Abstract
Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected.
An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.
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References
Carter, P. J. (2006) Potent antibody therapeutics by design. Nat. Rev. Immunol. 6, 343–357.
Hoogenboom, H. R. (2005) Selecting and screening recombinant antibody libraries. Nat. Biotechnol. 23, 1105–1116.
Foote, J., and Winter, G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J. Mol. Biol. 224, 487–499.
Lee, C. V., Liang, W. C., Dennis, M. S., Eigenbrot, C., Sidhu, S. S., and Fuh, G. (2004) High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J. Mol. Biol. 340, 1073–1093.
Lee, C. V., Hymowitz, S. G., Wallweber, H. J., Gordon, N. C., Billeci, K. L., Tsai, S. P., et al. (2006) Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells. Blood 108, 3103–3111.
Johnson, G., and Wu, T. T. (2000) Kabat database and its applications: 30 years after the first variability plot. Nucleic Acids Res. 28, 214–218.
Eigenbrot, C., Randal, M., Presta, L., Carter, P., and Kossiakoff, A. A. (1993) X-ray structures of the antigen-binding domains from three variants of humanized anti-p185HER2 antibody 4D5 and comparison with molecular modeling. J. Mol. Biol. 229, 969–995.
Chothia, C., and Lesk, A. M. (1987) Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901–917.
Smith, G. P., and Scott, J. K. (1993) Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol. 217, 228–257.
Lee, C. V., Sidhu, S. S., and Fuh, G. (2004) Bivalent antibody phage display mimics natural immunoglobulin. J. Immunol. Methods 284, 119–132.
Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, NIH, US Department of Health and Human Services, Washington, DC.
Gallop, M., Barrett, R., Dower, W., Fodor, S., and Gordon, E. (1994) Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial libraries. J. Med. Chem. 37, 1233–1251.
Weiss, G. A., Watanabe, C. K., Zhong, A., Goddard, A., and Sidhu, S. S. (2000) Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc. Natl. Acad. Sci. USA 97, 8950–8954.
Vajdos, F. F., Adams, C. W., Breece, T. N., Presta, L. G., de Vos, A. M., and Sidhu, S. S. (2002) Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis. J. Mol. Biol. 320, 415–428.
Sidhu, S. S., Lowman, H. B., Cunningham, B. C., and Wells, J. A. (2000) Phage display for selection of novel binding peptides. Methods Enzymol. 328, 333–363.
Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382.
Sidhu, S. S., Lowman, H. B., Cunningham, B. C., and Wells, J. A. (2000) Phage display for selection of novel binding peptides. Methods Enzymol. 328, 333–363.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Bostrom, J., Lee, C.V., Haber, L., Fuh, G. (2009). Improving Antibody Binding Affinity and Specificity for Therapeutic Development. In: Dimitrov, A. (eds) Therapeutic Antibodies. Methods in Molecular Biology™, vol 525. Humana Press. https://doi.org/10.1007/978-1-59745-554-1_19
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DOI: https://doi.org/10.1007/978-1-59745-554-1_19
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