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Biotechnology

Polyclonals: A Third Generation of Therapeutics Recombinant polyclonal overcome many of the challenges faced by, first, immunoglobulin therapy, and second, monoclonal antibodies to offer a third generation of antibody therapeutics.

By Kaja Tengbjerg and John S Haurum at Symphogen

Kaja Tengbjerg, MS, has recently taken up a position as Executive Assistant to John Haurum, CSO, Symphogen. She received a BS in Biochemistry and an MS in Human Biology at the University of Copenhagen, Denmark. She has eight years’ research experience in tumour immunology and antibody discovery. Before joining Symphogen as Scientist in 2001 she worked at the Danish Cancer Society and the University Hospital of Maastricht, the Netherlands.

John Haurum, MD, DPhil, is co-founder and Chief Scientific Officer of Symphogen. Dr Haurum received a DPhil in Immunology from the University of Oxford, England, and holds a Scandinavian International Management Institute diploma in Managing Medical Product Innovation. Before founding Symphogen, he took up a position as Assistant Professor at the Danish Cancer Society and completed his medical training. He has extensive research experience in molecular immunology and biochemistry and has published more than 30 articles in international scientific journals.

Antibodies have been used as therapeutics in various forms recognised more than a century ago, when it was discovered for over a century. Traditional immunoglobulin therapy that protection against certain toxins was conveyed by has the advantage of reflecting the diversity of the natural substances in the blood – referred to as antitoxins or immune response, but has limited clinical applications. antibodies. Since the first administration by von Behring (1) During the past 10 years, many monoclonal antibodies of antibodies in the form of animal-derived sera in the have been successfully introduced to the market. The 1890s, antibody therapeutics have come a long way. approach has the advantage of specificity, but often lacks efficacy in the treatment of Traditional Immunoglobulin Therapy diseases caused by complex antigens. Recombinant Early antibody therapy involved purification of the are expected to have the ability to immunoglobulin fraction of animal and later human tackle complex and highly mutagenic targets, and hold donor plasma, and subsequent infusion in patients. much promise for the future – but their large-scale Plasma-derived immunoglobulin from normal healthy industrial production has, until recently, remained elusive. donors offers the advantage of the polyclonal natural However, new discovery and expression technology immune response, with both a diverse and specific platforms – as discussed in the present review – have repertoire, and remains a preferred choice in the treatment provided a means for the consistent and robust of selected conditions (2). manufacturing of recombinant polyclonal antibody Deriving immunoglobulin from human plasma that compositions, and thus constitutes a third generation of reflects the multitude of binding specificities in the antibody therapeutics to enter clinical development. natural immune response implies that only a small fraction of all the immunoglobulin injected is actually ANTIBODY THERAPEUTICS targeting the particular antigen of interest. One way of A key aspect of the body’s reaction to infection is the enhancing the amount of relevant antibodies is to use activation and clonal expansion of many different antigen- hyperimmune immunoglobulin. These are derived from reactive B lymphocytes. Once these have matured into individuals who have, for instance, recovered from an plasma cells (antibody-producing cells), each clone of cells infection and have developed a high titre of antibodies will secrete its own unique specificity of antibody – thus, the against certain disease-related antigens. The products are invading pathogen will be met by a barrage of antibody therefore highly dependent on donor blood availability, molecules capable of binding at many different sites on its both in terms of quantity and suitability, resulting in surface. The range of specificities and affinities of such a considerable variation between batches. In addition, polyclonal response can shift with time making it ideal for screening technologies may fail to detect donor-derived combating infection. The importance of antibodies was pathogens, and thus immunoglobulin products carry a

