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European Biopharmaceutical Review

Novel Libraries

The identifications of therapeutic antibodies often requires several rounds of further development and fine-tuning. However, current methods and technologies can accelerate antibody optimisation to generate superior therapeutic and diagnostic candidates.

Only 10 of the 29 FDA approved monoclonal antibodies (mAb) account for over $36 billion of annual sales. The market forecast predicts the number of approved mAbs will further increase and clearly demonstrates that it will remain one of the fastest growing segments within biotech and pharmaceutical research (1).

In the past, the identification and development of mAbs was a tedious and cumbersome process. The only methods available were immunisation and murine hybridoma technologies. Muromonab, the first therapeutic mAb that received approval by the US Food and Drug Administration (FDA) in 1986, was derived from murine hybridoma. Muromonab is an immunosuppressant that targets CD3 and is used as a therapeutic for acute allograft rejection in patients with organ transplants. Mouse mAbs often cause a reaction called human anti-mouse response (HAMA) where the patient’s immune system recognises these therapeutic antibodies as foreign and causes an immune response.

Besides these problematic and undesirable immune responses to therapeutic antibodies that can neutralise their effect, mouse-derived mAbs can also cause serious adverse events, such as hypersensitivity. To circumvent these problems, the next generation of antibodies were chimeric mAbs – mouse-human antibodies made up of two-thirds human sequence homology – followed by humanised mAbs, which have 95 per cent human sequence homology. The goal was to develop antibodies that were as human as possible. In 1990 only 11.5 per cent of all antibodies in clinical development were fully human; by 2000, this number had increased to 45 per cent (2). Today, several human mAbs have been approved in the US and they account for the majority of antibodies in clinical development. The complete elimination of murine-derived protein structures will lead to less immunogenic mAbs as compared to humanised or chimeric mAbs. It is noteworthy that two-thirds of the monoclonal antibodies currently on the US market are either chimeric or humanised monoclonal antibodies (2).

In vitro selection methods have set a new standard and have become one of the most important tools for screening and identifying mAbs that complement a variety of targets/antigens, while bypassing hybridoma technology that relies on the immunisation of animals. The development of new selection methods enables fast and cost-efficient screening of these large libraries, basing conclusions on the antibody-antigen behaviour of individual clones. Antibody selection platforms include ribosome, mRNA, yeast cell and phage display (3). In particular, the phage display technology has paved the way for the development of fully humanised mAbs.

Antibodies

Antibodies, also known as immunoglobulins, are characterised by their specific ‘Y’ shaped structure. The specificity of antibodies is determined by the amino acid sequence on the tips of the Y shape. The constant region (Fc domain) – at the stem of the antibody – can have only a limited number of forms, and plays a role in modulating the effector function of an antibody, which usually requires the prior binding of an antigen.Manipulations in the Fc regions can influence the pharmacokinetic properties of mAbs as well as improve the antibodydependent cellular cytotoxicity (ADCC). Additionally, the molecular structure of antibodies includes a specified variable region at the end of the light and heavy chains.

The antigen-binding activity of mAbs is determined by the conformation of its amino acids in its complementary determining regions (CDRs). Three CDRs are located in the variable region of both the light and the heavy chains of the antibody. The human immune system has the ability to create millions of different antibodies with high affinity to the target molecules. One of the greatest challenges in biomedical research on antibodies is to mimic the screening process of the human immune system as closely as possible in order to identify antibodies with the highest target/antigen specificity (3). Additionally, therapeutic antibodies have to be safe in humans and must have the ability to be manufactured on a large scale.

Phage Display
The most dominant platform today remains the 20 year-old phage display technology. Phage display is based on the infection of bacteria with viruses called bacteriophages (or phages). The quality of an in vitrolibrary is characterised by its ability to generate large populations of highly diverse and functional antibodies. Phage display enables in vitro selection, rapid identification and optimisation of proteins based on their functional and structural behaviour. Each phage in the display library displays a unique antibody, protein or peptide.

Two types of phage systems can be used: the phage vector containing the entire phage genome including the antibody genome, and the phagemid consisting of the phage surface proteinantibody fusion and a helper phage, which is essential for the replication and assembly of phagemid particles during the library production. The design of the phage vector guarantees that the library antibody is expressed as a fusion protein with the coat protein exposed on the surface of the phages that are released from the bacterial cells.

Phages containing specific mAbs can be isolated based on their binding affinity to the desired antigen during the screening process (4). Single-chain Fv (scFv) fragments and fragment antigen-binding (Fab) can be produced with phage display. A novel and efficient technology enables the isolation of specific binders independent of their affinity to the antigen and ensures highthroughput screening in an automated way. The antibody fragments are linked to the specific phage coat protein via an engineered intramolecular disulfide bridge, allowing affinity-independent elution by the disruption of this disulfide bridge through use of a reducing agent.

Yeast Cell Display
Another display method is the yeast surface display. This method was established in 1997 by Boder and Wittrup and further developed by Feldhaus and colleagues (5,6). The yeast system is a eukaryotic expression system that enables the expression of full length IgGs. The antibodies are expressed via fusion protein to the α- agglutinin yeast adhesion protein which is located on the yeast cell wall. The surface display is compatible with fluorescent-activated cell sorting (FACS) and allows quantitative library screening.

Ribosome and mRNA Display
The ribosome display is an in vitro system that is limited to the expression of single-chain proteins such as Fv, but this method does not require any transformation and is very amenable to mutagenesis. The use of nonproofreading polymerases in the polymerase chain reaction (PCR) during the mRNA synthesis ensures a high mutagenesis/recombination rate in a short time. Furthermore, eliminating the transformation reaction allows for the screening of very large libraries. Nevertheless, the method is more technically challenging because of the instability of mRNA and being limited to single chain proteins (3,7).

Finding the Right Antibody

Antibody libraries are one of the most important tools for screening and identifying mAbs against a specific target. Antibody libraries are distinguished by their source and design. Naïve libraries originate from primary B-cells of non-immunised donors, whereas immune libraries are derived from source B-cells of an immunised animal and naturally immunised or infected humans.

Understanding the structural and functional characteristics of antibodies has led to precisely designed and highly-functional synthetic and semisynthetic libraries. Semi-synthetic libraries reflect natural and synthetic diversity, and thus they contain CDRs from both natural and synthetic sources. Synthetic libraries are constructed completely in vitro (7,8).

Immune Libraries
Immune libraries are derived from immume donors who are already exposed to certain antigens. These libraries are typically small and not appropriate for the screening of a large panel of antigens. However, many more antibodies can be derived from a recombinant immume library made from one immunised donor compared to the hybridoma technology. Additionally, in vitro selection enables the screening for antibodies with certain specificities (3,7).

Naïve Libraries
Naïve libraries originate from primary B-cells of non-immunised donors. These libraries are obtained from unimmunised, rearranged V genes to reduce antigen-induced bias (3). The quality of an in vitro library is characterised by its ability to generate a large population of highly diverse and functional antibodies. This depends on the principle of in vivo recombination of the light and heavy chain genes of an antibody. These libraries have been constructed using either the entire Fab, the scFv that consists of the light- and heavy-chain variable domains, or just consist of the smallest antibody domain able to recognise antigens. Several groups have constructed these naïve libraries to yield high-affinity antibodies (8).

Semi-Synthetic Libraries
Semi-synthetic libraries include both CDRs from natural and syntheticallydesigned sources. The ratio of both varies in different libraries. The semisynthetic scFv-antibody phage display library of Hoogenboom and Winter includes 49 germline variable heavy chain (VH) sequences and a single V-lambda light chain sequence, and is one of the first semi-synthetic libraries (9).

