Browse > Article
http://dx.doi.org/10.1007/s10059-009-0028-9

Construction of a Large Synthetic Human scFv Library with Six Diversified CDRs and High Functional Diversity  

Yang, Hye Young (Division of Life and Pharmaceutical Sciences, Ewha Womans University)
Kang, Kyung Jae (Division of Life and Pharmaceutical Sciences, Ewha Womans University)
Chung, Julia Eunyoung (Division of Life and Pharmaceutical Sciences, Ewha Womans University)
Shim, Hyunbo (Division of Life and Pharmaceutical Sciences, Ewha Womans University)
Publication Information
Molecules and Cells / v.27, no.2, 2009 , pp. 225-235 More about this Journal
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 ${\beta}$-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5'-end of the ${\beta}$-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of $7.6{\times}10^9$, 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;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
Times Cited By Web Of Science : 7  (Related Records In Web of Science)
연도 인용수 순위
1 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-determiningregion diversity. Nat. Biotechnol. 23, 344-348   DOI   ScienceOn
2 Hoogenboom, H.R., and Chames, P. (2000). Natural and designer binding sites made by phage display technology. Immunol. Today 21, 371-378   DOI   ScienceOn
3 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   DOI   ScienceOn
4 Kim, S.J., Park, Y., and Hong, H.J. (2005). Antibody engineering for the development of therapeutic antibodies. Mol. Cells 20, 17-29   PUBMED
5 Riechmann, L., Clark, M., Waldmann, H., and Winter, G. (1988). Reshaping human antibodies for therapy. Nature 332, 323-327   DOI   ScienceOn
6 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
7 Sidhu, S.S., and Fellouse, F.A. (2006). Synthetic therapeutic antibodies. Nat. Chem. Biol. 2, 682-688   DOI   ScienceOn
8 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   DOI   ScienceOn
9 Zacchi, P., Sblattero, D., Florian, F., Marzari, R., and Bradbury, A.R. (2003). Selecting open reading frames from DNA. Genome Res. 13, 980-990   DOI   ScienceOn
10 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
11 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   DOI   ScienceOn
12 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
13 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 Hoogenboom, 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   DOI
14 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   DOI   ScienceOn
15 Rader, C., and Barbas, C.F., 3rd (1997). Phage display of combinatorial antibody libraries. Curr. Opin. Biotechnol. 8, 503-508   DOI   ScienceOn
16 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   DOI   ScienceOn
17 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 subnanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14, 309-314   DOI   ScienceOn
18 Verhoeyen, M., Milstein, C., and Winter, G. (1988). Reshaping human antibodies: grafting an antilysozyme activity. Science 239, 1534-1536   DOI
19 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   DOI   ScienceOn
20 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   DOI   ScienceOn
21 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   DOI   ScienceOn
22 Rothe, C., Urlinger, S., Lohning, C., Prassler, J., Stark, Y., Jager, U., Hubner, B., Bardroff, M., Pradel, I., Boss, M.I=et alK (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   DOI   ScienceOn
23 Lee, C.V., Liang, W.C., Dennis, M.S., Eigenbrot, C., Sidhu, S.S., and Fuh, G. (2004). High-affinity human antibodies from phagedisplayed synthetic Fab libraries with a single framework scaffold. J. Mol. Biol. 340, 1073-1093   DOI   ScienceOn
24 Loset, G.A., Lobersli, I., Kavlie, A., Stacy, J.E., Borgen, T., Kausmally, 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   DOI   ScienceOn
25 Sidhu, S.S. (2001). Engineering M13 for phage display. Biomol. Eng. 18, 57-63   DOI   PUBMED   ScienceOn
26 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   DOI   ScienceOn
27 Lutz, S., Fast, W., and Benkovic, S.J. (2002). A universal, vectorbased system for nucleic acid reading-frame selection. Protein Eng. 15, 1025-1030   DOI
28 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   DOI   ScienceOn
29 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   DOI   ScienceOn