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Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library

Nature Biotechnology volume 21, pages 163–170 (2003)Cite this article

Abstract

A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.

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Figure 1: ScFv libraries expressed on the yeast surface.
Figure 2: Germline gene family usage for heavy (A) and light chains (B) in the scFv library.
Figure 3: Characterization of library expression and propagation stability.
Figure 4: Affinity of isolated scFvs.

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References

  1. Schweitzer, B. et al. Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 20, 359–365 (2002).

    Article  CAS  Google Scholar 

  2. MacBeath, G. & Schreiber, S.L. Printing proteins as microarrays for high-throughput function determination. Science 289, 1760–1763 (2000).

    CAS  Google Scholar 

  3. Gura, T. Therapeutic antibodies: magic bullets hit the target. Nature 417, 584–586 (2002).

    Article  CAS  Google Scholar 

  4. Clark, M. Antibody humanization: a case of the 'Emperor's new clothes'? Immunol. Today 21, 397–402 (2000).

    Article  CAS  Google Scholar 

  5. Mendez, M.J. et al. Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat. Genet. 15, 146–156 (1997).

    Article  CAS  Google Scholar 

  6. Winter, G., Griffiths, A.D., Hawkins, R.E. & Hoogenboom, H.R. Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455 (1994).

    Article  CAS  Google Scholar 

  7. Marks, J.D. et al. Bypassing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597 (1991).

    Article  CAS  Google Scholar 

  8. Sheets, M.D. et al. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci. USA 95, 6157–6162 (1998).

    Article  CAS  Google Scholar 

  9. Vaughan, T.J. et al. Human antibodies with subnanomolar affinities isolated from a large nonimmunized phage display library. Nat. Biotechnol. 14, 309–314 (1996).

    Article  CAS  Google Scholar 

  10. 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 

  11. Hanes, J., Schaffitzel, C., Knappik, A. & Plückthun, A. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotechnol. 18, 1287–1292 (2000).

    Article  CAS  Google Scholar 

  12. Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M. & Wittrup, K.D. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754–759 (2000).

    Article  CAS  Google Scholar 

  13. Holler, P.D. et al. In vitro evolution of a T-cell receptor with high affinity for peptide/MHC. Proc. Natl. Acad. Sci. USA 97, 5387–5392 (2000).

    Article  CAS  Google Scholar 

  14. Boder, E.T., Midelfort, K.S. & Wittrup, K.D. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. USA 97, 10701–10705 (2000).

    Article  CAS  Google Scholar 

  15. Daugherty, P.S., Iverson, B.L. & Georgiou, G. Flow cytometric screening of cell-based libraries. J. Immunol. Methods 243, 211–227 (2000).

    Article  CAS  Google Scholar 

  16. VanAntwerp, J.J. & Wittrup, K.D. Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnol. Prog. 16, 31–37 (2000).

    Article  CAS  Google Scholar 

  17. Yeung, Y.A. & Wittrup, K.D. Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture. Biotechnol. Prog. 18, 212–220 (2002).

    Article  CAS  Google Scholar 

  18. Sblattero, D. & Bradbury, A. A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology 3, 271–278 (1998).

    Article  CAS  Google Scholar 

  19. Ignatovich, O., Tomlinson, I.M., Jones, P.T. & Winter, G. The creation of diversity in the human immunoglobulin V(λ) repertoire. J. Mol. Biol. 268, 69–77 (1997).

    Article  CAS  Google Scholar 

  20. Foster, S.J., Brezinschek, H.P., Brezinschek, R.I. & Lipsky, P.E. Molecular mechanisms and selective influences that shape the κ-gene repertoire of IgM+ B cells. J. Clin. Invest. 99, 1614–1627 (1997).

    Article  CAS  Google Scholar 

  21. Brezinschek, H.P. et al. Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5+/IgM+ and CD5−/IgM+ B cells. J. Clin. Invest. 99, 2488–2501 (1997).

    Article  CAS  Google Scholar 

  22. Ellgaard, L., Molinari, M. & Helenius, A. Setting the standards: quality control in the secretory pathway. Science 286, 1882–1888 (1999).

    Article  CAS  Google Scholar 

  23. Field, C. & Schekman, R. Localized secretion of acid phosphatase reflects the pattern of cell surface growth in Saccharomyces cerevisiae. J. Cell Biol. 86, 123–128 (1980).

    Article  CAS  Google Scholar 

  24. Shusta, E.V., Raines, R.T., Plückthun, A. & Wittrup, K.D. Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat. Biotechnol. 16, 773–777 (1998).

    Article  CAS  Google Scholar 

  25. Huang, W., McKevitt, M. & Palzkill, T. Use of the arabinose p(bad) promoter for tightly regulated display of proteins on bacteriophage. Gene 251, 187–197 (2000).