The Dyax libraries are human antibody libraries that combine immunoglobulin sequences from human donors with synthetic diversity in the key antigen contact sites in heavy-chain complementarydetermining regions CDR1 and CDR2. The donors included healthy donors and patients with various autoimmune diseases. The latter helped to extend the libraries’ repertoire beyond that of healthy individuals. Additionally,mutations are designed according to related VH germline genes and the incorporation of hot spot mutations (10).

Synthetic Libraries
Fellouse and colleagues developed highly functional minimalistic phagedisplayed libraries for high-throughput generation of synthetic antibodies (11). This approach to engineering antibodies uses a single, highly stable Fab framework to support CDR diversity and is restricted to a binary code for only tyrosine (Tyr) and serine (Ser): two amino acids that are highly abundant in natural antigen-binding sites. These libraries effectively generated specific antibodies against a wide range of antigens and revealed a dominant role for tyrosine in antigen recognition (12). Using the most complex library combined with highthroughput technologies, they were able to produce high-affinity antibodies similar to those produced by the natural immune system (11). The Human Combinatorial Antibody Library (HuCAL®) is another fully synthetic human Fab library with structural diversity similar to the human antibody repertoire. The combination of the seven VHs and seven variable light chain (VL) region genes gives rise to 49 frameworks in the master library. These regions comprise the six CDRs, resulting in a collection of several billion distinct fully human antibodies. The most recent library is based on 45 billion different fully human antibodies (13).




Antibody Optimisation

Antibodies, especially therapeutic mAbs, require properties such as high affinity, functionality (for example, the induction of cell death or blocking of receptor ligand interaction), good expression rate, high solubility to enable high concentration formulation and low immunogenicity. Several rounds of further development and fine-tuning are often needed to achieve the complete set of desired properties required before they can enter the clinic. This is a long and costly process and makes great demands on antibody technology platforms.

Antibody affinity maturation is a process that happens naturally during the adaptive immune response to an antigen and increases the affinity of an antibody to its antigen. The in vitro affinity maturation process involves artificial and controlled mutagenesis and selection steps. The most commonly used method for antibody affinity maturation is error-prone PCR. Thermus aquaticus polymerase (Taq-Pol), a polymerase with a relatively high error rate, is used for the PCR amplification of the whole V gene or the CDRs. The errorrate is further increased by modifying the reaction conditions. Less common mutagenesis methods include DNA shuffling, a method based on digestion of the target gene with DNase I and pooling random DNA fragments. Another method is CDR walking mutagenesis (14-16).

Affinity maturation can also be achieved by replacing the natural structure of the VH and VL CDR3 region, the antibody’s most important region, with CDR3 library cassettes generated from mixed trinucleotides. This trinucleotide mutagenesis (TRIM) technology facilitates the synthesis of any desired combination of amino acids at each single position of the variable region in a ratio reflecting exactly that found in humans (8).

A more recent solid phase technology for high-throughput gene synthesis further accelerates and speeds up the antibody optimisation process by generating high quality and ‘intelligent mutant’ libraries and sub-libraries. This DNA engineering platform is based on the synthesis and ligation of stable double-stranded oligonucleotides with complementary regions. Two distinct hairpin double-stranded oligonucleotides are used as starting materials for the generation of small sub-fragments, called the elongation blocks. One of the oligonucleotides has a variable region of six nucleotides including three nucleotides 5’- overhangs (single-stranded), and, in addition, a biotin modification in the loop region. The biotin is used to bind the oligonuculotide to a streptavidin-coated surface during the immobilisation process. The second oligonucleotide has three nucleotides 5’-overhangs that also represent all possible permutations. The first step is the ligation of the complementary single-stranded overhangs of the two oligonucleotides. Repeating reaction cycles of immobilisation, washing, restriction and ligation results in blocks with 18 independent definable base pairs. These blocks are then assembled to form larger fragments of 462 base-pairs constructs, reflecting every possible permutation. The double-stranded structure of the oligonucleotides allows for stable, accurate, reliable and fast synthesis (see Figure 1) (17). Currently, this technology is used in combination with a synthetic human Fab library and enables the individual design of each CDR cassette for a variety of properties, such as high affinity and low immunogenicity, and it is applicable to all antibodies within the library.

