Summary
Diversity—the variability carried by the amino acid sequences of a synthetic antibody library—can be generated by synthetic degenerate oligonucleotides. One can experiment with different diversity designs in the variable domains of light and heavy chains (VH and VL) to generate antibody libraries with different properties. The ability to precisely define the final diversity of a library facilitates the process of isolating, characterizing, and optimizing an antibody lead. Here we describe detailed protocols for the design and construction of phage-displayed synthetic antibody libraries in which diversity is generated in the complementarity determining regions (CDRs) of the VH of a single humanized bivalent Fab scaffold. The example used in the protocol provides a general methodology for generation of libraries with engineered CDR diversity that can be applied to a template antibody sequence of choice.
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References
Liang, W. C., Dennis, M. S., Stawicki, S., Chanthery, Y., Pan, Q., Chen, Y. M., Eigenbrot, C., Yin, J. P., Koch, A. W., Wu, X. M., Ferrara, N., Bagri, A., Tessier-Lavigne, M., Watts, R. J., and Wu, Y. (2007) Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library. J. Mol. Biol. 366, 815–29.
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, 1073–93.
Fellouse, F. A., Wiesmann, C., and Sidhu, S. S. (2004) Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc. Natl. Acad. Sci. USA 101, 12467–72.
Fellouse, F. A., Li, B., Compaan, D. M., Peden, A. A., Hymowitz, S.G., and Sidhu, S. S. (2005) Molecular recognition by a binary code. J. Mol. Biol. 348, 1153–62.
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.
Krebs, B., Rauchenberger, R., Silke, R., Rothe, C., Tesar, M., Thomassen, E., Cao, M. Q., Dreier, T., Fischer, D., Hoss, A., Inge, L., Knappik, A., Marget, M., Pack, P., Meng, X. Q., Schier, R., Sohlemann, P., Winter, J., Wolle, J., and Kretzschmar, T. (2001) High-throughput generation and engineering of recombinant human antibodies. J. Immunol. Methods 254, 67–84.
Hanes, J., Schaffitzel, C., Knappik, A., and Pluckthun, A. (2000) Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotech. 18, 1287–92.
Rothe, C., Urlinger, S., Lohning, C., Prassler, J., Stark, Y. Jager, U., Hubner, B., Bardroff, M., Pradel, I., Boss, M., Bittlingmaier, R., Bataa, T., Frisch, C., Brocks, B., Honegger, A., and Urban, M (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, 1182–200.
Carter, P., Kelley, R. F., Rodrigues, M. L., Snedecor, B., Covarrubias, M., Velligan, M. D., Wong, W. L. T., Rowland, A. M., Kotts, C. E., Carver, M. E., Yang, M., Bourell, J. H., Shepard, H. M., and Henner, D. (1992) High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Biotechnology 10, 163–7.
Kelley, R. F., O’Connell, M. P., Carter, P., Presta, L., Eigenbrot, C., Covarrubias, M., Snedecor, B., Bourell, J. H., and Vetterlein, D. (1992) Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185HER2 antibody Fab fragments. Biochemistry 31, 5434–41.
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, 969–95.
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, 333–63.
Lasky, L. A., and Dowbenko, D. J. (1984) DNA sequence analysis of the type-common glycoprotein-D genes of herpes simplex virus types 1 and 2. DNA 3, 23–9.
O’Shea, E. K., Rutkowski, R., and Kim, P. S. (1989) Evidence that the leucine zipper is a coiled coil. Science 243, 538–42.
Lee, C. V., Sidhu, S. S., and Fuh, G. (2004) Bivalent antibody phage display mimics natural immunoglobulin. J. Immunol Methods 284, 119–32.
Sidhu, S. S., Li, B., Chen, Y., Fellouse, F. A., Eigenbrot, C., and Fuh, G. (2004) Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J. Mol. Biol. 338, 299–310.
Nissim, A., Hoogenboom, H. R., Tomlinson, I. M., Flynn, G., Midgely, C., Lane, D., and Winter, G. (1994) Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents. EMBO J. 13, 692–8.
Garrard, L. J., and Henner, D. J. (1993) Selection of an anti-IGF-1 Fab from a Fab phage library created by mutagenesis of multiple CDR loops. Gene 128, 103–9.
de Kruif, J., Boel, E., and Logtenberg, T. (1995) Selection and application of human single chain Fv antibody fragments from a semi-synthetic phage antibody display library with designed CDR3 regions. J. Mol. Biol. 248, 97–105.
Chothia, C., Lesk, A. M., Tramontano, A., Levitt, M., Smithgill, S. J., Air, G., Sheriff, S., Padlan, E. A., Davies, D., Tulip, W. R., Colman, P. M., Spinelli, S., Alzari, P. M., and Poljak, R. J. (1989) Conformations of immunoglobulin hypervariable regions. Nature 342, 877–83.
Padlan, E. A. (1994) Anatomy of the antibody molecule. Mol. Immunol. 31, 169–217.
Wilson, I. A., and Stanfield, R. L. (1994) Antibody-antigen interactions: new structures and new conformational changes. Curr. Opin. Struct. Biol. 4, 857–67.
Xu, J. L., and Davis, M. M. (2000) Diversity in the CDR3 region of VH is sufficient for most antibody specificities. Immunity 13, 37–45.
Kabat, E. A., Wu, T. T., Redi-Miller, M., Perry, H. M., and Gottesman, K. S. (1987) Sequences of proteins of immunological interest. 4th edition, National Institutes of Health, Bethesda, MD.
Johnson, G., and Wu, T. T. (2000) Kabat database and its applications: 30 years after the first variability plot. Nucleic Acids Res. 28, 214–8.
Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–82.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Bostrom, J., Fuh, G. (2009). Design and Construction of Synthetic Phage-Displayed Fab Libraries. In: Aitken, R. (eds) Antibody Phage Display. Methods in Molecular Biology, vol 562. Humana Press. https://doi.org/10.1007/978-1-60327-302-2_2
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DOI: https://doi.org/10.1007/978-1-60327-302-2_2
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