Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity

Abstract

Combinatorial libraries of rearranged hypervariable VH and VL sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of VHs and VLs. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries1,2. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies3,4,5,6. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.

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Figure 1: Design of Dyax libraries.
Figure 2: Fab dissociation constants (Kd) in nM for selected Fabs.
Figure 3: Diversity observed in the new libraries compared to diversity seen in the PCR library.

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References

  1. 1

    Souriau, C. & Hudson, P.J. Recombinant antibodies for cancer diagnosis and therapy. Expert Opin. Biol. Ther. 3, 305–318 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Pancook, J.D. et al. In vitro affinity maturation of human IgM antibodies reactive with tumor-associated antigens. Hybrid. Hybridomics 20, 383–396 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Cardarelli, P.M. et al. Binding to CD20 by anti-B1 antibody or F(ab')(2) is sufficient for induction of apoptosis in B-cell lines. Cancer Immunol. Immunother. 51, 15–24 (2002).

    CAS  Article  Google Scholar 

  4. 4

    Santora, L.C., Kaymakcalan, Z., Sakorafas, P., Krull, I.S. & Grant, K. Characterization of noncovalent complexes of recombinant human monoclonal antibody and antigen using cation exchange, size exclusion chromatography, and BIAcore. Anal. Biochem. 299, 119–129 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Knight, D.M. et al. Construction and initial characterization of a mouse-human chimeric anti-TNF antibody. Mol. Immunol. 30, 1443–1453 (1993).

    CAS  Article  Google Scholar 

  6. 6

    Goldstein, N.I., Prewett, M., Zuklys, K., Rockwell, P. & Mendelsohn, J. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin. Cancer Res. 1, 1311–1318 (1995).

    CAS  PubMed  Google Scholar 

  7. 7

    de Wildt, R.M., Tomlinson, I.M., van Venrooij, W.J., Winter, G. & Hoet, R.M. Comparable heavy and light chain pairings in normal and systemic lupus erythematosus IgG(+) B cells. Eur. J. Immunol. 30, 254–261 (2000).

    CAS  Article  Google Scholar 

  8. 8

    Virnekas, B. et al. Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 22, 5600–5607 (1994).

    CAS  Article  Google Scholar 

  9. 9

    Fan, Z.C. et al. Three-dimensional structure of an Fv from a human IgM immunoglobulin. J. Mol. Biol. 228, 188–207 (1992).

    CAS  Article  Google Scholar 

  10. 10

    Sidhu, S.S. et al. Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J. Mol. Biol. 338, 299–310 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Li, Y., Li, H., Yang, F., Smith-Gill, S.J. & Mariuzza, R.A. X-ray snapshots of the maturation of an antibody response to a protein antigen. Nat. Struct. Biol. 10, 482–488 (2003).

    CAS  Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

    Johnson, G. & Wu, T.T. Kabat database and its applications: 30 years after the first variability plot. Nucleic Acids Res. 28, 214–218 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Zemlin, M. et al. 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 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Knappik, A. et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57–86 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Lee, C.V. et al. High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J. Mol. Biol. 340, 1073–1093 (2004).

    CAS  Article  Google Scholar 

  17. 17

    Kretzschmar, T. & von Ruden, T. Antibody discovery: phage display. Curr. Opin. Biotechnol. 13, 598–602 (2002).

    CAS  Article  Google Scholar 

  18. 18

    Rauchenberger, R. et al. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3. J. Biol. Chem. 278, 38194–38205 (2003).

    CAS  Article  Google Scholar 

  19. 19

    de Haard, H.J. et al. 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 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Partanen, J. et al. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Mol. Cell. Biol. 12, 1698–1707 (1992).

    CAS  Article  Google Scholar 

  21. 21

    Netzel-Arnett, S. et al. Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer. Cancer Metastasis Rev. 22, 237–258 (2003).

    CAS  Article  Google Scholar 

  22. 22

    Johnson, G. & Wu, T.T. Preferred CDRH3 lengths for antibodies with defined specificities. Int. Immunol. 10, 1801–1805 (1998).

    CAS  Article  Google Scholar 

  23. 23

    Edwards, B.M. et al. The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS. J. Mol. Biol. 334, 103–118 (2003).

    CAS  Article  Google Scholar 

  24. 24

    Jostock, T. et al. Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries. J. Immunol. Methods 289, 65–80 (2004).

    CAS  Article  Google Scholar 

  25. 25

    van den Beucken, T. et al. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288–294 (2003).

    CAS  Article  Google Scholar 

  26. 26

    Marks, J.D. Antibody affinity maturation by chain shuffling. Methods Mol. Biol. 248, 327–343 (2004).

    CAS  PubMed  Google Scholar 

  27. 27

    Marks, J.D. et al. By-passing immunization: building high affinity human antibodies by chain shuffling. Bio/Technology 10, 779–783 (1992).

    CAS  PubMed  Google Scholar 

  28. 28

    Dower, W.J., Miller, J.F. & Ragsdale, C.W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16, 6127–6145 (1988).

    CAS  Article  Google Scholar 

  29. 29

    Adey, N.B., Sparks, A.B., Beasley, J. & Kay, B.K. in Phage Display of Peptides and Proteins: a laboratory manual. (eds. Kay, B.K., Winter, J. & McCafftery, J.) 67–77, (Academic Press, San Diego, 1996).

  30. 30

    Barbas, C.F., III, Kang, A.S., Lerner, R.A. & Benkovic, S.J. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88, 7978–7982 (1991).

    CAS  Article  Google Scholar 

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Acknowledgements

We are very grateful to Ed Madison and colleagues at Dendreon for the provision of the DESC1 and MSPL antigens. In addition, we are indebted to Kari Alitalo for our collaboration on TIE-1. We thank all our colleagues at Dyax, in both Cambridge and Liege, for many contributions and discussions throughout the course of this work.

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Correspondence to Robert Charles Ladner.

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All of the authors worked for Dyax Corp. during the work on the library and were paid by Dyax. Dyax licenses the library for financial consideration.

Supplementary information

Supplementary Fig. 1

Schematic representation of the novel ONCL technology used to clone the CDR3-heavy chain repertoire from autoimmune donors. (PDF 339 kb)

Supplementary Table 1

Human Fabs that bind human protein targets (PDF 375 kb)

Supplementary Table 2

t/c (XLS 25 kb)

Supplementary Table 3

Oligonucleotides (PDF 117 kb)

Supplementary Discussion (PDF 124 kb)

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Hoet, R., Cohen, E., Kent, R. et al. Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat Biotechnol 23, 344–348 (2005). https://doi.org/10.1038/nbt1067

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