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Construction of Semisynthetic Antibody Libraries

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Recombinant Antibodies for Cancer Therapy

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 207))

Abstract

Antibody libraries expressed on the surface of filamentous phage are proven to be a valuable tool in isolating antibodies specific for a wide variety of antigens (for review, see ref. 1). As it is assumed that the probability of isolating high affinity binders is related to the initial library size (2), the construction of large libraries representing a high diversity of molecules is a central goal of recombinant antibody technology. In addition to generate antibody libraries from naïve B-cell repertoires (3–6), germline sequences (7) or immunized donors (8), it is also possible to make use of the available information on antibody structure to generate diversity by including short stretches of random sequences in carefully chosen parts of the antibody.

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References

  1. Hoogenboom H. R., De Bruine A. P., Hufton S. E., Hort R. M., Arends J. W., and Roovers R. C. (1998). Antibody phage display technology and its applications. Immunotechniques 4, 1–20.

    Article  CAS  Google Scholar 

  2. Perelson A. S. (1989). Immune network theory. Immunol. Rev. 100, 5–36.

    Article  Google Scholar 

  3. Vaughan T. J, Williams A. J., Pritchard K., Osbourn J. K., Pope A. R., Earnshaw J. C., et al. (1996). Human antibodies with sub-nanomolar affinities from a large non-immunized phage display library. Nature Biotechnol. 14, 309–314.

    Article  CAS  Google Scholar 

  4. Sheets M. D., Amersdorfer P., Finnern R., Sargent P., Lindqvist E., Schier R., et al. (1998). Efficient construction of a large non-immune phage antibody library — The production of high-affinity human single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci. USA 95, 6157–6162.

    Article  PubMed  CAS  Google Scholar 

  5. De Haard H. J., Van Neer N., Reurs A, Hufton S. E., Roovers R. C., Henderikx P., et al. (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, 18,218–18,230.

    Article  PubMed  Google Scholar 

  6. Little M., Welschof M., Braunagel M., Hermes I., Christ C., Keller A., et al. (1999). Generation of a large complex antibody library from multiple donors. J. Immunol. Methods 231, 3–9.

    Article  PubMed  CAS  Google Scholar 

  7. Griffiths A. D., Williams S. C., Hartley O., Tomlinson I. M., Waterhouse P., Crosby W. L., et al. (1994). Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13, 3245–3260.

    PubMed  CAS  Google Scholar 

  8. Barbas C. F., Bain J. D., Hoekstra D. M., and Lerner R. A. (1992). Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Proc. Natl Acad. Sci. USA 89, 4457–4461.

    Article  PubMed  CAS  Google Scholar 

  9. Poljak R. J., Amzel L. M., Avery H, Chen B. L., Phizackerley R. P., and Saul F. (1973). Three-dimensional structure of the Fab-fragment of a human immunoglobulin on 2,8A resolution. Proc. Natl. Acad. Sci. USA 70, 3305–3310.

    Article  PubMed  CAS  Google Scholar 

  10. Wu T. and Kabat E. (1971). Attempts to locate complementarity-determining regions residues in the variable positions of light and heavy chains. Ann. NY Acad. Sci. 190, 382–393.

    Article  PubMed  Google Scholar 

  11. Hoogenboom H. R. and Winter G. (1992). By-passing immunisation: Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J. Mol. Biol. 227, 381–388.

    Article  PubMed  CAS  Google Scholar 

  12. Barbas C. F., Bain J. D., Hoekstra D. M., and Lerner R. A. (1992). Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Proc. Natl Acad. Sci. USA 89, 4457–4461.

    Article  PubMed  CAS  Google Scholar 

  13. Barbas C. F., Amberg W., Simoncsits A., Jones T. M., and Lerner R. A. (1993). Selection of human anti-hapten antibodies from semi-synthetic libraries. Gene 137, 57–62.

    Article  PubMed  CAS  Google Scholar 

  14. Hayashi N., Welschof M., Zewe M., Braunagel M., Dubel S., Breitling F., and Little M. (1994). Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR. Biotechniques 17, 310, 312, 314–315.

    Google Scholar 

  15. Nissim A., Hoogenboom H. R., Tomlinson I. M., Flynn G., Midgley C., Lane D., and Winter G. (1994). Antibody fragments from a —single pot—phage display library as immunochemical reagents. EMBO J. 13, 692–698.

    PubMed  CAS  Google Scholar 

  16. 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.

    Article  PubMed  CAS  Google Scholar 

  17. Braunagel M. and Little M. (1997). Construction of a semisynthetic antibody library using trinucleotide oligos. Nucleic Acids Res. 25, 4690–4691.

    Article  PubMed  CAS  Google Scholar 

  18. Barbas C. F., Hu D., Dunlop N., Sawyer L., Cababa D., Hendry R. M., et al. (1994). In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity. Proc. Natl. Acad. Sci. USA 91, 3809–3813.

    Article  PubMed  CAS  Google Scholar 

  19. Hemminki A., Niemi S., Hoffren A. M., Hakalahti L., Soderlund H., and Takkinen K. (1998). Specificity improvement of a recombinant anti-testosterone Fab fragment by CDRIII mutagenesis and phage display selection. Protein Eng. 11, 311–319.

