins with the foreign sequence in the middle, which was displayed on the virion surface. Smith showed that, after transfection in Escherichia coli, each of the ‘‘fusion phage’’ clones that were produced contained the insert, which encoded part of Eco R1 endonuclease, and could be aﬃnity puriﬁed from a library of random inserts using antibody to Eco R1 endonuclease. Cwirla et al. [25] further developed the technology by creating a large and diverse oligonucleotide library using inserts with randomly synthesized residues representing many variations. In this case, the phage were aﬃnity selected using a monoclonal antibody speciﬁc for the N-terminus of b-endorphin (YGGF). Almost all clones selected displayed YG on the N-terminus of the variable peptide, in agreement with the known speciﬁcity of the monoclonal antibody. Of importance for the acceptance of the technique, the screening method required no previous knowledge of the structure of the peptide, or its antibody speciﬁcity. The above studies pointed to the utility of the technology for identifying antibody epitopes on a wide range of antigens. Although for Smith’s initial study, the DNA insert was a restriction digest of plasmid DNA, and so in fact represented a gene-speciﬁc library, the possibility of selecting ligands from large libraries of random peptides with very diverse amino acid sequences created immediate interest. There are now various phage-displayed random peptide libraries commercially available, and phage display technology has become an economical and practical approach to ascertain the epitopes for a wide range of antibodies, both monoclonal and polyclonal [26–33]. Antibody screening readily discloses sequences that mimic linear, discontinuous, and even nonpeptide epitopes of antigens, such as DNA or carbohydrates [34–36]. Moreover, phage display technology has been extended to the study of diverse types of receptor–ligand interaction, using the techniques generally applicable to antibody–epitope interactions.

Characteristics of phage particles Foreign polypeptides have been displayed on viruses [37], eukaryotic cells [38], bacteria [39], as well as on bacteriophage l [40], T4 [41–43], T7 [44–46] and

155 P4 [47]. However most published work cites ﬁlamentous phage, a class of single stranded bacteriophage that infect only male bacteria. Filamentous phage have several properties that make them attractive for use as peptide display vectors, being well characterized, easy to work with, and having a surface of low complexity. Also, the phage grow to high titres, and are resistant to pH 2 or 12, which simpliﬁes the breakdown of phage–antibody complexes. The ﬁlamentous phage M13, closely related to ﬁlamentous phage fd and f1, has been used most extensively; it is a nonlytic phage, so that the problem of contamination from E. coli host proteins is much reduced, with less time spent isolating the phage. By electron microscopy, the wild-type M13 virus particle appears as a ﬂexible rod, about 1 mm in length, and 6 nm in diameter, depending on the strain used. The single-stranded circular genome is stretched along the entire particle length and is coated by the helically arranged molecules of the 50-residue major coat protein pVIII (about 0.42 copies of pVIII per nucleotide or for the wild-type 6408 nucleotides, about 2670 copies of pVIII) [48]. At one tip of the virus particle there are ﬁve copies each of the pIII and pVI proteins (genes III and VI, respectively) that are involved in host cell binding and termination of the assembly process, and the minor coat proteins pVII and pIX (genes VII and IX) are at the other tip (Fig. 1). The phage receptor on the bacterial surface is the tip of a thread-like structure, the sex pilus, encoded by the F episome in male strains, and thus referred to as the F pilus; phage infect strains of E. coli that display the F pilus. Infection is initiated by attachment of the N-terminal domain of pIII (about 200 amino acids) to the tip of the pilus, which is the end of the particle that ﬁrst enters the cell [49]. After the virion is brought to the cell surface and the single stranded genome (or ( þ ) strand) is delivered to the cytoplasm, host polymerases employ the ( þ ) strand as a template for synthesis of a complementary () strand, yielding a double-stranded phage genome [50]. Unlike lytic phage, which are released by cell lysis after assembly in the host cell cytoplasm, M13 phage are continuously extruded through the host cell envelope in a process that couples assembly with export. A feature of

Fig. 1. Schematic diagram of the structure of ﬁlamentous phage.

156 ﬁlamentous phage of importance for their use in phage display technology is their ability, merely by further addition of pVIII subunits, to package longer genomes than the wild type [51]. pIII is required not only for F-pilus absorption but also for terminating virion assembly and stabilising the viral particle; deﬁciency of pIII leads to the production of multilength viral particles (polyphage) containing two or more unit-length phage genomes [52–54]. Display systems in filamentous phage Phage libraries in ﬁlamentous phage have been generated by fusion of foreign peptides into three coat proteins, pIII, pVIII and, less often, pVI, with each having particular advantages and disadvantages [55]. The most widely used phage display vectors, in which peptides are displayed close to the signal peptide in the pIII coat protein, (type 3, type 3 þ 3, type 33), or the pVIII coat protein (type 8, type 8 þ 8 or type 88) are shown in Table 1 [49,56]. The ﬁrst phage system used was a type 3 vector in which the phage genome coded also for a fusion protein [24], but every copy of a coat protein containing a peptide has disadvantages: the size of peptides that can be inserted in every copy of a coat protein without disrupting function is limited; and a lower density of recombinant peptide on the surface of a bacteriophage should allow for better discrimination of peptides that bind with high aﬃnity [57]. The need to limit the number of peptide-displaying coat proteins prompted the development of phagemid vectors (type 3 þ 3 or type 8 þ 8 vectors) [58,59]; phagemids are plasmids that possess the usual plasmid origin of replication and selectable antibiotic-resistance markers together with all the bacteriophage elements required for single-strand synthesis and encapsidation. If a cell that is harboring a phagemid is co-infected with a helper phage, there is generated a mixture of particles that contain either the phagemid or helper-phage genome, with a combination of wild-type and hybrid molecules on the surface. Through selection for their antibiotic-resistance markers, phagemid virions can be selected from the helper virus. Alternative systems have been developed based on Table 1. Most common phage display vectors developed for use in ﬁlamentous phage. System

Coat protein

Type

No. of peptides

References

3 33 3þ3 8 88 8þ8

pIII pIII pIII pVIII pVIII pVIII

Single recombinant gene III with DNA insert Two genes III, one wild type, one with insert Two genes III, recombinant gene on plasmid Single recombinant gene VIII with DNA insert Two genes VIII, one wild type, one with insert Two genes VIII, recombinant gene on plasmid

5 <5 <5 2700 <2700 <2700

[24,188] [92,102] [189,190] [191] [60,61,192] [58,59]

157 phage in which the genome bears a second copy of a gene that is not homologous to the wild type gene in nucleotide sequence, but there is near identity in the encoded amino acid sequences [56,60]. Thus the second copy of the coat protein gene used for peptide display is located on the phage genome, rather than on a plasmid, with no requirement for helper phage (type 33 or 88) [61]. Peptide display in the pIII coat protein The pIII coat protein comprises 406 amino acids with an 18 amino acid leader sequence, and two domains. The N-terminal region protrudes outwards, forming a knob-like platform ideal for the display of foreign peptides, whilst the C-terminal region is buried in the particle [51]. The N-terminus of pIII mediates virion adsorption and the C-terminus determines virion assembly and structure [53,55,62]. In most libraries the displayed peptides are located in the N-terminus of mature pIII, 2–3 amino acids downstream from the signal peptide [63]. Peptides extending to hundreds of amino acids have been successfully displayed on pIII [34]; however, pIII is important in phage infectivity, and phage with inserts which disrupt pIII function (infection or morphogenesis) are disadvantaged during replication [64]. In practice, the use of pIII to present peptide sequences is limited by the small number of fusion proteins that can be displayed on each particle. Peptide display in the pVIII coat protein pVIII is the most abundant capsid protein, as approximately 2700 molecules are needed to form the protein cylinder around the DNA. It is synthesized as a 73-amino acid precursor containing a 23-residue N-terminal signal sequence [65]. Conditions for peptide insertion into pVIII are more stringent than for pIII and, in contrast to the pIII libraries, the pVIII inserts generally cannot exceed 10 amino acids if present on every subunit of the capsid [59], since the length of the peptide is prejudicial to phage viability [66]. This problem has been overcome by mixing both wild-type and chimeric pVIII molecules on the same particle, using a two gene or phagemid system, thereby allowing peptides displayed on pVIII to be increased to more than 100 amino acids in length, although only a fraction of the pVIII molecules will contain the insert. Peptide libraries in pVIII compared with pIII may produce clones with higher aﬃnity for the antibody, possibly as a result of cooperative binding of a number of identical peptides on the phage surface. Peptide display in the pVI coat protein Although expression in pIII or pVIII has been most widely exploited, fusion of inserts to the N-terminus of these proteins is unsuitable for the expression of inserts encoded by cDNA bearing stop codons. However such peptides can be displayed as a direct C-terminal fusion to the minor coat

158 protein VI [67]. Such libraries are more useful for the expression of full-length cDNA clones that include stop codons, and are rarely necessary when phage display is used for mapping of epitopes on antigenic molecules. Other bacteriophage display systems Filamentous phage have been used preferentially as display systems, because nonlytic phage vectors such as M13 simpliﬁes puriﬁcation of phagotopes; however all components of the phage particle need to be exported through the bacterial inner membrane before phage assembly. Successful export requires a signal peptide, and a particular length, sequence and folding characteristics of the protein to be encoded, so that only a subset of proteins encoded by a cDNA library will be capable of display. To overcome this, display systems in lytic phage have been developed, of which several allow the display of much larger proteins, >35 kD [41]. These include display in bacteriophage T4 [41,68], T7 [44–46,69–72], or l [73–78]. Such systems have been used particularly to extend the applicability of phage display, to screen tissue or organism-speciﬁc cDNA libraries [46,71,72,77,79], to identify RNA-binding proteins [44], DNA-binding proteins [75,77], to isolate mutant proteins with altered properties using gene-speciﬁc libraries developed by error-prone polymerase chain reaction [45], amongst other applications. However, they have been only rarely used for epitope mapping [76]. Epitiope mapping using phage display Standard procedures for the mapping of epitopes of protein antigens by phage display have been reviewed in detail previously [49,80–82]. The technique involves four particular steps: aﬃnity selection of peptides displayed on phage; identiﬁcation of antibody-speciﬁc peptide motifs; structural localization of the motifs on the antigen to identify the epitope; and validation of the epitope. Affinity selection of peptides displayed on phage There are a number of factors that are important for success using phage display for epitope mapping. In particular, the nature of the library will aﬀect characteristics of the peptides selected. Epitope mapping has been successfully performed using either gene-speciﬁc libraries or random peptide libraries, and both techniques have their particular applications. Gene-fragment libraries In these libraries small fragments of a particular protein of interest are presented within the bacteriophage coat protein. The ﬁrst such libraries were

159 constructed in the pIII protein, and allowed for the display on the surface of epitopes of up to 100 amino acids in their native structural context [64]. The libraries were constructed by generating gene fragments of approximately 50–200 base pairs in length using a randomly cutting endonuclease such as DNase 1, and cloning these fragments into the 50 end of the gene encoding pIII by using oligonucleotide linkers [32,83–85]. Gene-fragment libraries were the ﬁrst that were used for epitope mapping [24], and their use provided a ‘‘high throughput’’ procedure for epitope mapping using truncation mutants (see above). These libraries can contain large epitope structures that would not be represented in random peptide libraries [85] (see below). Also, just a single round of aﬃnity selection, or panning, usually suﬃces to identify unequivocally a linear epitope. Several applications of gene fragment libraries have used direct screening by plaque lifts [86] or employed only one or two rounds of aﬃnity enrichment followed by screening [32]. Moreover, genespeciﬁc libraries have a further utility when polyclonal sera are used to map epitopes, in that such sera will contain antibodies to many antigens beyond the one of interest, yet the phage selected will contain sequences of just one antigen. Although gene-fragment libraries have been quite successfully used for epitope mapping studies, they share the diﬃculty of all studies that attempt to deﬁne conformational epitopes based on linear peptides, namely the deﬁnition of the critical contact residues within a long primary sequence. Also, their use is limited to peptide epitopes that can be encoded in the genome, and so do not deﬁne mimotopes, or alternative ligand structures. Hence such libraries cannot be used for mapping non-peptide epitopes, such as carbohydrate antigens, nor DNA antigens. Finally, unless extensive studies are planned with a range of antibodies to one particular antigen, the need to develop a new antigen library for every study is labor intensive. Accordingly, random peptide libraries have been developed to allow for identiﬁcation of mimotopes for many antibodies. Random peptide libraries The major rationale for random peptide libraries is the generation of a diverse repertoire of molecular shapes, from which ligands for a given target molecule can be isolated. The development and design of such libraries have been reviewed previously [56,87]. Particular concerns in the construction of random peptide libraries are the completeness of the library and amino acid bias. The number of variants is limited by transformation eﬃciency. Since it is practically diﬃcult to assemble peptide collections that contain more than 108–1010 clones, the sequence and structural space that may be searched is limited [88]. The display of all possible combinations of just seven amino acids would require a library size of 207, i.e., 109 clones; this represents the upper limit of the current cloning techniques, and libraries that display more than seven amino acids are

160 incomplete [89]. However there are several advantages in using long peptide libraries, despite this limitation. A long peptide library can increase structural diversity, since it would represent a series of overlapping smaller peptides with variable adjacent amino acids [90]. For example, a 38-mer peptide library will contain 32 overlapping 7-mer sequences, each with diﬀerent ﬂanking regions; thus all possible 7-mer peptides can now be represented in a 38-mer library with a size of only 108, 10-fold smaller than that required for a normal 7-mer library [91]. Another advantage is that, as a folded structure may be required for binding to a target molecule, long peptides are more likely to fulﬁll this. The ideal unbiased library should comprise a collection of molecules whose sequences have an equal chance of containing any of all 20 amino acids at each position. However the degeneracy of the genetic code, with diﬀerent numbers of triplets encoding the 20 natural amino acids results in variation in representation of the single amino acids in the peptide sequence [88]. The importance of such variations depends on the aﬃnity and speciﬁcity of the binding interactions involved between peptides and antibody. An unbiased library can be achieved by assembly of the degenerate DNA sequence with trinucleotide precursors and using a mixture of 20 trinucleotides, each encoding a diﬀerent amino acid at each condensation step [92]. A further consideration is the biological bias towards particular codon usage in diﬀerent strains of bacteria. For example, the most common arginine codon in E. coli (CGU) tends to be avoided in Bordetella pertussis, whereas B. pertussis employs other arginine codons that are rare in E. coli [50,93]. E. coli also avoids the AGG codon for reasons possibly related to translation, as well as the AG_G triplet, where _ denotes a boundary between codons [94,95], and the ACG codon adversely aﬀects recombinant gene expression in E. coli [96]. Thus, codons used for particular peptides expressed on pVIII or pIII may aﬀect the ampliﬁcation, and hence the frequency of expression of phage bearing those peptides in the library [50,93].

Structural constraints of the displayed peptides Linear peptides with fewer than about 30 amino acids are unlike natural proteins, in that they do not fold into well-deﬁned three dimensional structures, and in solution most linear peptides can adopt a range of conformations that may bind with only low aﬃnity to the antibody [97], whereas insertion of the peptide within the coat-protein of the phage may limit the range of conformations that can be assumed by the peptide, as illustrated by the study of the hexapeptide GPGRAF displayed in the pVIII protein [98]. This peptide represents a major epitope on the envelope glycoprotein gp120 of HIV-1, and a crystal structure of the peptide complexed with Fab from a monoclonal antibody to gp120 is available [99]. By NMR the peptide within the pVIII coat-protein adopted a well-deﬁned double turn structure that resembled the

161 structure of the peptide when bound to antibody, whereas the peptide itself has no consistent conformation in solution [98]. Several phage displayed libraries have been developed in which artiﬁcial constraints are imposed on the peptide to limit the conformations available to it, and to increase binding aﬃnity of selected peptides. In particular, many have ‘‘looped’’ peptides comprising cysteine residues ﬂanking random amino acid sequences of varying lengths. This looped structure has been reported to increase aﬃnity with the antibody in some cases, but not invariably. This is illustrated by studies comparing binding of monoclonal antibodies to random peptide libraries, either unconstrained or constrained by disulphide bonds; high aﬃnity binding peptides were selected from only one of the libraries [34,100,101]. Moreover, using a ‘‘double loop’’ expression system, biopanning yielded a variety of diﬀerent sequences, in contrast to only a single, unrelated consensus sequence from linear and cysteine-constrained libraries [102]. The eﬀect of cysteine-constraint on the characteristics of peptide selection was studied particularly by Bonnycastle et al. [61] wherein 11 pVIII-displayed random peptide libraries were screened with polyclonal and monoclonal antibodies against peptides, proteins and carbohydrates, in order to elucidate structural requirements for peptide binding. The libraries were designed to display unconstrained peptides of various lengths, or peptides constrained by ﬁxed cysteine residues placed at diﬀerent sites within a randomized sequence of varying length. The outcome was that the peptide framework signiﬁcantly aﬀected the sequences selected from a particular biopanning, with no particular library preferred by all antibodies; individual antibodies had a speciﬁc preference for several types of peptide constraints, varying with each antibody. The recommendation was that multiple libraries be used for optimal results [61]. The context in which a particular peptide is displayed on the phage is also important for selection since the same antibody may select peptides with diﬀerent motifs, or aﬃnity, according to whether the peptide is expressed in pIII or pVIII [103]. For example, when a cysteine-constrained nonameric phagemid library in pVIII was biopanned with the monoclonal 3A9 to the CCR5 chemokine receptor, all phage selected contained the single sequence, HASIYDFGS; such phage were highly reactive with 3A9, and the peptide blocked binding of 3A9 to cells expressing CCR5 [104]. However using the same mAb 3A9 to biopan linear and constrained heptameric phage libraries in pIII, there were selected various other reactive phage that contained quite diﬀerent motifs [103]. A noteable ﬁnding was that the most reactive pIII-selected phage that displayed the peptides WHWTSAT and HHWASSN, strongly cross-inhibited the reactivity with mAb 3A9 of the pVIII-selected phage HASIYDFGS, and vice versa. Overall, the preference of an antibody for a particular library (or libraries) cannot be predicted, so that optimal selection of peptides with high aﬃnity

162 would be achieved by screening a panel of libraries [61,105]. Cysteineconstrained peptides may be more useful for structural determination by NMR or X-ray crystallography, but the peptides may be held in the incorrect conformation for interaction with the ligand [34]. Biopanning Phage that display peptides able to bind to a particular antibody may be puriﬁed from a library of sequences by the technique of biopanning [63] which, in its simplest form involves the binding of elements of a phage-displayed peptide library (ligand) to an antibody (receptor) linked to a solid support (Fig. 2). Those phage that display a peptide ligand that can bind the antibody are captured on the surface, and unbound phage are washed away. The bound phage are then eluted in a solution that loosens receptor–ligand bonds, yielding

Fig. 2. Isolation and identiﬁcation of phage clones containing reactive peptides. The method of biopanning is illustrated in which phage that bind to the selecting antibody are captured using paramagnetic beads to capture bound phage. Speciﬁcity of binding is increased by the use of rounds of positive selection with antibody, in which phage bound to the beads are retained, and negative selection without antibody, or using an irrelevant antibody, in which phage which bind non-speciﬁcally to the beads are discarded. The phage insert from phage clones identiﬁed by ELISA as reactive with the antibody can be sequenced to identify the reactive peptide on the coat protein.

163 an ‘‘eluate’’ consisting of phage that are greatly enriched for receptor-binding clones, which can be ampliﬁed by infecting fresh bacterial host. Many phage clones (phagotopes) selected will not necessarily be related to the receptor of interest, having bound to other components within the selection mix, including the solid support, or the secondary antibody in the biopanning mix used to identify speciﬁc phagotopes. A ‘‘negative’’ selection step needs then be included, for which biopanning is performed as previously but the receptor of interest is not included, so that phage speciﬁc for other ligands are captured and discarded. After several rounds of each selection, positive and negative, phage clones from the ﬁnal eluate are propagated, and amino acid sequences of the peptides bound speciﬁcally to the receptor are ascertained from the corresponding coding sequence in the viral DNA. Various biopanning procedures are available [49,106]. Aﬃnity selection has been accomplished by minor modiﬁcations of standard aﬃnity puriﬁcation techniques in common use in biochemistry. Antibodies may be adsorbed to plastic ware, tubes, petri dishes or microtiter plates [24,107–109], paramagnetic beads [108,110–112], or to BIACORE sensor chips [113]. The selecting antibody may be attached directly to a surface, or biotinylated and immobilized on streptavidin-coated plates [63,109]. Antibody-binding may also take place in solution, with subsequent selection of the antibody–phage complexes using beads coated with an appropriate second antibody [111,114]. Each of these techniques has given satisfactory results, but a diﬀerent phage may be selected according to the method used. Thus, in a comparison of phage selected using the mAb MA 18/7 to the pre-S1 protein epitope of the hepatitis B virus that contains the motif DPAF, biopanning with mAb attached to polystyrene plates via a streptavidin-biotin bridge, or bound directly to polystyrene or latex beads gave diﬀerent results [115]. All phage selected using the polystyrene beads contained the motif DPAF or close variants of it, whereas only 30% of phage isolated with the latex beads, and 2% of phage isolated using the streptavidin-coated plates contained that motif. Similarly, in our laboratory, we see clear diﬀerences between motifs of phagotopes selected by mAbs reacted in solution and selected using beads coated with antimouse Ig, and motifs of phagotopes selected using polystyrene plates [109], although there was not a clear advantage to either technique. In general, bound phage are eluted by changing pH (e.g., glycine-HCL, pH 2; ethanolamine, pH 12) which results in phage–antibody dissociation without loss of phage infectivity, since ﬁlamentous phage are resistant to these conditions. However such biopanning and elution can result mainly in the selection of phage with medium to low aﬃnity binding [25,116]. The various techniques used to increase the aﬃnity of the phage clones selected include extensive washing to allow the dissociation of low aﬃnity binding, decreasing the concentration of the antibody used for selection in later rounds of positive selection [116], or inclusion of a stepwise pH elution process during the ﬁnal round of biopanning to remove sequentially phage with increasing aﬃnity of

164 binding [115]. However, not all phage are released by acid elution, and residual high aﬃnity binding phage particles may remain attached to the matrix according to Balass et al. [117] who used immobilization on nitrostreptavidin, a form of streptavidin in which binding is reversible, followed by elution with biotin to isolate phage particles with higher binding aﬃnity. Phage may also be eluted with 6 M guanidine–HCl, 2 M urea, or 10 mM DTT, although these reagents must be removed before the phage are ampliﬁed, and direct elution of phage ssDNA by treatment with phenol can be performed, but this requires DNA transformation to regenerate infectious phage particles. When receptors other than antibodies are studied, alternative methods may be applicable, depending on the binding properties of the receptor. For example, the receptor may require metal ions for binding activity, in which case phage could be eluted with EDTA or EGTA. Other reagents for elution include natural or synthetic ligands that competitively displace the selected ligand [118]. Also, phage bound to a receptor can be recovered without prior elution by directly infecting a bacterial culture [117]; here, the eluted phage remain infective and can be propagated simply by infecting fresh bacterial host cells to yield an ‘‘ampliﬁed’’ eluate as input to another round of aﬃnity selection.

Characterizing phage clones that contain contact residues for an antibody epitope The aﬃnity between phage and antibody will vary widely, so that various methods have been developed to conﬁrm the speciﬁcity of binding of an antibody to a phage-borne peptide (see Sparks et al. [118]). In brief, these include dot-blotting [112], plaque lifts using the phage clone as antigen [119], micropanning [63], ELISA [104,111,114,120,121], inhibition of binding of antibody to phage using either antigen (or peptide), or antibody as inhibitor [104,111,112,121], immunoprecipitation of phage with antibody [122], but ELISA-based techniques have been most widely used. Such ELISAs vary in style but the main formats are a direct ELISA in which the phage are ﬁrst coated to the plate, and the antibody that was used for screening is then added, or a capture ELISA, in which the antibody is coated onto the plate and then phage is added, followed by an anti-phage antibody [120]. Direct ELISA is convenient and simple, and serves well for high aﬃnity interactions [104,111,120,121]. However the direct ELISA is rather insensitive, such that speciﬁcally aﬃnity-selected phage can give a feeble signal by direct ELISA that is indistinguishable from the background, and probably reﬂects predominantly the reaction of the antibody with non-phage bacterial impurities [63]. Capture ELISA is more sensitive and allows for semi-quantitative analysis of binding constants, but has the disadvantage of being inapplicable when biopanning is performed using antibody bound to microtiter plates.

165 Localization of linear epitopes Identiﬁcation of linear epitopes is usually uncomplicated, as families of peptides that contain consensus sequences and are highly homologous to the amino acid sequence of the original protein can be selected. Epitope mapping has been successfully performed for a range of linear epitopes, whether using synthetic peptides [123–125], or phage-displayed peptide libraries [61,119,126]. The epitopes so identiﬁed are characterized by regions that are ﬂexible [125,127–129], surface exposed [123,130] and usually hydrophilic [131]. Such epitopes have usually corresponded to sites of turns and loops in folded proteins [128]. Craig et al. [132] discuss in detail the use and applicability of various techniques that can be applied to the identiﬁcation of linear epitopes using phage display. In an exemplary study, they used polyclonal antibodies against the familiar and well-characterized antigens, hen egg-white lysozyme (HEL), and worm myohemerythrin, to biopan a panel of phage displayed peptide libraries. They identiﬁed consensus sequences among the selected peptides that identiﬁed linear epitopes for each of these antigens, and were able to nominate ‘‘critical contact residues’’ based on particular amino acids that were always present, or could only be conservatively replaced among reactive peptides. The epitopes for hen egg lysozyme, with contact residues underlined, were 119 DVQ121, 87DITASV92, 106NAWV109, 68RTPGS72 and 40TQATNR45, and for myoerythrin were 66KYSEV70 or 67YSEV70, 5PEPYV9, 10WDESF14, 86 GLSAPVDA93 and 41APNLA45. The epitopes were mapped to the appropriate region within the protein sequence by matching conserved residues in each consensus sequence group to a sequence within the cognate protein antigen. The epitopes were well exposed on the surface of the protein, and most were contained within turns or loops between regions of regular secondary structure. Although discontinuous epitopes predominate in anti-protein responses [8,9], and the structures for HEL-mAb complexes revealed by high resolution X-ray crystallography do identify conformational epitopes [133–135], all of the peptides obtained by Craig et al. [132] could be aligned to linear sequences on the two antigens. In the case of myohemerythrin, four of ﬁve epitopes were also identiﬁed by Geysen et al. [125] using a panel of overlapping synthetic peptides to test the reactivity of polyclonal antibodies. Craig et al. [132] concluded that peptide-mimics of linear epitopes dominate in aﬃnity selections, possibly because the critical residues in mimotopes for conformational epitopes are more numerous and more dispersed than those in direct linear epitopes, and may be less well represented in the libraries. They suggested that the phage clones selected with monoclonal antibodies against discontinuous epitopes often bind only weakly, and require optimization of residues ﬂanking the consensus sequences to obtain moderate binding aﬃnities. It is notable however, that the ‘‘linear’’ epitopes identiﬁed above, using polyclonal antibodies, were within the

166 1

11

21

31

41

HEL D1.3 HyHEL-5 HyHEL-10 Polyclonal

KVFGRCELAA -------------------------------------

AMKRHGLDNY -------DN-------------HG---Y ----------

RGYSLGNWVC RGYSL-N-----------R------------------

AAKFESNFNT ------------------------------------T

QATNRNTDGS ---------Q--NRNTDG---------QATNRXTD--

HEL D1.3 HyHEL-5 HyHEL-10 Polyclonal

51 TDYGILQINS -----------Y-------------------------

61 RWWCNDGRTP ---------------GRTP --W-------------RXP

71 GSRNLCNIPC --------------------R-L----GS--------

81 SALLSSDITA ------------L-------------T------DITX

91 SVNCAKKIVS --------------------N---KKI-XV--------

HEL D1.3 HyHEL-5 HyHEL-10 Polyclonal

101 DGNGMNAWVA ------------------DG------------NAW--

111 WRNRCKGTDV -----KGTDV --------------------------DV

121 QAWIRGCRL Q--I-----L ------------------Q---------

Fig. 3. Alignments on the sequence of hen egg-white lysozyme of contact amino acids from X-ray crystal structures of lysozyme-mAb for the three monoclonal antibodies D1.3, HyHEL-5 and HyHEL-10 [4], and also ﬁve peptide motifs for linear epitopes derived by biopanning phage libraries with rabbit polyclonal antibodies [132]. Critical contact residues are underlined. Four of the ﬁve phage-derived linear epitopes correspond to regions of contact for the conformational epitopes identiﬁed for the monoclonal antibodies.

three previously described monoclonal antibody-deﬁned conformational epitopes for which crystal structures are available [4] (Fig. 3), favoring the idea that apparent linear epitopes may in fact represent parts of a complex conformational epitope. Localization of conformational epitopes Compared with linear epitopes, the structural localization of discontinuous epitopes poses additional problems, since peptides may be true mimotopes in lacking any sequence homology, or may contain amino acids from disparate regions of the molecule. Even in the study described above, in which linear epitopes were identiﬁed [132], not all peptides could be aligned to a single consensus group in that there were residue patterns common to two consensus groups, or the peptide had only two residues in common with a consensus group. No single technique has proved to be universally successful for localizing such mimotopes, and hence epitope identiﬁcation has remained as much an art as a science. Nonetheless, other than crystallography, epitope mapping by phage display is the most informative technique for identifying conformational epitopes and is more widely applicable than crystallography. For large proteins, success usually requires some antecedent knowledge of the structure of the

167 protein, derived either from crystallography, NMR or homology modeling, and is further enhanced by any prior information that localizes the epitope within a particular region. Nuisance peptides A problem particular to localization of discontinuous epitopes that are mimotopes without direct sequence homology with the antigen, is the need to distinguish irrelevant ‘‘nuisance peptides’’ from peptides that truly represent partial mimotopes. In cases where there is no obvious homology between the amino acid sequences of phage-displayed peptides and the sequence of the original antigen [101], and where such peptides may have only low aﬃnity for the selecting antibody, it is often diﬃcult to identify relevant sequences. Even with extensive rounds of biopanning and negative selection, there may be selected peptide sequences that bind to components of the biopanning process itself, including plastic, the antibody immobilization system, or blocking agents [136,137]. Public databases of peptides derived from biopanning phage-displayed libraries have been developed to allow investigators to view and compare phage-displayed peptide sequences [138]. Alternatively, alignment algorithms such as PILEUP or ClustalW [139,140] can be used that cluster similar sequences in the form of dendrograms or guide trees. Davies et al. [141] describe the use of the PILEUP algorithm [140] combined with the Tudos matrix that scores amino acid substitutions on the basis of physicochemical properties of amino acid sidechains [142], to distinguish nuisance peptides from valid peptides selected using various antibodies. Their alignments showed that peptides selected by diﬀerent sources of IgG, or selected in the absence of any antibody, aligned in unique branches of the guide tree such that the alignments could be used to identify nuisance peptides selected with each biopanning. Short motifs that localize epitopes In instances where particular motif(s) are recognizable, various techniques have been applied to identify these. In several studies, it was possible to localize an epitope based on a unique amino acid or a short motif. For example, Luzzago et al. [143] used phage display to locate a discontinuous epitope recognized by a monoclonal antibody to the H-subunit of human ferritin. In that study there were derived two populations of sequences, of which the ﬁrst was characterized by the presence of Y and W, spaced by ﬁve amino acids, and the second by the consensus sequence G-S-X-F. The only tryptophan residue (W93) in the sequence of the H subunit was located at the end of the loop connecting the B and C helices, and this was provisionally used to localize the epitope, based on the ﬁrst group of peptides. Notably, S95 is located a short distance from F41in the native structure of ferritin, so allowing the prediction of a discontinuous epitope with speciﬁc contacts with antibody involving Y40

168 and F41on one helix, and W93 and S95 on the adjacent loop; these predictions were subsequently conﬁrmed by mutagenesis. Similarly, in our analysis of the well-studied conformational epitope for autoantibody on the inner lipoyl domain of the E2 subunit of the pyruvate dehydrogenase complex [144], an epitope was deﬁned by phage display according to the presence of two motifs MH and F(E)V in the selected peptides. In that study, there was only a single histidine (H132) within the inner lipoyl domain of PDC-E2 in which the epitope was known to exist; on the NMR structure of the inner lipoyl domain the sequence 178FEV180 was located just adjacent to that region (Fig. 4). Here also, the location and nature of the predicted epitope was conﬁrmed by mutagenesis [145]. Use of algorithms and guide trees to localize epitopes In each of the above-mentioned instances, the peptides revealed by phage display could be readily assigned to two separate groups based on their sequences, and these groups could be localized to separate regions of the primary structure. However the problem becomes more diﬃcult when particular peptide motifs cannot be readily identiﬁed, or when the selected sequences either have no obvious sequence homology with the antigen, or react only weakly with the antibody by conventional screening techniques. We have used the multiple sequence alignment algorithm PILEUP, together with the Tudos matrix, to align related peptides into clusters that can be depicted visually on a guide tree [141]. The alignments allow the objective identiﬁcation of groups of peptides that contain particular motifs, which can then be aligned to the sequence of the antigen. Using this approach, a human monoclonal antibody MICA3, that is reactive with an autoantigen glutamic acid decarboxylase (GAD) was used to screen phage-displayed libraries, and the derived peptides were localized to three particular surface exposed loops on the GAD molecule that constituted a conformational epitope [114]. Reversed sequences in phage peptides Although the alignment technique can usually be used successfully to provide an objective measure of the similarity of peptides, alignments to the antigen sequence is not always successful, and visual alignments may be more useful. This was illustrated for the peptides derived using human antibodies to PDC-E2, described above. Alignment of the phage-derived peptides allowed the identiﬁcation of particular groups of peptides, that were reactive with aﬃnity puriﬁed antibodies to PDC-E2, and were distinguished from nuisance peptides [141] (Fig. 4A). However, in this case the peptide sequences did not give meaningful alignments with the PDC-E2 sequence using the computer-based alignment algorithms. In particular, the alignment algorithms were unable to identify the motif MH in the phage-derived peptides as corresponding to the

169

Fig. 4. (A) Alignment of peptides from phage clones selected with polyclonal IgG containing antibodies to PDC-E2, showing that the peptides grouped to four major clusters using the Tudos Matrix [141]. Those phage-displayed peptides that were positive in a capture ELISA with antibodies to PDC-E2 are marked by þ to þ þ þ according to the level of reactivity. These clustered together, and contained the motifs MH and FV(E). (B) The motif MH was localized to the sequence 132 HM133 containing the only histidine within the inner lipoyl domain of PDC-E2 where the epitope is located. The sequence 178FEV179 is located just adjacent to that region on the surface of the molecule according to the NMR structure, and the surrounding area contains various amino acids that occur frequently in the selected peptides [144].