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potential risk of infectious disease transmission. Today, humanised antibodies, where only the antigen-binding hyperimmune immunoglobulin is used for the parts of the antibody are of animal origin, whereas the prophylaxis or treatment of a number of infectious remaining regions of the antibody are replaced by human diseases caused by, for example, hepatitis B virus, counterparts. Another way has been to completely avoid cytomegalovirus, and tetanus or botulinum intoxication, non-human antibodies through the development of new as well as prophylaxis of Rhesus D allo-immunisation. technologies, such as phage display and human antibody Animal-derived immunoglobulins essentially overcome transgenic animals. the shortage of supply for human plasma-derived Phage display allows target-specific screening of large products. Thymoglobulin (Genzyme) – a hyperimmune so-called combinatorial antibody libraries for the immunoglobulin purified from blood of rabbits identification of potentially useful antibodies, which immunised with human T-lymphocytes – is successfully subsequently can be produced in large quantities. The used in the treatment or prevention of solid organ technology uses bacteria and bacterial viruses (phages) to transplant rejection. However, the animal origin of these express and select recombinant antibodies that have the products constitutes a manufacturing challenge, since the target recognition qualities of natural human antibodies presence of even small amounts of otherwise innocuous (6). However, this method relies on random pairing of the animal protein – such as animal albumin – would add to antibody heavy and light chains, which disrupts the the inherent risks of anti-animal responses associated with original pairing of the antibody chains, potentially leading their use in humans. Like human immunoglobulin to new reactivity as well as cross-reactivity patterns. products, animal-derived immunoglobulins also contain a Transgenic animals, on the other hand, are majority of irrelevant antibody molecules not binding to engineered to replace their endogenous antibody genes the intended target, and potentially carry the risk of with genes encoding human antibody sequences. transmitting infectious pathogens (including prions) which Following immunisation with the appropriate target considerably limit their clinical applications (3). antigen, these transgenic animals develop target-specific immune responses and can thereby act as a source for Monoclonal Antibodies antibodies homologous to human antibodies (3). In the 1980s, Nobel Prize-winners Kohler and Milstein developed a method of producing highly specific POLYCLONALS VERSUS MONOCLONALS antibodies: monoclonal antibodies (mAb). Hybridoma A natural antibody-mediated immune response involves cells secreting antibodies of the appropriate a series of direct and indirect effector mechanisms. These characteristics could be obtained by fusing antibody- are triggered by the synergistic action of antibodies, with producing cells with immortalised cells, and then a plurality of specificities binding several . The subsequent single cell cloning (4). -specific nature of monoclonal antibodies does Monoclonal antibodies have revolutionised the use of not generate such a concerted action. Another inherent pharmaceuticals of biological origin in all aspects of feature of monoclonal antibodies, which might have medicine (5). They provide the ability to have an implications for their clinical use, is the fact that all the unlimited supply of a single antibody that is clearly molecules compete for the same antigenic epitope and defined, and of reproducible affinity and specificity. In epitope density consequently becomes a limiting factor. theory, mAb technologies allow the development of an The only way to improve efficacy is to increase the dose antibody against any target of choice, whereas plasma- of drug; however, this also increases the risk of side effects derived immunoglobulin products are highly dependent resulting from the excess of unbound antibodies binding on sufficient and suitable blood donors. to tissues other than the target. Nonetheless, mAb products have been successfully The Development of Recombinant Antibodies introduced into the clinical management of cancer, for Adverse immune reactivity against non-human proteins example: Rituxan (Genentech/Biogen Idec), an anti-CD20 after repeated use of hybridoma antibodies, which are antibody approved for treatment of various B cell mainly derived from rodents, are a concern in a clinical malignancies; and Herceptin (Genentech/Roche), an anti- setting as these might lead to hyper-responsiveness and Her2 antibody used in certain breast cancer indications. – in severe cases – anaphylaxis in immunocompetent Both antibodies have improved the clinical response rates of individuals. These issues have been addressed by different anti-cancer treatment, but problems with significant relapse approaches. One way has been to produce genetically rates and drug resistance remain. Thus, it is believed by engineered antibodies, for example, chimeric or many that a mixture of antibodies may provide improved

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Figure 1: The SymplexTM technology. Antibody- Figure 2: The SympressTM technology comprising eliciting a range of secondary effector producing cells are isolated from the blood of immune establishment of a polyclonal master cell bank and individuals by single cell sorting using flow cytometry production of a polyclonal antibody composition functions including activation of

Antibody heavy and light chain mRNAs are reverse-transcribed, amplified and linked Constructs expressing each of the selected antibodies are transfected into mammalian complement, opsonisation and antibody- by the Symplex PCR, preserving the natural heavy and light chain pairing. Following cells, selected for stable integration and stored. Vials of each clone producing the high throughput screening for antigen specificity, antibodies can be selected and selected antibodies are mixed together to constitute a polyclonal master cell bank, dependent cellular cytotoxicity. expressed – mirroring the natural immune response. Adapted from (14). which is subsequently used as inoculation material for production. Production can be scaled up in a reproducible manner without altering the representation in the final The first description of methods Lymphocyte Discarded cells product of the clones in the master bank. Adapted from (14). isolation using phage display to isolate and Production of Manufacturing scale production Antibody-producing cells Transfection polyclonal generate antibody-encoding libraries – are single cell-sorted and selection cell bank Lab-scale Blood donation production which can be utilised as a template for