    Article  CAS  Google Scholar 

  26. Krebber, A., Burmester, J. & Plückthun, A. Inclusion of an upstream transcriptional terminator in phage display vectors abolishes background expression of toxic fusions with coat protein g3p. Gene 178, 71–74 (1996).

    Article  CAS  Google Scholar 

  27. Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng. 12, 613–621 (1999).

    Article  CAS  Google Scholar 

  28. Lou, J. et al. Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries. J. Immunol. Methods 253, 233–242 (2001).

    Article  CAS  Google Scholar 

  29. Boder, E.T. & Wittrup, K.D. Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol. 328, 430–444 (2000).

    Article  CAS  Google Scholar 

  30. Chen, G. et al. Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS). Nat. Biotechnol. 19, 537–542 (2001).

    Article  CAS  Google Scholar 

  31. Sblattero, D. & Bradbury, A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat. Biotechnol. 18, 75–80 (2000).

    Article  CAS  Google Scholar 

  32. 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 

  33. Griffiths, A.D. et al. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13, 3245–3260 (1994).

    Article  CAS  Google Scholar 

  34. Soderlind, E. et al. Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat. Biotechnol. 18, 852–856 (2000).

    Article  CAS  Google Scholar 

  35. Pini, A. et al. 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 (1998).

    Article  CAS  Google Scholar 

  36. Kieke, M.C. et al. High affinity T cell receptors from yeast display libraries block T cell activation by superantigens. J. Mol. Biol. 307, 1305–1315 (2001).

    Article  CAS  Google Scholar 

  37. Llorente, M. et al. Natural human antibodies retrieved by phage display libraries from healthy donors: polyreactivity and recognition of human immunodeficiency virus type 1gp120 epitopes. Scand. J. Immunol. 50, 270–279 (1999).

    Article  CAS  Google Scholar 

  38. Nieba, L., Honegger, A., Krebber, C. & Plückthun, A. Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng. 10, 435–444 (1997).

    Article  CAS  Google Scholar 

  39. Bothmann, H. & Plückthun, A. Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nat. Biotechnol. 16, 376–380 (1998).

    Article  CAS  Google Scholar 

  40. de Bruin, R., Spelt, K., Mol, J., Koes, R. & Quattrocchio, F. Selection of high-affinity phage antibodies from phage display libraries. Nat. Biotechnol. 17, 397–399 (1999).

    Article  CAS  Google Scholar 

  41. de Wildt, R.M., Mundy, C.R., Gorick, B.D. & Tomlinson, I.M. Antibody arrays for high-throughput screening of antibody–antigen interactions. Nat. Biotechnol. 18, 989–994 (2000).

    Article  CAS  Google Scholar 

  42. Braun, P. et al. Proteome-scale purification of human proteins from bacteria. Proc. Natl. Acad. Sci. USA 99, 2654–2659. (2002).

    Article  CAS  Google Scholar 

  43. Gavin, A.C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002).

    Article  CAS  Google Scholar 

  44. Gietz, R.D., Schiestl, R.H., Willems, A.R. & Woods, R.A. Studies on the transformation of intact yeast cells by the LiAc/SS– DNA/PEG procedure. Yeast 11, 355–360 (1995).

    Article  CAS  Google Scholar 

  45. Boder, E.T. & Wittrup, K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

    Article  CAS  Google Scholar 

  46. Krebs, B. et al. High-throughput generation and engineering of recombinant human antibodies. J. Immunol. Methods 254, 67–84 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded in part by NSF BPEC, the Hereditary Disease Foundation, and by the Laboratory Directed Research and Development program of the US Department of Energy (DOE). The p53 phosphopeptides were provided by Ettore Appella, Shari Mazur, and Yuichiro Higashimoto (NCI). The authors are grateful for comments on the manuscript by David Kranz, Michael Roguska, and Eric Shusta.

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Authors and Affiliations

  1. Biological Sciences Division, Pacific Northwest National Laboratories, 902 Battelle Blvd., P.O. Box 999, Richland, 99352, WA

    Michael J. Feldhaus, Robert W. Siegel, Lee K. Opresko, James R. Coleman, Jane M. Weaver Feldhaus & H. Steven Wiley

  2. Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, MIT 66-552, 25 Ames Street, Cambridge, 02139, MA

    Yik A. Yeung, Jennifer R. Cochran, Peter Heinzelman, David Colby, Jeffrey Swers, Christilyn Graff & K. Dane Wittrup

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Correspondence to Michael J. Feldhaus or K. Dane Wittrup.

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K.D.W. is an inventor listed on US patents 6423538, 6300065, and related filings that pertain to yeast surface display methodology, and receives a portion of any royalty income paid to the University of Illinois for licensing of these patents.

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Feldhaus, M., Siegel, R., Opresko, L. et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21, 163–170 (2003). https://doi.org/10.1038/nbt785

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