170 sequence 132HM133 in the antigen. From our experience, the partial reversal of sequences of amino acids in peptides derived by biopanning is not unusual. Interestingly, one of the phagotopes derived with antibodies to PDC-E2 included the sequence AMDPPYS which represents a ﬁve amino acid direct reversal of the epitope sequence 128SYPPHM133, although this phagotope did not react with anti-PDC-E2 by capture ELISA [141]. Similarly, in a study (unpublished) of phagotopes derived by biopanning constrained and nonconstrained 7-mer libraries with the human monoclonal antibody b78 to GAD65, 14 of 26 sequences contained the motif LRS. These amino acids were localized to the sequence 522RMSRLKV529 in the C-terminal region of GAD65, and conﬁrmed as part of the epitope by mutagenesis. Scrambled residues in phage peptides A further problem can be that amino acids in the phage peptides selected may be ‘‘scrambled,’’ so that amino acids that are true contact residues for antibody binding may be over-represented in derived peptides, but do not necessarily appear as motifs, nor in the order found in the primary sequence of the antigen to which the screening antibody is directed. This is illustrated by sequences derived by the biopanning with the monoclonal antibody b78 to GAD described above, since, in addition to the 14 phage peptides that contained the reversed motif LRS, 7 further sequences contained at least two of the amino acids SRL, often as pairs, SL or LS, SR or LR. We can deduce that peptides displayed at the N-terminus of a phage peptide have much greater conformational ﬂexibility than the same sequence within a protein. Accordingly, they are likely to be susceptible to selection by an induced-ﬁt binding mechanism. Favoring this, we failed, using pepscan analysis of 8-mer overlapping peptides from PDC-E2, to identify an epitope recognized by autoantibodies, yet many of the most reactive peptides from the pepscan analysis contained dimers, VF, FV, MQ, MF, HY, that contained amino acids consistent with the sequences in the predicted epitope from the two regions, 132HM133 and 178FEV180, identiﬁed by phage display [146]. The repeated identiﬁcation of such amino acid combinations may provide valuable ancillary evidence to support the location of an epitope in a particular region.

Confirmation of the epitope by localization on a 3D structure Phage display is of great value in identifying critical contact residues for antibody-binding within the linear sequence of an antigen, but the validity of the alignment does need to be tested against the known structure of that antigen. As described hitherto, epitopes of antigenic proteins contain regions that are ﬂexible [125,127–129], usually hydrophilic [131], and surface exposed

171 [123,130], and tend to correspond to sites of turns and loops in folded proteins [128]. Amino acids that have been identiﬁed so far as contact regions for epitopes fulﬁll these properties in being surface exposed and contained within a diameter of 23 A˚ which is the approximate size of an epitope. In cases where motifs of only 2–3 amino acids can be identiﬁed by phage display, and such amino acid motifs occur in several places on the antigenic sequence, such structural localization may become essential. Furthermore, with epitope deﬁnition, the amino acids surrounding the critical contact residues are likely to be well represented in the phage-displayed peptides selected, as illustrated for the epitope of human H ferritin [143], described earlier. For H ferritin the 3D structure of the molecule was used to localize a tyrosine (Y) in the peptide motif to one of two positions, Y39 or Y40 on the A helix close to W93 with Y40 being designated as the contact residue based on its surface exposure; ﬁnally, the likely epitope was not only localized, but also there could be predicted additional amino acids that made up part of the epitope, since D, G, S, F that occurred in combinations in the most reactive phage peptides, were surface exposed on the 3D structure, in close conjunction to W93 and Y40. Confirmation by mutagenesis Notwithstanding increasing evidence for the meaningful mapping of conformational epitopes by aligning phage peptide sequences on the surface of an antigen molecule [112,114,143,144], validation is required. This is usually achieved by site-directed mutagenesis of those amino acids predicted as critical sites for antibody binding. In each of the studies referenced above, the amino acids predicted to be part of the epitope were mutated, and the reactivity of the mutant and wild-type antigen was compared. Such techniques have likewise been extensively used in various other epitope mapping studies to identify the amino acids involved in antibody binding [16,147,148], and need not be described in detail here. However, although site-directed mutagenesis provides the best independent corroboration that a particular amino acid is part of the epitope, the nature of the mutation may aﬀect the results obtained for several reasons. First, the binding energy of the interaction may depend on antibody binding to eight or more critical contact residues [8], so that mutation of just a single residue could have too slight an eﬀect to be measurable by standard antibody assays; hence mutagenesis at several sites would be required, as demonstrated in our own studies [145]. Second, the nature of the amino acid substituted may also aﬀect the binding, noting that Geysen [149] has found diﬀerences in the eﬀects of replacement of particular amino acids in linear peptide epitopes in relation to antibody binding. In fact, the allowable replacements varied considerably from peptide to peptide. Although charge or hydrophobicity, and bulk of the side chains of the amino acids were both of importance,

172 their contribution in the case of linear peptides varied according to the antibody; this is very likely to be true also for amino acids in conformational epitopes. Thus replacement of a small residue by a more bulky amino acid within the region of the epitope could well result in steric inhibition of binding to the antibody, to a much greater degree than would be observed with mutagenesis to alanine. Conversely, mutagenesis to alanine could underestimate the importance of a particular amino acid in the presence of other strongly binding residues, and careful measurement of changes in binding energy would be required to detect eﬀects. Third, mutations in regions away from the epitope that cause marked structural changes to the antigen may alter antibody binding to the epitope. Hence further evidence that the antigen structure has not been aﬀected by the mutant, e.g., retention of reactivity with other antibodies known to react with conformational epitopes, may be required [114,143]. Confirmation of mimotopes by immunization Our description of techniques for epitope mapping by phage display concludes with a particular caveat. Peptides derived by phage display that bind speciﬁcally with the selecting antibody might not necessarily represent a true mimotope. Thus, although the capacity of a peptide to bind with a particular antibody, and to block the reactivity of the cognate antigen with that antibody, usually indicates that a particular peptide is a mimotope of the antigen, this is not invariably so. In particular, peptides can bind strongly with the antibody such that the paratope (antigen-binding domain) of the antibody is blocked, yet the speciﬁc interactions between antigen and antibody may engage quite diﬀerent amino acids [150]. We can cite from our own studies [111] an interesting example on the selection of phage peptides using a monoclonal antibody CII-C1, which is to a highly conformational epitope of type II collagen. The epitope had been precisely deﬁned to ﬁve amino acids, ARGLT, on the collagen triple helix by use of chimeric collagen molecules [151]; most of the phagotopes we derived contained variations on both the motifs RRL, and FGxQ, and were strongly reactive with CII-C1 by direct ELISA. However from molecular modelling, it seemed unlikely that the third complementarity determining region (CDR3) of CII-C1 contributed to antibody binding with collagen, and we therefore proposed that the glutamine (Q), which was present in most of the selected phage-displayed peptides but did not occur in the C1 epitope for CII-C1, may bind to the CDR3 of CII-C1. In this case, the strong motif FGxQ derived from phage display was misleading for epitope localization. It is interesting to note that prior to epitope mapping with chimeric collagens that retain the intact triple helix, the C1 epitope had been incorrectly localized to amino acids 316–333 of the protein that contained the sequence FPGQ based on reactivity with peptide digests that contained linear peptides [152], whereas CII-C1 reacts only with the

173 intact triple helical collagen molecule, and not with individual a-chains. Ultimate conﬁrmation that a phage-displayed peptide represents a true mimotope requires immunization either with phagotopes or with the actual peptide. This has proven successful in many of the studies described in this review, to the extent that peptide mimotopes are being examined closely as new vaccine candidates.

Epitope mapping using polyclonal sera The mapping of epitopes of antigens by biopanning random peptide libraries is clearly simpler using monoclonal antibodies. However, in the case of a deﬁned antigen, the techniques have been adapted successfully for polyclonal sera, particularly by use of antibodies that have been aﬃnity puriﬁed on the deﬁned antigen as a source of monospeciﬁc antibodies. Examples are provided by the deﬁnition of a dominant epitope RQHPKM, residues 15–20 of the human lymphotoxin sequence, using polyclonal antibodies puriﬁed on recombinant lymphotoxin [153], the identiﬁcation of a 15-mer peptide epitope of Chlamydia pneumoniae using polyclonal antibodies from patients with chlamydial infections puriﬁed on immobilized C. pneumoniae elementary bodies [154], or identiﬁcation of a peptide mimotope of double-stranded DNA using polyclonal antibodies from patients with systemic lupus erythematosus aﬃnity puriﬁed on dsDNA [155], with the latter example illustrating the use of aﬃnity puriﬁcation to obtain antibodies to a non-protein antigen. As an alternative strategy, when high levels of one particular antibody are present in serum, the initial biopanning can be performed with use of puriﬁed IgG that contains the antibody and negative selection with nonimmune IgG; the selected phagotopes would then be tested for reactivity using aﬃnity puriﬁed antibodies. The latter approach was used to identify phagotopes reactive with the very high titer autoantibodies to the antigen PDC-E2 in sera of patients with primary biliary cirrhosis [144]. Folgori et al. [119] used a procedure of sequential screening of a nonapeptide phage library, ﬁrst with polyclonal IgG from the serum of an individual immunized with hepatitis B virus envelope protein (HBsAg), and then with IgG from normal individuals; this enabled selection of phagotopes that were mimotopes of two distinct HbsAg epitopes. The original biopanning was performed with the serum from just one individual, but the phagotopes selected reacted with a panel of sera from other individuals immunized with HBsAg and, moreover, mice immunized with the selected phagotopes developed a strong, speciﬁc response to the HbsAg protein. The technique was seen to provide a means of identifying common features of an immune response of diﬀerent individuals with the one particular disease, what in fact was called the ‘‘immunofootprint’’ of the disease, leading to a proposed protocol for that purpose [156].

174 Selection of disease-specific phagotopes for the development of diagnostic assays Peptides selected by biopanning with IgG from disease sera, that reﬂect the proﬁles of disease-speciﬁc antibodies, have been identiﬁed for various viral diseases, including those due to infection with hepatitis C virus (HCV) [157,158], human immunodeﬁciency virus-1 (HIV-1) [159], and human papillomavirus (HPV) [160]. These studies illustrate the applicability of ‘‘immunofootprinting,’’ as described above, for the development of more discriminatory diagnostic reagents, particularly for diseases in which an appropriate test antigen is not readily available, or a disease for which the current diagnostic assay is based on the use of a crude antigenic preparation that gives high non-speciﬁc background readings, and in which quality control is diﬃcult. Examples include Lyme disease, in which existing ELISAs depend on the use of crude extracts of Borrelia burgdorferi [161], and parasitic infestations due to Taenia solium or Taenia crassiceps [162,163]. As an interesting divergence from the same technique, there has been scrutiny of the repertoire of antibodies in the serum of patients with cancer, whether occurring in response to a viral antigen such as HPV in cervical cancer [160], or in response to a novel tumor-speciﬁc antigen. Thus, in the exemplary study by Mintz et al. [164], screening of a cysteine-constrained random peptide library with sera from patients with prostate cancer yielded a peptide, with a consensus motif, that bound strongly and speciﬁcally with antibodies to that peptide in sera of a further large panel of patients with prostate cancer. The peptide was identiﬁed as a constituent of a protein that was only weakly expressed in the normal prostate gland, but ampliﬁed in metastases. Strikingly, serum reactivity with that peptide correlated with the occurrence of more severe and androgen-independent metastatic disease.

Immunoscreening of phage libraries to provide clues to etiology of autoimmune diseases A further application of ‘‘immunofootprinting’’ is the identiﬁcation of diseasespeciﬁc phagotopes in diseases for which initiating events are unknown. Thus phage libraries have been immunoscreened using various sources of polyclonal antibodies including sera, synovial ﬂuids, or cerebrospinal ﬂuids, from patients with a range of autoimmune diseases including type I diabetes mellitus [165,166], rheumatoid arthritis [167], multiple sclerosis [168–171], and autoimmune cholangitis [172], or with isolated myeloma proteins from patients with multiple myeloma [173]. In several of these studies there have been identiﬁed particular peptide motifs that might represent mimotopes of a stimulating antigen; sequence alignments and homology searches have identiﬁed homologies with various viral or other antigens. However the actual signiﬁcance of

175 such homologies is usually uncertain because these homologies may be ‘‘coincidental,’’ or the peptide mimotopes need not show any recognizable homology to the selecting antigen. The study of Lunardi et al. [174] is one that provides encouraging evidence that phage library-derived peptides indeed can provide useful clues to pathogenesis. A random peptide library displayed on the major ﬂagellar protein of E. coli (rather than on phage) was biopanned with pooled IgG from patients with systemic sclerosis to yield an immunodominant peptide that had homology both with various autoantigens including heterogenous ribonucleoproteins (hnRNP), cytochrome C and ﬁbrillarin, and also with the late protein UL94 of human cytomegalovirus (CMV). When antibodies from patients’ sera were aﬃnity puriﬁed on this peptide, there was cross-reactivity with the autoantigens and also with CMV-derived protein. Moreover, these cross-reactive anti-viral antibodies could be linked to the pathogenesis of the disease in that aﬃnity puriﬁed anti-peptide antibodies from the patients reacted with a surface component of endothelial cells and induced apoptosis. We draw attention to this study because, hitherto, there has been a singular diﬃculty in linking the obliterative vascular and the immunological expressions of systemic sclerosis, and because no primary pathogenic event has been convincingly visualized. Applications As the techniques described in this review are becoming better established, and the inﬂuences that aﬀect results are being deﬁned, epitope mapping using phage display is becoming recognized as a valuable procedure in numerous settings, exempliﬁed by the following. Vaccines utilizing peptide mimotopes of carbohydrate antigens Many of the most important antigens that elicit protective immunity against bacteria are nonprotein surface antigens, usually polysaccharides. Among these are the capsular polysaccharide of Neisseria meningitidis that causes bacterial meningitis [175], the lipopolysaccharide (LOS), a virulence factor for nontypeable Haemophilus inﬂuenzae that causes otitis media in children [176], and capsular polysaccharides for gram negative enteric bacteria including shigella, salmonella, and enterohemorrahagic E. coli [177]. Also, many tumor antigens are carbohydrates. Most polysaccharides are thymus-independent antigens that do not require ‘‘help’’ from mature T cells to elicit an antibody response. Hence such antigens are usually poorly immunogenic, fail to elicit a lasting memory response, and antibodies do not undergo aﬃnity maturation. However since the organisms are important pathogens, particularly in infants, young children, and in immuno-compromised individuals, considerable

176 interest has arisen in the use of peptide mimotopes as candidate vaccines to stimulate stronger and durable thymus-dependent antibody responses. Phage display provides the means to isolate appropriate peptide mimotopes of carbohydrate antigens, by library screening with appropriate antibodies [175,176], allowing new candidate vaccines to be developed that combine both B and T cell epitopes in the same formulation. The review by MonzaviKarbussi et al. [177] is pertinent to this question. The full potential of peptide mimotopes as vaccines for carbohydrate antigens has been shown by DNA immunization of mice with a plasmid vector that encoded a peptide mimotope of the Lewis Y (LeY) carbohydrate antigen, together with a secretory leader sequence and a T cell epitope from HIV-1 gp120. This construct induced a strong IgG response to LeY, that was highly amenable to boosting with the LeY carbohydrate [178], even though the immunizing antigen was a DNAencoded peptide. Inhibitory peptides to block harmful interactions with toxins Two particular snake neurotoxins are crotoxin which is a b-neurotoxin that blocks acetylcholine release from nerve endings, and a-bungarotoxin which is an a-neurotoxin that blocks the function of acetylcholine at the neuromuscular junction by blockage of the nicotinic acetylcholine receptor in muscle. For each of these, phage display has been used to develop peptides that inhibit the activity of the toxins. In one study [112], phage display was used to characterize the epitope recognized by a monoclonal antibody A-56.36 that can neutralize the eﬀects of crotoxin, a lethal component of rattlesnake toxin, and to evaluate mimicking peptides as possible vaccine candidates. Crotoxin is a phospholipase A2 (PLA2) formed by the noncovalent association of two subunits, CA and CB, that act as neurotoxin by blocking acetylcholine release from nerve endings. CA is a nontoxic nonenzymatic subunit derived from a PLA2-like subunit, and CB is a weakly toxic PLA2. The toxicity of crotoxin results from the syngergistic action of the two subunits, and its toxicity depends on the stability of the interaction between CA and CB. The mouse mAb A-56.36 acts by binding to CA, and thereby dissociating the complex between the two subunits; antibodies with such speciﬁcity could be expected to be potent antitoxins, but the epitope recognized by A-56.36 is conformational and has not been deﬁned. Demangel et al. [112] screened phage-displayed random peptide libraries with mAb A-56.36, and derived two families of phagotopes that had high aﬃnity for the mAb, and induced polyclonal antibodies to CA in mice immunized with the phagotopes. These mouse antibodies had the same crotoxin-dissociating eﬀect as did mAb A-56.36. The two families of peptides were used to predict, on a model of CA, an epitope region that was proposed to interact with CB; the phagotopes mimicking the epitope on CA were shown to bind to CB, so conﬁrming that the mAb does act at the

177 site of interaction of CA and CB. Although the antibody induced by the peptide had lower reactivity than did the parent mAb, the study provided important structural information to localize the zone of interaction between CA and CB, and to design ‘‘second generation’’ peptides as better vaccine candidates. In the other study [179], phage display was used to identify peptides that could block the binding of a-bungarotoxin to the acetylcholine receptor. In this case a 15-mer random peptide phage-displayed library was biopanned not with antibody, but rather with a biotinylated a-bungarotoxin, and phage peptides that bound to the bungarotoxin were selected on streptavidin-coated plates. The derived phagotopes were tested by ELISA for binding to the toxin, and the most reactive were selected for synthesis of peptides that might inhibit the binding of a-bungarotoxin to the acetylcholine receptor. A 3-dimensional structure was derived for one high aﬃnity reactive peptide [180], and the results obtained were used to design further peptides that could bind with higher aﬃnity as candidate antidotes to a-bungarotoxin poisoning [181].

Epitope mapping using phage display as a paradigm for other receptor–ligand interactions The techniques described above are far from being limited to epitope mapping since there is applicability to a very wide range of receptor–ligand interactions. As one example, for development of peptide antidotes to a-bungarotoxin poisoning as described above, biopanning was performed using biotinylated a-bungarotoxin rather than an antibody, although the procedures used were directly analogous to those used in other studies cited herein of antibody–antigen interactions using phage display. Random peptide phage-displayed libraries have been biopanned with ICAM-1 to identify peptides that interfere with the ligand receptor interaction of ICAM-1 and b2-integrins [182], with TNF-a to identify peptides that block TNF-a mediated cytotoxicity [183], with taxol to identify peptides that could be used to identify Bcl-2 as a taxol-binding protein [184], or with the insulin receptor to identify the interactions between insulin and the insulin receptor [185]. In each case, the techniques used were directly analogous to those used successfully to analyze antibody–antigen interactions. We can add that the scope of phage display is evolving very rapidly and in many directions. New libraries are being developed in which peptides can be displayed in deﬁned conﬁgurations, such as knottins that contain a core of four cysteine residues [186], or as the Z-domain of protein A [92,187]. Moreover, phage libraries in which an entire cDNA library is displayed can be used and screened for binding partners in a manner analogous to the yeast two hybrid system [44,71,72]. As the principles that govern such interactions become increasingly clear, applications of phage display for analysis of

178 molecular interactions should continue to expand far beyond the already broad perimeter described in this review.

Acknowledgments We thank Ian Mackay for carefully reviewing the manuscript and many helpful comments, and Daniela Martino Roth for assistance in compiling the references.

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DNA vaccines and their application against parasites – promise, limitations and potential solutions Peter M. Smooker1, Adam Rainczuk2, Nicholas Kennedy2, and Terry W. Spithill3,* 1 Department of Biotechnology and Environmental Biology, RMIT University, Bundoora 3083, Australia 2 Department of Biochemistry and Molecular Biology, Monash University, Australia and the Cooperative Research Centre for Vaccine Technology, Brisbane, Australia 3 Institute of Parasitology and Centre for Host–Parasite Interactions, McGill University, Ste. Anne de Bellevue, Canada H9X 3V9

Abstract. DNA or nucleic acid vaccines are being evaluated for eﬃcacy against a range of parasitic diseases. Data from studies in rodent model systems have provided proof of principle that DNA vaccines are eﬀective at inducing both humoral and T cell responses to a variety of candidate vaccine antigens. In particular, the induction of potent cellular responses often gives DNA vaccination an immunological advantage over subunit protein vaccination. Protection against parasite challenge has been demonstrated in a number of systems. However, application of parasite DNA vaccines in large animals including ruminants, primates and humans has been compromised by the relative lack of immune responsiveness to the vaccines, but the reasons for this hyporesponsiveness are not clear. Here, we review DNA vaccines against protozoan parasites, in particular vaccines for malaria, and the use of genomic approaches such as expression library immunization to generate novel vaccines. The application of DNA vaccines in ruminants is reviewed. We discuss some of the approaches being evaluated to improve responsiveness in large animals including the use of cytokines as adjuvants, targeting molecules as delivery ligands, electroporation and CpG oligonucleotides. Keywords: DNA vaccine, nucleic acid vaccine, prime-boost vaccine, protozoa, parasite, malaria, expression library immunization, mice, ruminants, human vaccine.

Section 1: Features of DNA vaccines The age of DNA vaccines The key set of experiments that ushered in the age of DNA vaccines were performed over a decade ago. The trio of papers began with Wolﬀ et al. [1] who demonstrated expression of plasmid-encoded protein in muscle. These authors were, in fact, performing gene therapy experiments, and noted that they obtained expression of marker protein within myocytes. This observation was followed by that of Tang et al. [2], who demonstrated that an immune response was generated to an encoded protein after biolistic (gene gun) delivery to the epidermis of mice. The ﬁnal key paper truly set the scene and generated the dogma that underpins the promise of DNA vaccination [3]. Ulmer and *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10007-0

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

190 colleagues demonstrated that vaccination of mice with the nucleoprotein (NP) gene of a mouse inﬂuenza virus induced NP-speciﬁc antibodies and cytotoxic T lymphocytes (CTLs). These mice were protected against viral challenge with a heterologous strain. Such cross-protection is not generally observed when the corresponding protein is used as the immunogen and, indeed, mice vaccinated with puriﬁed NP, while raising high-titer antisera against the protein, were not protected. The assumption was made that the CTLs are largely responsible for the protective response, as protection from antibodies induced to virus is normally strain-speciﬁc. In this way the two basic tenets of DNA vaccines were born; ﬁrstly, that vaccination with plasmid DNA encoding antigen can induce a broad immune response, including CTL responses to the antigens; secondly, such vaccination may aﬀord protection where other strategies do not. In the following decade there was substantial activity applying DNA vaccine technology, as a large proportion of research groups investigating antigenspeciﬁc vaccination have taken at least a passing interest in DNA vaccination. A large number of model pathogen/host systems have been evaluated, some of which we will detail in this review. DNA vaccination has been used not only for prophylactic vaccination but also for therapeutic vaccination against infectious disease [4] or cancer [5], to raise antisera to a protein where no native or recombinant protein is available [6] and as a tool for antigen discovery (see Section 3). In 10 years, DNA vaccination had progressed from the laboratory into Phase I and II clinical trials [7]. There are, however, some limitations and early optimism fuelled by promising results in rodents has not yet been fulﬁlled in target species. While very eﬀective in rodent models, especially against viral infections, hurdles arise when attempting vaccination against larger complex pathogens, particularly parasites, and in large non-rodent species, such as ruminants and humans. A vigorous area of investigation is to develop strategies to increase the eﬀectiveness of DNA vaccination. We will detail some of these studies throughout this review. Advantages of DNA vaccines Several features of DNA vaccines make them an attractive proposition for vaccination and, in particular, for mass immunization programs. Firstly, as DNA vaccines are subunit vaccines, there is no prospect of reversion to virulence due to incomplete attenuation of a living organism, as may occur with attenuated or killed bacterial or viral vaccines. Another important advantage is the ease of manufacture of DNA subunit vaccines compared to a recombinant protein subunit vaccine. With respect to manufacture, every DNA vaccine is similar to every other DNA vaccine. This is obviously not the case for recombinant protein vaccines (this also has real beneﬁts for multi-valent vaccination, see Section 3). Finally, plasmid DNA is a most stable entity and, when stored dry, can be transported at ambient temperatures. Thus, DNA vaccines are ideal

191 candidates for application in developing countries where vaccines are sorely required but distribution costs associated with a cold-chain for a conventional vaccine are often prohibitive. Notwithstanding the practical considerations, it is the ability to induce both CD4 þ and (particularly) CD8 þ T-cell responses against encoded antigens that oﬀer the promise of DNA vaccination. For example, the induction of CD8 þ T cells and antibodies induced by malaria circumsporozoite DNA vaccination has been found to be superior to CD8 þ T cell levels induced by irradiated sporozoites [8,9]. One of the most important features of DNA vaccines is that immunization results in protein expression of the encoded gene within the host. This is important for vaccine eﬃcacy from two angles. Firstly, conformational epitopes contained within the antigen will most likely be preserved [9]. This is often not the case for proteins made in vitro using recombinant expression. For example, prokaryotic host cells do not consistently fold proteins of interest with the correct disulﬁde-bonding pattern due to the reducing environment in the cytosol, and this can inﬂuence the induction of a protective response [10]. Eukaryotic expression systems such as yeast may refold proteins with the correct conformation but modiﬁcation of the gene (or expressed protein) may be required to prevent or to remove the products of hyperglycosylation [11]. Secondly, the expression of the encoded protein within the host cell aﬀords the possibility of MHC class I presentation, essential for the activation of the aforementioned CD8 þ T cells. The mechanism of this induction will be discussed shortly. Safety considerations for DNA vaccines As with any new technology, eﬃcacy must be balanced with safety and, as such, a number of safety issues have been addressed early in the development of DNA vaccination. One concern is that plasmid DNA may integrate into the host chromosome, resulting in somatic mutation that may inactivate suppressor genes and activate oncogenes [12,13]. Another safety issue is the potential for adverse immunological consequences arising from long-term expression of a foreign gene, such as the induction of anti-DNA antibodies and possible autoimmune disease [12] These fears have largely been allayed. In human malaria DNA vaccine trials, it has been established that a P. falciparum CSP DNA vaccine was safe and well tolerated after intramuscular (IM) DNA vaccination [14]. Human trials using a HIV DNA vaccine have also shown that patients did not develop local or systemic reactions, and no anti-DNA antibodies were detected [15]. A quantitative assay for investigating the tissue distribution and integration into chromosomes of plasmid DNA vaccines has been developed, where genomic DNA is assayed for integrated plasmid using PCR [16]. After IM vaccination of mice and guinea pigs, it was found that there was no evidence of DNA integration, to a sensitivity of about one copy per microgram of DNA,

192 approximately three orders of magnitude below the spontaneous mutation frequency [16]. Features of a DNA vaccine vector

Pr o

-r ic

C

m ot er

An ti

B

bi ot

Most plasmids used for vaccination share the basic attributes of vectors developed for in vitro expression of genes in transfected cell lines [17]. Figure 1 depicts the functional components of a DNA vaccine vector, which includes an origin of replication and an antibiotic resistance gene for producing the plasmid in E. coli, a strong enhancer/promoter (such as the human cytomegalovirus immediate-early promoter), an intron to enhance expression of mammalian genes and an mRNA transcription termination/polyadenylation sequence for terminating transcription in mammalian cells. The gene to be expressed is cloned into the vector at a site downstream of the intron sequences. There are variations on this general theme. For example, signal peptide sequences within the vector may be used to direct expression of the encoded

D

i Or s

t

A

Intron

DNA Vaccine Vector

Poly A

e Ge n

e er Int f o

E

F Fig. 1. The functional components of a DNA vaccine vector. The plasmid vector includes (A) an origin of replication for producing plasmid in E. coli; (B) an antibiotic resistance gene for selective E. coli growth; (C) a strong enhancer/promoter (such as the human cytomegalovirus immediateearly promoter); (D) an intron to assist expression of mammalian genes; (E) a gene of interest; and (F) an mRNA transcription termination/polyadenylation sequence for terminating transcription in mammalian cells. Signal peptide sequences within the vector may also inﬂuence the type of immune response generated. A nonsecreted antigen may be optimal for CD8 þ mediated protection, as intracellular expression may be preferential for the endogenous pathway. Alternatively, a secretion signal sequence may also be added to the vector. This may preferentially stimulate CD4 þ cellular and humoral responses by directing antigen presentation through the exogenous pathway.