    Article  PubMed  CAS  Google Scholar 

  20. Lamminmaki U., Pauperio S., Westerlund-Karlsson A., Karvinen J., Virtanen P. L., Lovgren T., and Saviranta P. (1999). Expanding the conformational diversity by random insertions in CDRH2 results in improved anti-estradiol antibodies. J. Mol. Biol. 291, 589–602.

    Article  PubMed  CAS  Google Scholar 

  21. Yang W. P., Green K., Pinz-Sweeney S., Briones A. T., Burton D. R., and Barbas C. F. (1995). CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J. Mol. Biol. 25, 392–403.

    Article  Google Scholar 

  22. Schier R., McCall A., Adams G. P., Marhall K. W., Merritt H., Yim M., et al. (1996b) Isolation of picomolar affinity anti-c-erbb-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J. Mol. Biol. 263, 551–567.

    Article  PubMed  CAS  Google Scholar 

  23. Wu H., Beuerlein G., Nie Y., Smith H., Lee B. A., Hensler M., et al. (1998). Stepwise in vitro affinity maturation of Vitaxin, an alpha(v)beta(3)-specific humanized Mab. Proc. Natl Acad. Sci. USA 95, 6037–6042.

    Article  PubMed  CAS  Google Scholar 

  24. Knappik A., Ge L., Honegger A., Pack P., Fischer M., Wellnhofer G., et al. (2000). Fully synthetic human combinatorial libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57–86.

    Article  PubMed  CAS  Google Scholar 

  25. Chothia C., and Lesk A. M. (1987). Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901–917.

    Article  PubMed  CAS  Google Scholar 

  26. Chothia C., Lesk A. M., Tramontano A., Levitt M., Smith-Gill S. J., Air G., et al. (1989). Conformations of immunoglobulin hypervariable regions. Nature 342, 877–883.

    Article  PubMed  CAS  Google Scholar 

  27. Chothia C., Lesk A. M., Gherardi E., Tomlinson I. M., Walter G., Marks J. D., et al. (1992). Structural repertoire of the human VH segments. J. Mol. Biol. 227, 799–817.

    Article  PubMed  CAS  Google Scholar 

  28. Rees A. R., Martin A. C. R., Pedersen J. T., and Searle S. M. J. (1992)ABM/YTM, A Computer Program for Modelling Variable Regions of Antibodies. Oxford Molecular Ltd., Oxford, UK.

    Google Scholar 

  29. Virnekas B., Ge L., Pluckthun A., Schneider K. C., Wellnhofer G., and Moroney S. E. (1994). Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 22, 5600–5607.

    Article  PubMed  CAS  Google Scholar 

  30. Lyttle M. H., Napolitano E. W., Calio B. L., and Kauvar L. M. (1995). Mutagenesis using trinucleotide betacyanoethyl phosphoramidites. Biotechniques 19, 274–281.

    PubMed  CAS  Google Scholar 

  31. Kayushin A. L., Korosteleva M. D., Miroshnikov A. I., Kosch W., Zubov D., and Piel N. (1996). A convenient approach to the synthesis of trinucleotide phosphoramiditessynthons for the generation of oligonucleotide/peptide libraries. Nucleic Acids Res. 24, 3748–3755.

    Article  PubMed  CAS  Google Scholar 

  32. Marks J. D., Griffiths A. D., Malmqvist M., Clackson T. P., Bye J. M., and Winter G. (1992). By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (NY) 10, 779–783.

    Article  CAS  Google Scholar 

  33. Welschof M., Terness P., Kipriyanov S. M., Stanescu D., Breitling F., Dörsam H., et al. (1997). The antigen-binding domain of a human IgG-anti-F(ab′)2 autoantibody. Proc. Natl. Acad. Sci. USA 94, 1902–1907.

    Article  PubMed  CAS  Google Scholar 

  34. Dorsam H., Braunagel M., Kleist C., Moynet D., and Welschof M. (1996). Screening of phage-displayed antibody libraries in Methods in Molecular Medicine, vol. 13: Molecular Diagnosis of Infectious Diseases, (Reischl U., ed.), Humana Press Inc., Totowa, NJ, 605–614.

    Google Scholar 

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Authors and Affiliations

  1. National Cancer Institute at Frederick, Frederick, MD

    Michael Braunagel

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  1. Michael Braunagel
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Editors and Affiliations

  1. Axaron Bioscience AG, Heidelberg, Germany

    Martin Welschof

  2. National Institutes of Health, National Cancer Institute, Frederick, MD

    Jürgen Krauss

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© 2003 Humana Press Inc.

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Braunagel, M. (2003). Construction of Semisynthetic Antibody Libraries. In: Welschof, M., Krauss, J. (eds) Recombinant Antibodies for Cancer Therapy. Methods in Molecular Biology™, vol 207. Humana Press. https://doi.org/10.1385/1-59259-334-8:123

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  • DOI: https://doi.org/10.1385/1-59259-334-8:123

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-918-6

  • Online ISBN: 978-1-59259-334-7

  • eBook Packages: Springer Protocols

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