Immune individual the development of recombinant - Vaccination - Natural immunity polyclonal antibodies – was in 1994 Cognate pairs of antibody genes Antibody constructs Storage of Storage of are linked by Symplex PCR individual polycolonal clones master cell by Sarantopoulos et al (10). These bank Polyclonal antibody expression concepts have subsequently been used High-throughput screening, for * Site-specific integration (Flpln) Antigen-specific example by Elisa for antigen-specifity * Single integration event per cell to develop libraries for the identification antibodies * Homogenous expression and growth of polyclonal antibodies against, for potency due to greater antigen coating, as well as reduced example, colorectal cancer (7), Cryptosporidium parvum susceptibility to immune escape of the cancer (7). Likewise, (11) and Rhesus D-expressing erythrocytes (12). the ability of pathogens to evade a polyclonal response is less probable, and recombinant polyclonal antibodies might Mirroring Nature Using SymplexTM Technology very well offer a better alternative for the treatment of A new discovery platform, SymplexTM (see Figure 1), is a infectious diseases, where monoclonal antibodies often lose novel, high-throughput technology that allows direct their effectiveness because of the antigenic drift presented by cloning, screening and identification of antibody drug leads highly mutagenic targets as, for example, viruses (8). from human individuals generating disease-specific Finally, the immunogenicity of therapeutic antibodies antibodies (through vaccination or natural immunity). is a concern, not only as mentioned earlier because of Antibody-producing cells are isolated from the blood of adverse reactions leading to hypersensitivity and in severe immune individuals by single cell sorting. Antibody heavy cases anaphylaxis, but also because anti-drug antibodies and light chain mRNA are then reverse-transcribed, may impact negatively on the pharmacodynamics of even amplified and linked by SymplexTM PCR. Unlike phage fully human antibody drugs. Interestingly, an animal study display-based approaches, the key feature of this technology performed in our laboratory has shown that the use of is the ability to capture immune antibody repertoires while polyclonal antibody compositions generally evoked fewer preserving the original pairing of the antibody heavy and blocking antibodies (anti-idiotypic antibodies) in the light chain (‘cognate pairing’). Using model antigens, we recipient than the respective monoclonal antibodies have been able to isolate a wide spectrum of unique administered alone at the same dose range (9). antibodies which represent the genetic and functional diversity of the natural antibody response in the immune RECOMBINANT POLYCLONAL ANTIBODIES individuals (13). Furthermore, the technology preserves the The third generation of antibody therapeutics, recombinant diversity, affinity and specificity of the natural repertoire, polyclonal antibodies, aims to tackle and can thereby provide detailed insight into the nature and Figure 3: Analysis of Sym001 purified the shortfalls of the first two diversity of the natural human antibody response. from bioreactor cultures generations of antibody therapeutics by Three different recombinant polyclonal antibody preparations were produced to a 5L scale Sym001:5/21, Sym001:5/23 & 24, and Sym001:5/03 and mimicking nature’s way. Recombinant Manufacturing Polyclonal Recombinant Antibodies one to a 400L scale. Elution profiles from cation exchange chromatography were used to visualise compositional reproducibility (13). polyclonal antibodies binding in close Constructs expressing selected antibodies, for example proximity to multiple target epitopes identified by SymplexTM technology, are sub-cloned into will accordingly provide neutralisation the mammalian expression platform, SympressTM (see 450 Sym001: 5/21 by steric hindrance (antigens coated Figure 2) for manufacturing antigen-specific recombinant Sym001: 5/23 & 24 400 Sym001: 5/03 350 with antibodies are prevented from human polyclonal antibodies (14). Different from Sym001: 400/01 TM 300 attaching to host cells or mucosal conventional expression technologies, Sympress 250 employs a site-specific recombinase recognition system, 200 surfaces), and agglutination or 150 precipitation (antibodies binding ensures that only one copy of a plasmid is integrated into 100 several soluble antigens cause any one cell at the same genomic site in the producer 50 0 aggregation and subsequent clearance). cells. Thus, genomic position effects are minimised and 0 102030405060 They are also believed to be capable of manufacturing consistency and robustness are further