193 protein to the extracellular compartment, which will inﬂuence the presentation of the antigen to the host immune system. The inﬂuence of such variations on the immune responses will be discussed after a generalized overview of antigen presentation following DNA vaccination. Immune mechanisms induced following DNA vaccination In the original experiments performed by Wolﬀ et al. [1], it was shown that after the deposition of a plasmid expressing a marker protein into mouse muscle, a proportion of the myocytes was observed to express the protein. Thus it was proposed (correctly) that the myocyte takes up the plasmid DNA by an unknown mechanism, and the plasmid translocates to the nucleus and the gene is transcribed. In later experiments demonstrating that an immune response [2] and protective immunity [3] was induced, it was assumed that the protein produced by the myocyte is in fact responsible for this response. The mechanism by which this occurred was, however, debated. It is known that myocytes express low levels of MHC class I on their surface, and will present peptides to T-cells, however no MHC class II presentation occurs and therefore there is no direct presentation to CD4 þ cells. Furthermore, there are few co-stimulatory molecules expressed on the surface of myocytes: in particular, B7.1 and B7.2 (CD80 and CD86), that are required for the ligation of CD28 on T cells, are not present on myocytes. Hence, activation of naı¨ ve T cells is not possible via MHC class I presentation by myocytes. In a key set of experiments, Doe et al. [18] and Fu et al. [19] used chimeric mice to demonstrate that the cells presenting foreign antigen after DNA vaccination were in fact derived from bone marrow, rather than being myocytes. Hence, the question as to how these APCs gain antigen after intramuscular (IM) vaccination was posed. There are several possibilities. Firstly, the muscle cell may be considered as a simple factory, producing foreign protein that then actively (using a secretion signal) or passively gains access to the extracellular compartment. This protein is then endocytosed by APCs and processed in the normal way, presented with MHC class II, triggering the activation of naı¨ ve CD4 T cells. A second possibility is that transfected myocytes periodically undergo apoptosis and are taken up by phagocytic cells, particularly naı¨ ve dendritic cells [20]. These APCs then cross-present the antigen with MHC class I, resulting in the induction of CTLs. Another possibility is that the production of protein within myocytes is a blind alley – it may be that this is incidental to the generation of an immune response. If this is the case, it can be assumed that it is the resident APCs surveying the muscle that take up the plasmid and express the encoded protein, with presentation either occurring directly or by crosspresentation to bystander APCs. However, the observation by Doe et al. [18] and Fu et al. [19] that transplanted myocytes, expressing a foreign protein, can induce an immune response that is restricted to the recipient bone-marrow derived APCs indicates that direct transfection of APCs is not necessary.

194 Further conﬁrmation of this comes from the use of a DNA vaccine driven by a muscle-speciﬁc promoter whereby the full range of immune responses (including CTLs) was induced [21]. Hence there is a transfer of protein from the myocyte to the APC. Transfection of antigen presenting cells is a mechanism that probably operates in some circumstances, particularly after biolistic vaccination of the skin. In this case, resident APCs, particularly Langerhans cells, are transfected and are able to present peptide to CTLs [for example, see Ref. 22]. In a recent study, Timares et al. [23] showed that gene gun immunized mice harbored transfected Langerhans cells that produced detectable transgene mRNA and protein and, when transferred to naı¨ ve mice, were able to induce comparable CTLs, but lower humoral responses, compared to mice vaccinated in the normal manner. Hence direct transfection of skin dendritic cells appears suﬃcient for the induction of CTL responses, but is not suﬃcient for the induction of humoral responses. An early intriguing observation of the eﬀects of plasmid DNA vaccination was the adjuvant eﬀect, which is antigen-independent. This was shown to be due to the presence of unmethylated CpG motifs, which are present in bacterial DNA [reviewed in Ref. 24]. Many experiments have shown that the CpG motifs can be used as an adjuvant, and that this is not restricted to DNA vaccines but can also be employed with protein vaccines. The explanation for this eﬀect was revealed with the identiﬁcation of the target of CpG; toll-like receptor 9 [TLR9, reviewed in Ref. 25]. Thus the CpG motifs present a danger signal to the immune system via this TLR. These observations do point to one possible reason for the general low level of immune responses to DNA vaccines, particularly in large animals. In order for there to be full activation of APCs, particularly dendritic cells, some form of danger signal must be present in order for the cell to mature and upregulate co-stimulatory molecules. CpGs are one such signal but it may be that in the form of plasmid DNA there are simply not enough of these signals to fully commit an APC to maturation. Supplying a stronger signal by engineering more CpG motifs into a plasmid is one solution, however supplying these signals in another form may be a viable alternative. This may be the rationale behind the successes of using attenuated bacterial vaccines as DNA plasmid carriers. This will be further discussed later in this review. Immune responses are dependent on the route of vaccine delivery The compartment of expression has been shown to inﬂuence the quality and magnitude of humoral immune responses and the frequency of induction of CD8 þ T cells, where intracellular expression may be preferential for presentation via MHC class I (for example, Boyle et al. [26]; Smooker et al. [27]). Indeed, it may be that one of the most useful properties of DNA vaccines is the ability to tailor the immune response to an encoded antigen. The immune responses

195 generated after DNA vaccination are dependent on many factors including the route of delivery, type of vaccine vector used, antigen used and dose of vaccine plasmid. There are two main forms of DNA vaccine delivery used in the basic research environment. Immunization can be either in the form of an intramuscular or intradermal injection of plasmid in physiological saline, or the DNA can be bound to gold microparticles and delivered to the dermis of the skin using a helium-propelled ‘‘gene gun.’’ The advantage of gene-gun vaccination is the amount of DNA required to achieve comparable immune responses is 100–1000 fold less than saline injection. As little as 16 ng of DNA has been found to induce an immune response in mice using a gene-gun, while the injection of 5000-fold more DNA (both ID, and IM) was required to achieve comparable immunogenicity [28]. After 13 years of DNA vaccine studies, we have some knowledge of the mechanism of induction of immune responses after DNA vaccination, and have observed that diﬀerent routes of delivery alter the quality and magnitude of the responses. The general sentiment that improved immune responses will be required for vaccination both against complex pathogens, and of large organisms (including humans), leads to the speciﬁc sections of this review. Section 2: DNA vaccines for intracellular protozoan parasites Although DNA vaccination against intracellular protozoan parasites has been tested using a variety of antigens, the vaccines have mainly been evaluated using mouse models (Table 1) [29–64]. DNA vaccination against malaria has been the most extensively tested, in hosts ranging from mice to humans (discussed below). Other protozoan parasites for which DNA vaccination has been tested include: Leishmania, Toxoplasma, Trypanosoma, Entamoeba and Cryptosporidium (Table 1). All DNA vaccines tested have resulted in varying degrees of success (particularly in mouse models); however, there has been no successful DNA vaccine against a protozoan parasite that is yet available for human use. Leishmania Leishmania is endemic in 88 countries across ﬁve continents (excluding Australia) where a total of 350 million people are at risk, with cutaneous leishmaniasis representing 50–75% of the 1.5–2 million new cases annually (www.who.int/inf-fs/en/fact116.html). Leishmania is second to malaria as the protozoan most studied for DNA vaccine development. Immunity to Leishmania in murine models using L. major has been found to be dependent on a CD4 þ Th1 type response, while CD4 þ Th2 type responses are associated with susceptibility to infection [65]. The availability of NK cells has also been found to be a requirement for the maintenance of protection after L. major

196

Table 1. Parasite

Host

Leishmania

Mice

Toxoplasma

Antigens tested as DNA vaccine

TSA/LmSTI1 [30] p36(LACK) (DNA prime/vaccinia boost) [40] p36(LACK) (DNA alone) [46] PSA2 [42] cDNA expression library [45] p36(LACK), LmSTI1, TSA [47] Genomic expression library [50] CPa/CPb (DNA prime/protein boost) [52] meta-1 [58] p36(LACK), IL-12, IL-18 (DNA prime) (p36/LACK vaccinia boost) [59] Lm-gp63 [61] IL-12 (and soluble antigen) [63] Hamster papLe22 [35] Dogs DNA/LACK prime (vaccinia/LACK boost) [53] Mice SAG1/IL-2 [31] GRA4 [34] ROP1/IFN-g [41] ROP2 [44] SAG1 [49] GRA1 [56] GRA1/GRA7/ROP2 [60] HSP70/HSP30/SAG1 [48]

Route

Eﬀectiveness

IM ID (IP boost) ID or SC IM SC or IV ID, IM or SC IM IM (IP boost) IM ID (IP boost)

Reduced footpad lesions 70% lesion reduction, 1000 fold-less parasite load No protective eﬀect Reduced footpad lesions Reduced hepatic parasite burden ID combination reduced ear lesions Reduced footpad lesions Reduced footpad lesions No protective eﬀect Reduced footpad lesion

ID ID IM SC IM IM IM IM IM IM IM ID, IM, IP

30% of mice with reduced footpad lesions Complete healing of lesions and long term immunity 50% reduction in peripheral blood 60% protection against visceral leishmaniasis Sig. delay in death after lethal challenge 62% survival Not determined Sig. delay in death after lethal challenge 80–100% protection after lethal challenge 75–100% protection after lethal challenge 50–90% protection after lethal challenge Sig. reduction in parasite burden

Trypanosoma

Mice

Entamoeba

Mice

Gerbil Cryptosporidium Goat Mice

ASP2 [29] TS [32,33] TS and CD4 þ /CD8 þ epitopes [36] ASP1/ ASP2/TSA1 [37] TSSA [43] KMP11/HSP70 [51] CRP [57] TSA1 [62] Gal-lectin [38,39]

IM IM IM IM IM IM IM IM ID

SREHP [64] SREHP [64] CP15 [54] CP15 [55]

IM IM IN IN

Sig. reduction in parasitemia and 100% survival Sig. reduction in parasitemia and 100% survival Sig. reduction in parasitemia and 100% survival Sig. reduction in parasitemia and 30-80% survival 100% protection after lethal challenge 50% protection after lethal challenge Sig. reduction in parasitemia and 100% survival 64–89% protection after lethal challenge Immunogenic and 29% inhibition of trophozoite adherence 80% protection against liver abscesses 60% protection against liver abscesses Kids born from vaccinated goats shed sig. less oocysts Immunogenicity study

197

198 infection [66]. Several major vaccine candidate antigens have been tested against Leishmania infection as DNA vaccines including the LACK antigen (Table 1) [40,46,59,67,68]. It was initially shown that vaccination of BALB/c mice (highly susceptible to L. major infection) using a LACK DNA vaccine was superior to vaccination with recombinant LACK protein [68]. The use of recombinant IL-12 and soluble L. amazonensis antigen in alum adjuvant, has been shown to protect primates after L. amazonensis challenge [69]. Vaccination of mice with recombinant IL-12 and LACK protein is highly protective against L. major infection, whereas the administration of LACK DNA vaccine alone induces a level of protection comparable to that observed using the recombinant protein [68]. Recently it was shown that DNA priming with LACK, IL-12, and IL-18 followed by boosting with recombinant vaccinia virus expressing LACK is also highly protective against L. major infection in mice [59]. DNA priming and vaccinia boosting using LACK, followed by challenge with L. infantum, has been shown to protect 60% of dogs against visceral leishmaniasis [53]. DNA priming alone using this large animal model did not induce any protection against L. infantum challenge. More large animal trials using DNA priming against Leishmania are required. Toxoplasma Infection with T. gondii in immunosuppressed humans may result in toxoplasma encephalitis, and transmission during pregnancy may result in miscarriage or severe health risks to the fetus [reviewed in Ref. 70]. Infection with T. gondii also subverts macrophage function, preventing critical IL-12 and TNF-alpha production early in infection to avoid immune elimination [reviewed in Ref. 71]. It has been established that T-lymphocytes and IFN-g play an important role in host resistance during chronic and acute stages of infection [70]. Major vaccine candidates tested against T. gondii used in DNA vaccination studies are shown in Table 1. Immunity after DNA vaccination using these antigens was associated with a Th1 response, characterized by elevated levels of IFN-g. Mice have been the only host tested using DNA vaccines against Toxoplasma, with most studies resulting in a high degree of protection after infection. Trypanosoma DNA vaccine development against Trypanosomes has focussed upon T. cruzi, the etiologic agent of Chagas’ disease; DNA vaccine studies involving T. brucei (African trypanosomiasis) are yet to be reported (Table 1). Infection of humans with T. cruzi extends from Mexico to the south of Argentina, aﬀecting 16–18 million people with 25% of the population of Latin America at risk of acquiring Chagas’ disease (www.who.int/ctd/chagas/disease.htm).

199 Antigens tested in DNA vaccine studies range from the trypomastigote to amastigote stages, with vaccination of mice resulting in survival against lethal T. cruzi infection (Table 1). The activation of CD8 þ T-cells and CD4 þ Th1 subsets as a result of DNA vaccine priming is the primary mechanism by which such high levels of protection have been observed (Table 1). Activation of both T-cell subsets are important for protective eﬃcacy of T. cruzi DNA vaccines, as abrogation of the CD4 þ Th1 or CD8 þ response in vivo abolished the protective eﬀect of TSSA vaccination of mice [43]. DNA vaccination with both CD4 þ Th1 and CD8 þ T-cell epitopes derived from the T. cruzi TS gene is required for protective immunity of mice from lethal T. cruzi challenge [36]. A direct comparison of recombinant CRP protein versus CRP DNA vaccination of mice resulted in the production of lytic DNA vaccine-induced antibodies and protection against lethal challenge, which was not found in mice vaccinated with recombinant CRP protein [57]. The elicitation of potent CD8 þ and CD4 þ Th1 immune responses after IM DNA vaccination seem to make DNA vaccination an ideal method of disease prevention. However, no studies have yet reported the eﬃcacy of DNA vaccination (priming or prime/boost) against Trypanosomes in large animal models.

Entamoeba Entamoeba histolytica is responsible for up to 100,000 deaths per annum, placing it second only to malaria in mortality due to protozoan parasites (www.who.int/docstore/wer/pdf/1997/wer7214.pdf). Reported DNA vaccination studies against this protozoan parasite have been limited, with only two antigens tested and one (SREHP) being successful in reducing liver abscesses in mice and gerbils (Table 1).

Cryptosporidium Cryptosporidium exists in over 40 countries on six continents, infects both immunocompetent as well as immunocompromised patients and is a cause of severe diarrhoeal illness [reviewed in Ref. 72]. Cryptosporidium parvum infects the intestinal epithelial cells of a wide variety of mammals, including humans [reviewed in Ref. 73]. To date, there is no therapeutic or preventive treatment approved for cryptosporidiosis, and mechanisms of chemotherapeutic resistance are not yet fully understood [reviewed in Ref. 74]. Table 1 shows the only reported attempts at DNA vaccine development against C. parvum. Nasal immunization of goats using the CP15 antigen resulted in signiﬁcantly less oocyts in kids from vaccinated goats than control animals. The demonstration of vaccine eﬃcacy in a large animal model may reduce economic losses due to cryptosporidiosis in ruminants [54].

200 Malaria DNA vaccines have been extensively tested for control of malaria using mice and primate as models, as well as in humans (described below). Although a large body of work has involved the testing of DNA vaccines against malarial challenge, there is still no successful malaria vaccine (DNA or protein) accepted for human use. Malaria is estimated to kill between 1.5 and 2.7 million people every year, with 300–500 million people having the disease, and one third of humans living in malaria endemic regions [75,76]. Drug resistant malaria parasites and insecticide resistant Anopheles mosquitos aﬀect almost every country where malaria is endemic, further emphasizing the need for new control measures such as a malaria vaccine [77]. Four species of the genus Plasmodium, a unicellular protozoan parasite, infect humans: P. malariae, P. vivax, P. ovale, and P. falciparum. P. falciparum accounts for 90% of all infections [76]. Each year, approx. 10–30,000 tourists visiting malaria endemic areas are infected, and US military campaigns conducted in malaria endemic regions over the past century have experienced more casualties to malaria than hostile ﬁre [78]. During the blood meal of a Plasmodium infected Anopheles mosquito, sporozoites contained within the salivary glands are injected into the blood of the host, then migrate to the liver and invade the cytoplasm of hepatocytes. This phase of the life cycle is termed the ‘‘pre-erythrocytic’’ stage of malaria infection. The hepatocyte is the ﬁrst stage of parasite proliferation within the host. An eﬀective vaccine directed against the sporozoite stage of the malaria cycle would prevent progression to the next stage of the malaria life cycle, the ‘‘erythrocytic stage’’ which is associated with clinical illness [79]. The erythrocytic stage of malaria is characterized by the rupture of infected hepatocytes, which release merozoites that enter the blood circulation and invade erythrocytes. The merozoite enters the erythrocyte and undergoes a stage of asexual ampliﬁcation. Within 72 h, the erythrocyte ruptures releasing an average of 16 merozoites which continue the erythrocytic cycle [80]. This stage is responsible for clinical illness, and may lead to anaemia, cerebral malaria and possible death. The ‘‘sexual-stage’’ completes the malaria cycle, occurring when haploid merozoites within erythrocytes diﬀerentiate into male and female gametocytes, which then undergo gametocytogenesis following ingestion by a mosquito [80]. It is believed that the ultimate malaria vaccine will require the delivery of multiple antigens from diﬀerent stages of the complex life-cycle, that is, a ‘‘multivalent’’ malaria vaccine is required [9,81]. The uniformity of DNA vaccines makes this a much easier task than the co-vaccination of a number of diﬀerent recombinant proteins. An early classical malaria DNA vaccine study ﬁrst demonstrated that vaccination of mice with two

201 pre-erythrocytic stage antigens can enhance protection over the use of either antigen alone, as well as overcome genetic restriction in diﬀerent mouse strains [82]. Combinations of malarial antigens delivered as DNA vaccines in primates have induced antigen-speciﬁc cytotoxic T lymphocytes (CTLs) in pre-erythrocytic stage vaccines [83]. In primates it has also been shown that enhanced antibody responses to combinations of malarial antigens can be generated by erythrocytic stage malarial DNA vaccines [84]. The ‘‘First Generation’’ DNA vaccines (i.e., delivery of only plasmid/antigen DNA) are not optimal for inducing protection against malaria in humans and the possible reasons for this are discussed below. Immune enhancement strategies for DNA vaccination alone are required for this method of vaccination to be practical [reviewed in Refs. 9,81]. Experimental strategies in malaria vaccine design have attempted to incorporate multiple malarial antigens from diﬀerent stages of the life cycle [9,85–89]. The most recent multi-stage malarial vaccine eﬀort is the ‘‘MuStDO’’ (Multi-Stage Malaria DNA-based Vaccine Operation) program [reviewed in Refs. 78,89]. The vaccine comprises 15 P. falciparum antigens: ﬁve from the pre-erythrocytic stage and ten from the erythrocytic stage. However, as yet, this vaccine formulation has not led to a vaccine for humans and is still in the testing phase [89]. Problems after expression of antigens from the encoded DNA, such as antigenic competition between antigens leading to suppression (or complete abrogation) of immune responses, have occurred when vaccinating with candidate malarial antigens in mice [90]. Generation of antibodies to the pre-erythrocytic stage candidate antigen, the circumsporozoite protein (CSP), could not be detected in the ﬁrst two malaria DNA vaccine clinical trials in humans, although a cellular response was detected [14,91]. These problems are yet to be resolved. The asymptomatic pre-erythrocytic stage has been a major focus in attempting to design a vaccine against malaria [78]. This pre-erythrocytic vaccine aims to prevent the development of all clinical symptoms associated with the erythrocytic stage of malaria by eradicating sporozoites from the liver. However, the successful growth and replication of a single malaria parasite within an erythrocyte will result in a viable erythrocytic stage infection, which may cause severe disease or death in a malaria naı¨ ve individual [reviewed in Ref. 81]. Erythrocytic stage vaccine design aims to prevent invasion and infection of erythrocytes by merozoites, and to reduce morbidity and mortality by decreasing the parasite load [79,81]. Sexual-stage ‘‘transmission blocking’’ vaccines aim to reduce malaria within a community as a whole, by preventing the re-infection of the mosquito vector by malaria parasites contained within infected humans. These ‘‘altruistic’’ vaccines confer no beneﬁt for a malaria-infected individual but will reduce the overall parasite load within a community [92].

202 Malaria DNA vaccines encoding single antigens: ‘‘First generation’’ DNA vaccines Early malaria DNA vaccine trials in humans using the pre-erythrocytic stage CSP have not been optimal in inducing protection against sporozoite challenge [78]. In mice, it has been shown that DNA vaccination using single antigens alone does not result in complete protection, and does not withstand high parasite challenge doses regardless of the mouse model used [reviewed in Ref. 78]. The type of T-cell response generated by a DNA vaccine is important for the stage of the malaria life-cycle that is being targeted. It has been shown that IM immunization of both mice [82] and monkeys [93] using CSP plasmid DNA induces IFN-g and CD8 þ CTLs, indicative of a cellular type response perhaps driven by Th1 CD4 þ cells. However CD4 þ T-cell responses after CSP immunization have been found to be poor in mouse, monkey, and human DNA vaccine trials [reviewed in Ref. 9]. It has since been shown that a CD4 þ T-cell response to a conserved CSP epitope correlated with protection from P. falciparum infection in adults living in malaria endemic regions [94]. This emphasizes the importance of including antigenic epitopes in a vaccine that will activate a broad immune response against malaria infection. Sedegah et al. [8] ﬁrst reported protection after sporozoite challenge in 64% of mice upon IM injection of the P. yoelii CSP gene incorporated into plasmid DNA. Immunization induced CSP speciﬁc antibodies and CD8 þ T cells against infected hepatocytes. However, this protection was observed using a single mouse strain (BALB/c). It was subsequently found that combining P. yoelii CSP and HEP17 pre-erythrocytic stage antigens could enhance protection against sporozoite challenge in three of ﬁve strains of mice, while protection after immunization with CSP alone was restricted to a single mouse strain [82]. A DNA vaccine that includes a single antigen will not be suﬃcient to protect the outbred human population. Human malaria DNA vaccine trials have mainly concentrated on delivery of a single P. falciparum CSP DNA vaccine construct to assess safety, tolerability and immunogenicity [89,91,95]. These studies have shown the generation of CD8 þ CTL and IFN-g after CSP immunization, but no antibody responses were detected. For a malaria vaccine to be eﬀective in a heterogeneous human population, a diverse range of class I and II HLA types must be included in the vaccine to overcome the problems of genetic restriction [96]. DNA vaccines encoding multiple malarial antigens DNA vaccines encoding multiple malarial antigens from diﬀerent stages of the life-cycle have been shown to enhance immune responses in animal trials, as well as overcome genetic restriction between animal strains [9,78]. The early demonstration that synergy between malarial antigens can be achieved by co-immunization (using P. yoelii HEP17 and P. yoelii CSP plasmid DNA) [82]

203 was an important observation, and this knowledge has been widely applied to malaria vaccine design. Antibody levels against a P. falciparum MSP119 have been found to be enhanced when mice are injected with multiple plasmids encoding both pre-erythrocytic and erythrocytic P. falciparum antigens [85]. In primates, P. falciparum pre-erythrocytic DNA vaccines alone have been found to generate low levels of antibody, CD8 þ CTL, and IFN-g [83]. An erythrocytic stage vaccine comprised of three antigens, AMA-1, MSP142 and erythrocyte binding protein-175 (EBA-175), has also been tested in primates [84]. Antibody responses were generated to all three antigens, with the trivalent mixture generating antibody responses 3–12 fold higher than vaccination with a single plasmid alone. Again, this result demonstrates that the synergy between multiple malarial antigens can be used to advantage when vaccinating with DNA vaccines, which in turn may provide the knowledge for a future human malaria vaccine able to overcome genetic restriction. It has been shown that the use of a GM-CSF plasmid DNA as an adjuvant with MSP142 plasmid DNA can induce a rapid induction of antibody in Rhesus monkeys after the ﬁrst dose, when compared to MSP142 plasmid DNA alone [97]. In humans the MuStDO 5 pre-erythrocytic DNA vaccine is in the process of being tested by co-vaccination with a human granulocyte macrophagecolony stimulating factor (hGM-CSF) plasmid. It was found that the vaccine was safe and well tolerated, however eﬃcacy and immunogenicity results are not yet available [89]. Heterologous boosting of DNA vaccines Boosting with viral vectors after DNA priming primarily induces responses that generate enhanced CD8 þ T-cell responses (often with reduced antibody responses), while DNA priming followed by boosting with recombinant protein in adjuvant helps to generate enhanced antibody responses (but sometimes reduced CD8 þ T-cell responses) [78]. Complete protection against P. berghei sporozoite challenge has been demonstrated using heterologous prime/boost vaccines in diﬀerent strains of mice [98]. Mice were ﬁrst primed with a single dose of two plasmids each encoding a single P. berghei pre-erythrocytic stage protein. Mice were then administered a single boost with recombinant modiﬁed vaccinia virus Ankara (MVA), encoding the same antigens. Challenge with P. berghei sporozoites showed protection of 100% of mice, with protection associated with CD8 þ cells and secretion of IFN-g. It has been shown in many studies that boosting with viral vectors encoding pre-erythrocytic stage antigens results in enhanced CD8 þ T-cell responses, and that pre-erythrocytic immunity can be improved against sporozoite challenge [99–102]. Natural ‘‘priming’’ of humans by infected mosquitos results in low levels of naturally acquired CD8 þ T cells [103]. It may be possible to boost natural CD8 þ T-cell levels in humans by using recombinant MVA [98].

204 Boosting Aotus monkeys with pox virus, after priming with plasmids encoding two P. knowlesi pre-erythrocytic and two erythrocytic antigens (as well as combinations of GM-CSF, IL4 and TNF-a plasmids), resulted in sterile protection of 18% of monkeys, with 78% of monkeys resolving parasitemia spontaneously [104]. Control monkeys were treated for high parasitemias after all groups were challenged with a lethal dose of P. knowlesi sporozoites. This protocol, with pox virus boosting, is now being planned for testing in humans [89,105]. Although viral boosting is eﬀective in murine and primate systems, whether boosting humans on a large scale with pox virus will be accepted still remains to be seen. Attempts to enhance erythrocytic stage protection against malaria have involved DNA priming with antigens expressed at the erythrocytic stage followed by boosting with the homologous protein in adjuvant. Haddad et al. [106] have reported boosting of antibody titers in three diﬀerent mouse strains to the P. falciparum antigen Pf155/RESA. As the antigen used in this murine experiment was P. falciparum, no protection data was obtained. Boosting monkeys with recombinant malarial protein has been shown to enhance antibody levels, and protect against erythrocytic stage infection. Priming Rhesus monkeys with a MSP142 DNA vaccine, followed by boosting with recombinant MSP119 has lead to enhanced antibody responses that were reactive with infected P. falciparum erythrocytes in vitro [97]. Protection against the erythrocytic stage has also been demonstrated in Aotus monkeys using P. falciparum EBA-175 DNA priming and recombinant boosting [107]. Vaccination with EBA-175 plasmid DNA three times, followed by a single boost with recombinant EBA-175, produced an antibody response equivalent to four doses of recombinant EBA-175. Three out of four monkeys receiving the EBA-175 prime/boost did not require treatment, while all monkeys receiving DNA alone and recombinant protein alone required treatment. This demonstrates the power of a single recombinant protein boost after focussing the immune system using DNA vaccine priming. Use of DNA vaccines alone to prevent malaria DNA vaccines on their own may be adequate to enhance the immune responses of individuals already exposed and at risk of malaria in endemic regions. A model of malaria vaccine development by Hoﬀman and Doolan [105] proposes two vaccine types: (1) a vaccine to prevent all clinical manifestations (for malaria naı¨ ve individuals); and (2) a vaccine to reduce mortality and severe disease. Results from the ﬁrst human trial for the ﬁrst type of vaccine have recently been published. The trial involved priming with plasmid DNA encoding the pre-erythrocytic antigen thrombospondin-related adhesion protein (TRAP) linked to a codon-optimized string of epitopes (derived from pre-erythrocytic stage antigens covering multiple HLA types) and boosting with recombinant

205 vaccinia virus [108]. Signiﬁcant delays in parasitemia were found after challenge with P. falciparum sporozoites as well as enhanced T-cell responses. Human T-cell responses were found against multiple epitopes contained within the 20 selected sequences, indicating that they were successfully processed and presented after DNA vaccination. DNA vaccines alone may be most suitable for reducing mortality and disease due to malaria. In mice infected with Mycobacterium tuberculosis, it has been shown that a DNA vaccine initially designed to prevent infection can switch the immune response from Th2 to Th1, and kill the bacteria [4]. DNA vaccination of humans with previous exposure to malaria could re-program the immune response to important antigens or epitopes and induce responses that will reduce parasite burden and morbidity. Future of DNA vaccination against intracellular parasites DNA vaccines, when used in their present form against protozoans, particularly in non-rodent host models, are not eﬀective at preventing disease. However, eﬀectively ‘‘priming’’ the immune system using DNA vaccines is an immediate practical application. Studies from malaria in particular, as described above, have shown that DNA vaccines in their present forms are not adequate to prevent disease. Boosting the immune system after the DNA immunizing antigen has been used to prime the immune system is the most practical outcome of DNA vaccines in their present forms. The enhancement of antigen recognition and augmentation of the immune system against invading parasites by DNA vaccination are now discussed further. Section 3: Expression library immunization: A genomic approach to vaccine discovery Expression Library Immunization (ELI) is a true multivalent expression system, as it is possible to utilize the entire genome (as genomic DNA) or speciﬁc stages of a pathogen (as cDNA) to generate the vaccine. The identiﬁcation of more eﬀective multivalent malarial vaccines may be possible through the construction of ‘‘DNA libraries’’ containing DNA sequences from a pathogen’s genome. ELI has been applied to bacterial [109,110] and parasitic infections [27,45,50,111], and recently against simian immunodeﬁciency virus [112]. Genomic ELI libraries encode antigens from all stages of the life-cycle, and potentially allow the discovery of antigens from all stages of a parasite’s lifecycle, while cDNA ELI targets speciﬁc stages of a life-cycle. ELI involves digestion of DNA from an organism, fusing fragments into a DNA vaccine vector and immunizing animals (Fig. 2) [reviewed in Ref. 113]. Sequential partitioning of protective libraries into smaller subsets of the genome may result in protective antigen discovery, although the discovery of a single antigen using genomic ELI has not yet been reported.

Sequence Gene Yes

No

Malaria Genome

Protection from single plasmid? Vaccinate

Fragment

Make Plasmids

Divide into Pools

Challenge with parasite infected RBC

Fig. 2. The steps involved in ELI toward eventual antigen discovery (using a murine malarial model as an example). The genome is fragmented with a restriction enzyme, followed by insertion into a DNA vaccine expression vector. The plasmids are then divided into ‘‘pools’’ of known number, followed by vaccination into groups of animals. Protective plasmid groups are selected for, and the whole process is repeated until protection from a single plasmid or group of plasmids is achieved.

206

Divide into smaller pools

207 ELI against Trypanosoma cruzi Two expression library studies have been reported using the T. cruzi model as either a genomic library [111] or a pool of amastigote stage cDNAs [114]. Using a T. cruzi genomic library, immunization of BALB/c mice resulted in expression of antigens from muscle (detected using indirect immunoﬂuorescence), and the detection of T. cruzi speciﬁc antibody and cellular responses to T. cruzi soluble antigens [111]. However, no protective data or subdivision of the expression library was investigated. A recent study has demonstrated that vaccination of BALB/c mice with a pool of amastigote-stage trans-sialidase genes protected mice against lethal T. cruzi infection [114]. A pool of 10 transsialidase genes were ampliﬁed from amastigote cDNA based upon conserved sequences found in the trans-sialidase gene family. Vaccination of BALB/c mice resulted in 75% protection against lethal T. cruzi challenge, and expanded the repertoire of potential T. cruzi vaccine candidates [114]. ELI against Leishmania The ﬁrst genomic expression library reported against L. major by Piedraﬁta et al. resulted in signiﬁcant protection against challenge and enrichment of library pools [50]. Three libraries of 105 clones, with each library in a diﬀerent reading frame were generated. One of the 105 clone libraries (from one reading frame) resulted in a signiﬁcant protective eﬀect after IM vaccination of BALB/c mice. Further subdivision of the protective pool to ﬁve sets of 2 104 clones resulted in an enhanced protective eﬀect of one of the subpools. This study demonstrates that genomic expression library immunization can be used as a tool to enhance the protective eﬀects and eventually identify protective sets of genes using this parasite model. Further success using a cDNA expression library taken from the amastigote stage of L. donovani was later reported by Melby et al. [45]. Immunization of mice with plasmid DNA from 15 cDNA pools (approximately 2000 cDNAs per pool) signiﬁcantly reduced hepatic parasite burden. It was found that one protective cDNA pool encoded a set of nine novel cDNAs and another protective pool of ﬁve cDNAs encoded L. donovani histone proteins. This result demonstrated two important points: ﬁrstly, that the ELI approach could actually identify protective clones derived from much larger subpools; secondly, that this approach could identify novel sets of protective sequences, such as histones and unknown sequences, that would not have been predictable candidate vaccines. ELI against Toxoplasma gondii Genomic expression library immunization using T. gondii has also been reported by Fachado et al. [115]. Vaccination with a T. gondii genomic

208 expression library resulted in signiﬁcant survival of BALB/c mice when challenged 24 weeks after the ﬁnal vaccination [115]. It was also shown that CD4 þ and CD8 þ splenocytes were responsive to T. gondii antigen stimulation 24 weeks after vaccination. Although this study described signiﬁcant survival when vaccinating with the entire T. gondii library, further studies describing devolution and protective eﬃcacy of sub-pools are yet to be reported.

