Therapeutics


References 1 Reichert, JM. Antibody-based therapeutics to watch in 2011. MAbs. 11;3(1), 2010. 2 Nelson, AL, Dhimolea, E, Reichert, JM. Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov. 9(10):767-74, 2010. 3 Hoogenboom, HR. Selecting and screening recombinant antibody libraries. Nat Biotechnol. (9):1105-16, 2005. 4 Bratkovic, T. Progress in phage display: evolution of the technique and its application. Cell Mol Life Sci.67(5):749-67, 2010. 5 Sidhu, SS, Fellouse, FA. Synthetic therapeutic antibodies. Nat Chem Biol. 2(12):682-8, 2006. 6 Fellouse, FA, Wiesmann, C and Sidhu, SS. Synthetic antibodies from a four-amino- acid code: a dominant role for tyrosine in antigen recognition. Proc. Natl. Acad. Sci. USA 101, 12467–12472, 2004. 7 Hoet, RM et al. Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining- region diversity. Nat. Biotechnol. 23, 344–348, 2005. 8 Knappik, A, Ge, L, Honegger, A, Pack, P, Fischer, M, Wellnhofer, G, Hoess, A, Wölle, J, Plückthun, A, Virnekäs, B. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol. 296(1):57-86, 2000. 9 Stemmer, WP. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci U S A. 25;91(22):10747-51, 1994. 10Yang, WP, Green, K, Pinz- Sweeney, S, Briones, AT, Burton, DR, Barbas, CF 3rd. CDR Walking Mutagenesis for the Affinity Maturation of a Potent Human Anti-HIV-1 Antibody into the Picomolar Range. J Mol Biol.1;254(3):392- 403. 1995.


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bypassing the hybridoma technology, which relies on immunisation of animals. Today, new selection meth- ods enable the timely and cost-efficient screening of these large libraries basing conclusions on the anti- body-antigen behaviour of individual clones. Antibody selection platforms include ribosome and mRNA display, yeast cell display and phage display3. The most dominant platform today remains the


20-year-old phage display technology. Phage dis- play enables in vitro selection and the rapid identi- fication and optimisation of proteins based on their functional and structural behaviour. Bacteriophages (phages) are viruses that can infect bacteria. Each phage in the display library displays a unique antibody, protein or peptide. The design of the phage vector ensures that the library pro- tein/antibody is expressed as a fusion protein with the coat protein and exposed on the surface of the phages that are released from the bacterial cells. Phages containing specific mAbs can be isolated upon their binding affinity to the desired antigen4. A novel and efficient approach to display tech- nology combines the advantage of the convention- al phage display technology with the additional feature of a cleavable disulfide bridge. This allows for the elution of the antibody that displays phage binding to an antigen by the disruption of this disulfide bridge, a consequence of the bridge’s sen- sitivity to reducing agents. This technology enables the specific isolation of phage particles independ- ent of their affinity to the antigen and ensures high- throughput screening in an automated way.


Sophisticated antibody libraries – the best antibody possible


At the heart of modern antibody screening tech- nologies is the antibody library. The quality of an in vitro library is characterised by the ability to gener- ate a large population of highly diverse and func- tional antibodies. Recombinant antibody libraries can be based on gene pools that originate from source B-cells of immunised animal, naturally immunised humans or infected humans. Synthetic libraries are constructed completely in vitro5. The understanding of the structural and func- tional characteristics of antibodies has led to pre- cisely designed and highly-functional synthetic and semi-synthetic libraries. Semi-synthetic libraries reflect natural and synthetic diversity. Several groups have constructed these synthetic libraries to yield in high-affinity antibodies5.


Fellouse and colleagues developed libraries with solvent-accessible CDR positions that were ran- domised with a degenerated codon encoding for only four amino acids6. The authors showed a


dominant role for tyrosine in antigen recognition and demonstrated that synthetic libraries can be used to study the role of different amino acids for antigen recognition. The donors included normal donors and patients with various autoimmune dis- eases in an effort to extend the library’s repertoire beyond that of healthy individuals. Therefore, these human Fab libraries represent immunoglobulin sequences captured from human donors combined with synthetic diversity in the key antigen contact sites found in the heavy-chain complementary- determining regions CDR1 and CDR2. Also known as Dyax libraries, they include CDR1 and CDR2 repertoires that are designed based on analysis of germline VH genes. Furthermore, mutations are designed according to related VH germline genes and the incorporation of hot spot mutations7. One example of a synthetic human Fab library that represents the structural diversity of the human antibody repertoire is Human Combinatorial Antibody Library (HuCAL®). The combination of the seven variable heavy chain (VH) and seven vari- able 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 antibodies8.


The goal of any antibody technology platform is the identification of antibodies with specific prop- erties such as high affinity, functionality (eg induc- tion of cell death, or blocking of receptor ligand interaction), good expression rate, high solubility to enable high concentration formulation and low immunogenicity. Therapeutic antibodies in partic- ular often require several rounds of further devel- opment and fine-tuning to achieve the complete set of desired properties – a long and costly process – before they can enter the clinic.


Antibody affinity maturation is a technique used to increase the affinity of an antibody to its antigen, a process that happens naturally during the adaptive immune response to an antigen. In vitro affinity maturation involves artificial and controlled muta- genesis and selection steps. The most commonly used method for antibody affinity maturation is the error-prone PCR. Thermus aquaticus polymerase (Taq-Pol) is used for PCR amplification of the whole V gene or the CDRs. This polymerase is known to introduce errors and the error-rate is increased by modifying the reaction conditions. Other mutagene- sis methods include DNA shuffling9, a method based on digestion of the target gene with DNase I and pooling random DNA fragments and CDR walking mutagenesis10 (reviewed in 11).


Drug Discovery World Winter 2010/11


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