Abstract
The use of combinatorial libraries displayed on the surface of filamentous bacteriophage offers an efficient route to obtain a diverse set of monoclonal antibodies (MAbs) with desired specificity (1,2). However, following selection of such libraries, certain epitope specificities may dominate the cloned Abs because, for example, the epitope is immunodominant or because Abs to that epitope have a selective advantage in the panning process arising from a higher affinity. Although these attributes may be desirable, other selection conditions are required to recover Abs with different characteristics. In this chapter, an epitope-masking strategy is described by which Abs to an extended range of epitopes can be retrieved from phage-display libraries. The strategy uses antigen (Ag)-specific Fabs or single-chain Abs retrieved by an initial selection of the phage libraries to block the corresponding immunodominant epitope. Reselection of the phage library on the masked Ag refocuses the selection against other epitopes on the Ag (see Fig. 1).
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Burton, D. R. and Barbas, C.F. (1994) Human antibodies from combinatorial libraries. Adv. Immunol. 57, 191–280.
Ditzel, H. J. (1997) Combinatorial libraries: an approach for generating human monoclonal antibodies, in Immunotechnology (Gosling, J. P. and Reen, D. J., eds.), Portland, London, UK, pp. 27–38.
Ditzel, H. J., Binley, J. M., Moore, J. P., Sodroski, J., Sullivan, N., Sawyer, L. S. W., et al. (1995) Neutralizing recombinant human antibodies to a conformational V2-and CD4-binding site-sensitive epitope of HIV-1 gp120 isolated by using an epitope-masking procedure. J. Immunol. 154, 893–906.
Burton, D. R., Barbas, C. F., Persson, M. A. A., Koenig, S., Chanock, R. M., and Lerner, R. A. (1991) A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. NatlAcad. Sci. USA 88, 10,134–10,137.
Barbas, C. F., Collet, T. A., Amberg, W., Roben, P., Binley, J. M., Hoekstra, D., et al. (1993) Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries. J. Mol. Biol. 230, 812–823.
Moore, J. P. and Ho, D. D. (1993) Antibodies to discontinuous or conformationally sensitive epitopes on the gp120 glycoprotein of human immunodeficiency virus type 1 are highly prevalent in sera of infected humans. J. Virol. 67, 863–875.
LaRosa, C. J., Davide, J. P., Weinhold, K., Waterbury, J. A., Profy, A. T., Lewis, J. A., et al. (1990) Conserved sequence and structural elements in HIV-1 principal neutralizing determinant. Science 249, 932–935.
Lamers, S. L., Sleasman, J. W., and Goodenow, M. M. (1996) A model for alignment of Env V1 and V2 hypervariable domains from human and simian immunodeficiency viruses. AIDS Res. Hum. Retroviruses 12, 1169–1178.
Ditzel, H. J., Parren, P. W. H. I., Binley, J. M., Sodroski, J., Moore, J. P., Barbas, C. F., and Burton, D. R. (1997) Mapping the protein surface of human immunodeficiency virus type 1 gp120 using human monoclonal antibodies from phage display libraries. J. Mol. Biol. 267, 684–695.
Ping, T., Tornetta, M. A., Ames, R. S., Bankosky, B. C., Griego, S., Silverman, C., et al. (1996) Isolation of a neutralizing human RSV antibody from a dominant, non-neutralizing immune repertoire by epitope-blocked planning. J. Immunol. 157, 772–780.
Williamson, R. A., Peretz, D., Smorodinsky, N., Bastidas, R., Serban, A., Mehl-horn, I., et al. (1996) Circumventing tolerance in order to generate autologous monoclonal antibodies to the prion protein. Proc. Natl. Acad. Sci. USA 93, 7279–7282.
Williamson, R. A., Peretz, D., Pinilla, C., Ball, H., Bastidas, R. B., Rozenshteyn, R., et al. (1998) Mapping the prion protein using recombinant antibodies. J. Virol. 72, 9413–9418.
Martin, F., Toniatti, C., Salvati, A. L., Ciliberto, G., Cortese, R., and Sollazzo, M. (1996) Coupling protein design and in vitro selection strategies: improving specificity and affinity of a designed beta-protein IL-6 antagonist. Mol. Biol. 255, 86–97.
Ames, R. S., Tornetta, M. A., Jones, C. S., and Tsui, P. (1994) Isolation of neutralizing anti-C5a monoclonal antibodies from a filamentous phage monovalent Fab display library. J. Immunol. 152, 4572–4581.
Lowman, H. B., Bass, S. H., Simpson, N., and Wells, J. A. (1991) Selecting high-affinity binding proteins by monovalent phage display. Am. Chem. Soc. 30, 10,832–10,838.
Meulemans, E. V., Slobbe, R., Wasterval, P., Ramaekers, F. C. S., and van Eys, G. J. J. M. (1994) Selection of phage-displayed antibodies specific for a cytoskel-etal antigen by competitive elution with a monoclonal antibody. J. Mol. Biol. 244, 353–360.
Sanna, P. P., Williamson, A., De Logu, A., Bloom, F. E., and Burton, D. R. (1995) Directed selection of recombinant human monoclonal antibodies to HSV glycoproteins from phage display libraries. Proc. Natl Acad. Sci. USA 92, 6439–6443.
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Ditzel, H.J. (2002). Rescue of a Broader Range of Antibody Specificities Using an Epitope-Masking Strategy. In: O’Brien, P.M., Aitken, R. (eds) Antibody Phage Display. Methods in Molecular Biology™, vol 178. Humana Press. https://doi.org/10.1385/1-59259-240-6:179
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DOI: https://doi.org/10.1385/1-59259-240-6:179
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