ELI against malaria Genomic expression library immunization using malaria as a model was ﬁrst reported by Smooker et al. [27] using the highly virulent Plasmodium chabaudi adami DS strain of rodent malaria. A pool of 30,000 plasmids containing P. c. adami DS genomic DNA was partially protective in mice against DS challenge: when this pool was segregated to a subpool of 3000 plasmids vaccination of mice resulted in an increase in protective eﬃcacy against lethal challenge (from 50 to 63%). Stimulation of splenocytes taken from BALB/c mice, previously primed with the 30,000 plasmids, resulted in signiﬁcant levels of proliferation and IFN-g secretion after exposure to parasitised erythrocytes [116]. Subdivision of plasmids leading to the discovery of new malarial vaccine candidates is in progress. Using P. berghei, Shibui et al. [117] constructed an erythrocytic stage cDNA library. After initial vaccination of mice directly into the spleen, followed by two IM boost injections, survival in the empty plasmid DNA control groups exceeded that of the cDNA library vaccinated groups suggesting that splenic delivery of this vaccine (which in itself is an impractical approach) was inappropriate in this model. Existing candidate antigens may be suﬃcient to protect against malaria in humans with corresponding HLA types, although it is possible that more antigen sequences will be required to protect across diverse genetic backgrounds. The discovery of new full-length antigens or epitopes by gradual elimination of non-protective pools is the ultimate goal of ELI. Support for a multivalent vaccine comes from studies of Doolan who showed that it is the combination of malarial genes acting synergistically which have been shown to provide the greatest protection after challenge [e.g., 82–84]. The body of evidence already discussed suggests, at least for malarial vaccination studies, that the removal of antigens acting synergistically is detrimental to protection. It has also been shown that vaccination of mice with combinations of 10 aa peptide epitopes derived from MSP133 can enhance protection of mice over vaccination with individual peptides [118]. Therefore, maximizing protection by including all combinations of diverse antigens/epitopes encoded within a genomic DNA vaccine library itself cannot be ignored, since this may lead to improved multivalent vaccines. The use of ELI can be best described as a ‘‘bottom-up’’ approach to the discovery of protective epitopes; from the genomic level to the protein level.

209 Epitopes contained in erythrocytic stage antigens from malaria are being investigated by others, and have the potential to be exploited as malaria vaccines in the future [118]. This approach, utilizing known antigens, can be described as ‘‘top-down,’’ with the end point being the elucidation of protective epitopes. The common feature of both methods is the discovery and use of epitopes to aid in malaria vaccine design. These would make attractive vaccine targets in the future. The idea of including epitopes derived from known antigens in malaria vaccine design is not a new concept. Identiﬁcation of protective epitopes on pre-erythrocytic stage antigens such as the circumsporozoite protein have been investigated [119–122]. An example of the potential for epitopes to be exploited for erythrocytic stage vaccine design is a region contained within the variant erythrocytic stage antigen PfEMP1, that is highly conserved between Plasmodium strains [123]. Monoclonal antibodies directed at a functionally conserved region of PfEMP1 (the cysteine-rich interdomain region 1, mediating adhesion of infected erythrocytes to CD36), have been found to cross-react with epitopes on multiple parasite strains [124]. This region of PfEMP1 contains highly conserved residues (particularly cysteine) between parasite strains that form a conserved CD36 binding domain [123]. The CD36 binding domain of PfEMP1 is poorly immunogenic in natural human P. falciparum infections [125], and this would lead to less immune pressure and therefore possibly less variation in these epitopes, making this region a potentially eﬀective epitope vaccine target. Multiple T-cell epitopes have also been found in MSP133, and peptides derived from these epitopes are able to protect mice against lethal P. yoelii YM erythrocytic stage challenge [118]. It has also been shown that DNA vaccination of single epitopes (derived from a mutant p53 sequence) was found to induce anti-tumor immunity and protective CTL responses in mice, showing that delivery of small epitopes as DNA vaccines is feasible [126]. These results help to justify the use of ELI for the discovery of protective epitopes for use in the development of malaria vaccines.

The Vaccinome approach to vaccine discovery Another genomic approach to malaria vaccine discovery proposed by Hoﬀman et al. [127] was to use the malaria genome sequence to predict genes likely to encode important candidates, such as merozoite surface proteins, and to pool such sequences into a combination vaccine (the Vaccinome approach). The concept is analogous to ELI in that genomic sequences (exons) are engineered into a pool of DNA vaccine plasmids and delivered as a multivalent vaccine. The key diﬀerence is that this approach relies on the judicious selection of a pool of candidate antigens rather than the random testing of pools of sequences. The Vaccinome approach assumes of course that our selection process is robust, that is, we have chosen the ‘‘best candidates.’’ In the case of

210 merozoite surface proteins the approach is a reasonable one. However, this approach is unlikely to be chosen for testing previously unknown or hypothetical antigens that could be good vaccines, but which are missed because either they do not meet certain criteria (abundance in merozoites, presence of predicted signal sequences) or because our knowledge of what is actually a ‘‘good’’ candidate is blinkered by dogma. This is not a trivial point: as discussed above, Melby et al. [45] showed that housekeeping sequences such as histones and unknown hypothetical sequences were actually protective in the Leishmania mouse model. We feel that application of ELI or a targeted Vaccinome approach are both viable strategies for identifying multivalent vaccine candidates for malaria since it is clear that the ﬁnal human vaccine is likely to require multiple antigens that elicit cross protection against a variety of parasite genotypes. Two of the leading malaria vaccine candidates (MSP1 and AMA1) do not elicit cross protection in mouse studies [128,129] and antibodies to AMA1 do not give good cross inhibition of parasite growth in vitro when tested against certain heterologous strains of P. falciparum [130,131]. Such observations raise concerns about the likely eﬃcacy of MSP1 and AMA1 when tested in ﬁeld studies where individuals can be exposed to multiple parasite genotypes [132]. Since discovery of multivalent vaccines is inherently a tedious process any approach that allows rapid screening of candidate sequences from the genome is of strategic interest. Section 4: The challenge of vaccinating large animals and humans with DNA vaccines As previously detailed, DNA vaccine technology has been extensively used in murine models against viruses, bacteria and parasites. While the results from the murine work are very promising, very little work has been done in large animals. Considering that large animals (and humans) will be the primary beneﬁciaries of such vaccines it is imperative that immunogenicity studies are carried out in the target species. However, the expense, absence of inbred large animal populations, poor diversity of immunological reagents and the diﬃculty of housing and handling large animals have been signiﬁcant obstacles to the generation of such data. DNA vaccination of ruminants against parasitic disease Most of the early work carried out in large animals using DNA vaccines has concerned its application to viral pathogens in cattle, pigs and a variety of other veterinary species [reviewed in Ref. 133]. These original observations highlighted the ability of DNA vaccines to generate modest cell mediated immune (CMI) responses in large animal models where, in some instances, signiﬁcant levels of protection were generated [134–138]. However, the accompanying

211 humoral responses were quite often low or absent and in the studies where signiﬁcant levels of antibody were generated, high dose repeated vaccinations were required. Similar immune responses to DNA vaccines encoding parasite antigens have been obtained in large animals. The ﬁrst reported DNA vaccine study applied to a parasitic disease in a large animal model was performed in sheep using a DNA vaccine encoding sporozoite antigens from the protozoan parasite Cryptosporidium parvum [139]. Intramuscular DNA vaccination resulted in a highly variable IgG response where up to 3 doses of 1 mg of DNA failed to seroconvert some animals. Similarly, the IM vaccination of sheep with a plasmid encoding the 45W antigen from the cestode parasite Taenia ovis resulted in the generation of variable, low level antibody responses that were unable to be boosted by repeated DNA vaccinations [140]. Encouragingly, the vaccination of pigs with a DNA vaccine expressing the antigen B gene from Cysticercus cellulosae induced antibody and protected pigs on average by 90% against challenge infection with T. solium eggs [141]. Recently, DNA vaccines encoding Schistosoma japonicum antigens Sj28 and Sj23 were evaluated in sheep and water buﬀalo under laboratory and ﬁeld conditions [142]. Sheep vaccinated IM three times with 100 mg of either construct were partially protected from experimental challenge, with signiﬁcant worm reductions of up to 65% seen in Sj28 vaccinated animals. Signiﬁcant protection levels (mean 39%) following ﬁeld infection were also observed in buﬀalo vaccinated with Sj28 or Sj23, which were equivalent to protection levels aﬀorded by vaccinating with recombinant Sj28GST in FCA/ FIA. Similarly, when cattle were vaccinated as above and exposed to infection in the ﬁeld, signiﬁcant protection levels of up to 44% were seen in the absence of a detectable IgG response [143]. To our knowledge, this represents the ﬁrst report where signiﬁcant protection levels have been aﬀorded by DNA vaccination alone in a large animal model against a parasitic disease. These results have oﬀered encouragement that DNA vaccination may in some instances be capable of inducing protective responses against parasite infection in production animals. Direct comparisons in animal models, using the same DNA construct expressing the same antigen, highlight the vast diﬀerences between rodents and large animals in their ability to mount an immune response to DNA vaccination. In one study, mice and sheep were vaccinated with DNA vaccines encoding the 16k, 18k and 45W antigens of T. ovis [144]. While these DNA vaccines were all immunogenic in mice, only the 45W DNA vaccine was capable of generating an antigen-speciﬁc humoral response, but only two out of ﬁve sheep seroconverted. Similarly, while mice vaccinated with a DNA vaccine encoding the M. bovis antigen MPB83 developed antibody and were protected against intravenous challenge [145], the DNA vaccination of cattle induced strong proliferative responses in the absence of a detectable humoral response. The vaccination of mice and calves with a DNA vaccine encoding BHV-1 glycoprotein IV stimulated antigen-speciﬁc antibody responses [146]. However, while a titer of 1/9000 was generated in mice after two 50 mg IM injections, ﬁve 500 mg

212 IM injections were needed to generate a mean titer of just 1/1200 in DNA vaccinated calves. Ineﬃcient induction of immune responses is not limited to veterinary species, as studies evaluating the safety and immunogenicity of DNA vaccines in humans have yielded similar results. Vaccination of humans has indicated that multiple injections of up to 2.5 mg of DNA are well tolerated and capable, in some instances, of inducing CTL and/or humoral responses [15,91,147–150], [reviewed in Ref. 151]. Collectively, these studies indicate that immune responses in humans to DNA vaccination are generally poor and usually show high inter-individual variability. Encouragingly, a recent report has shown that gene gun vaccination was capable of generating seroprotective titers combined with strong cell-mediated responses in 12 out of 12 healthy adult volunteers vaccinated with a DNA vaccine encoding HBV surface antigen [152]. Similarly, biolistic vaccination has been used to induce antibody responses in 12 of 16 human subjects who had previously responded sub-optimally to conventional vaccination [153]. It may be that the unique eﬃciency of biolistic vaccination is a feature that will make this the method desirable for immunization of humans. The reasons underlying the poor immunoresponsiveness in large animals (and humans) to DNA vaccines are currently not known, but it is possibly related to transfection eﬃcacy. It has been shown that the levels of gene expression following transfection correlates with the magnitude of the resultant immune response [154,155]. Thus, a decrease in the transfection eﬃciency would result in a concomitant decrease in the amount of antigen expressed and subsequently presented to APCs, thus limiting the immune response. In fact, reporter gene expression in non-human primates has been shown to be considerably less than that observed in rodents [156]. This reduced eﬃcacy could be in part related to physical barriers such as the higher proportion of connective tissues present in the muscles of larger animals. In addition to weak immune responses an added problem is the high inter-individual variability that is often associated with the vaccination of large out-bred species. Finally, there is the issue of dose. Although it is never practical to deliver a vaccine dose to a large animal that is in proportion to that delivered in model systems such as mice, the discrepancy in relative amounts is rather extreme. For example, a 100 mg dose in a 20 g mouse is equivalent to a 350 mg dose in a 70 kg human, or a 2.5 g dose in a 500 kg cow. These relative amounts are 100–1000 times more than is typically administered in DNA vaccine experiments. Although enhancements in immune responsiveness in large animals (and humans) can be aﬀorded by altering the route, method and interval of delivery of a DNA vaccine [157,158], the overall improvements in immunogenicity have been relatively minor and, in some instances, species-speciﬁc. For example, ID immunization seems to be most eﬀective in cattle [136] and pigs [159] while IM immmunization seems to be more eﬀective in sheep [160]. Diﬀerences in immune responsiveness between delivery methods have similarly been observed

213 in non-human primates where ID and gene gun administration appears more eﬀective than IM immunization [161,162]. Today, the poor immunogenicity of DNA vaccines in large animals/humans remains a major immunological hurdle to be overcome. Although the reasons for this remain to be deﬁned, it is the advantages oﬀered by DNA vaccines over conventional vaccination that are the driving force behind the continual development of this technology. Thus, in recent years there has been a large body of work, largely in mice, concentrating on optimizing transfection eﬃciency and gene expression [reviewed in Ref. 163]. A number of these techniques have been utilized in large animals with varying degrees of success. Heterologous prime/boost vaccines The heterologous prime/boost approach has been successfully used in several animal and disease models to enhance immune responses, where a primary DNA vaccination is boosted by a secondary injection with attenuated vectors or protein in adjuvant [164]. Rothel and colleagues observed that when sheep are primed twice with a DNA plasmid expressing 45W and boosted with 45W protein in Quil A adjuvant, the resultant antibody levels are signiﬁcantly greater than those obtained in sheep that only received the protein/Quil A vaccine [165]. A follow up study looked at the eﬀect of DNA, adenovirus and protein formulations, delivered in various combinations [166]. The most eﬀective combinations were priming with a DNA vaccine or recombinant protein followed by a booster injection with the recombinant adenovirus vaccine. In fact the DNA primed animals mounted IgG1 responses 65 fold higher than sheep receiving either vaccine alone, and were protected against experimental challenge [166]. When cattle were vaccinated with a DNA vaccine encoding HSP65 from Mycobacterium tuberculosis then boosted with recombinant HSP65, both lymphocyte proliferation and IFN-g production were signiﬁcantly increased compared to protein and DNA immunization alone [167]. However, DNA priming prior to protein boosting did not enhance the antigen-speciﬁc IgG production compared to animals vaccinated with recombinant HSP65 protein alone. Loehr et al. [168] immunized cattle via the gene gun into the genital mucosa with a DNA vaccine encoding the bovine-herpes virus 1 glycoproteins B and D. Analysis of immune responses following boost with modiﬁed live virus (MLV) showed that DNA priming signiﬁcantly enhanced T-cell responses, but had no eﬀect on the antibody titres. There were no diﬀerences in the levels of protection aﬀorded by the MLV vaccine alone compared to the DNA prime/MLV boost regime. Recently, a DNA prime/Mycobacterium bovis BCG boost strategy was tested in cattle. Cattle were primed with DNA vaccines coding for the Hsp 65, Hsp 70, or Apa antigens followed by a boost with BCG. Following challenge the prime/boost regime enhanced protection in six of the parameters tested compared to only two parameters in the cattle vaccinated with BCG alone [169].

214 Vaccination of pigs with DNA expressing the gp55/E2 gene from classical swine fever virus (CSFV) followed by boosting with a recombinant porcine adenovirus expressing the gp55 gene protected pigs from challenge. Although all vaccinated animals were protected, DNA primed animals showed fewer clinical symptoms of disease following challenge [170]. Not surprisingly, pigs boosted with an inactivated inﬂuenza virus vaccine 4 weeks after priming with a HA DNA vaccine displayed signiﬁcantly higher levels of virus-speciﬁc serum antibodies and protection than pigs that received two doses of DNA vaccine alone. Interestingly, the observed protection levels in these pigs were equivalent to those seen after 2 vaccinations with inactivated inﬂuenza virus, suggesting that DNA vaccination was just as eﬀective at priming the immune response as the inactivated virus [171]. The acceptability of applying DNA and MVA vaccines in humans has stimulated a signiﬁcant amount of pre-clinical work concerning the eﬃcacy of the DNA prime/MVA boost approaches in non-human primates. This strategy has been particularly eﬀective in inducing cellular immune responses [172–176]. Enhanced immune responses, and in some cases protection, have also been observed in non-human primates by DNA priming followed by boosting with adenovirus [177–179], Sendai virus [180,181] and poxvirus [104,182]. Together, these studies have highlighted the eﬀectiveness of DNA prime/ recombinant virus regimes in generating potent cell mediated responses in preclinical models. A discussed in Section 2, the ﬁrst human evaluation of a heterologous prime/ boost regime has recently been conducted [108]. Human volunteers were primed IM or ID by DNA vaccination followed by an ID boost with recombinant MVA, expressing a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP) fused to a string of B and T cell epitopes. This vaccination regime induced high frequencies of IFN-g secreting, antigen-speciﬁc T-cell responses that were capable of conferring partial protection against a malaria challenge. Preliminary data from DNA/MVA human trials being conducted in England and Kenya for HIV and AIDS has also generated some promising results [183].

Cytokines as DNA vaccine adjuvants Molecular adjuvants such as cytokines, chemokines and co-stimulatory molecules have been shown to eﬀectively modulate immune responses in rodents [184], and some of these molecules are now beginning to be tested in large animals. Mwangi et al. [185] tested the ability of fetal liver tyrosine kinase 3 ligand (Flt3L) and GM-CSF to enhance antigen-speciﬁc immune responses in DNA vaccinated calves. Ftl3L and GM-CSF are important growth factors for dendritic cells (DC) that are known to expand DC numbers upon administration to mice [186] and humans [187,188]. This makes them ideal

215 adjuvants for DNA vaccines, as previous studies have shown that increases in DC recruitment and activation potentiate vaccine immunogenicity in mice [189], rabbits [190] and humans [191]. Calves were injected intradermally on day 1 with DNA constructs encoding Flt3L and GM-CSF, or vector alone, followed 9 days later by a DNA vaccine encoding MSP1a of Anaplasma marginale, the most prevalent tick born pathogen of cattle [192]. Co-vaccination of DNA encoding Flt3L and GM-CSF signiﬁcantly enhanced DC recruitment compared to control calves. The MSP1a speciﬁc CD4 þ T cell proliferation, IFN-g secretion and the population of memory/eﬀector cells in PBMC were also signiﬁcantly enhanced. Whether this immunization strategy can augment protection against challenge remains to be seen. De Rose et al. [193] looked at the ability of cytokine co-administration in the presence/absence of protein boosting to induce protective immune responses in sheep using a DNA vaccine encoding the concealed antigen Bm86 from the tick Boophilus microplus. Intramuscular DNA vaccination in the presence of plasmids encoding the ovine genes for GM-CSF or IL-1b resulted in low-level antibody and cell mediated responses, although co-vaccination with GM-CSF did result in a signiﬁcant reduction in the fertility of ticks. Antibody titers following boosting with Bm86 protein indicated that a priming eﬀect was aﬀorded by DNA priming, however this response was signiﬁcantly less than prime/boosting with protein alone. A similar study investigated whether co-delivery of the cytokines IL-4, IL-5, IL-15, GM-CSF or IFN-g could enhance the immune response to a DNA vaccine coding the EG95 antigen from Echinococcus granulosis, the causative agent of hydatid disease, using a DNA prime/protein boost strategy in sheep [194]. While vaccination with a plasmid expressing EG95 alone and in combination with IL-5, IL-15 or GM-CSF signiﬁcantly primed the humoral response, co-vaccination with IL-4 and IFN-g reduced this priming eﬀect. Only the co-delivery of DNA encoding GM-CSF signiﬁcantly enhanced the antibody response compared to animals that received the EG95 DNA vaccine alone prior to boosting with protein. Similarly, when pigs were immunized with a single injection of a pseudorabies virus (PRV) DNA vaccine along with various combinations of plasmid coding for porcine GM-CSF, IL-2 and IFN-g, only those pigs receiving the GM-CSF expressing plasmid displayed signiﬁcantly enhanced humoral responses combined with improved protection [195]. Co-delivery of IL-2 and IFN-g secreting plasmids had no adjuvant eﬀects. The ineﬀectiveness of IFN-g to enhance immune responses in pigs to PRV DNA vaccines has been observed by others [196]. Interestingly, the co-administration of the conventional adjuvant, dimethyldioctadecylammonium bromide (DDA) with the same PRV DNA vaccine enhanced immune responses and signiﬁcantly enhanced protection against viral challenge [196]. The co-delivery of an IL-2 expressing plasmid with a DNA vaccine, encoding two foot-and-mouth disease virus (FMDV) VP1 epitopes has been eﬀective in augmenting T cell responses and protection in pigs [197].

216 A number of studies have looked at the potential of cytokines to augment antigen-speciﬁc immune responses in non-human primates. Adjuvanting a DNA prime/virus-like particle (VLP) protein boost strategy with plasmids coding for IL-12/GM-CSF was found to enhance the protection of rhesus macaques against challenge with simian immunodeﬁciency virus (SIV) [198]. In contrast, while monkeys immunized with ELI libraries from simian immunodeﬁciency virus (SIV) cDNA showed a trend indicating enhanced protection, the co-administration of plasmids expressing IL-12 or GM-CSF had a negative eﬀect on protection [112]. Similar studies have shown that the co-administration of IL-12 seems to have a positive eﬀect on T-cell proliferative responses and an inhibitory eﬀect on antibody responses in non-human primates models [199,200]. The co-vaccination of a GM-CSF expressing plasmid induced higher speciﬁc antibody responses following the ﬁrst immunization with a DNA vaccine encoding MSP1(42) [97]. However, there were no discernable diﬀerences aﬀorded by GM-CSF co-vaccination, either in antibody titers or T cell responses, following the third dose. The co-administration of IL-2 has been shown to enhance the antibody and T cell mediated responses in non-human primates [200,201]. Further, the co-vaccination of rhesus monkeys with a plasmid that drives the expression of a fusion protein consisting of IL-2 fused to the Fc portion of IgG2, markedly enhanced the humoral and cellular immune response [202], where the level of immunity generated was capable of controlling viremia and preventing clinical disease [203]. Other cytokines that augment immune responses in non-human primate models include IL-4, Il-18 and IFN-g [204,205]. Recently, potent antibody, helper T cell and cytotoxic T lymphocyte responses have been generated in rhesus macaques by priming with a Gag expressing DNA vaccine followed by boosting with Gag protein adsorbed to cationic poly (lactide-co-glycolide) (PLG) microparticles [206]. The biodegradability, eﬀectiveness as a delivery system and a history of safe use in humans makes boosting with PLG microparticles an attractive approach [207]. Use of targeting molecules to improve efficacy of DNA vaccines Given that increasing the numbers of immune cells at the site of antigen expression potentiates vaccine immunogenicity, traﬃcking of antigen directly to immune cells or particular cellular locations via antigen–ligand fusions is a well-reasoned strategy of enhancing vaccine potency [208–210]. A number of these targeting molecules have been used in large animal models. CTLA4 on activated T cells binds to the B7 (CD80/86) molecules present on the surface of antigen presenting cells, such as DCs, and has been eﬀective in enhancing immune responses to both protein [211] and DNA vaccines [208] in mice. Intramuscular vaccination of sheep with the phospholipase D (PLD) antigen from Corynebacterium pseudotuberculosis fused to CTLA4 enhanced the speed, magnitude and longevity of the antibody response and generated the highest

217 levels of protection against challenge compared to animals vaccinated with the non targeted construct [212]. In a similar study, we vaccinated sheep with 500 mg of DNA at weeks 0 and 4 using a plasmid expressing ovine CTLA4 fused to the proCathepsin B antigen from Fasciola hepatica [Kennedy et al., unpublished observations]. Targeting with CTLA4 signiﬁcantly enhanced the speed of the antigen-speciﬁc IgG titre at weeks 2 and 4 following primary DNA vaccination, compared to sheep vaccinated with the non-targeted construct. In fact, similar IgG titres induced in animals given the non-targeted vaccine were not observed until 2 weeks following the secondary immunisation (week 6). Thus, CTLA4 targeting appears to have enhanced the speed of the antigen-speciﬁc IgG response in sheep, but, in this case, has had little eﬀect on the magnitude of the IgG response. In contrast, another study showed that although sheep vaccinated intramuscularly with a DNA vaccine encoding CTLA4 fused to the 45W antigen from T. ovis developed detectable antibodies, they were not signiﬁcantly diﬀerent from those generated by the non-targeted constructs [213]. Recent work in pigs using the model antigen ovalbumin fused to CTLA4 markedly enhanced the speed and magnitude of the immune response following gene gun mediated vaccination [214]. In this case targeting enhanced the IgG, IgA, IgG1 and IgG2 antibody responses. Further, all pigs receiving the targeted vaccine seroconverted with antibody titres >1/100 compared to a 40% seroconversion rate in the non-targeted control group. CD154 is another targeting ligand, present on activated T cells, that binds to the CD40 receptor present on the surface of B cells, DCs, macrophages and Langerhans cells. CD154-CD40 signaling has been shown to stimulate the activation and maturation of DCs [215]. In fact co-administration of mice with separate plasmids encoding the antigen of interest and CD154 enhanced the humoral and cellular immune response [216,217]. To test the adjuvanting ability of CD154 in a large animal model, sheep were vaccinated ID with a plasmid expressing CD154 fused to the truncated secreted form of glycoprotein D (tgD) from BHV-1 [218]. While there was little diﬀerence in the primary antibody response induced by CD154 fusion vaccine, signiﬁcantly higher antigen-speciﬁc and virus-neutralizing antibody titres were seen following the second immunization, compared to those animals that received the non-targeted plasmid. The fusion of porcine IgG to the VP1 capsid protein of foot-and-mouth disease virus (FMDV) induced T cell responses and protection following the gene gun vaccination of pigs [219]. Use of electroporation to improve efficacy of DNA vaccines Electroporation has been shown to increase the transfection eﬃciency of DNA vaccines in both skin [220] and muscle [221] and when used in combination with DNA vaccination can enhance the cellular and humoral immune responses in mice [154,222–224]. Similarly, increases in gene expression aﬀorded by electroporation has been shown to correlate with increased antibody responses in

218 pigs [155]. Babiuk et al. [155] vaccinated pigs IM with plasmids encoding hepatitis B surface antigen (HbsAg) and BHV glycoprotein D (gD) using a single needle and a six-needle electrode. Two weeks after the secondary vaccination a signiﬁcant enhancement of the HBsAg IgG titre was seen in pigs vaccinated with the six-needle electrode, compared to the single needle electrode and the nonelectroporated controls. In fact, the IgG response was 10-fold higher in the animals vaccinated using the six-needle electrode compared to the single needle vaccinates. The number of responders also increased following vaccination with the six-needle electrode. Enhancement of immune response to the more immunogenic BHV gD antigen aﬀorded by electroporation were not as evident. DNA vaccination of goats with the MPB70 and Ag85B mycobacterial antigens, in combination with electroporation, enhanced the humoral response after primary immunization compared to nonelectroporated controls. However, electroporation led to no enhancement of the IgG response following the 2nd and 3rd immunizations, although there was some evidence suggesting that cellular responses were enhanced. While humoral responses were absent, electroporation improved T cell responses in cattle vaccinated with a DNA vaccine expressing hsp65 [225]. Use of CpG motifs to improve efficacy of DNA vaccines As described in the Introduction, it is well known that unmethylated CpG motifs present in bacterial DNA are inducers of innate and adaptive immune response in vertebrates. The recognition of CpG motifs through Toll-like receptor 9 (TLR9) induces a broad range of immunological eﬀects, including potent stimulation of DCs, macrophages, monocytes, B cells and NK cells, that are strongly Th-1 biased [reviewed in Ref. 24]. Adjuvant eﬀects of CpG oligonucleotides (ODN) on antigen-speciﬁc immune responses have been shown in mice, humans and a range of veterinary species [reviewed in Ref. 226]. Although some studies have shown minor enhancements [227–229], the eﬀects of CpG ODN co-delivery on immune responses to DNA vaccination has not been as promising [230,231]. It is thought that competition between the DNA plasmid and the CpG ODNs for entry into the cell results in reduced transfection eﬃcacy and thus antigen expression. In contrast, studies in mice have shown that immune responses can be enhanced when CpG motifs are incorporated into the DNA vaccine backbone [232,233]. Recently, the optimal CpG motif for outbred veterinary species was identiﬁed [234] and incorporated into a DNA vaccine expressing BHV-1 tgD [235]. When DNA vaccines containing 0, 40 or 88 copies of the ruminant speciﬁc CpG motif were vaccinated ID into calves, the antigen-speciﬁc lymphocyte proliferation and IFN-g secretion positively correlated with the CpG content of the plasmids. However, no diﬀerences in the resultant IgG response were observed, although a signiﬁcantly lower IgG1/IgG2 ratio was seen in animals vaccinated with the construct containing 88 CpG motifs. Although no signiﬁcant diﬀerences were observed between the CpG

219 modiﬁed and the unmodiﬁed vectors there was a clear dose dependent augmentation of the cellular immune response aﬀorded by the CpG enhanced plasmids. It was recently observed that the CpG motifs contained within a DNA vaccine expressing the HIV-1 tat gene can enhance immune responses in vaccinated cynomolgus monkeys [236]. While the DNA vaccine containing CpG motifs conferred complete protection, animals vaccinated with the empty plasmid alone were aﬀorded partial protection, suggesting a possible role for CpG mediated stimulation of innate antiviral immunity. The above-mentioned studies have illustrated that the eﬃcacy and potency of DNA vaccines in large animals (and humans) can be augmented via a variety of approaches each of which have their own advantages depending on the required immunological outcome. However, as the ﬁeld of DNA vaccine technology stands now, it is clear that further enhancements are needed before it can reach its full potential in veterinary and/or clinical applications. Concluding remarks DNA vaccination has certainly evolved signiﬁcantly since 1990 and, having been already deployed in clinical trials, may be well on the way to becoming a product. As detailed in this review, DNA vaccination has been tested on many model systems, mainly in rodents, and there is a vigorous research community applying the technique to larger animals and humans. However, at this stage of development, there would seem to be some distance to go before DNA vaccination will become the standard for the prevention and treatment of infectious and neoplastic disease. The main limitation for this is that, generally speaking, DNA vaccines do not induce potent immune responses in large animals, including humans. The immune responses that are induced can be broad, encompassing antibody, CD4 þ and CD8 þ responses, and as demonstrated by the initial observations utilizing immunization with the mouse inﬂuenza hemagglutinin gene, it is the property of DNA vaccines to induce broad responses (particularly in this case CD8 þ ) that may be the most important contributor to protective eﬃcacy. However, irrespective of the quality of the immune response, magnitude matters! Therefore, much eﬀort is being expended to enhance the immune responses to DNA vaccines. Some recent observations indicate possible ways that this may be achieved. The understanding of the mechanism of action of stimulation by CpG motifs is one such example. These motifs are recognized by Tolllike receptor 9 (TLR9), which is abundantly expressed by B cells and dendritic cells, particularly plasmactoid dendritic cells [237,238]. Much of the early work seeking to exploit CpG-containing ODNs was performed in mice-transferring this to target species has been diﬃcult, as (1) stimulatory ODN appear to be species-speciﬁc [239], and (2) the cells of the immune system that are activated are dependent on the sequence context of the ODN [reviewed in Ref. 240]. Nevertheless, the potential of CpG-containing ODN to stimulate appropriate