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supported by selection of stably transfected producer References cells. A polyclonal cell bank is subsequently generated by mixing the individual antibody-producing cell lines. Cells 1. Gavilondo JV and Larrick JW (2000). Antibody from the polyclonal cell banks are then used for seeding engineering at the millennium. BioTechniques, 29, the bioreactors at the production facility. As exemplified 128-6, 138. by the manufacture of Sym001, production can be scaled 2. Farrugia, A and Poulis, P (2001) Intravenous up in a reproducible manner without altering the immunoglobulin: regulatory perspectives on use and representation in the final product of the clones in the supply. Transfus. Med. 11, 63-74 master bank (see Figure 3). We have analysed several 3. Kellermann, SA and Green LL (2002) Antibody production runs of Sym001 anti-RhD antibodies, and discovery: the use of transgenic mice to generate they all show batch-to-batch consistency in respect of human monoclonal antibodies for therapeutics. Curr their protein chemistry characteristic, as well as their Opin. Biotechnol 13, 593-597 antigen-binding properties and ability to elicit effector 4. Kohler G and Milstein C (1975) Continuous cultures function in vitro (14). of fused cells secreting antibody of predefined specificity. Nature 256, 495-497 CONCLUSION 5. Brekke, OH and Sandlie, I (2003) Therapeutic Antibodies have been around as therapeutics for more antibodies for human diseases at the dawn of the than a century in various forms. First they were twenty-first century. Nat. Rev. Drug Discov. 2, 52-62 administered as immunoglobulins after extraction from 6. Marks, JD et al. (1991) By-passing immunization. suitable donor plasma. This first generation of antibody Human antibodies from V-gene libraries displayed on therapeutics which reflects the full diversity of the phage. J. Mol. Biol. 222, 581-597 natural immune response, but also presents several 7. Sharon, J et al. (2005) Recombinant polyclonal disadvantages such as safety issues, lack of specificity and antibodies for cancer therapy. J Cell Biochem. 96, limited clinical applications. Nevertheless, plasma- 305-313 derived immunoglobulin products are still the best 8. Bregenholt, S et al. (2006) Recombinant human treatment available for many clinical conditions. polyclona antibodies: A new class of therapeutic The second generation of antibody therapeutics antibodies against viral infections. Curr. Pharm. constitutes monoclonal antibodies and fragments Design 12, 2007-2015 thereof. These antibodies offer numerous advantages 9. Klitgaard JL et al. Reduced susceptibility of recombinant over plasma-derived immunoglobulins including polyclonal antibodies to inhibitory anti-variable domain better safety, reproducibility and defined specificity. antibody responses. J. Immunol. In press Monoclonal antibodies constitute a substantial 10. Sarantopoulos, S. et al. (1994) A method for linking VL commercial success, especially in the last 10 years, and and VH region genes that allows bulk transfer between they will continue to do so with a promising pipeline of vectors for use in generating polyclonal IgG libraries. more than 100 molecules in development. However, J. Immunol. 152, 5344-5351 their monospecific nature limits their clinical potential in 11. Baecher-Allan, C.M. et al. (1999) Generation of a the treatment of diseases caused by complex antigens. polyclonal Fab phage display library to the protozoan Recombinant polyclonal antibodies – the most recent parasite Cryptosporidium parvum. Comb. Chem. High antibody therapy innovation – provide an answer to most Throughput. Screen. 2, 319-325 of the challenges faced by the previous two generations of 12. Andersen, PS et al. (2006) Extensive restrictions in the antibody therapeutics (15). Until now, the large-scale VH sequence usage of the human antibody response industrial production of polyclonal compositions has against the Rhesus D antigen. Mol Immunol. In press remained elusive. However, the development of an 13. Meijer, PJ et al. (2006) Isolation of human antibody expression platform (SympressTM) has made it feasible to repertoires with preservation of the natural heavy and consistently manufacture recombinant polyclonal light chain pairing. J Mol Biol 358, 764-772 antibodies originating from immune human individuals, 14. Wiberg, FC et al. (2006) Production of target-specific thereby opening the way for third-generation antibody recombinant human polyclonal antibodies in therapeutics to progress to clinical development. mammalian cells. Biotechnol Bioeng. 94, 396-405 15. Haurum, JS (2006) Recombinant polyclonal antibodies: The authors can be contacted at the next generation of antibody therapeutics? Drug [email protected] and [email protected] Discov Today 11, 655-60

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