Conclusion

Therapeutic antibodies remain a major focus of the biotech and pharmaceutical industry. Combinatorial antibody libraries have been established and are proven to be fast and successful for antibodies. Improved technologies and methods for design and screening of antibody libraries have matured, and in vitro antibody libraries have had a major impact on drug discovery and development. Human mAbs will remain a promising and rapidly growing category of targeted agents. Advances in technology will help to engineer mAbs with the desired properties required to target a variety of diseases.

References
  1. Reichert JM, Antibody-based therapeutics to watch in 2011, MAbs 3(1): pp76-99, 2011
  2. Nelson AL, Dhimolea E and Reichert JM, Development trends for human monoclonal antibody therapeutics, Nat Rev Drug Discov 9(10): pp767-774, 2010
  3. Hoogenboom HR, Selecting and screening recombinant antibody libraries, Nat Biotechnol (9): pp1,105-1,116, 2005
  4. Bratkovic T, Progress in phage display: evolution of the technique and its application, Cell Mol Life Sci 67(5): pp749-767, 2010
  5. Boder ET and Wittrup KD, Yeast surface display for screening combinatorial polypeptide libraries, Nat Biotechnol 15: pp553-557, 1997
  6. Feldhaus MJ et al, Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library, Nat Biotechnol 21: pp163-170, 2003
  7. Ponsel D, Neugebauer J, Ladetzki- Baehs K and Tissot K, High affinity, developability and functional size: the holy grail of combinatorial antibody library generation, Molecules 16(5): pp3,675-3,700, 2011
  8. Sidhu SS and Fellouse FA, Synthetic therapeutic antibodies, Nat Chem Biol 2(12): pp682-688, 2006
  9. Hoogenboom HR and Winter G, By-passing immunisation: human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro, J Mol Biol 227(2): pp381-388, 1992
  10. Hoet RM et al, Generation of high-affinity human antibodies by combining donorderived and synthetic complementaritydetermining- region diversity, Nat Biotechnol 23: pp344-348, 2005
  11. Fellouse FA et al, High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries,J Mol Biol 373(4): pp924-940, 2007
  12. Fellouse FA, Wiesmann C and Sidhu SS, Synthetic antibodies from a fouramino- acid code: a dominant role for tyrosine in antigen recognition, Proc Natl Acad Sci USA 101: pp12,467- 12,472, 2004
  13. Knappik A et al, Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomised with trinucleotides, J Mol Biol 296(1): pp57-86, 2000
  14. Stemmer WP, DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution, Proc Natl Acad Sci USA 91(22): pp10,747-10,751, 1994
  15. Yang WP, Green K, Pinz-Sweeney S, Briones AT, Burton DR and Barbas CF, CDR Walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range, J Mol Biol254(3): pp392-403, 1995
  16. Almagro JC and William R Strohl, Therapeutic Monoclonal Antibodies: From Bench to Clinical, John Wiley & Sons Inc, New Jersey: pp311-334, 2009
  17. Van den Brulle J et al, A novel solid phase technology for high-throughput gene synthesis, Biotechniques 45(3): pp340-343, 2008



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Markus Enzelberger is a chemist by training and received his PhD from the University of Stuttgart on the directed evolution of industrial enzymes. Markus did his postdoctoral studies with Stephen Quake at the California Institute of Technology where he worked on new microfludic systems and its application in high-throughput screening of biologics. He continued this work at Mycometrx Inc, South San Francisco. He has been with MorphoSys for nine years and currently holds the position of a Vice President of R&D. He is responsible for all partnered antibody discovery projects and technology development. Email:markus.enzelberger@morphosys.com
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