220 immune cells (particularly B cells and dendritic cells) has obvious advantages for DNA vaccination. The growing realization that dendritic cells play a central role in antigen processing and presentation to naı¨ ve T cells has prompted research into how dendritic cells and DNA vaccines can be best bought together. In an excellent early review on the topic, Coombes and Mahony [241] describe how the increasing knowledge of dendritic cell biology can explain the observed immune responses after DNA vaccination. The ability to target dendritic cells with DNA vaccines would appear to have obvious advantages; as an example You et al. [242] fused the coding sequences for the hepatitis B virus e protein to an IgG Fc fragment. After vaccination, somatic cells secrete the fusion protein and it was targeted to DCs via receptor-mediated endocytosis. Enhancement of CD4 þ , CD8 þ and humoral responses were obtained. This is rather similar in concept to the ﬁndings of Boyle et al. [208], who targeted APCs by fusing the coding region of cytotoxic T-lymphocyte antigen 4 (a ligand of B7) to a model antigen. A mechanism of delivering DNA vaccines that predates detailed knowledge of dendritic cell biology and TLRs is to use attenuated bacteria as carriers. These vaccines are very attractive as they are cost-eﬀective to manufacture and can be delivered orally. Several species have been evaluated, including Salmonella, Shigella and Listeria [reviewed in Ref. 243]. Salmonella species, in particular, have been rigorously evaluated, and it appears that the transfer of plasmid to host cells occurs at very high eﬃciency [reviewed in Ref. 244]. This has obvious beneﬁts in terms of delivering a higher dose of DNA vaccine to APC that occurs using naked plasmid vaccination. Many authors have demonstrated the utility of Salmonella species to transfer plasmid DNA to host cells and induce an immune response and there are prospects that this vaccination strategy may be applicable in humans [245]. In our laboratory we have recently shown that STM1, an attenuated Salmonella enterica serovar Typhimurium vaccine strain [246] can deliver epitopes of the model antigen ovalbumin through the MHC class 1 pathway after infection in vitro of dendritic cell-enriched bone marrow or after oral vaccination of mice [247]. Furthermore, this MHC Class I presentation occurred irrespective of whether the ovalbumin gene was expressed from a prokaryotic promoter, and therefore expressed within STM1, or from a CMV promoter (i.e., a DNA vaccine), which must translocate to the host nucleus for expression transferred to host cells (Fig. 3). Recently, the role of dendritic cells in infection by Salmonella has become clearer [248]. Dendritic cells are the ﬁrst to take up Salmonella after oral vaccination [249] and in fact this may occur in the gut lumen, as dendritic cells were recently shown to penetrate the gut epithelium, sample bacteria and shuttle them across the epithelium [250]. Cheminay et al. [251] have shown that dendritic cells harboring Salmonella up-regulate expression of surface markers such as MHC II and CCR7, migrate to local lymph nodes and present antigen to naı¨ ve T cells. In attenuated bacterial strains carrying plasmid DNA, the maturation of the dendritic cells is induced by the Salmonella itself, rather than

221 Relative Stimulation of T cells

0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 Empty vector

Prokaryotic

Eukaryotic þ

Fig. 3. The in vitro triggering of an ovalbumin- speciﬁc CD8 cell line by mouse bone-marrow derived dendritic cells. Cells were incubated with STM1 carrying a plasmid encoding ovalbumin. Prokaryotic and eukaryotic refers to the promoter driving expression of the ovalbumin gene. Adapted from Bachtiar et al. [247].

the plasmid DNA. This is probably via TLR4, although there is still some uncertainty as to the pathway [252,253]. Of course, within the bacterial carrier there will be many CpG motifs (far more than in a single plasmid DNA molecule), and this DNA probably signals TLR9 once the vaccine is taken into the endosome. TLR9 is unusual amongst TLRs as it is not present on the cell surface, but rather localizes to the endosomal membrane and internalization of the ODN-containing CpG is required for activation [254,255]. The future of DNA vaccination is unclear, with many avenues of inquiry being undertaken. Whether the plasmid is used as a priming vaccine in a primeboost strategy or targeted in some way to appropriate host cells, two things remain clear. Firstly, there is a need to increase the immunogenicity of DNA vaccines, particularly for vaccination of target species, be they veterinary animals or humans. Finally, the utility of DNA vaccination (ease of manufacture, ability to induce CD8 þ responses among a broad immune response, and success at vaccinating against disease in animal models where conventional vaccines are ineﬀective) ensures that research and development aimed at developing commercial, life-saving DNA vaccines will continue. Abbreviations IM ID IP IN SC IV

intramuscular intradermal intraperitoneal intranasal sub-cutaneous intravenous

222 Antigens Leishmania TSA STI1 LACK PSA2 CP meta1 Lm-gp63 papLe22 HSP

thiol-speciﬁc-antioxidant stress-inducible protein 1 Leishmania homolog of receptors for activated C kinase Parasite Surface Antigen 2 cysteine proteinase metacyclic stage-expressed gene 1 L. major cell surface glycoprotein 63 22-kDa potentially aggravating protein of Leishmania Heat Shock Protein

Toxoplasma SAG1 GRA GRA ROP

membrane associated Surface Antigen 1 excreted-secreted dense Granule Protein excreted-secreted dense Granule Protein Rhoptry Protein

Trypanosoma ASP TS TSA1 TSSA KMP11 CRP

Amastigote Surface Protein Trans-Sialidase Trans-Sialidase Antigen 1 Trans-Sialidase Surface Antigen Kinetoplastid Membrane Protein 11 Complement Regulatory Protein

Entamoeba SREHP

Serine Rich Entamoeba histolytica Protein

Cryptosporidium CP15

Cryptosporidium parvum 15 kDa surface sporozoite protein

Acknowledgments A. Rainczuk is a recipient of an Australian Postgraduate Award scholarship and a scholarship from the Cooperative Research Centre for Vaccine Technology. N. Kennedy is a recipient of a scholarship from the Australian Research Council

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Drug-induced and antibody-mediated pure red cell aplasia: A review of literature and current knowledge Ralph Smalling, MaryAnn Foote*, Graham Molineux, Steven J. Swanson, and Steve Elliott Amgen Inc, One Amgen Center Drive, M/S 24-1-C, Thousand Oaks, CA 91320-1799, USA Abstract. Anti-erythropoietin (EPO)-induced pure red cell aplasia (PRCA) is an uncommon, potentially life-threatening condition in which the bone marrow stops manufacturing red blood cells. In the past few years, reports of drug-induced, anti-EPO antibody-mediated PRCA have increased substantially, with most cases attributed to the use of one erythropoiesis-stimulating protein, Eprex. A literature review was undertaken to document the reports of drug-induced PRCA, with all drugs and drug regimens. The sudden increase in reports of antibody-mediated PRCA is discussed. Keywords: adverse drug reaction reporting systems, anemia, erythropoietin, markers, laboratory.

Introduction Drug-induced pure red cell aplasia (PRCA) was such an uncommon occurrence that it was not mentioned in comprehensive reviews of hematologic adverse drug reactions until 1980 [1]. A variety of drugs have been reported to be associated with sporadic cases of generally reversible PRCA, including antimicrobial drugs, antidiabetic drugs, anticonvulsants, immunosuppresants, and recently, recombinant human erythropoietin (rHuEPO). The drugs most commonly associated with PRCA, excluding rHuEPO, are antibacterials, immunosuppressants, and antiretroviral/antivirals (Table 1). rHuEPO was ﬁrst used clinically in 1985, and reports of antibody-mediated PRCA in humans initially were rare [26–29]. Reports of antibody-mediated PRCA associated with rHuEPO increased substantially starting in 1999. These reports have been primarily in patients receiving one brand of rHuEPO therapy (Eprex) for the treatment of chronic renal failure [30,31] and are associated with the induction of antibodies that neutralize both rHuEPO and endogenous EPO, a condition described as antibody-induced PRCA [32,33]. This chapter reviews the role of endogenous EPO in the production of red blood cells, basic information about recombinant erythropoietic proteins, the published reports concerning the problem of PRCA associated with rHuEPO, and current methods for testing for anti-EPO antibodies and highlights some of the concerns about the recent increase in cases of antibody-mediated PRCA. *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10008-2

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

238 Table 1. Summary of most commonly cited drugs from literature reports, excluding erythropoietic-stimulating products. Class of drug

No. of patients

Reference

Immunosuppressant Azathioprine FK506 ATG

4 1 4

25 6 7

Antibacterial Linezolid Isoniazid Rifampicin Chloramphenicol

1 4 1 1

8 9–11 12 13

Antiviral/antiretroviral Interferon-a Lamivudine Zidovudine Fludarabine

1 5 4 2

14 15 16,17 18,19

Anticonvulsant Diphenyldrantoin Carbamazepine Valproic acid

1 1 2

20 21 22,23

Antiparasitic Chloroquine

3

24

Arthritic treatment Gold

1

25

Role of erythropoietin EPO is an endogenous protein produced primarily by the adult human kidneys (i.e., >90%) [34,35], but some EPO is produced by the adult liver [36,37] and adult brain [38]. The amount of EPO is increased when oxygen tension is low because of increased amounts of hypoxia inducible factor (HIF). Increased amounts of HIF result in more EPO protein because of the increased transcription of the EPO gene. When oxygen tension is high, amounts of HIF are low because of oxygen-dependent degradation. At low oxygen concentrations, the amount of HIF increases. In this manner, an oxygen sensor within the kidney detects the oxygen content of the blood that controls the amounts of active HIF protein, thereby regulating the amount of EPO synthesized and released into the blood. EPO stimulates the proliferation and maturation of red blood cells into the peripheral circulation, increasing oxygen transport and resulting in hemostasis, which maintains the appropriate oxygen concentration.

239 Recombinant human erythropoietic agents The cloning of the human EPO gene was a diﬃcult task; interested readers should consult any one of a number of original papers, review articles, or books on the topic [39–46]. The cloning of the EPO gene and its subsequent expression as rHuEPO was an important milestone in the treatment of anemia caused by chronic kidney disease, chemotherapy, or other diseases and disease settings. Several recombinant erythropoietic proteins are commercially available worldwide (Table 2). Eprex, a brand of epoetin alfa, is sold in Europe, Australia, and Canada by Ortho Biotech/Janssen-Cilag and is manufactured by Ortho Biologics LLC. Eprex was ﬁrst marketed in 1988; other brand names for this formulation of epoetin alfa include Erypo, Epopen, and Globuren. Epogen and Procrit are two other brands of epoetin alfa. Both Epogen and Procrit are manufactured by Amgen Inc. and are formulated diﬀerently than Eprex. Epogen is sold by Amgen and Procrit is distributed by Johnson and Johnson/Ortho Biotech Products. Epogen and Procrit were ﬁrst marketed in 1989. Epogen is marketed for the treatment of anemia in patients receiving dialysis, and Procrit is marketed for the treatment of anemia in patients not on dialysis (e.g., anemia associated with cancer chemotherapy or zidovudine therapy for human immunodeﬁciency virus [HIV] infection). Espo is another brand of epoetin alfa, which is manufactured by Kirin Brewery and is sold in Japan and China. It was ﬁrst marketed in 1990. NeoRecormon, a brand of epoetin beta, is marketed by Roche. NeoRecormon was ﬁrst marketed in 1990. Approved indications for NeoRecormon include the treatment of anemia in patients with chronic renal failure, patients with Table 2. Erythropoietic proteins currently available. Brand names (manufacturer)

Availability

Epoetin alfa

EprexÕ (Johnson & Johnson) ErypoÕ (Ortho Biotech/Janssen-Cilag) EpopenÕ (Ortho Biotech/Janssen-Cilag) GloburenÕ (Ortho Biotech/Janssen-Cilag) EpogenÕ (Amgen) EspoÕ (Kirin) ProcritÕ (Amgen, Ortho Biotech)

Canada, EU Australia, Canada Australia, Canada Australia, Canada US Japan, China US

Epoetin beta

NeoRecormonÕ (Roche) RecormonÕ (Roche) RecopenÕ (Roche)

EU EU EU

Darbepoetin alfa

AranespÕ (Amgen)

Australia, Canada, EU, ROW, US

EU ¼ European Union; ROW ¼ rest of world; US ¼ United States.

240 anemia due to chemotherapy, predonated autologous blood (PAD) programs, or for increasing hemoglobin concentrations before elective surgery (Table 3). Darbepoetin alfa, a new generation erythropoietic protein, has a 3-fold longer half-life in the blood than epoetin alfa, which means that it does not need to be injected as often as epoetin alfa. Darbepoetin alfa is marketed as the brand Aranesp and was introduced in 2001. It is manufactured by Amgen and is sold in Europe, the United States, Canada, and Australia. Darbepoetin alfa is a rHuEPO glycosylation analog with ﬁve N-linked carbohydrate chains, two more than rHuEPO. The increased serum half-life results in increased in vivo potency. Darbepoetin alfa activates the same EPO receptor (EPOR) by the same mechanism as rHuEPO, resulting in the same biologic eﬀects [47,48]. Darbepoetin alfa, like the Epogen and Procrit brands of epoetin alfa and epoetin beta, can be administered either as a subcutaneous injection or an intravenous infusion according to approved labelling in all regions and is approved for treating anemia associated with chronic kidney disease and chemotherapy. (Note: regulatory authorities in Europe, Canada and Australia state that Eprex should not be administered by subcutaneous injection.) In addition to the advantage associated with less-frequent administration, patients and their caregivers may beneﬁt from reduced time missed from work for physician visits and better patient compliance. Antibody-mediated pure red cell aplasia PRCA is an erythroid aplasia in which, the bone marrow is otherwise normocellular. The clinical presentation of PRCA includes severe anemia, low reticulocyte count, absence or reduced numbers of erythroid blast cells, normal white cell and platelet counts, and little or no detectable circulating endogenous EPO activity. Antibody-mediated PRCA should be suspected in patients being treated with rHuEPO who have a clinical history of declining hemoglobin concentrations despite increasing dose of rHuEPO. Hyporesponsiveness to rHuEPO therapy (e.g., iron deﬁciency, vitamin B deﬁciency, systemic autoimmune disease, parvovirus infection, hemolysis) and exclusion of other typical causes of PRCA must be completed as part of the diagnosis of antibodymediated PRCA. A bone marrow biopsy is required to conﬁrm a diagnosis of PRCA. Antibody-mediated PRCA is conﬁrmed when neutralizing anti-EPO antibodies are detected in the serum. Early reports of antibody-mediated PRCA were primarily in animals administered rHuEPO. In several studies with dogs, cats, and rats administered rHuEPO, a high frequency of antibody-mediated PRCA was observed; 60–100% of animals in diﬀerent studies developed this condition over a period of 1 to 6 months of rHuEPO administration [49–52]. This result was not surprising given the 20–30% diﬀerence in amino acid sequence and interspecies variation in protein structure between animal EPOs and rHuEPO. Neutralizing antibodies in the animal sera cross-reacted with their endogenous EPO, depleting

241 circulating erythropoietic activity. Animals frequently developed severe anemia and died if not given treatment. Patients administered rHuEPO in the early years after commercial introduction of rHuEPO had an extremely rare incidence of antibody-mediated PRCA [31,53]. Occasionally, rare reports of patients with antibody-mediated PRCA were published for patients who had not been administered rHuEPO, suggesting that that incidence approached background rates [54,55]. The low incidence was attributed to the similarity, if not identity, in amino acid sequence and structure between rHuEPO and endogenous EPO as well as the high quality of manufactured rHuEPO products.

Increased numbers of reported cases of PRCA The number of cases of PRCA increased after 1998, particularly with one brand of epoetin alfa (Eprex) [30,31] (Table 4). Antibody-mediated PRCA was rarely reported for patients treated with other brands of epoetin alfa and epoetin beta (Epogen/Procrit and NeoRecormon, respectively). The incidence with these products was substantially lower and was reported at levels similar to that of earlier historical levels. The fact that the rHuEPO amino acid sequences are identical for these diﬀerent products and that the antibodies in patients with PRCA targeted the peptide and not the carbohydrate component of rHuEPO [30] suggested that other diﬀerences were responsible. Possible factors considered have included changes in clinical practice or change in formulation or manufacturing methods that resulted in a more immunogenic Eprex product. Antibody-mediated PRCA has not been reported for patients who have received darbepoetin alfa exclusively. The few cases of antibody-mediated PRCA reported were in patients treated with other epoetin products before they were administered darbepoetin alfa. In addition, these patients had EPO resistance and clinical indications of PRCA (e.g., declining hemoglobin concentrations) before they started receiving darbepoetin alfa. The fact that darbepoetin alfa’s amino acid sequence diﬀers by ﬁve amino acids from that of rHuEPO indicates further that the immunogenicity associated with PRCA is not due to amino acids or structural diﬀerences of the products per se. The increase in the reports of antibody-mediated PRCA associated with Eprex treatment resulted in the issuance of an urgent safety restriction from the manufacturer of the drug along with a position statement from the Department of Health in the United Kingdom and other countries [56–58]. The cause of the increase in reports of PRCA since 1998 remains obscure, but one manufacturer (Ortho Biotech/Janssen-Cilag) [59] believes this increase may be multifactorial and may be due to the greater use of the subcutaneous route of administration, change in formulation, or improper storage and handling of the protein product. As a result of data suggesting that the increase in PRCA is associated with the

242 Table 3. Comparison of commercially available erythropoietic agents. Not all indications are approved in all countries. The package insert should be consulted for licensed indications, dosages, and precautions. Agent

Indications

Dosage range

Chemotherapy-induced anemia: usual starting Epoetin alfa* Chemotherapy-induced anemia in dose is 150 U/kg SC TIW, and dose may patients with nonmyeloid cancers; be doubled to 300 U/kg SC TIW if anemia in AZT-treated patients with hematocrit does not increase or if HIV; anemia associated with chronic transfusion requirement does not decrease renal failure; anemia in patients in 4–8 weeks; anemia in AZT-treated HIV scheduled for elective, noncardiac, patients: 100 U/kg SC TIW; chronic renal nonvascular surgery to reduce the need for allogeneic blood transfusions failure: 50–100 U/kg SC TIW; surgery: 300 U/day for 10 days before surgery, day of surgery, and for 4 days after surgery Epoetin beta

Chemotherapy-induced anemia: recommended Chemotherapy-induced anemia in starting dose of 450 U/kg/wk, which can be patients with solid tumors; anemia divided into 3–7 single doses; chronic renal of multiple myeloma, low-grade nonfailure: 20 U/kg TIW; anemia of multiple Hodgkin’s lymphoma, or chronic myeloma, low-grade non-Hodgkin’s lymphocytic leukemia; anemia lymphoma, or chronic lymphocytic leukemia: associated with chronic renal failure; Recommended starting dose of 450 U/kg/wk; anemia in patients scheduled for autologous blood predonation: calculated by elective, noncardiac, nonvascular reference to a nomogram and conversion surgery to reduce the need for formula in SmPC; anemia of prematurity: allogeneic blood transfusions; 250 U/kg SC TIW starting day 3 of life and prevention of anemia of prematurity continued for 6 wk

Special considerations Contraindicated in patients with uncontrolled hypertension and/or cardiac disease and in patients with known hypersensitivity to mammalian-derived products or human albumin; use with caution in patients with porphyria. If the hematocrit is >40%, the dose of epoetin alfa should be held until the hematocrit decreases to 35%. The dose then should be reduced by 25% upon restarting therapy Contraindicated in patients with uncontrolled hypertension or with known hypersensitivity to any of the constituents or benzoic acid

Darbepoetin alfa

Chemotherapy-induced anemia in patients with nonmyeloid cancers; anemia associated with chronic renal failure

Contraindicated in patients with Chemotherapy-induced anemia: recommended uncontrolled hypertension. Use with starting dose is 2.25 mg/kg/week. The dose caution in patients with history of should be adjusted to maintain target Hgb hypertension and/or cardiovascular concentration. Dose should be increased to disease. Contraindicated in patients with 4.5 mg/kg/week if Hgb concentration increase is known hypersensitivity to mammalian<1.0 g/dL after 6 weeks. Darbepoetin alfa derived products or human albumin should be administered less frequently than epoetin alfa because of its longer serum half life. Chronic renal failure: recommended starting dose is 0.45 mg/kg/week. The dose should be adjusted to maintain target Hgb concentration. Dose should be increased by 25% if Hgb concentration increase is <1.0 g/dL after 6 weeks

AZT ¼ zidovudine; Hgb ¼ hemoglobin; HIV ¼ human immunodeﬁciency virus; SC ¼ subcutaneously; SmPC ¼ summary of product characteristics; TIW ¼ 3 times per week; U ¼ units; wk ¼ week. *EPOGEN/PROCRIT

243

244 Table 4. Cases of antibody-mediated pure red cell aplasia after treatment with a single rHuEPO brand in patients with chronic renal failure. The data were current as of 31 March 2003 for epoetin alfa (Epogen, Procrit) and darbepoetin alfa (Aranesp); and 31 December 2002 for epoetin alfa (Eprex, Erypro, Epopen, and Globuren) and epoetin beta (NeoRecormon, Recormon). Epoetin alfa: Epoetin alfa: Epoetin beta: Darbepoetin alfa: Eprex Epogen NeoRecormon Aranespb Erypro Procritb Recormonc Epopen Epoxitin Globurena Cases (number) Suspected or conﬁrmed

163 Ab pos 28 Ab neg Ab unk NR Ab-mediated, one product used 142 Ab-mediated, two or more 21 products used

4 Ab pos 6 Ab neg 2 Ab unk 4 0

21 Ab pos Ab neg NR Ab unk NR 8 13

4 Ab pos 1 Ab neg 0 Ab unk 0 4

Ab neg ¼ antibody negative; Ab pos ¼ antibody positive; Ab unk ¼ antibody status unknown, NR ¼ not reported. a Johnson & Johnson website posting of 24 March 2003; http://www.jnj.com_news/1021024_095632.htm; accessed 24 March 2003. b Amgen data on ﬁle. c Swissmedic Medical Update letter, 25 March 2003; http://www.swissmedic.ch/cgi/news; accessed 24 March 2003.

use of subcutaneous administration, one of the manufacturers of epoetin alfa (Ortho Biotech/Janssen-Cilag) recommended that their brand (Eprex) be administered only intravenously to patients with chronic renal failure [57,58]; however, an analysis published by Macdougall [60] suggests that route of administration may not be the sole reason for the increase.

Testing for anti-EPO antibodies Therapeutic proteins have a theoretical risk of producing neutralizing antibodies [61,62], and appropriate testing methods should be in place to detect their presence. Immunoassays to directly detect anti-protein-based drug antibodies that are validated and well characterized can be used to monitor the development of antibodies when it occurs in patients. Typically, radioimmunoprecipitation (RIP) or enzyme-linked immunosorbent assays (ELISA) are used; however, these methods may give false-negative results because of failure to detect either all classes of antibodies or low-aﬃnity antibodies. The BIACORE 3000 (BIACORE Inc., Piscataway, NJ), one analytic approach for monitoring antibodies, has several advantages over other assays [63,64], since it detects all

245 antibody isotypes and subclasses in real time without the need for secondary signaling molecules. The real-time detection provides optimal detection of rapidly dissociating antibodies. Discussion Non-erythropoietic-stimulating drugs A review of the literature suggests that PRCA due to use of non-erythropoieticstimulating drugs is, indeed, rare (Appendix). It should be noted that of all the reports, only one [11] seemed to ﬁrmly establish causality between drug and PRCA. In this report, the patient was rechallenged with drug and the PRCA returned with the rechallange. Most other reports merely describe patients who were diagnosed with PRCA but recovered over a variable period of time when certain drugs were discontinued. In addition, other aspects or conditions occurred in the same time period after discontinuation of the suspect drug, such as resolution of viral infection, improvement in underlying medical condition, changes in drug metabolism, or recovery of the bone marrow from other causes of insult. None of the reports appeared to have established an immunologic etiology for the diagnosis of PRCA. Confounding interpretation of these cases was the fact that approximately 50% of them occurred in patients who were immunocompromised (i.e., patients with AIDS, leukemia, or autoimmune or rheumatic diseases), or patients who were malnourished, elderly, or the recipient of a transplant. Many of these conditions are themselves associated with PRCA in the absence of drug; immunosupression might predispose these patients to an infectious etiology for PRCA. In all but one case report, the PRCA was reversible. The one case that was not, an elderly patient using chloramphenicol eye drops, may be questionably drug related. In general, the reports are so few that commonalities could not be found among the cases. Pure red cell aplasia was reported at all ages, young children to elderly, and in both sexes. The duration of drug treatment before the onset of PRCA varied greatly, from a few weeks to several years; the time to resolution was equally broad. Diagnostic tests were not uniformly reported. The results of this literature search suggest that cases of PRCA are few and among those reported, association to drug may be tenuous, and that drug-induced PRCA is both a rare event and rarely reported in the literature. Erythropoietic-stimulating proteins In the case of PRCA associated with the use of recombinant human erythropoietic products, however, literature review and company websites suggest a substantial increase in the number of cases reported since 1999. This increase has been associated primarily with the use of Eprex [65]. Consistent with this

246 association is the fact that the number of new cases reported has decreased as patients are converted from Eprex to other erythropoietic proteins, such as epoetin beta or darbepoetin alfa [59,66]. The increase cannot be declared a class eﬀect of erythropoietic proteins because the incidence of antibody-mediated PRCA between Eprex and other erythropoietic proteins are not comparable. For example, as of 30 September 2003, more than 350,000 patients with more than a cumulative 200,000 patient-years of experience have been exposed to darbepoetin alfa (Aranesp), with no reported cases of antibody-mediated PRCA caused by the product [66]. The data suggest that the cause of the increase is multifactorial. The change in product formulation (i.e., removal of human serum albumin and addition of polysorbate) is temporally associated with the increase in case reports. Additionally, the route of administration and patient handling of the product may also contribute. One theory is that the change in formulation resulted in a somewhat less stable product. Improper handling may cause the formation of altered proteins (e.g., aggregate formation), which may be immunogenic. The immunogenicity may be enhanced by subcutaneous administration; however, route of administration alone cannot explain the increase in the number of cases of antibody-mediated PRCA reported with the use of Eprex. Changing the route of administration may reduce the number of cases, but it does not resolve the underlying cause. Regulatory authorities have limited the subcutaneous administration of Eprex in patients with chronic renal failure. European authorities have contraindicated the subcutaneous route of administration [56,57]; Australian authorities state that Eprex should be administered only by the intravenous route [67]; and Canadian authorities have recommended that patients undergoing hemodialysis should use the intravenous route of administration for Eprex when feasible [58]. No similar regulatory actions have been taken for other erythropoietic proteins. Patients who have been diagnosed with antibody-mediated PRCA should not be converted to other brands of erythropoietic proteins. Casadevall et al. [30] reported that neutralizing antibodies cross-react with other erythropoietic proteins, and patients with antibody-mediated PRCA do not beneﬁt from treatment with a diﬀerent erythropoietic protein. It is also imperative that a uniform standard for the testing of antibodies to erythropoeitic products be adopted. The BIACORE assay currently is the best assay for such testing.

Note Added in Proof It is comforting, however, that data available since the writing of this paper suggest that the overall incidence in cases of PRCA attributable to Eprex has substantially declined, as per the sponsor’s website

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249 49. Weiss TL, Kavinsky CJ and Goldwasser E. Characterization of a monoclonal antibody to human erythropoietin. Proc Natl Acad Sci USA 1982;79:5465–5469. 50. Kato M, Miura K, Kamiyama H, et al. Immunological response to repeated administration of recombinant human erythropoietin in rats: biphasic eﬀect on its pharmacokinetics. Drug Metab Dispos 1997;25:1039–1044. 51. Cowgill LD, James KM, Levy JK, et al. Use of recombinant human erythropoietin for management of anemia in dogs and cats with renal failure. J Am Vet Assoc 1998;212: 521–528. 52. Randolph JF, Stokol T, Scarlett JM and MacLeod JN. Comparison of biological activity and safety of recombinant human erythropoietin in clinically normal dogs. Am J Vet Res 1999;60: 636–642. 53. Indiveri F and Murdaca G. Immunogenicity of erythropoietin and other growth factors. Rev Clin Exp Hematol 2002;1:7–11. 54. Peschle C, Marmont AM, Marone G, Genovese A, Sasso GF and Condorelli M. Pure red cell aplasia: studies on an IgG serum inhibitor neutralizing erythropoietin. Br J Haematol 1975;30: 411–417. 55. Casadevall N, Dupuy E, Molho-Sabatier P, Tobelem G, Varet B and Mayeux P. Autoantibodies against erythropoietin in a patient with pure red-cell aplasia. N Engl J Med 1996;334:630–633. 56. UK Medicines Control Agency. Eprex (epoetin alfa) and pure red cell aplasia – contraindication of subcutaneous administration to patients with chronic renal disease. http://www.mca.gov.uk (accessed 18 August 2003). 57. Agence Francaise de Securite´ Sanitaire des Produits de Sante´ Website Letter from JanssenCilag to French Healthcare Professionals of July 19th 2002. http://agmed.sante.gouv.fr/htm/ 10/10000.htm (accessed 8 August 2002). 58. Health Canada. Important Drug Safety Update. http://www.hc-sc.gc.ca/hpfb-dgpsa/tpd-dpt/ eprex_e.html (accessed 6 August 2002). 59. Johnson & Johnson Website posting of July 14th 2003. http://www.jnj.com/news/jnj_news/ 1021024_095632.htm (accessed 15 July 2003). 60. Macdougall IC. Pure red cell aplasia with anti-erythropoietin antibodies occurs more commonly with one formulation of epoetin alfa than another. Curr Med Res Opin (published on line 28 October 2003). 61. Porter S. Human immune response to recombinant human proteins. J Pharm Sci 2001;90: 1–11. 62. Koren E. From characterization of antibodies to prediction of immunogenicity. Dev Biol 2002;109:87–95. 63. Mason S, La S, Mytych D, Swanson SJ and Ferbas J. Validation of the BIACORE 3000 platform for detection of antibodies against erythropoietic agents in human serum samples. Curr Med Res Opinion 2003;19:651–659. 64. Swanson SJ, Ferbas J, Mayeux P and Casadevall N. Evaluation of methods to detect and characterize antibodies against recombinant human erythropoietin. Nephron Clin Pract 2004; 96:88–95. 65. Locatelli F, Aljama P, Barany P, et al. Erythropoiesis stimulating agents and antibodymediated pure red cell aplasia: where are we now and where do we go from here? Nephrol Dial Transplant 2004;19:288–293. 66. Amgen Inc. Information for Healthcare Providers. http://wwwext.amgen.com/clinicians/ prca.html (accessed 24 November 2003). 67. ADRAC. Epoetin alfa and pure red cell aplasia. Available at: http://www.health.gov.au/tga/ adr/aadrb/aadr0208.htm#4 (accessed 24 November 2003).

Appendix: Literature Search Methods Two literature searches were done. On 26 September 2002, a preliminary search was done on Medline (1 January 1996 to 26 September 2002) and on Embase (1 January 1974 to 26 September 2002). The search terms used were red cell aplasia, pure or pure red cell aplasia. Two hundred ﬁfty-seven unique citations were retrieved. Only full articles of primary research on drug-induced pure red cell aplasia were used. Six references were read and summarized. A second search was done on 27 September 2002. This search was done on Medline (1 January 1966 to 27 September 2002) and Embase (1 January 1988 to 27 September 2002). This search was for articles concerning iatrogenic, druginduced, or immunogenic pure red cell aplasia. Medline and Embase were searched using database-speciﬁc indexing for pure red cell aplasia. The pure red cell aplasia concept was combined with available subheadings suggestive of exogenous etiology, and with other indexing addressing iatrogenic or immunogenic origin of disease. Results were limited to human, English language, 1988 or newer. The earlier strategy was rerun using ‘‘NOT’’ to exclude previously retrieved references. The resulting set consists of 230 references. Twenty-six references were read and summarized. Between the 2 searches, 32 papers were found to be relevant.

