Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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. 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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  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. 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).

    Article  CAS  Google Scholar 

  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. 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).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Robert Charles Ladner.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1067

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing