Abstract
Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Antibody clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a β-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5′-end of the β-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of 7.6 × 109, greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.
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Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, C.F., III (2001). generation of antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. In Phage Display: A Laboratory Manual, C.F. Barbas, III, D.R. Burton, J.K. Scott, and G.J. Silverman, eds. (Cold Spring Harbor, USA: Cold Spring Harbor Laboratory Press), pp. 9.1–9.111.
de Haard, H.J., van Neer, N., Reurs, A., Hufton, S.E., Roovers, R.C., Henderikx, P., de Bruine, A.P., Arends, J.W., and Hoogen-boom, H.R. (1999). A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J. Biol. Chem. 274, 18218–18230.
de Wildt, R.M., Mundy, C.R., Gorick, B.D., and Tomlinson, I.M. (2000). Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat. Biotechnol. 18, 989–994.
Faix, P.H., Burg, M.A., Gonzales, M., Ravey, E.P., Baird, A., and Larocca, D. (2004). Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection. Biotechniques 36, 1018–1029.
Gerth, M.L., Patrick, W.M., and Lutz, S. (2004). A second-generation system for unbiased reading frame selection. Protein Eng. Des. Sel. 17, 595–602.
Hoet, R.M., Cohen, E.H., Kent, R.B., Rookey, K., Schoonbroodt, S., Hogan, S., Rem, L., Frans, N., Daukandt, M., Pieters, H., et al. (2005). Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat. Biotechnol. 23, 344–348.
Hoogenboom, H.R., and Chames, P. (2000). Natural and designer binding sites made by phage display technology. Immunol. Today 21, 371–378.
Jirholt, P., Ohlin, M., Borrebaeck, C.A., and Soderlind, E. (1998). Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework. Gene 215, 471–476.
Jones, P.T., Dear, P.H., Foote, J., Neuberger, M.S., and Winter, G. (1986). Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321, 522–525.
Kim, S.J., Park, Y., and Hong, H.J. (2005). Antibody engineering for the development of therapeutic antibodies. Mol. Cells 20, 17–29.
Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., and Virnekas, B. (2000). Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57–86.
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.
Loset, G.A., Lobersli, I., Kavlie, A., Stacy, J.E., Borgen, T., Kaus-mally, L., Hvattum, E., Simonsen, B., Hovda, M. B., and Brekke, O.H. (2005). Construction, evaluation and refinement of a large human antibody phage library based on the IgD and IgM variable gene repertoire. J. Immunol. Methods 299, 47–62.
Lutz, S., Fast, W., and Benkovic, S.J. (2002). A universal, vector-based system for nucleic acid reading-frame selection. Protein Eng. 15, 1025–1030.
Pini, A., Viti, F., Santucci, A., Carnemolla, B., Zardi, L., Neri, P., and Neri, D. (1998). Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel. J. Biol. Chem. 273, 21769–21776.
Rader, C., and Barbas, C.F., 3rd (1997). Phage display of combinatorial antibody libraries. Curr. Opin. Biotechnol. 8, 503–508.
Riechmann, L., Clark, M., Waldmann, H., and Winter, G. (1988). Reshaping human antibodies for therapy. Nature 332, 323–327.
Rothe, C., Urlinger, S., Lohning, C., Prassler, J., Stark, Y., Jager, U., Hubner, B., Bardroff, M., Pradel, I., Boss, M., et al, (2008). The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J. Mol. Biol. 376, 1182–1200.
Scott, J.K., and Barbas, C.F., III (2001). Phage Display Vectors. In phage display: A Laboratory Manual, C.F. Barbas, III, D.R. Burton, J.K. Scott, and G.J. Silverman, eds. (Cold Spring Harbor, USA: Cold Spring Harbor Laboratory Press), pp. 2.1–2.19.
Seehaus, T., Breitling, F., Dubel, S., Klewinghaus, I., and Little, M. (1992). A vector for the removal of deletion mutants from antibody libraries. Gene 114, 235–237.
Sidhu, S.S. (2001). Engineering M13 for phage display. Biomol. Eng. 18, 57–63.
Sidhu, S.S., and Fellouse, F.A. (2006). Synthetic therapeutic antibodies. Nat. Chem. Biol. 2, 682–688.
Silacci, M., Brack, S., Schirru, G., Marlind, J., Ettorre, A., Merlo, A., Viti, F., and Neri, D. (2005). Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics 5, 2340–2350.
Soderlind, E., Strandberg, L., Jirholt, P., Kobayashi, N., Alexeiva, V., Aberg, A.M., Nilsson, A., Jansson, B., Ohlin, M., Wingren, C., et al, (2000). Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat. Biotechnol. 18, 852–856.
Vaughan, T.J., Williams, A.J., Pritchard, K., Osbourn, J.K., Pope, A.R., Earnshaw, J.C., McCafferty, J., Hodits, R.A., Wilton, J., and Johnson, K.S. (1996). Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14, 309–314.
Verhoeyen, M., Milstein, C., and Winter, G. (1988). Reshaping human antibodies: grafting an antilysozyme activity. Science 239, 1534–1536.
Winter, G., Griffiths, A.D., Hawkins, R.E., and Hoogenboom, H.R. (1994). Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455.
Zacchi, P., Sblattero, D., Florian, F., Marzari, R., and Bradbury, A.R. (2003). Selecting open reading frames from DNA. Genome Res. 13, 980–990.
Zemlin, M., Klinger, M., Link, J., Zemlin, C., Bauer, K., Engler, J.A., Schroeder, H.W., Jr., and Kirkham, P.M. (2003). Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures. J. Mol. Biol. 334, 733–749.
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Yang, H.Y., Kang, K.J., Chung, J.E. et al. Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. Mol Cells 27, 225–235 (2009). https://doi.org/10.1007/s10059-009-0028-9
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DOI: https://doi.org/10.1007/s10059-009-0028-9