251

Using the biologic license application or new drug application as a basis for the common technical document MaryAnn Foote* Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA Abstract. With the introduction of the common technical document (CTD), many writers in the biotech and pharmaceutical industries are now required to submit dossiers in this format. The format of the CTD is not extremely diﬃcult from the familiar documents of the Biologic License Application (BLA) or New Drug Application (NDA). The CTD can be mapped to existing areas of the BLA or NDA. The components of the CTD are discussed and references to the current guidance worldwide are provided to assist the writer. Keywords: guidance documents, International Conference on Harmonisation, marketing applications, regulatory interactions.

Introduction The Common Technical Document (CTD) is a format for the preparation of a well-structured presentation for marketing applications to be submitted to regulatory authorities in the three International Conference on Harmonisation (ICH) regions of Europe, the United States, and Japan. Submission in the CTD format is encouraged in Australia, New Zealand, Canada, Switzerland, and other countries. The intention of the CTD is to save time and resources on behalf of the drug sponsor and to facilitate review and communication on behalf of regulatory agencies. The CTD does not address all aspects of the content of the ﬁle. Many regional requirements aﬀect the contents of the dossiers submitted in each region. Thus, the CTD may not be identical for all regions of the world. Documentation for the CTD does not provide any information about the content of a dossier, the studies, or the data required for a successful approval. The CTD was designed to be applicable to all categories of medicinal products, including generics, herbals, radiopharmaceuticals, and vaccines. At the time of this writing (May 2003), the CTD format is ‘‘encouraged’’ (but not mandated) by the United States Food and Drug Administration (US FDA). United States law must be changed before the CTD can be mandated for submissions, and some believe this step is unlikely to occur. The European Agency for the Evaluation of Medicinal Products (EU EMEA), the Japanese Ministry of Health, Labour and Welfare (MHLW), and other regulatory agencies, such as Canada’s Health Products and Food Branch (HPFB) and Australia’s Therapeutic Goods Administration (TGA) are implementing the *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10009-4

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252 CTD. Many of these agencies are publishing CTD-related information on their websites to help sponsors understand speciﬁc regulatory authority expectations of the CTD, and they are also working to train their application reviewers about the CTD. Several agencies, including EMEA, US FDA, and Health Canada, have speciﬁc staﬀ whose job it is to facilitate the transition to the CTD and who can be approached by sponsors with questions. The CTD replaces documents in three of the previous marketing application formats: the US FDA application summary, Integrated Summary of Eﬃcacy (ISE), and Integrated Summary of Safety (ISS); the three EU EMEA expert reports, and the Japanese Gaiyo. Although these documents are no longer required by name, the information contained in them is largely still part of marketing applications in the CTD format, as will be discussed in this paper. We oﬀer suggestions on writing the CTD, particularly in mapping the current Biologic License Application (BLA) and New Drug Application (NDA) formats to the CTD, and discuss some current controversies and interactions with regional health authorities. The appearance of the CTD will diﬀer among drug sponsors and regions, but I hope that the reader ﬁnds the paper useful. Implementation dates The CTD was acceptable as of July 2001 by regulatory authorities in the EU EMEA, the US FDA, Japan, Canada, and Switzerland. In July 2003, the CTD became mandatory in the European Union, Japan, and Switzerland, and highly recommended for applications to the US FDA and Canadian HPFB. In July 2004, the CTD will be required in Australia. The CTD is not mandated for US FDA ﬁlings because to do so, laws would need to be changed by an Act of Congress. Nonetheless, FDA is actively encouraging sponsors to submit CTDs and will work with them to ensure a smooth transition from the old BLA/NDA to the new CTD format. Guidance The CTD guidance, or ICH topic M4, was issued as a guidance document (i.e., it reached step 4, or became ﬁnal) in November 2000. Once an ICH guidance reaches step 4, the individual ICH regions (European Union, United States, and Japan) endorse the guidance and it is published by the three signatory regulatory agencies. US FDA draft ICH guidance is available on the FDA website; draft and ﬁnal guidances are published in the Federal Register. In the EU, ICH guidance is published by EMEA and endorsed by the Committee for Proprietary Medicinal Products (CPMP), the committee within EMEA that reviews applications for marketing approval and then makes recommendations to EMEA as to whether approval should be granted. In Japan, notiﬁcations on

253 Table 1. Guidance documents for writing the Common Technical Document. All sites accessed 26 May 2003. Agency/Information document

Web address

Australian TGA CTD information EMEA notice to applicants, Vol 2B (general information about ﬁling a CTD in Europe) EMEA safety advice EMEA eﬃcacy advice Japan MHLW guidance Switzerland Swissmedic

http://www.health.gov.au/tga/index.htm

US US US US

FDA FDA quality advice FDA safety advice FDA eﬃcacy advice

http://pharmacos.eudra.org/F2/eudralex/vol-2/home.htm

http://www.emea.eu.int/pdfs/human/ich/554902en.pdf http://www.emea.eu.int/pdfs/human/ich/555102en.pdf http://www.nihs.go.jp/dig/ich/m4index-e.html http://www.swissmedic.ch (type CTD in the ‘‘search with keywords’’ window) need a web address here http://www.fda.gov/cder/guidance/1215dft.doc http://www.fda.gov/cder/guidance/5495fnl.doc http://www.fda.gov/cder/guidance/5433fnl.doc

guidelines are available from the Pharmaceutical and Medial Safety Bureau of the MHLW. General information about submitting a CTD in Europe can be found at the EMEA website. Information about EMEA submissions is also available in guidance documents and in question and answer format for safety and eﬃcacy sections. Table 1 provides the web addresses for these documents. Most questions and issues are handled during the presubmission meetings between applicants (sponsors) and health authorities or during the validation of the application. Applicants are urged to discuss the format of their applications with the EMEA assessor before submission. For US FDA applications, the Agency has issued guidance for the industry concerning general considerations (Table 1). In this guidance, the Agency explains the speciﬁcs of voluntary submissions of applications in the CTD format. In Japan, the MHLW has general information on its website (Table 1).

Components of the CTD The CTD has a structure similar to the EU Marketing Authorisation Application (MAA) and the US BLA/NDA in that it has a hierarchical structure that includes written summaries, tabular formats, and individual study data. The CTD includes high-level summary documents that provide critical assessment of data from each technical discipline. Like the BLA/NDA, MAA, and Japanese NDA (JNDA), the CTD can be considered to be an overview document that is accompanied by longer summaries of data across studies. The CTD is typically illustrated as a pyramid (Fig. 1). The parts of the CTD are given in Table 2 and details about the contents of each module are given in Table 3.

254

Module 1

Not part of

CTD Regional Administrative information

Nonclinical Overview

Clinical Overview

Quality Overall Summary

Module 2

Nonclinical Summary

Module 3 Quality

Clinical Summary

CTD

Module 4

Module 5

Nonclinical Study Reports

Clinical Study Reports

Fig. 1. The parts of the common technical document.

Table 2. Components of the common technical document. Module 1 Regional administrative information and product labeling; is not part of the Common Technical Document (CTD) Module 2 Introduction Nonclinical overview, clinical overview, quality overview Nonclinical summary, clinical summary

Module 3 Quality summary

Module 4 Nonclinical study reports

Module 5 Clinical study reports

Module 2 contains a brief clinical overview (approximately 30 pages and very similar to the old MAA Clinical Expert Report) and a more detailed clinical summary (50 to 400 pages, excluding tables) and very similar to the former ISE/ ISS. The US FDA now publicly states that the name ‘‘summary’’ in the name of

255 Table 3. Details of composition of components of the common technical document. Module 1: Regional information for US ﬁling is illustrated Cover letter Table of contents Financial disclosure Prescribing information Labeling Annotated package insert User fee sheet Form 356h Patent information (not needed for Biologics Licensing Application) Debarment certiﬁcation Field copy certiﬁcation Module 2: Replaces documents currently in US FDA application summary, EU EMEA expert report, and Japanese Gaiyo Part 2.1 – overall CTD table of contents Part 2.2 – introduction Part 2.3 – quality overall summary Part 2.4 – nonclinical overview Part 2.5 – clinical overview Part 2.6 – nonclinical written and tabulated summaries Part 2.7 – clinical summaries Module 3: Technical data to support the Chemistry, Manufacturing, and Control [CMC] section in the US ﬁling and the quality section in EU/Japan/Other ﬁlings Module 4: Nonclinical study reports Module 5 Individual clinical study reports Region-speciﬁc reports s case report forms s case report tabulations s meta-analyses

the ISE/ISS was regrettable, because these sections should be an analysis rather than a summary. (If the integrated analysis of eﬃcacy and safety are longer than 400 pages, these analyses should be placed in Module 5.) Although the clinical summary has all of the sections of an ISS, it is not a complete ISS. The reports of individual studies in the clinical summary section should contain the essential safety conclusions and analyses.

Comparing the CTD with NDA/BLA Like the CTD, the NDA and BLA formats are always pictured as pyramids, to illustrate the progression of data presentation (Fig. 2). The components of an NDA or BLA are given in Table 4. Much of what is in an NDA/BLA can be mapped to a section in the CTD (Table 5).

256 NDA/BLA Pyramid

Overall NDA Summary Integrated Clinical Reports (ISS/ISE) Biostatistical Report Raw Data (SAS data sheets, case report tabulations) Case Report Forms Fig. 2. New Drug Application/Biologic License Application pyramid.

Table 4. Content and format of New Drug Applications and Biologic License Applications. Form FDA 356h Item 1 Comprehensive index Item 2 Labeling

Item 3 50- to 200-page summary, an overview

Item 4 CMC/Quality

Item 5 Nonclinical pharmacology/toxicology data

Item 6 Human pharmacokinetics and bioavailability studies

Item 7 Clinical microbiology (only needed if microbial agent) – note that there currently is no place speciﬁed in a CTD for this information, although it is still needed. Until the CTD guidance can be amended, FDA advises putting this information ‘‘in the most logical place in the CTD, given the indication and product’’ and discussing the placement at the presubmission meeting for concurrence by FDA.

Item 8 Clinical data

Item 9 Safety update (submitted after ﬁle is accepted for review)

(Continued)

257 Table 4. (Continued) Item 10 Statistical section Item 11 Case report tabulations

Item 12 Case report forms, including those for patients who died or withdrew because of adverse events; other case report forms as required

Item 13 Patent information

Item 14 Patent certiﬁcations

Item 15 Establishment description

Item 16 Debarment certiﬁcation

Item 17 Field copy certiﬁcation

Item 18 User fee cover sheet

Item 19 Financial information

Item 20 Other information

Table 5. Mapping sections of New Drug Application and Biologic License Application to the Common Technical Document. Module 1 of the CTD

NDA/BLA

Regional administrative information Prescribing information and labeling In the US, contains: Cover letter Table of contents Financial disclosure Prescribing information Labeling Annotated package insert User fee sheet etc.

Module 2 of the CTD Summaries Table of contents (overall for the CTD) Introduction Quality overall summary

NDA/BLA Table of contents Item 3 – summary (overview) Item 5 – nonclinical pharmacology/toxicology

(Continued)

Form 356h Item 1 – Comprehensive index Item 2 – Labeling Item 18 – User fee cover sheet Item 19 – Financial disclosure Item 20 – Other

258 Table 5. (Continued) Nonclinical overview Clinical overview Nonclinical summaries (written and tabulated) Clinical summaries

Item 6 – clinical pharmacokinetics Item 11 – tabular data ISS/ISE

Module 3 of the CTD Quality summary

NDA/BLA Item 4 – CMC/Quality Item 14 – Establishment description, if applicable Literature and references

Module 4 of the CTD Nonclinical study reports Pharmacology, pharmacokinetics, and toxicology

NDA/BLA Item 5 – Nonclinical pharmacology/ toxicology study reports

Module 5 of the CTD Clinical study reports

NDA/BLA Item 8 – Clinical study reports Item 10 – Statistical section Item 11 – Tabular data Item 12 – Case report forms

Discussion When ﬁrst faced with the prospect of submitting a drug application in the form of a CTD, many medical writers and their employers become anxious. While some of the physical layout diﬀers between the BLA/NDA format and the CTD format, closer examination shows that information contained within each format is quite similar, as would be expected. Perhaps the most noticeable diﬀerence between the BLA/NDA and CTD formats is the inclusion of high-level summaries. The format we have included is only a suggestion of how the CTD might be arranged. When I have presented at national and international meetings, I invariably learn of other ways to arrange the CTD. The drug sponsor is encouraged to work with the health authority to whom it is submitting should questions arise about placement of certain information.

259

Guidelines and policies for medical writers in the biotech industry: An update on the controversy MaryAnn Foote Amgen Inc., Thousand Oaks, California, USA Abstract. Papers reporting the results of clinical trials written by medical writers employed by the biotech and pharmaceutical industries have been criticized for possible bias in presentation and failure to adhere to authorship guidelines. Several groups have attempted to address the concerns of journal editors, academics, regulators, and the general public by issuing guidelines and policies for the preparation of such material. Keywords: biotechnology–medical biotechnology, clinical research–publication of trials, good publication practices, PhRMA guidelines, uniform requirements for biomedical journals.

The scientiﬁc method consists of several steps: observation, questioning, formulation of an hypothesis, testing and possibly retesting of the hypothesis, and publication or communication of the results. ‘‘Science’’ is not complete until the results of an experiment/trial, the hypothesis testing whether successful or not, are published or otherwise presented to other scientists. The use of medical writers employed by biotech and pharmaceutical companies (herein referred to as ‘‘drug sponsors’’) and the ‘‘ownership’’ of data from clinical trials has been controversial for more than a decade, and several groups have attempted to address various aspects of these issues [1]. This paper reviews some history of the controversy, from a writer’s point of view, and summarizes guidelines and changes in guidelines, since the inital report [1], that have been oﬀered to standardize reporting of clinical trial results, acknowledgment of assistance from medical writers, authorship, and other issues that may concern the medical writer. Table 1 summarizes the basic points of each of the guidelines. International Council of Medical Journal Editors (ICMJE) The International Council of Medical Journal Editors (ICMJE) is best known for formulating the Uniform Requirements for Manuscripts Submitted to Journals [2], often called the ‘‘Vancouver Conventions.’’ Most medical journals adhere to the tenets of the Uniform Requirements and most writers will cite the Requirements if asked about authorship. The Uniform Requirements state: ‘‘Each author should have participated suﬃciently in the work to take public responsibility for the content. Authorship credit should be based only on substantial contributions to (a) conception and design, or acquisition of data, or Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10010-0

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260 Table 1. Summary of guidelines for medical writers, with emphasis on manuscripts and educational materials. Organization

Main points

Reference

ICMJE

Authors need to be actively involved in the concept, writing, and reviewing of manuscripts; 2001 editorial critical of role of drug sponsor in preparing and publishing clinical trial data No discussion of role of medical writers Position statement encourages acknowledgement of services of writer Encourages publication of clinical trial results Supports Uniform Requirements in determining authorship; commits to timely communication of clinical trial results that are of medical or scientiﬁc importance in one primary paper; supports data review by investigators of multisite studies Supports use of professional writers to facilitate timely publication; guidelines are compatible with Uniform Requirements and PhRMA guidelines; encourages use of unique identiﬁers for all studies

N Engl J Med 1997;336:309–315; Ann Intern Med 2001;135:463–466

FDA AMWA ICH PhRMA

GPP

Federal Register 1997;62:64073–64100 http://www.amwa.org/ positionstatement.html http://www.ifpma.org/pdﬁfpma/e6.pdf http://www.phrma.org

http://www.gpp-guidelines.org/

AMWA ¼ American Medical Writers’ Association; FDA ¼ Food and Drug Administration; GPP ¼ Good Publication Practice for Pharmaceutical Companies; ICH ¼ International Conference on Harmonisation; ICMJE ¼ International Council of Medical Journal Editors; PhRMA ¼ Pharmaceutical Research and Manufacturers of America.

analysis and interpretation of data; and to (b) drafting the article or revising it critically for important intellectual content; and on (c) ﬁnal approval of the version to be published. Conditions (a), (b), and (c) must all be met.’’ Before the release of the Uniform Requirements, it was a routine practice to add the name of the laboratory head or section head to all papers, a practice known as ‘‘guest’’ authorship. The Uniform Requirements further states that people making contributions but not meeting the criteria for authorship, such as medical writers, should be acknowledged. It should be noted that the tradition of guest authorship has not disappeared with the Uniform Requirements. The issue of guest authorship remains controversial and surveys, done primarily in academic institutions, suggests that while guest authorship is not unique to industry, it is far more common in the academic enviroment [3,4]. In September 2001, however, 11 journals, all of whose editors belong to ICMJE, simultaneously published a paper [5] calling for more involvement of physicians and academic centers in the research required for gaining marketing

261 approval for drugs. The authors of this paper were concerned with knowing who is an author on papers submitted to their journals, and want to be sure that authors did the work (i.e., were not guest authors or the beneﬁciaries of ghost authorship). The journal editors also stated that they wanted investigators to have full and unfettered access to the data and to control the decision to publish. This issue has lead to confusion in the publication of clinical trial data. While each site ‘‘owns’’ its own patient data, the consolidated electronic database for the clinical trial is generally assumed to be the property of the drug sponsor [6,7]. Some writers report confusion and misunderstanding of data ‘‘ownership’’ to be quite a problem in writing the manuscripts. The Vancouver Conventions were revised in 2001 to include the category of data acquisition. American Medical Writers Association (AMWA) In the autumn of 1991, the United States Food and Drug Administration (FDA) drafted guidelines entitled, ‘‘Regulation of Drug-Company-Sponsored Activities in Scientiﬁc or Educational Contexts’’ [8]. These draft guidelines were oﬀered for public comment, and proposed severe restrictions of the roles of medical writers employed by drug sponsors. The guidelines proposed that ‘‘. . .articles about a company’s drug or directly competing drugs should not be written by medical writers employed by the ﬁrm, including freelance writers hired by the ﬁrm for speciﬁc projects. In addition, medical writers employed by the ﬁrm should not ghostwrite, edit or otherwise inﬂuence the content of articles, purporting to be independent . . . .’’ The American Medical Writers Association (AMWA), in conjunction with other groups, including Pharmaceutical Research and Manufacturers of America (PhRMA), met with the FDA to explain the role of medical writers, with much success. In 1997, the FDA issued the ‘‘Guidance for Industry: Industry-Supported Scientiﬁc and Educational Activities’’; medical writers were not discussed in the ﬁnal guidance [9]. It must be noted, however, that writers must follow all guidelines issued by the Agency in terms of use of materials, such as manuscripts, in promotional activities; use is regulated, but not the actual act of writing. Although the ﬁnal guidance omitted any reference to medical writers, the controversy over the use of medical writers continued to be an issue for AMWA members. Over the past decade, numerous articles have been published concerning the use of ‘‘ghostwriters’’ [e.g., 10–16]. In most reports, ghostwriters and ghostwriting are perceived negatively. In 2003, AMWA published a position statement about the use of medical writers (AMWA eschews the name ‘‘ghostwriters’’) [17]. The statement aﬃrms that AMWA ‘‘recognizes the valuable contributions of biomedical communications to the publication team’’ and states that they ‘‘should adhere to AMWA’s Code of Ethics.’’ This code stresses the need for writers to ‘‘recognize and observe statutes and regulations,’’ ‘‘apply objectivity, scientiﬁc accuracy and rigor, and fair balance,’’ and ensure that their work meets ‘‘the highest

262 professional standards’’ [18]. The position statement states that writers ‘‘who contribute substantially to the writing or editing of a manuscript should be acknowledged with their permission and with disclosure of any pertinent professional or ﬁnancial relationships.’’ International Conference on Harmonisation (ICH) Publication of clinical trial results is not solely the concern of drug sponsors and journal editors. The International Conference on Harmonisation (ICH) has addressed the issue, also, but from the science as communication and patients as volunteers points of view [19]. The ICH guidelines state that a publication policy should be included in the study protocol or as a separate agreement to ensure that clinical trial results are disseminated. Such a policy requires that clinical trial results, whether positive or negative, become part of the public domain and are available for other researchers to use in designing their clinical trials. Many journal editors strongly believe that people who have volunteered to be research subjects should have their clinical trial data published to fortify the attitude that human trials are worthwhile and valuable. Pharmaceutical Research and Manufacturers of America In 2002, PhRMA issued guidelines on the conduct of clinical trials and publication of results [20]. In these guidelines, PhRMA members commit to the timely publication (or communication) of clinical trials results, but do not commit to publishing results of all trials (i.e., exploratory studies) unless medically important, and state that some communications may be delayed to protect intellectual property; this delay is generally a matter of months, at most. Data from all sites of a multicenter clinical trial are to be published ﬁrst as one paper, and data from individual study sites (that are thought to be of severely limited scientiﬁc validity) are to be published only after multicenter composite data are published. Drug sponsors retain the right to review all publications in a timely manner and to work with the investigator-authors if opinions or interpretation of data diﬀer. Drug sponsors cannot, however, refuse to allow the data to be published at some reasonable point in time. Good Publication Practice for Pharmaceutical Companies (GPP) A group of writers and editors attempted to codify how results from clinical trials are published. This group formulated the guidelines on Good Publication Practice for Pharmaceutical Companies, commonly called GPP [21]. The GPP attempt to minimize publication bias and to strengthen the relationship between drug sponsors and academic investigators. The GPP endorse the use of unique trial identiﬁers in all publications to make clear the number of patients actually treated with a given drug in a given trial (multiple publications of the same

263 clinical data may give the appearance that more patients have been exposed to a compound than actually have). The GPP endorses the use, and acknowledgment of, professional writers as a means of generating timely publications. The GPP were designed to be used in conjunction with other guidelines, such as the Uniform Requirements, and complement the PhRMA guidelines. The GPP recommends complete transparency in terms of a writer’s involvement in the drafting or editing of manuscripts.

Discussion The use of medical writers in developing papers based on clinical trial results, the ownership of the clinical trial data, the timing of the publications and the authorship of them, and the reuse of clinical trial data are all controversial subjects that have been addressed by one or more professional organizations. Very often the medical writer is asked to determine who should be an author or how authors will interact with the drug sponsor, and in such a position, the writer has the opportunity to encourage adherence to the Uniform Requirements and other guidelines. Actually, writers employed by the drug sponsor, in addition to being trained communicators, may be very familiar with the design and results of a trial. Therefore, they may be the best persons to draft the Material and Methods and Results sections of manuscripts. Through the use of professional medical writers, the data may be published more quickly in peer-reviewed journals, thus fulﬁlling the PhRMA and ICH guidelines for publication of all clinical trial results in a timely manner. Writers would be the ideal keepers of unique publication numbers (as per GPP) for given studies, as they would most likely be involved in writing secondary papers based on the clinical trial results. Acknowledgment of the contributions of medical writers is consistent with GPP, PhRMA, and AMWA guidelines. It is not anticipated that the controversy about writers, authorship, data, and such will be instantly resolved with the recent publication of PhRMA, AMWA, and GPP guidelines. Nevertheless, these guidelines, in conjunction with the Uniform Requirements and appropriate regulatory guidance, are useful to writers, allowing them to guide their work and anchor their positions within generally accepted norms.

Acknowledgments Cindy Hamilton, PharmD, ELS; Lawrence J Hirsch, MD; Mary G Royer, MS, ELS; Valerie Siddall, PhD, ELS; and Liz Wager, MA critically reviewed the manuscript. Their suggestions were invaluable and I thank them for their help, but I retain full responsibility for the paper.

264 References 1. Foote MA. Review of current authorship guidelines and the controversy regarding publication of clinical trial data. Biotech Ann Rev 2003;9:303–313. 2. International Committee of Medical Journal Editors. Uniform requirements for manuscripts submitted to biomedical journals. N Engl J Med 1997;336:309–315. 3. Bhopal R, Rankin J, McColl E, et al. The vexed question of authorship: views of researchers in a British medical faculty. BMJ 1997;314:1009–1012. 4. Goodman NW. Survey of fulﬁllment of criteria for authorship in published medical research. BMJ 1994;309:1482. 5. Davidoﬀ F, DeAngelis CD, Drazen JM, et al. Sponsorship, authorship, and accountability. Ann Intern Med 2001;135:463–466. 6. Griﬃn J. Clinical trial data: Ownership, access and publication. Clin Res 2002;2:12–22. 7. Hirsch LJ. Conﬂicts of interest in drug development: The practices of Merck & Co., Inc. Sci Engineer Ethics 2002;8:429–442. 8. Food and Drug Administration. Regulation of drug-company-sponsored activities in scientiﬁc or educational contexts (draft proposed policy, October 8, 1991). Division of Drug Marketing, Advertising, and Communications (HFD-240), Rockville, Maryland. 9. Food and Drug Administration. Guidance for industry: Industry-supported scientiﬁc and educational activities published by FDA. Federal Register 1997;62:64073–64100. 10. Altman LK. Some authors in medical journals may get paid by ‘‘spin doctors’’. The New York Times October 4, 1994;C3. 11. Brennan TA. Buying editorials. N Engl J Med 1994;331:673–675. 12. Rennie D and Flanagin A. Authorship! Authorship! Guests, ghosts, grafters, and the twosided coin. JAMA 1994;271:469–471. 13. DeBakey L and DeBakey S. Ghostwriters: not always what they appear. JAMA 1995;274: 870–871. 14. Flanagan A and Rennie D. Acknowledging ghosts. JAMA 1995;273:73. 15. Levy D. Ghostwriters: a hidden resource for drug makers. USA Today September 25, 1996; 1A:2A. 16. Reidenberg JW. Unmasking ghost writers. Clin. Pharmacol. Ther 2001;70:208–209. 17. Hamilton CW and Royer MG. for the AMWA 2002 Task Force on the Contributions of Medical Writers to Scientiﬁc Publications. AMWA Position Statement on the Contributions of Medical Writers to Scientiﬁc Publications. AMWA J 2003;18:13–16. 18. American Medical Writers Association. Code of Ethics. http://www.amwa.org/about/ethics (assessed 28 May 2003). 19. International Conference on Harmonisation, Guidance for Industry, E6 Good Clinical Practice: Consolidated Guidance, April 1996, http://www.ifpma.org/pdﬁfpma/e6.pdf (accessed 28 May 2003). 20. Pharmaceutical Research and Manufacturers of America. PhRMA adopts principles for conduct of clinical trials and communication of clinical trial results. http://www.phrma.org/ mediaroom/press/releases/20.06.2002.427.cfm (accessed 28 May 2003). 21. Wager E, Field EA and Grossman L. Good publication practice for pharmaceutical companies. Curr Med Res Opin 2003;19:149–154.

265

Radioimmunotherapy of non-Hodgkin’s lymphoma: Clinical development of the Zevalin regimen Charles P. Theuer, Bryan R. Leigh, Pratik S. Multani, Roberta S. Allen, and Bertrand C. Liang* IDEC Pharmaceuticals Corporation, 10996 Torreyana Road, San Diego, CA 92121, USA Abstract. ZevalinÕ (ibritumomab tiuxetan; IDEC Pharmaceuticals Corporation, San Diego, CA, USA) was approved by the United States Food and Drug Administration on February 19, 2002, following 9 years of clinical development. Six clinical studies supported the Zevalin Biologics License Application. The Zevalin regimen is indicated for the treatment of patients with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin’s lymphoma (NHL), and for those with follicular NHL refractory to RituxanÕ (rituximab, MabTheraÕ ; IDEC Pharmaceuticals Corporation, San Diego, CA and Genentech, South San Francisco, CA). In the year following FDA approval, approximately 1300 patients were treated in clinical trials or with the commercially available product. Keywords: ibritumomab tiuxetan, rituximab, NHL, antibodies, B cells, CD20, imaging, dosimetry, clinical trials.

Introduction Non-Hodgkin’s lymphoma The non-Hodgkin’s lymphomas (NHLs) comprise a heterogeneous group of lymphoid neoplasms that range from predominantly indolent to highly aggressive malignancies. The majority of NHLs (approximately 85%) originate from B cells [1]. More than 53,400 new cases of NHL and 23,400 deaths were estimated in the United States (US) for the year 2003, representing about 4% of new cancer cases and cancer deaths for both men and women [2]. Collectively, the NHLs rank ﬁfth in cancer incidence and mortality. The incidence increases with advancing age, and patients’ median age at diagnosis is 55 to 60 years [3,4]. SEER data (Surveillance, Epidemiology, End Results Program of the National Cancer Institute to provide incidence, mortality, and survival statistics) indicate that age-adjusted incidence rates of NHL rose faster than rates for the majority of cancers between 1973 and 1997. During that period, cumulative rates increased nearly 80%, with a 3% annual increase; mortality increased 45% overall, with a 1.5% annual percentage increase [5]. Although largely unexplained, the increase is partially due to the growth of the aging population, AIDS-related NHL, and environmental factors [6]. From 1996 to 2000, incidence rates were highest among Caucasians, and age-adjusted rates *Correspoding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10011-2

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

266 were higher among men (23.5 and 15.6 per 100,000 men and women, respectively), though rates rose more signiﬁcantly in women (p < 0.05) [7]. Low-grade or follicular lymphomas account for approximately 65% of lymphoma cases in the population at a given time [8]. Most of these patients (>80%) present with advanced disease, and the median survival time has been estimated at 6.2 years [8,9]. The incidence of transformation from low-grade or follicular NHL to a more aggressive histology may reach 40 to 70% in patients with progressive disease [10–12]. Transformation is a major event that changes the course of the disease, wherein follicular Grade I (follicular small-cell), Grade II (mixed-cell), or Grade III (large-cell) subtypes transform into diﬀuse large-cell lymphoma (diﬀuse mixed cell, diﬀuse large cell, and immunoblastic subtypes according to the Revised European-American Lymphoma/World Health Organization [REAL/WHO] classiﬁcation) [13–16]. Transformation occurs in 15 to 50% of patients at 5 years, in 60% at 8 years, and may occur in as many as 90% of patients at the time of death [11,17]. The prognosis for patients with transformed NHL is uniformly poor: the estimated median survival ranges from 7 to 22 months from the date of transformation [18,19]. Patients with low-grade or follicular NHL generally respond to initial therapy. However, a pattern of remission and relapse usually occurs, and the duration of remission becomes shorter with repeated therapy [20]. In time, patients invariably become refractory to treatment. In the absence of survival beneﬁt, extending the time that a patient is in remission and oﬀ therapy is clinically meaningful. An estimated 30 to 50% of patients with low-grade or follicular NHL are aged 60 years or older [21,22]. Age 60 years is 1 of 5 variables in the multifactorial International Prognostic Index (initially established for aggressive NHL), which identiﬁes patients with diﬀerent risks for death based on speciﬁc baseline characteristics. Age 60 years has been identiﬁed as an important prognostic factor that negatively aﬀects the survival of NHL patients [23]. In the US, the elderly population will continue to grow signiﬁcantly, as those 65 þ years of age were projected to comprise 13% of the population in 2000 (34.7 of 267 million people; US Census Bureau data). As the ‘‘baby boom’’ generation reaches age 65 beginning in 2010, 20% of Americans (69.4 million) are expected to attain age 65 þ in the next 30 years [24]. Because elderly NHL patients may suﬀer from other chronic, debilitating conditions, eﬀective therapies with tolerable toxicity proﬁles are critically needed to minimize complications, as adverse events are common in elderly patients who undergo chemotherapy. Therapy for indolent NHL Chemotherapy A number of cytotoxic agents (alone or in combination) are active in patients with indolent NHL. However, no single treatment approach has proven to be

267 superior, since conventional-dose therapy has not been shown to be curative or to prolong survival. Conventional chemotherapy is often accompanied by side eﬀects ranging from unpleasant to life-threatening. Approximately 70 to 80% of patients who receive chemotherapy experience nausea and vomiting [25], which is often persistent. Similarly, alopecia is a signiﬁcant and distressing side eﬀect considered as the most bothersome aspect of treatment in 88% of women who received chemotherapy [26]. Treatment of low-grade NHL with antineoplastic agents may cause neurotoxicity, nephrotoxicity, and cardiotoxicity [27], and the use of multidrug regimens may compound toxicity. Commonly used chemotherapeutic agents include oral alkylating agents [28,29] such as chlorambucil and cyclophosphamide. These agents oﬀer the advantage of oral administration and ease of dose titration with a relatively modest toxicity proﬁle. Although these agents are used frequently for the treatment of relapsed low-grade and follicular NHL, their eﬃcacy has been established primarily in ﬁrst-line therapy. Thus, response rates for chlorambucil or cyclophosphamide for relapsed disease are not well represented in the literature. The purine analogs ﬂudarabine [30–37] and cladribine [38–44] are also used frequently as single agents in relapsed or refractory NHL patients. Published studies have reported an overall response rate (ORR) of 31 to 66%. Toxicity is primarily hematologic, although prolonged lymphopenia presents an increased risk of opportunistic infection. Other active single agents include ifosfamide [45], mitoxantrone [46], and paclitaxel [47]. More aggressive combination regimens have also been explored. In general, these regimens produce higher response rates without causing myeloablation; however, the cost is greater toxicity and there is no apparent diﬀerence in longterm outcome [48–51]. Most such combination regimens were developed initially as treatment for relapsed aggressive NHL. Published response rates to ESHAP (etoposide, methylprednisolone, cytarabine) range from 36% to as high as 82%, but this regimen is associated with signiﬁcant toxicity. The rate of febrile neutropenia in one ESHAP series [49] was 30% with ﬁve deaths due to infectious complications. Similarly, the combination of ﬂudarabine, mitoxantrone, and dexamethasone achieved an impressive 94% response rate with a 47% complete response (CR) rate, but infections (half of which were opportunistic) were associated with 12% of courses [52]. Given that no overall survival beneﬁt has been demonstrated for this aggressive approach, the role of these combination regimens in the treatment of low-grade follicular NHL is questionable. In contrast with chemotherapy regimens for low-grade NHL, standard chemotherapy cures about 50% of patients with diﬀuse large B-cell lymphoma (DLBCL). Upon ﬁrst relapse, young patients in good health are candidates for salvage chemotherapy regimens; however, combination treatments are not well tolerated, the median DR is only 3 to 5 months, and median survival time is less than 8 months. Myeloablative therapy with transplant of bone marrow or peripheral blood stem cells may produce long-term remission. However, high-dose regimens usually beneﬁt only those patients who respond to salvage

268 chemotherapy. Furthermore, many patients with relapsed or refractory DLBCL are not candidates for high-dose therapy due to poor health or advancing age. Myelosuppression is the major toxic eﬀect associated with most chemotherapy treatments, and it is primarily associated with the intensity of the cytotoxic drugs administered [53]. Myelosuppression is cumulative for many chemotherapeutic agents, and repeated courses worsen toxicity. Moreover, the cumulative time at nadir for blood cell counts increases 4- to 6-fold with repeated cycles of chemotherapy. Intensive chemotherapy has no advantage over less intensive treatments in altering the natural history of low-grade NHL [51,54–56]. Long-term side eﬀects of NHL therapy include an increased risk of secondary malignancies. High-dose chemotherapy with autologous stem-cell support confers a signiﬁcant risk of myelodysplasia (MDS), with actuarial 5-year incidence rates ranging from 6 to 15%, depending on the series [57]. Also, a cumulative incidence of MDS of 4 to 8% was reported in NHL patients who had not undergone high-dose therapy, but who had been exposed to alkylator-based therapies [58]. In a series of 602 NHL patients reported by Pedersen-Bjergaard et al., 9 patients developed MDS or leukemia, resulting in a 6.3% estimated cumulative probability at 7 years after the start of treatment [59]. These authors quoted a general risk of MDS following alkylator therapy of 1% to 1.5% per year, from 2 years to at least 9 years after initiation of therapy.

