Molecules and Cells

, Volume 27, Issue 2, pp 225–235 | Cite as

Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity

  • Hye Young Yang
  • Kyung Jae Kang
  • Julia Eunyoung Chung
  • Hyunbo Shim
Article
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Accepted:

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.

Keywords

Beta-lactamase selection functional diversity phage display scFv Synthetic antibody library 
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References

  1. 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.Google Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 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.PubMedGoogle Scholar
  5. 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.PubMedCrossRefGoogle Scholar
  6. 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.PubMedCrossRefGoogle Scholar
  7. Hoogenboom, H.R., and Chames, P. (2000). Natural and designer binding sites made by phage display technology. Immunol. Today 21, 371–378.PubMedCrossRefGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 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.PubMedCrossRefGoogle Scholar
  10. Kim, S.J., Park, Y., and Hong, H.J. (2005). Antibody engineering for the development of therapeutic antibodies. Mol. Cells 20, 17–29.PubMedGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. 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.PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 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.PubMedCrossRefGoogle Scholar
  16. Rader, C., and Barbas, C.F., 3rd (1997). Phage display of combinatorial antibody libraries. Curr. Opin. Biotechnol. 8, 503–508.PubMedCrossRefGoogle Scholar
  17. Riechmann, L., Clark, M., Waldmann, H., and Winter, G. (1988). Reshaping human antibodies for therapy. Nature 332, 323–327.PubMedCrossRefGoogle Scholar
  18. 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.PubMedCrossRefGoogle Scholar
  19. 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.Google Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. Sidhu, S.S. (2001). Engineering M13 for phage display. Biomol. Eng. 18, 57–63.PubMedCrossRefGoogle Scholar
  22. Sidhu, S.S., and Fellouse, F.A. (2006). Synthetic therapeutic antibodies. Nat. Chem. Biol. 2, 682–688.PubMedCrossRefGoogle Scholar
  23. 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.PubMedCrossRefGoogle Scholar
  24. 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.PubMedCrossRefGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. Verhoeyen, M., Milstein, C., and Winter, G. (1988). Reshaping human antibodies: grafting an antilysozyme activity. Science 239, 1534–1536.PubMedCrossRefGoogle Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. Zacchi, P., Sblattero, D., Florian, F., Marzari, R., and Bradbury, A.R. (2003). Selecting open reading frames from DNA. Genome Res. 13, 980–990.PubMedCrossRefGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar

Copyright information

© The Korean Society for Molecular and Cellular Biology and Springer Netherlands 2009

Authors and Affiliations

  • Hye Young Yang
    • 1
  • Kyung Jae Kang
    • 1
  • Julia Eunyoung Chung
    • 1
  • Hyunbo Shim
    • 1
    • 2
  1. 1.Division of Life and Pharmaceutical SciencesEwha Womans UniversitySeoulKorea
  2. 2.Department of Life ScienceEwha Womans UniversitySeoulKorea

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