Immunotherapy: Rituxan Prior to 2002, RituxanÕ (rituximab, MabTheraÕ ; IDEC Pharmaceuticals Corporation, San Diego, CA and Genentech, South San Francisco, CA), a chimeric anti-CD20 monoclonal antibody given as a single agent, was the only US Food and Drug Administration (FDA)-approved therapy for relapsed or refractory low-grade or follicular NHL. Rituxan was also approved for use in European Union countries for Stage III/IV, follicular, chemoresistant, or relapsed ( 2 relapses) NHL. The FDA subsequently approved an expanded label that included information regarding treatment of patients with bulky disease; retreatment of Rituxan responders; and an extended treatment schedule of 8 infusions. Since its approval in 1997, Rituxan has been evaluated in more than 100 trials in a variety of indications and in combination with diﬀerent agents. Rituxan is a chimeric immunoglobulin (Ig) G1 kappa monoclonal antibody with mouse variable and human constant regions, and is derived from a parent murine antibody, ibritumomab. Both Rituxan and ibritumomab speciﬁcally recognize the CD20 antigen [60]. Rituxan and ibritumomab have been shown to have antiproliferative eﬀects in tritiated thymidine incorporation assays and to induce apoptosis in vitro [61,62]. In addition, in vitro studies demonstrated that Rituxan binds human complement and lyses lymphoid B-cell lines through complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity [60].

269 Radioimmunotherapy Radioimmunotherapy utilizes radionuclides conjugated to monoclonal antibodies to target and destroy tumor cells, by combining radiolytic and biologic mechanisms of action. The penetrating eﬀect of radiation allows treatment of poorly vascularized or bulky tumors, since malignant cells inaccessible to a targeting antibody can be killed at a distance by the ionizing radiation. Because NHLs are inherently radiation sensitive, this crossﬁre eﬀect makes radioimmunotherapy an excellent treatment modality. The target antigen, speciﬁc radionuclide emission properties, and chemical stability of radioimmunoconjugates are important factors that contribute to the eﬀectiveness of radioimmunotherapy. B-lymphocyte diﬀerentiation antigens are appealing targets for antibody therapy of NHL. B-cell tumors express a wide range of relatively lineage-speciﬁc cell surface diﬀerentiation antigens, and many are displayed on normal lymphocytes only during discrete stages of diﬀerentiation. The CD20 B-lymphocyte diﬀerentiation antigen provides an exceptional target for NHL immunotherapy for several reasons [63,64]. The CD20 antigen is expressed by more than 90% of B-cell tumors, and is present exclusively on mature B cells and most B-cell lymphomas [65]. It is absent from hematopoietic stem cells, pro-B cells, normal plasma cells, and other non-lymphoid normal tissues; and does not circulate as free protein that might interrupt anti-CD20 antibody targeting. Also, the antigen does not shed from the cell surface when bound by the anti-CD20 antibody. Radioiodinated (131I) antibodies have been studied extensively for the treatment of B-cell lymphomas; however, their use is complicated by several factors [66–71]. The 8-day half-life of 131I is signiﬁcantly longer than that of a murine antibody, and dehalogenation (separation of the radionuclide from the antibody) can result in rapid excretion or accumulation of the free radionuclide in the patient’s thyroid, or both. Optimal localization of an intact monoclonal antibody to its antigen requires 44 to 72 h [72], and 131I clearance varies signiﬁcantly among individuals [73]. It has been reported that 46 to 90% of 131 I is excreted in the urine within 48 h following administration of an 131Iradiolabeled immunoconjugate [72–75]. Variable rates of dehalogenation require the use of whole-body clearance to calculate individually administered doses. Iodine-[131] presents safety issues for patients and for their close contacts. Penetrating gamma emissions of 131I irradiate distant organs and increase wholebody radiation exposure, and expose others in close proximity [76,77]; therefore, hospitalization and/or shielding [68,75,78] may be required. Finally, 131I contamination of body ﬂuids presents a further risk to close contacts. Since methods to stabilize radiolabels have improved by attachment of novel metal chelating groups, the radiometals 90Y and 67Cu have been used to label therapeutic antibodies [79,80]. Yttrium-[90] can deliver higher beta energy (2.3 MeV) to tumor compared with 131I (0.6 MeV), resulting in a longer mean path length over which 90% of the emitted energy is absorbed (5 mm for 90Y versus 0.8 mm for 131I) [77,81–84]. A number of investigators using various

270 radioimmunoconjugates have reported that 90Y delivers radioactivity to tumors more eﬀectively than 131I and is associated with a better therapeutic index [85,86], and that 90Y-labeled antibodies appear to be more clinically active in a human xenograft model [87,88]. Copper-[67] emits beta particles of moderate energy (0.577 MeV) and lower-energy gamma photons (0.185 MeV) compared with 131I. Its half-life (2.6 days) is similar to that of 90Y (2.7 days). Clinical trials with 67Cu-labeled antibodies suggest that better copper chelators are needed [80]. Also, although 67Cu can be administered on an outpatient basis, its radiopharmaceutical availability has been limited. Chelator-linkers have been developed to attach radioactive metals to antibodies, as direct incorporation of metals into antibodies is not feasible. The radionuclide-antibody complex must be stable, because some free metals accumulate in bone. Improved methods for attaching metal chelating groups have enhanced the properties of 90Y radioimmunoconjugates in vivo by increasing both radionuclide retention time and tumor-to-nontumor ratios. First-generation chelates developed for use with radioimaging and radioimmunotherapy include the polyaminocarboxylic acids, DTPA, and EDTA [89,90]. A second-generation chelator, MX-DTPA (tiuxetan) (an isothiocyanatobenzyl derivative of DTPA), was developed to increase stability of the chelate without compromising antibody speciﬁcity, altering metabolism of antibody conjugates, or allowing measurable elution of 90Y [90,91]. A comparison of the MX-DTPA derivative with other chelates containing DTPA or its cyclic anhydride derivative demonstrated that the MX-DTPA derivative yielded conjugates with increased tumor-to-nontumor ratios, and resulted in greater in vivo retention of 90Y [92,93]. Since 90Y emits only pure beta radiation, high doses are easily managed and risk of exposure to others is minimal. Yttrium-[90]-labeled therapeutic agents can be administered on an outpatient basis, since no penetrating gamma-waves are produced during treatment, and no shielding of hospital personnel or hospital stay is required. Therefore, determination of activity limits or dose-rate limits is unnecessary. In contrast, due to the potential for environmental hazard, patients treated with radioiodinated compounds may require hospitalization with shielding. Following treatment, these patients often must restrict contact with family and the public to prevent exposing them to radiation [68,75,78]. Radioimmunotherapy may be conducted in conjunction with imaging and/or dosimetry. If imaging is desired as a component of 90Y-labeled antibody therapy, a surrogate radionuclide that emits gamma radiation such as 111In is optimal. Biodistribution proﬁles of 90Y- and 111In-labeled targeting antibodies are suﬃciently comparable to enable imaging and to predict dosimetry [94,95]. Typically, imaging doses are signiﬁcantly lower than those needed to achieve a therapeutic response. These characteristics were considered when choosing the radionuclide component for the Zevalin regimen. The goal of organ dosimetry is to assess estimated radiation absorbed doses to normal organs. Typically, antibodies are radiolabeled with a small quantity of

271 radionuclide before administration to patients, and then sequential imaging studies are performed using gamma camera and single photon emission computed tomography (SPECT) imaging techniques. However, studies have shown that dosimetry does not necessarily predict toxicity in deﬁned groups of patients such as those with bone marrow involvement with lymphoma, and it may not be necessary for certain radioimmunotherapy regimens. The Zevalin regimen Zevalin is a radioimmunoconjugate composed of a murine, IgG1 kappa antiCD20 monoclonal antibody (ibritumomab) covalently bound to tiuxetan, which strongly chelates the radionuclide 90Y for therapy or 111In for imaging. This murine antibody construct was selected based on its short half-life in serum, an important characteristic for reducing the possibility of bone marrow toxicity [96,97]. The tiuxetan linker-chelator [[N-[2-bis(carboxymethyl)amino]-3-(p-isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)-ethyl]]glycine provides a high-aﬃnity, conformationally restricted chelation site. This chelator provides stability in vivo between the 90Y radiometal and ibritumomab, and coupled with a predictable pharmacokinetic proﬁle, allows weight-based dosing with no requirement for determination of whole-body clearance [98]. For imaging, 111In Zevalin, a gamma emitter, is used as a substitute for the 90 Y-labeled antibody [94,99]. The physical half-life of 111In (2.8 days) is similar to that of 90Y (2.7 days), and the biodistribution of 111In-labeled Zevalin is thought to predict that of the 90Y-labeled antibody. Indium-[111] Zevalin and 90 Y Zevalin are the radiolabeled components of the Zevalin regimen, and Rituxan is the unlabeled component. The US FDA approved the Zevalin regimen on February 19, 2002, following 9 years of clinical development [100]. The Zevalin regimen is indicated for the treatment of patients with relapsed or refractory low-grade, follicular, or transformed B-cell NHL, and for those with follicular NHL refractory to Rituxan. Prior to 2002, no therapy was approved for the treatment of transformed NHL. The clinical development of the Zevalin regimen is described, including information about dose selection, safety, eﬃcacy, imaging, dosimetry, pharmacokinetics, and ongoing studies. Dose selection rationale Pretreatment antibody Rituxan is used as the nonradiolabeled (unlabeled) pretreatment antibody in the Zevalin regimen. Initial studies with the Zevalin regimen demonstrated that in the absence of unlabeled antibody, only 18% of the known disease sites visible with computed tomography (CT) scans had bound suﬃcient 111In Zevalin to generate a positive gamma camera image. When unlabeled ibritumomab was injected prior to 90Y Zevalin at 1 mg/kg (70 mg) or 2.5 mg/kg (175 mg), 56% and 92% of known disease sites, respectively, were imaged. In a subsequent study,

272 Rituxan was evaluated as the pretreatment antibody at 100 mg/m2 or 250 mg/m2 (approximately 170 mg and 425 mg, respectively). No diﬀerences were observed in biodistribution, imaging, or dosimetry between 100 mg/m2 and 250 mg/m2 pretreatment antibody doses. Based upon the potential for greater clinical activity with the higher dose, the 250 mg/m2 dose of Rituxan was selected as the pretreatment antibody given prior to administration of 111In Zevalin and 90 Y Zevalin in all patients. Therapeutic dose The therapeutic dose of 90Y Zevalin was determined in Phase I and Phase II clinical trials. Patients received 90Y Zevalin at ﬁxed single doses of 10 mCi (370 MBq) to 50 mCi (1.9 GBq) in a dose-escalation trial, with three patients receiving multiple doses leading to a cumulative exposure of 70 mCi (2.6 GBq). Doses 40 mCi (1.5 GBq) were not myeloablative. The duration of thrombocytopenia (deﬁned as <100,000 platelets (plt)/mm3) and of nadir platelet count correlated with the 90Y Zevalin dose. Calculation of Pearson correlation coeﬃcients demonstrated a signiﬁcant correlation between the duration of thrombocytopenia and weight-adjusted doses of 90Y Zevalin (p ¼ 0.038), but the correlation was not signiﬁcant for doses adjusted to body surface area (p ¼ 0.081) or for unadjusted doses (p ¼ 0.328). Weight-adjusted doses of 0.2 mCi/kg (7 MBq/kg) to 0.4 mCi/kg (15 MBq/kg) were evaluated in a subsequent Phase I/II trial. A separate analysis by dose group revealed that this mixed population of low-grade, intermediate-grade, and mantle cell NHL patients receiving 0.2 mCi/kg (7 MBq/kg), 0.3 mCi/kg (11 MBq/kg), or 0.4 mCi/kg (15 MBq/kg) 90Y Zevalin achieved response rates of 40%, 75%, and 67%, respectively. Time to progression (TTP) and duration of response (DR) were longer in the higher dose groups (Table 1). The nonmyeloablative maximum tolerated dose (MTD) of 90Y Zevalin was identiﬁed as 0.4 mCi/kg (15 MBq/kg); maximum 32 mCi (1.2 GBq). Imaging The purpose of imaging using 111In-labeled Zevalin is to assess whether biodistribution is acceptable prior to proceeding with an injection of the 90 Y Zevalin therapeutic dose. As part of the Zevalin regimen, 111In Zevalin (5 mCi; 185 MBq) is injected within 4 h following an infusion of Rituxan (250 mg/m2). Biodistribution is assessed by a visual evaluation of whole-body, planarview, anterior, and posterior gamma images at 2 to 24 h (scan 1) and 48 to 72 h (scan 2) after injection. To resolve ambiguities, a third image can be obtained at 90 to 120 h. The radiopharmaceutical is expected to be easily detectable in the blood pool areas on scan 1, with less activity in the blood pool on later images. Moderatelyhigh to high uptake is expected in the normal liver and spleen, and low uptake is expected in the lungs, kidneys, and urinary bladder. Localization to lymphoid

Table 1. Median time to progression (TTP) and duration of response (DR)* by dose in phase I/II clinical studies with the Zevalin regimen. Dose

0.2 mCi/kg (7 MBq/kg) 0.3 mCi/kg (11 MBq/kg) 0.4 mCi/kg (15 MBq/kg)

Responders

Complete responders

N (%)

TTP (Months)

DR (Months)

N (%)

TTP (Months)

DR (Months)

2/5 (40%) 12/16 (75%) 20/30 (67%)

12.5 13.3 15.4

10.8 11.7 14.4

1/5 (20%) 8/16 (50%) 4/30 (13%)

12.6 14.4 Rangey 28.3 – 75.5 þ

10.3 13.1 Rangey 27.1 – 74.2 þ

*Kaplan–Meier (K–M) estimate. y Median cannot be estimated for N ¼ 4. ‘‘ þ ’’ indicates K–M-estimated median not yet reached.

273

274 aggregates in the bowel wall has been reported. Tumor uptake is variable and may be visualized, but tumor visualization on the 111In Zevalin scan is not required to proceed to 90Y Zevalin therapy. If visual inspection of the gamma images reveals an altered biodistribution, the patient will not receive the therapeutic dose of 90Y Zevalin. The patient may be considered to have an altered biodistribution if the blood pool is not visualized on the ﬁrst image, which may indicate rapid clearance of the radiopharmaceutical by the reticuloendothelial system to the liver, spleen, and/or marrow. Other potential examples of altered biodistribution include diﬀuse uptake in the normal lungs or kidneys that is more intense than in the liver on the second or third image.

A course of the Zevalin regimen A standard course of the Zevalin regimen was deﬁned based on ﬁndings regarding the pretreatment antibody, therapeutic dose, and imaging studies. An initial intravenous (IV) infusion of Rituxan (250 mg/m2) is administered on Day 1 to deplete B cells from the peripheral circulation, and to optimize Zevalin biodistribution. Within 4 h after the Rituxan infusion, patients receive a tracer (imaging) dose of 111In Zevalin (5 mCi, 185 MBq; 1.6 mg) administered as a slow IV push over approximately 10 min to assess biodistribution. Patients receive a therapeutic injection of 90Y Zevalin on Days 7 to 9, unless inspection of the images reveals an altered biodistribution. On Days 7 to 9, patients receive a second infusion of Rituxan (250 mg/m2) and an IV injection of 90Y Zevalin (0.4 mCi/kg; 15 MBq/kg, not to exceed 32 mCi [1.2 GBq]) for patients with baseline platelet counts 150,000 plt/mm3. Thrombocytopenia at baseline is a recognized indicator of reduced marrow reserves and of potentially severe cytopenia in oncology patients undergoing treatment with chemotherapy [53,101]. Although patients with moderate or severe thrombocytopenia (<100,000 plt/mm3) were excluded from studies with the Zevalin regimen, patients with mild thrombocytopenia (100,000 to 149,000 plt/mm3) were allowed to enroll. During Phase I of a Phase I/II trial, 2 patients with thrombocytopenia at baseline developed nadir platelet counts <25,000 plt/mm3 (1 patient received 90Y Zevalin at 0.2 mCi/kg [7 MBq/kg] and 1 patient received 0.3 mCi/kg [11 MBq/kg]), whereas this did not occur in 8 of 8 patients with normal baseline platelet counts [102]. For this reason, patients with baseline thrombocytopenia were not dose-escalated beyond 0.3 mCi/kg (11 MBq/kg). Results from a later Phase II trial conﬁrmed the safety and eﬃcacy of 90Y Zevalin at the 0.3 mCi/kg (11 MBq/kg) dose level in 30 patients with mild thrombocytopenia: patients had an acceptable safety proﬁle, and the ORR was 83% [103]. The standard course of the Zevalin regimen is adjusted for patients with mild thrombocytopenia: patients with baseline platelet counts 100,000 to 149,000 plt/mm3 receive a reduced dose of 90Y Zevalin (0.3 mCi/kg; 11 MBq/kg, not to exceed 32 mCi [1.2 GBq]) [103,104].

275 Clinical studies Clinical development of the Zevalin regimen was initiated in 1993. Six clinical studies were conducted to support ﬁling of the Biologics License Application (BLA) in November 2000; two open-label studies were ongoing when the product received marketing approval in February 2002 (Table 2). No standardized response criteria existed for evaluating the eﬀectiveness of NHL therapies when protocols were designed for the Zevalin regimen and therefore, protocol-deﬁned response criteria were planned in consultation with the FDA. Subsequently, an International Workshop convened in 1998 at the US National Cancer Institute (NCI) to deﬁne standardized response criteria for NHL patients. The International Workshop Response Criteria (IWRC) for NHL were published [105] and were later adopted by the NCI cooperative study groups and academic oncology researchers. These criteria are used to present eﬃcacy results for the Zevalin regimen, except where noted. A panel of experts (Lymphoma Experts Conﬁrmation of Response; LEXCOR) assessed response to treatment. The panel comprised independent third parties (radiologists and oncologists with expertise in lymphoma treatment) who were not investigators for the Zevalin regimen studies. LEXCOR was blinded to patient identity, to treatment received, and to investigators’ assessments of response. The panel assigned a response category for each patient who received study treatment after applying a uniform set of criteria to clinical data and CT scans. Phase I/II dose-escalation study An open-label, single-arm, multicenter, Phase I/II, dose-escalation study was performed in 58 patients with relapsed or refractory, advanced low-grade, intermediate-grade, or mantle cell NHL to determine the MTD of 90Y Zevalin [102]. Patients had a poor prognosis: 43% had bone marrow involvement; 59% had bulky disease 5 cm and 37% 7 cm; nearly all received prior treatment with an anthracycline-based chemotherapy regimen; 20% had not responded to at least 1 prior chemotherapy regimen; and 27% had 2 extranodal disease sites. Patients were treated in 3 groups. Phase I (Group 1, 7 patients) received 100 mg/m2 or 250 mg/m2 Rituxan prior to administration of 111In Zevalin. Phase I (Group 2, 15 patients) received 2, once-weekly doses of 250 mg/m2 Rituxan prior to an imaging dose of 111In Zevalin (5 mCi; 185 MBq) and a therapeutic dose of 90 Y Zevalin at 0.2 mCi/kg (7 MBq/kg), 0.3 mCi/kg (11 MBq/kg), or 0.4 mCi/kg (15 MBq/kg) to determine the MTD. Phase II (Group 3, 36 patients) received the selected doses of Rituxan and 90Y Zevalin. Patients’ classiﬁcation as complete responders required that all lymph nodes on CT scans of neck, chest, abdomen, and pelvis were to have regressed to 1 cm 1 cm in size. The response rate was 73% (37/51 patients) in all patients and was 82% (38/34) in the low-grade population. Median TTP for responders

276

Table 2. Completed clinical studies with the Zevalin regimen. Type

Description

Phase I and I/II Dose-Escalation*; Dose-Finding; Safety; Dosimetry

Designed to determine the optimal biodistribution of ibritumomab tiuxetan and the maximum tolerated dose of 90Y Zevalin, and to obtain Phase II safety and eﬃcacy data. The study population included low-grade, intermediate-grade, and mantle cell NHL patients [102,126].

Phase III Randomized Controlled Comparison; 0.4 mCi/kg (15 MBq/kg) Phase III Nonrandomized; Rituxan-Refractory 0.4 mCi/kg (15 MBq/kg) Phase II Supportive; Mild Thrombocytopenia 0.3 mCi/kg (11 MBq/kg) Open label

Planned to compare the eﬃcacy and safety of the Zevalin regimen with Rituxan as the control in relapsed or refractory low-grade, follicular, or CD20 þ transformed NHL patients [108,122].

Compassionate Use

Internally controlled study in relapsed or refractory follicular NHL patients refractory to Rituxan treatment [110]. Safety and eﬃcacy of a reduced dose of 90Y Zevalin in mildly thrombocytopenic patients ( 150,000 plt/mm3) with relapsed or refractory low-grade, follicular, or CD20 þ transformed NHL [103,111]. Treatment access for patients with no other treatment alternatives; added to the overall safety experience. Histologies included low-grade, follicular, de novo diﬀuse large cell, transformed including Richter’s, and mantle cell NHL. Treatment for patients with relapsed, CD20 þ B-cell NHL who were ineligible for other IDEC Pharmaceuticals Corporation protocols because of prior myeloablative therapy. Five patients were treated at 5 clinical sites.

* 0.2 mCi/kg (7 MBq/kg), 0.3 mCi/kg (11 MBq/kg), or 0.4 mCi/kg (15 MBq/kg) dosimetry.

90

Y Zevalin; 5 mCi (185 MBq)

111

In Zevalin administered for imaging and

277 was 12.6 months (95% CI 9.3, 19.0) and median DR was 11.7 months (95% CI 8.1, 18.0). The median DR for patients achieving a CR/CRu was 23 months, and was 62.1 months (range 60 þ to 66 þ months) for ongoing responders [106]. The MTD was established as 0.4 mCi/kg (15 MBq) in patients with a baseline platelet count 150,000 plt/mm3, and 0.3 mCi/kg (11 MBq) in patients with pre-existing mild thrombocytopenia. Twelve patients in this trial had diﬀuse aggressive NHL histology: diﬀusemixed (IWF E, 3 patients) or diﬀuse large cell (IWF G, 9 patients). Seven of 12 (58%) responded (33% CR) [107]. Four of these had remissions longer than 3 years and 2/4 remain in remission after 5 years, indicating that further study is warranted in this population [106]. Phase III randomized controlled comparison study A Phase III, randomized, controlled multicenter study was designed to compare the eﬃcacy and safety of the Zevalin regimen (N ¼ 73) with a Rituxan control group (N ¼ 70) in patients with low-grade, follicular, or transformed NHL [108]. The Zevalin regimen treatment group received an infusion of Rituxan (250 mg/ m2) followed by an IV injection of an imaging and dosimetry dose of 111In Zevalin (5 mCi; 185 MBq). One week later, patients meeting dosimetry requirements received a second infusion of Rituxan (250 mg/m2) followed by an IV injection of 90Y Zevalin (0.4 mCi/kg, 15 MBq/kg; maximum dose 32 mCi, 1.2 GBq). Patients randomized to the Rituxan control group received a course of Rituxan consisting of a weekly infusion of 375 mg/m2 for 4 weeks. Patients were stratiﬁed by NHL histology (IWF A/follicular/transformed) at enrollment. The median number of prior therapies was 2 (range 1 to 6). Nearly half of all patients (45%) had tumors 5 cm, 20% 7 cm, and 8% 10 cm; and 56% did not respond to at least 1 prior chemotherapy regimen. A signiﬁcantly higher ORR was achieved in the Zevalin regimen treatment group compared with the Rituxan control group: 80% versus 56% (p ¼ 0.002). Complete response rates were 30% and 16%, respectively (p ¼ 0.04). An additional 4% in each group achieved an unconﬁrmed complete response (CRu). TTP appeared to be longer in Zevalin regimen-treated follicular NHL patients (Fig. 1) and in those with nontransformed NHL. For all patients, the Kaplan– Meier (K-M) estimated median DR was 14.2 months in the Zevalin regimen treatment group versus 12.1 months in the Rituxan control group (p ¼ 0.64), and TTP was 11.2 months versus 10.1 months (p ¼ 0.173); see Fig. 2. For patients with follicular NHL, estimated median DR was 18.5 months in the Zevalin regimen treatment group versus 12.1 months in the Rituxan control group (p ¼ 0.37). At latest follow up [109], the median duration for patients achieving a CR/Cru in the Zevalin regimen treatment group was 23 months, and was 42.2 months for ongoing responders. The time interval from study treatment to next antineoplastic therapy was evaluated. For patients with low-grade or follicular histology, the Zevalin regimen treatment group had a longer time interval before their next

% Progression Free

278 100 90 80 70 60 50 40 30 20 10 0

C

Ibritumomab Tiuxetan (n = 55) Rituximab (n = 58) C

CC

C C C C C

0

10

20

CC CC C C C CCC

30

CC C C

40

C

50

60

TTP (Months)

% Progression Free

Fig. 1. Time to progression (Kaplan–Meier) in patients with follicular NHL in the Phase III Randomized Controlled Comparison Study (reprinted with permission from the Japanese Society of Hematology, International Journal of Hematology [127], Copyright 2002).

100 90 80 70 60 50 40 30 20 10 0

- - - Ibritumomab Tiuxetan (n=73) Rituximab (n=70)

C

C

CC C C

C C C C CCCC C CC C

0

10

20

CCC C C C C

30

CCC

40

C

50

60

TTP (Months) Fig. 2. Time to progression (Kaplan–Meier) in all patients in the Phase III Randomized Controlled Comparison Study (reprinted with permission from the Japanese Society of Hematology, International Journal of Hematology [127], Copyright 2002).

antineoplastic treatment compared with the Rituxan control group. Time to next antineoplastic therapy was 17.6 months for patients treated with the Zevalin regimen versus 12.4 months in patients treated with Rituxan (p ¼ 0.1); see Fig. 3. Phase III rituxan-refractory study This non-randomized, controlled, open-label multicenter study was designed to evaluate the Zevalin regimen in follicular B-cell NHL patients who were refractory to Rituxan [110]. Eligible patients were treated previously with Rituxan (375 mg/m2 weekly for 4 weeks) and either did not obtain and objective

% of Patients Without Therapy

279 - - - Ibritumomab Tiuxetan (n=73) Rituximab (n=70)

100 90 80

C

70 60

CC

C

50 CC

40

C

30

C

CC CC CC CC C C C C C

C C

C CC C

CCC

C CCCC

C

C C

20 10 0 0

10

20

30

40

50

60

Time from Treatment to Next Anti-NHL Therapy (Months) Fig. 3. Time to next antineoplastic therapy (Kaplan–Meier) in all patients in the Phase III Randomized Controlled Comparison Study (reprinted with permission from the Japanese Society of Hematology, International Journal of Hematology [127], Copyright 2002).

1.

Diagnosis

1st Chemo

2nd Chemo

Last Chemo

Prior Rituxan

Zevalin Regimen

Time 2. Fig. 4. Response rates to the Zevalin regimen were compared with (1) prior Rituxan therapy and with (2) last chemotherapy in the Phase III Rituxan-Refractory Study (reprinted with permission from the American Society of Clinical Oncology, Journal of Clinical Oncology [110], Copyright 2002).

response to treatment or had a TTP <6 months. Patients enrolled in the Rituxan control group of the Phase III randomized controlled comparison study were allowed to participate if they had IWF A or transformed lymphoma histology and did not respond to Rituxan. Such patients (N ¼ 3) had safety assessments only. The primary eﬃcacy endpoint was ORR as determined by the LEXCOR panel. Secondary eﬃcacy endpoints were comparisons of ORR and DR to those achieved with prior Rituxan treatment and with last chemotherapy (Fig. 4). Patients’ median age was 54 years and the incidence of tumor bulk was high (74% had tumors 5 cm, 44% 7 cm, 19% 10 cm; 11.1% splenomegaly;

Maximum Percentage Lesion Change

280 100% 75% 50% 25% 0% −25% −50% −75%

−100% Patients

Fig. 5. Maximum reduction in lesion size assessed by the sum of perpendicular diameters (SPD) of lesions by patient in the Phase III Rituxan-Refractory Study.

31.5% bone marrow involvement; and 16.7% had 2 extranodal sites of disease). All patients were extensively pretreated (median 4 prior antineoplastic therapies; range 1 to 9); and 82% had not responded to at least 1 prior chemotherapy regimen. The ORR was 74% in 54 patients with follicular NHL (40/54 patients); 15% CR (8/54), and 59% PR (32/54). ORR was signiﬁcantly higher than the ORR to the last Rituxan therapy (74% versus 32%; p<0.001). Of 37 patients who did not respond to their last Rituxan therapy, 25 (68%) responded to the Zevalin regimen. The overall tumor burden, measured as the sum of perpendicular diameters (SPD) of lesions, was reduced in 94% of patients (51 of 54) following treatment (Fig. 5). ORR was not signiﬁcantly diﬀerent from the ORR to patients’ last chemotherapy (75% versus 67%; p ¼ 0.32). Of 17 patients who did not respond to their last chemotherapy, 10 (59%) responded to the Zevalin regimen. The ORR to the Zevalin regimen (median ﬁfth therapy) was similar to that achieved with prior chemotherapy (median third therapy). The result was better than expected, given that subsequent therapies are usually associated with successively lower response rates. As of October 1, 2002, K-M estimated median TTP was 6.8 months (1.1 to 50.9 þ months), and was 8.7 months for responders (1.7 to 50.9 þ months). The K-M estimated median DR was 6.4 months (0.5 to 49.9 þ months). Phase II supportive study in patients with mild thrombocytopenia Thrombocytopenic patients with relapsed or refractory low-grade NHL have an increased risk of chemotherapy-induced myelosuppression following treatment [101]. The safety and eﬃcacy of a reduced dose of 90Y Zevalin (0.3 mCi/kg, 11 MBq/kg; maximum 32 mCi, 1.2 GBq) was evaluated in this study of 30 patients with mild thrombocytopenia (100,000 to 149,000 plt/mm3) who

281 had advanced relapsed or refractory low-grade, follicular, or transformed B-cell NHL [103]. Indium-[111] Zevalin (5 mCi; 185 MBq) was injected for dosimetry evaluation after the initial Rituxan infusion; estimated radiation absorbed doses were well below the study-deﬁned maximum allowable for all 30 patients [111]. Patient characteristics included a median age of 61 years (range 29 to 85 years); 90% Stage III/IV at study entry; 83% follicular histology; 47% had tumors 5 cm, 17% 7 cm, and 7% 10 cm; 67% bone marrow involvement; and 20% had 2 extranodal sites of disease. Most patients (97%) had a prestudy WHO performance status of 0 or 1; 23% had splenomegaly, and 63% were resistant to at least 1 prior chemotherapy regimen. The ORR was 83% (37% CR, 6.7% CRu, and 40% PR). K–M estimated median TTP was 9.4 months (range 1.7 to 24.6 months). In responders, K–M estimated median TTP was 12.6 months (range 4.9 to 24.6 months), with 35% of data censored. Toxicity was primarily hematologic, transient, and reversible. The incidence of Grade 4 neutropenia, thrombocytopenia, and anemia was 33%, 13%, and 3%, respectively. The incidence of overall infection, Grade 3 and 4 infection, hospitalization, and treatment-related death (none in this study) was similar to that of patients with normal platelet counts, conﬁrming that the higher incidence of hematologic toxicity did not cause a greater number of clinically signiﬁcant events. The non-hematologic toxicity proﬁle was similar to that seen with the 0.4 mCi/kg (15 MBq/kg) dose in patients with normal platelet counts. Overall safety analysis An integrated safety analysis was performed on data for 349 patients treated with the Zevalin regimen in 5 clinical trials prior to approval of the product by the FDA [112]. Of these 349 patients, 345 (99%) completed treatment. The primary toxicity of the Zevalin regimen was reversible myelosuppression. Grade 4 neutropenia was observed in 30% of patients, Grade 4 thrombocytopenia in 10%, and Grade 4 anemia in 4%. Most nadirs of absolute neutrophil counts (ANC), platelet counts, and hemoglobin percentages occurred at 7 to 9 weeks. Although Grade 4 neutropenia occurred in nearly one-third of patients, only 7% were hospitalized with infections (2% with febrile neutropenia), which may reﬂect the low incidence of mucositis (<1%) associated with the Zevalin regimen. Bone marrow involvement with lymphoma at baseline was associated with a signiﬁcantly greater incidence of Grade 4 hematologic toxicity, including neutropenia (p ¼ 0.001), thrombocytopenia (p ¼ 0.013), and anemia (p ¼ 0.040). In addition, increasing percentages of bone marrow involvement at baseline correlated with increasing Grade 4 hematologic toxicity. Regarding prior chemotherapy and hematologic toxicity, patients treated with >2 prior chemotherapy regimens had 16% Grade 4 thrombocytopenia compared with 7% of those with 2 prior regimens (p ¼ 0.02) [113]. The number of prior chemotherapy regimens was not associated with a longer median duration of Grade 3 or 4 neutropenia, thrombocytopenia, or anemia. Patients treated

282 previously with ﬂudarabine had signiﬁcantly lower platelet counts (p ¼ 0.001) and percentages of hemoglobin (p ¼ 0.020) at baseline, and were more likely to develop Grade 3 or 4 neutropenia (p ¼ 0.050), thrombocytopenia (p ¼ 0.025), and anemia (p<0.001). The most frequent nonhematologic adverse events were consistent with those associated with Rituxan infusions: asthenia, nausea, chills, fever, and headache [114]. No acute dysfunction of any major organs was noted. Bleeding events occurred in 62 patients (18%) and were Grade 1 or 2 in 56 of these patients. The median absolute B-cell count declined after treatment initiation, but recovered by 6 to 9 months. Of 211 patients, 3 (1%) developed human antimouse antibodies, and 1 (0.05%) developed human antichimeric antibodies. No toxicity was associated with these immune responses. Serum immunoglobulin concentrations were largely unaﬀected, and T-cell counts remained normal. Although mild abnormalities in liver function tests occurred in a few patients, most could be attributed to other causes such as lymphoma, IV drug abuseassociated viral hepatitis, or Gilbert’s syndrome. Radiation absorbed doses to the liver in these patients were in the same range as those of the overall patient population. No pulmonary or renal toxicity related to the Zevalin regimen was observed. Analysis of the safety proﬁle in geriatric patient subsets ( 65; 65 to <75; and 75 years of age) revealed no clinically signiﬁcant age-related eﬀects compared to younger patients (<65 years of age). Also, treatment with the Zevalin regimen did not preclude relapsed patients from receiving additional therapy such as ESHAP; cisplatin, Ara-C, dexamethasone (DHAP); or prednisone, doxorubicin, cyclophosphamide, and etoposide followed by cytarabine, bleomycin, vincristine, and methotrexate with leucovorin rescue (ProMACECytaBOM). Of 84 patients who received antineoplastic therapy following relapse after treatment with the Zevalin regimen, 49 (58%) were responders. In addition, subsequent stem-cell mobilization and high-dose chemotherapy were performed safely. These observations were conﬁrmed in additional safety analyses of patients treated with the Zevalin regimen compared with those treated with chemotherapy: subsequent chemotherapy was tolerated equally in both groups as measured by Grade 4 neutropenia and thrombocytopenia, use of G-CSF, transfusions of red blood cells or platelets, neutropenic fever, and hospitalization for complications. In addition, response rates to subsequent therapy (chemotherapy, biologics, or radiation) were similar for patients treated with the Zevalin regimen compared with those treated with Rituxan [115]. Myelodysplasia or acute myelogenous leukemia (AML) was reported in 10 of 770 patients treated with the Zevalin regimen (1.3%) during the past 9 years, from 4 to 34 months following therapy, and from 1.5 years to 14 years following diagnosis [116]. Based on K–M estimates, this corresponds to an estimated annualized incidence of 0.21% from the date of NHL diagnosis and 0.62% from the date of the ﬁrst infusion of Rituxan in the Zevalin regimen. These patients had extensive prior exposure to alkylator-based therapy. Published reports cite

283 a cumulative incidence of about 4 to 8% of MDS for NHL patients who never received dose-intensive therapy [58], or a general risk of about 1 to 1.5% per year from 2 years to at least 9 years after the start of therapy [59]. Dosimetry and pharmacokinetics Dosimetry estimates radiation absorbed doses to individual organs and to tumor. The estimates to organs help determine whether a patient can be treated safely, and the estimated dose to tumors may be useful for predicting the therapeutic value of a radioimmunotherapy. During initial clinical development of the Zevalin regimen, radiation dosimetry was performed at the investigative site for 205 patients prior to treatment with 90Y Zevalin. Radiation absorbed dose was estimated following imaging with 111In Zevalin in order to support a decision for proceeding with 90Y Zevalin treatment. Estimates were obtained using quantitative imaging and blood sampling data, using MIRDOSE3.1 computer software [117]. All 205 patients met protocol-deﬁned criteria for proceeding with 90Y Zevalin treatment. Estimated radiation absorbed doses were below the allowed maximum of 2000 cGy for uninvolved major organs and 300 cGy for red marrow. Detailed dosimetry results have been published for several patient subsets [111,118–121]. Dosimetry At the clinical site, a tracer dose of 111In Zevalin (5 mCi; 185 MBq) was administered on Study Day 1 following an infusion of Rituxan (250 mg/m2). Organ 111In activity was measured from anterior and posterior gamma scans obtained at 5 to 8 time points over 1 week. A region-of-interest (ROI) method was used to calculate radiation absorbed dose for four organs (liver, lungs, kidney, spleen), and a remainder method was used for all other organs. Blood 111 In activity was measured at similar time points, and was decay-corrected to 90 Y activity and then converted to fraction of injected activity (FIA). Finally, residence times were calculated from the area under the FIA-versus-time curve (AUC) and analyzed with MIRDOSE3.1. Following 90Y Zevalin treatment, gamma images and blood samples were transferred to a central laboratory (Mayo Clinic, Rochester, MN, and Oak Ridge Associated Universities, Oak Ridge, TN) for a uniform review of the data. For 179 patients, the ROI method was used to calculate radiation absorbed dose for 5 organs (lungs, kidneys, liver, spleen, sacral marrow) and the remainder method was used for all other organs. For a subset of 15 patients, the ROI method was used for 10 organs: heart wall, small intestine, upper and lower large intestine, testes, liver, lungs, kidney, spleen, sacral marrow. The remainder method was used for all other organs. Central dosimetry was performed in 179 of the 205 patients receiving 90 Y Zevalin doses of 0.2 mCi/kg (7 MBq/kg), 0.3 mCi/kg (11 MBq/kg), or 0.4 mCi/kg (15 MBq/kg) [121]. For these 179 patients, median radiation absorbed doses from 90Y Zevalin were as follows: lungs, 211 cGy (range 41 cGy

284 to 527 cGy); kidneys, 23 cGy (0 cGy to 76 cGy); liver, 450 cGy (64 cGy to 1856 cGy); spleen, 742 cGy (24 cGy to 2448 cGy); blood derived red marrow, 62 cGy (7 cGy to 221 cGy); and sacral-derived red marrow, 97 cGy (6 cGy to 257 cGy). As reported by Wiseman [122], radiation absorbed dose factors (cGy/mCi) from 111In and 90Y to all organs and to total body were estimated for 15 randomly selected patients in the randomized controlled comparison study. Of the 10 organs measured using the ROI method, the highest median radiation absorbed dose was to the spleen, an organ often involved with NHL [122]. Radiation absorbed dose to tumor from 90Y Zevalin was determined for 57 tumors in 38 patients. The median radiation absorbed dose factor to tumor was 60 cGy/mCi (range 3 cGy/mCi to 778 cGy/mCi). The median total radiation absorbed dose to tumor was 1480 cGy (range 61 cGy to 24,274 cGy). The median radiation absorbed dose was 2352 cGy for tumors <15 g (N ¼ 29) and 873 cGy for tumors 15 g (N ¼ 28). No adverse event was attributed to radiation from an adjacent tumor, including four cases where radiation absorbed dose to tumor was >10,000 cGy. Pharmacokinetic analysis Pharmacokinetic data were derived from 111In activity measured in whole blood and plasma and not from direct measurement of antibody in serum, because serum concentrations were undetectable at the ibritumomab tiuxetan dose given (approximately 2.0 mg). The estimated median eﬀective half-life of 90Y Zevalin in blood was 27 h (range 14 to 44 h); median biologic half-life of the antibody was 46 h (range 18 to 140 h); and median residence time for 90Y Zevalin in blood was 25 h [121]. Similar results were obtained for analyses in plasma. Urinary excretion Urinary excretion is the main route of elimination of Zevalin, as determined by data that correlate urinary excretion and whole-body retention [123]. A consecutive 7-day, total urine collection was obtained in 2 studies from patients during the week following treatment with 111In Zevalin or 90Y Zevalin. The mean percentage of injected 90Y activity excreted in urine over 7 days was 5.8% and 7.9% when measured directly, and was 9.2% and 11.5% when estimated from 111In activity. Recommendations to patients during the ﬁrst 3 days following release after treatment with the Zevalin regimen include washing hands thoroughly after using the toilet, cleaning up spilled urine, and disposing of material contaminated by body ﬂuids. Patients are advised to use condoms for sexual relations during the ﬁrst week. Exposure to family members Radiation exposure following treatment with the Zevalin regimen was quantiﬁed in family members of patients. The family member with closest contact to the patient wore an electronic dosimeter (DoseGUARD Plus; AEA Technology QSA, Inc., Burlington, MA) for 7 days following treatment. Even though

285 patients were not isolated and no shielding was used, the total deep dose equivalent exposure was 0.035 mSv (median), which is within the range of normal background radiation. The only instructions to the family were to avoid contact with the patient’s body ﬂuids, including saliva, blood, urine, and stool [124]. Correlation analyses of dosimetry and pharmacokinetics with hematologic and non-hematologic toxicity Several analyses were performed to determine whether hematologic measures correlated with either dosimetric parameters (radiation absorbed dose to red marrow and to total body) or pharmacokinetic parameters (half-life and AUC of 90 Y estimated by 111In activity in blood and plasma). ANC and platelet counts were evaluated to assess hematologic toxicity, because irradiation of blood and bone marrow signiﬁcantly aﬀects the depth of these nadirs and the time to recovery of normal counts. However, no clinically meaningful correlations were noted for any of the analyses. This may be due to the interpatient variability in bone marrow reserve. Loss of marrow reserve is common in NHL patients as a result of bone marrow damage from prior chemotherapy, external beam radiation therapy, or from marrow involvement with NHL. In addition, bloodderived red marrow dosimetry is based on blood radioactivity counts and does not account for secondary irradiation of hematopoietic cells from radiopharmaceutical targeting of NHL within the marrow. Nearly half (49%) of patients in these studies had bone marrow involvement with NHL. Elimination of dosimetry The value of radiation dosimetry is restricted if the radiation absorbed dose to the dose-limiting organ does not correlate with toxicity. Results from studies of the Zevalin regimen indicate that radiation absorbed dose does not exceed safe limits and does not correlate with hematologic toxicity. Therefore, dosimetry has been eliminated from the Zevalin regimen. However, 111In Zevalin imaging to assess appropriate biodistribution will continue to be performed as a safety measure. Ongoing clinical studies Since the Zevalin regimen received marketing approval in 2002, approximately 1300 patients have been treated in clinical trials or with the commercially available product. Several clinical studies have been initiated in the US, Europe, and Japan. A Phase III, randomized, controlled, multicenter study comparing the Zevalin regimen plus Rituxan to Rituxan alone recently opened for enrollment at approximately 90 clinical sites. Planned enrollment for the study is approximately 400 patients with relapsed or refractory follicular NHL. Patients will be stratiﬁed and prospectively randomized to a control group (Rituxan therapy

286 alone) or to a comparison group (Zevalin regimen plus Rituxan regimens). The control group will receive Rituxan, 250 mg/m2 weekly for 2 weeks and then 375 mg/m2 weekly for 4 weeks. The comparison group will receive Rituxan (250 mg/m2) followed by 111In Zevalin (5 mCi; 185 MBq) to assess biodistribution, and then 1 week later, will receive another infusion of Rituxan (250 mg/m2) and an injection of 90Y Zevalin (0.4 mCi/kg; 15 MBq/kg). One week following the 90Y Zevalin dose, patients will receive Rituxan, 375 mg/m2 weekly for 4 weeks. The study is designed to conﬁrm that improvement in event-free survival and other endpoints is attributed only to the radioimmunotherapy (90Y) portion of the Zevalin regimen. Administration of Rituxan following the Zevalin regimen may have a maintenance eﬀect and may prolong time-to-event variables. Clinical beneﬁt will be measured by comparing time-to-event variables for each group. Two clinical studies with the Zevalin regimen were initiated in Europe by Schering AG (Berlin, Germany). A randomized, Phase III, multicenter study is ongoing in patients with Stage III or IV follicular NHL who achieved a partial or complete remission after ﬁrst-line chemotherapy. The eﬃcacy and safety of subsequent treatment with the Zevalin regimen will be compared with no further treatment in these patients. A total enrollment of 350 patients is planned. The second study is an open-label, Phase II study in patients with relapsed or refractory DLBCL who are not appropriate candidates for autologous stem-cell transplantation. The eﬃcacy and safety of a single dose of 90Y Zevalin (0.4 mCi/kg; 15 MBq/kg) will be evaluated in these patients. A total enrollment of 100 patients is planned. A Phase I, open-label, dose-escalation study of the Zevalin regimen (0.3 mCi/kg; 11 MBq/kg or 0.4 mCi/kg; 15 MBq/kg) has been initiated in Japan in patients with relapsed or refractory, indolent NHL. Patient enrollment is ongoing. Additionally, several physician-sponsored clinical studies of the Zevalin regimen were open for enrollment in the US during 2003; some representative studies are shown in Table 3.

Conclusions The Zevalin regimen is the ﬁrst radioimmunotherapy available commercially for clinical use, and it represents a clinically meaningful advance in therapy for patients with relapsed or refractory, low-grade, follicular, or CD20 þ transformed and Rituxan-refractory follicular NHL. Treatment is completed in 1 week with no requirement for hospitalization, isolation, or shielding. Patient-speciﬁc dosing is based simply on body weight and pretreatment platelet count; no determination of whole-body clearance is required. The speciﬁc targeting of tumor cells allows systemic therapy without such side eﬀects as hair loss, nausea

287 Table 3. Ongoing physician-sponsored clinical studies with the Zevalin regimen. Phase Description

Indication

I

Relapsed NHL, indolent or diﬀuse large-cell lymphoma Relapsed, low-grade or follicular NHL Relapsed, low- and intermediate-grade NHL Relapsed NHL

I I I I/II I/II I/II

I/II II II II II II

Rituxan /cyclophosphamide followed by dose-escalation of 90Y Zevalin with autologous stem cell support Two sequential doses of Zevalin in patients with relapsed low-grade follicular NHL Zevalin in children with recurrent/refractory NHL BEAM (BCNU, etoposide, cytarabine and melphalan) with dose-escalation of 90Y Zevalin and PBSC support Cyclophosphamide/etoposide with dose-escalation of 90Y Zevalin and peripheral blood stem cell (PBSC) support Zevalin in nonmyeloablative transplantation for lymphoid malignancies 90 Y Zevalin in post-transplant patients relapsed after prior high-dose therapy (autologous stem cell transplantation; ASCT) Zevalin in combination with high-dose BEAM followed by ASCT Zevalin in patients with relapsed CLL and residual disease after chemotherapy Zevalin for previously untreated patients with low-grade follicular NHL Zevalin in patients with relapsed mantle cell lymphoma Rituxan and short duration chemotherapy followed by Zevalin Zevalin in patients with relapsed/refractory classical Hodgkin’s disease

Relapsed NHL Relapsed NHL or chronic lymphocytic leukemia (CLL) Relapsed NHL

Relapsed low- and intermediate-grade NHL Relapsed CLL in PR or CR Untreated low-grade NHL Relapsed mantle cell NHL Untreated low-grade NHL Relapsed Hodgkin’s disease

and vomiting, cardiotoxicity, nephrotoxicity, or neurotoxicity. An ORR of 80% is achieved in patients with relapsed or refractory low-grade NHL. In addition, the Zevalin regimen is a favorable treatment option when aggressive therapy is being considered for indolent NHL, as Zevalin-treated patients can safely and eﬀectively receive a variety of subsequent antineoplastic therapy. Compared with indolent disease, aggressive NHL is notably diﬀerent. Aggressive NHL progresses rapidly and requires therapeutic intervention. Survival of patients with untreated DLBCL, the most common aggressive NHL histology, is measured in months [4]; chemotherapy cures about half of these patients. The majority of patients who relapse after initial therapy, however, are not eligible for transplant; have poor response rates to chemotherapy; and have poor survival rates [125]. DLBCL typically expresses the CD20 antigen, and results of treatment with the Zevalin regimen in relapsed patients with diﬀuse aggressive NHL histology suggest beneﬁcial clinical activity in this population. Further study will deﬁne the clinical beneﬁts of the Zevalin regimen in this disease.

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Biosimulation software is changing research Richard L.X. Ho1 and Lenore Teresa Bartsell2* 1

Johnson & Johnson Pharmaceutical Research & Development, L.L.C., La Jolla, CA, USA Medical Writer, Saratoga, CA, USA

2

Abstract. Biosimulation software is being used in pharmaceutical drug development to mimic diseases. Virtual clinical trials of new developing pharmaceutical drugs can be conducted on computers running disease simulations. Using virtual patients instead of clinical research patients can save both time and money for pharmaceutical companies in their search to discover new drugs and bring them to market. In the future, this type of research will be commonplace. Keywords: biosimulation, modeling, diabetes, obesity, asthma, arthritis, drug discovery, drug development, biotechnology, clinical trials.

Introduction In La Jolla, California, a new type of research is taking place that will change the process of pharmaceutical drug discovery and drug development. In these southern California research labs, ‘‘sick’’ computers suﬀer from Type 2 diabetes. Linux servers are being used to run biosimulation software of Type 2 diabetes. These virtual diabetic patients are created using a mathematical model of normal human metabolism that can be altered to mimic the problems that occur in diabetes. Simulations allow the scientists to virtually test new drugs for diabetes over a shorter time in relation to the amount of time that it takes to test these drugs on real patients with diabetes. A major beneﬁt is that some complications can be detected early. Mathematical models of disease The biosimulations are created by a team of biologists and engineers focused on the clinical endpoints of the disease on which medical decision-making is based. They use a collection of normal human health and physiology data, in addition to disease data. Each patient has a unique combination of genetic, environmental, and lifestyle factors, and because of these natural variations, it is diﬃcult to treat many chronic diseases. Even successful drugs only work in an estimated 30–40% of patients who are treated with them so it is important for the team to have as much individual data as possible from a wide range of patients under a variety of interventions. Virtual patients are created by selecting a set of these individual factors to run in the biosimulation, and each virtual patient has a diﬀerent set of factors *Corresponding author: E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 10 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(04)10012-4

ß 2004 ELSEVIER B.V. ALL RIGHTS RESERVED

298 Virtual Patients: Development and Response

Mild Disease Severe Disease

Physiologic State

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Variable Variable Response Disease State to Treatment

Fig. 1. Virtual patients – development and response.

causing their disease (Fig. 1). Like clinical trial patients, virtual patients respond diﬀerently depending on their lifestyle and reason for their disease. However, unlike in clinical trials, the scientists know these diﬀerences for each patient and can use the information to better understand the response to the treatment. Entelos, the company that created the biosimulation software called PhysioLab,* outlines its collaborative R&D technology process as shown in Fig. 2. They have used this approach to create PhysioLabs for asthma, rheumatoid arthritis, obesity, and diabetes. Developing digital diabetes To create their Type 2 Diabetes PhysioLab, Entelos ﬁrst identiﬁed the clinical variables thought to be important in the disease, that is, glucose, insulin, carbohydrates, proteins, fats, etc., and focused on how their homeostasis is maintained in normal individuals. This required describing important biochemical pathways in the liver, pancreas, muscle, and adipose tissue and understanding the physiological functions and feedbacks of the system as a whole. Then they examined the many ﬁndings that have been reported in patients with diabetes, such as levels of insulin sensitivity and eﬀects of fatty acids, to see if distrubing the model’s parameters could reproduce those ﬁndings. Building the ﬁrst version took about a year of intense eﬀort by Entelos (Fig. 3). In order to validate the diabetes model, the behavior of each subsystem and then of the whole virtual patient is compared to human data from oral glucose tolerance tests and euglycemic ‘‘clamp’’ studies where the glucose level is artiﬁcially kept constant. Mismatches between biosimulation results and clinical data indicate an insuﬃcient characterization of the supporting physiology and often highlight areas for informative experimentation. If putting all the *Entelos, the Entelos logo, PhysioLab, CytoLab, Entelos In Silico Insights, and the cone and arrow icon are registered trademarks, trademarks, or servicemarks of Entelos, Inc. in the United States and throughout the world. All other trademarks are the property of their registered owners.

299 1 Identify Customer Goals

2 Customize PhysioLab to include proprietary data as needed 3 Create Virtual Patients

4 Test Virtual Patients aginst public and proprietary data

5 Discuss and Refine

6 Simulate Experiments and Clinical Trials to determine best treatments and reserch paths 7 Share results. Plan future experiments. Fig. 2. Collaborative R&D technology process.

Fig. 3. Adipose tissue – fat storage and release.

300

Fig. 4. Integration of multiple organ physiology modules leads to the entire Diabetes PhysioLab.

underlying biochemical steps together yields good systems level behavior at multiple time scales from minutes to days, then there is more conﬁdence that the system and all of its feedbacks have been understood (Fig. 4). Results of drug testing on virtual patients The Type 2 Diabetes PhysioLab has now been used in diabetes projects at Johnson & Johnson Pharmaceutical Research & Development, L.L.C. (J&JPRD) from drug target validation to clinical trial optimization. In one such project, a drug target that several companies are pursuing has already been shown to have beneﬁcial eﬀects on glucose levels in acutely treated animal models of diabetes. However, in longer-term biosimulations regarding that target, the dynamics and feedback inherent in the system cause those beneﬁcial eﬀects to fade with chronic treatment. As a result, until contradictory data becomes available, that drug target has been assigned a low priority at J&JPRD thus saving time and resources for other projects. In the realm of clinical trials, results from the PhysioLab have been used to eliminate arms in a Phase I study. The clinical investigators were concerned that hypoglycemia was possible in normal volunteers undergoing an oral glucose tolerance test after treatment with their drug candidate. The biosimulation showed both the maximal eﬀect and during what time frame the glucose lowering was likely to occur. After careful examination of the model and results, the team decided to forgo two-thirds of the lower dose testing in the

301 Oral Glucose Tolerance Test - Normal Volunteers 180

Plasma Glucose (mg/dL)

160

Simulated Placebo Simulated 800 mg Trial Placebo Trial 800 mg

140 120 100 80 60 40 20 0 0

0.5

1

1.5

2

2.5

3

3.5

4

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Fig. 5. Oral glucose tolerance test – normal volunteers.

study. The clinical ﬁndings at the high dose conﬁrmed the model predictions, again saving time and resources while still providing the information needed to proceed (Fig. 5).

Summary Drug development is a costly process for pharmaceutical companies and is a major contributor to the ﬁnal market price. Developing one new drug takes an estimated 12–15 years and costs over $500 million. Approximately one-third of this amount can be attributed to drug and experiment failures. The failure rate in clinical trials is over 80%. Because of this failure rate in clinical trials, using virtual patients reduces both costs and years of development. Biosimulation will contribute to a better understanding of diabetes and other diseases, help drug companies improve the research and development process, and bring more eﬀective drugs to market with faster development.

Acknowledgments Thanks to the following for help with this paper. Seema Kumar, Vice President of Communications, J&J PRD, Raritan, New Jersey; James Karis, Entelos, Foster City, California; Andrea Braidman, Entelos, Foster City, Calﬁornia.

302 References 1. http://www.entelos.com/science/ 2. http://pf.fastcompany.com/magazine/74/5tech.html 3. News release on J&J PRD’s collaboration in June 2002: http://www.diabetes.org/community/ info_news/entelos_jnj.jsp 4. Gary Stix. ‘‘Reverse-Engineering Clinical Biology’’. Scientiﬁc American, 288(2): 28–30. Feb 2003. http://www.sciam.com/article.cfm?articleID ¼ 000C4F01-FABE-1E19-8B3B809EC588 EEDF&pageNumber ¼ 1&catID ¼ 2

303

Index of authors Allen, R.S. 265 Bartsell, L.T. 297 Bottomley, S.P. 31 Buchberger, B. 1 Cabrita, L.D. 31 Desiere, F. 51 Elliott, S. 237 Foote, M.A. 237, 251, 259 Fujitani, M. 123 Hasegawa, Y. 123 Ho, R.L.X. 297 Hoﬀmann, M. 1 Kennedy, N. 189 Leigh, B.R. 265 Liang, B.C. 265 Madin, K. 1

Molineux, G. 237 Multani, P.S. 265 Nemetz, C. 1 O’Connor, K. 151 Paparini, A. 85 Rainczuk, A. 189 Romano-Spica, V. 85 Rowley, M.J. 151 Smalling, R. 237 Smooker, P.M. 189 Spithill, T.W. 189 Swanson, S.J. 237 Theuer, C.P. 265 Wijeyewickrema, L. 151 Yamagishi, S. 123 Yamashita, T. 123

305

Keyword index adverse drug reaction reporting systems 237 agriculture 85 anemia 237 animals 85 anti-nutrients 85 antibiotic resistance 85 antibodies 265 antibody 151 apoptosis 123 arthritis 297 asthma 297 autoimmunity 151 axon 123 B cells 265 BAR 85 bioinformatics 51 biosimulation 297 biotechnology 85, 297 biotechnology–medical biotechnology 259 BT 85 CD20 265 carbohydrate antigens 151 cell survival 123 cell-free expression 1 cells 51 central nervous system 123 clinical research–publication of trials 259 clinical trials 265, 297 conformational epitope 151 critical contact residues 151 crops 85 cry 85 cysteine-constrained libraries 151 diabetes 297 diagnostics 51 diet 51, 85 discontinuous epitope 151 disease 51 DNA antigens 151 intake 85 uptake 85 vaccine 189 dosimetry 265

drug development 297 drug discovery 297 E. coli lysate 1 epigenetics 51 epitope 151 EPSPS 85 erythropoietin 237 expression library immunization 189 ﬁlamentous bacteriophage 151 food allergies 85 intolerance 85 safety 85 G protein 123 GE 85 gene speciﬁc library 151 genetic manipulation 85 genetic modiﬁcation 85 genetically engineered organisms 85 genetically manipulated organisms 85 genetically modiﬁed organisms 85 genomics 51 glia 123 GMO 85 good publication practices 259 growth factor 123 guidance documents 251 health 51 herbicide tolerance 85 high-throughput screening 1 homology modeling 151 horizontal transfer 85 human vaccine 189 ibritumomab tiuxetan 265 imaging 265 immunofootprinting 151 in vitro translation 1 inclusion body 31 insect-resistant 85 International Conference on Harmonisation 251 labeling 85 laboratory 237

306 malaria 189 markers 237 marketing applications 251 metabolism 51 metabolomics 51 mice 189 migration 123 mimotopes 151 modeling 297 molecular databases 51 myelin 123 networks 51 neurite 123 neuron 123 neurotrophin 123 NHL 265 novel food 85 nptII 85 nucleic acid vaccine 189 nutrigenomics 51 nutrition 51 obesity 297 oligodendrocyte 123 p75 123 pIII 151 pVI 151 pVIII 151 parasite 189 peptide 123 phage displayed libraries 151 pharmacogenetics 51 pharmacogenomics 51 PhRMA guidelines 259 plants 85 polymorphisms 51 prevention 51 prime-boost vaccine 189 protein aggregation 31

engineering 31 expression 31 folding 31 puriﬁcation 31 protein–protein interactions 1 proteomics 51 protozoa 189 public health 85 random peptide libraries 151 receptor 123 receptor–ligand interactions 151 recombinant 85 refolding 31 regeneration 123 regulations 85 regulatory interactions 251 rho 123 rituximab 265 ruminants 189 scale-up 1 signal 123 SNPs 51 solubility 1 synapse 123 systems biology 51 template optimization 1 transcriptomics 51 transgenic 85 tumor-speciﬁc antigens 151 unconstrained libraries 151 uniform requirements for biomedical journals 259 vaccines 151 X-ray crystallography 151 wheat germ 1