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Directed evolution of human T-cell receptors with picomolar affinities by phage display

Abstract

Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide–human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to 1–100 μM1. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond2. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax11–19 peptide–HLA-A*0201 complex3 and the NY-ESO-1157–165 tumor-associated peptide antigen–HLA-A*0201 complex4, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs.

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Figure 1: Biacore SPR binding of high-affinity TCRs compared to their parent wild-type TCRs.
Figure 2: High-affinity TCR targeting of cell-surface pHLAs.

References

  1. van der Merwe, P.A. & Davis, S.J. Molecular interactions mediating T cell antigen recognition. Annu. Rev. Immunol. 21, 659–684 (2003).

    CAS  Article  Google Scholar 

  2. Boulter, J.M. et al. Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng. 16, 707–711 (2003).

    CAS  Article  Google Scholar 

  3. Garboczi, D.N. et al. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384, 134–141 (1996).

    CAS  Article  Google Scholar 

  4. Jager, E. et al. Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes. J. Exp. Med. 187, 265–270 (1998).

    CAS  Article  Google Scholar 

  5. Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).

    CAS  Article  Google Scholar 

  6. Clackson, T., Hoogenboom, H.R., Griffiths, A.D. & Winter, G. Making antibody fragments using phage display libraries. Nature 352, 624–628 (1991).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  8. Smith, I.E. New drugs for breast cancer. Lancet 360, 790–792 (2002).

    Article  Google Scholar 

  9. 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).

    CAS  Article  Google Scholar 

  10. 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).

    CAS  Article  Google Scholar 

  11. Holler, P.D., Lim, A.R., Cho, B.K., Rund, L.A. & Kranz, D.M. CD8(-) T cell transfectants that express a high affinity T cell receptor exhibit enhanced peptide-dependent activation. J. Exp. Med. 194, 1043–1052 (2001).

    CAS  Article  Google Scholar 

  12. Holler, P.D. & Kranz, D.M. Quantitative analysis of the contribution of TCR/pepMHC affinity and CD8 to T cell activation. Immunity 18, 255–264 (2003).

    CAS  Article  Google Scholar 

  13. Holler, P.D., Chlewicki, L.K. & Kranz, D.M. TCRs with high affinity for foreign pMHC show self-reactivity. Nat. Immunol. 4, 55–62 (2003).

    CAS  Article  Google Scholar 

  14. Weidanz, J.A., Card, K.F., Edwards, A., Perlstein, E. & Wong, H.C. Display of functional alphabeta single-chain T-cell receptor molecules on the surface of bacteriophage. J. Immunol. Methods 221, 59–76 (1998).

    CAS  Article  Google Scholar 

  15. Lefranc, M-P.L.G. The T Cell Receptor Facts Book (Academic Press, London, 2001).

    Google Scholar 

  16. Garboczi, D.N. et al. Assembly, specific binding, and crystallization of a human TCR-alphabeta with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2. J. Immunol. 157, 5403–5410 (1996).

    CAS  PubMed  Google Scholar 

  17. Khalifa, M.B., Choulier, L., Lortat-Jacob, H., Altschuh, D. & Vernet, T. BIACORE data processing: an evaluation of the global fitting procedure. Anal. Biochem. 293, 194–203 (2001).

    CAS  Article  Google Scholar 

  18. Karlsson, R. Real-time competitive kinetic analysis of interactions between low-molecular-weight ligands in solution and surface-immobilized receptors. Anal. Biochem. 221, 142–151 (1994).

    CAS  Article  Google Scholar 

  19. Hutchinson, S.L. et al. The CD8 T cell coreceptor exhibits disproportionate biological activity at extremely low binding affinities. J. Biol. Chem. 278, 24285–24293 (2003).

    CAS  Article  Google Scholar 

  20. Altman, J.D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    CAS  Article  Google Scholar 

  21. Laugel, B. et al. Design of soluble recombinant T cell receptors for antigen targeting and T cell inhibition. J. Biol. Chem. 280, 1882–1892 (2004).

    Article  Google Scholar 

  22. Schirle, M. et al. Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach. Eur. J. Immunol. 30, 2216–2225 (2000).

    CAS  Article  Google Scholar 

  23. Cohen, C.J., Denkberg, G., Lev, A., Epel, M. & Reiter, Y. Recombinant antibodies with MHC-restricted, peptide-specific, T-cell receptor-like specificity: new tools to study antigen presentation and TCR-peptide-MHC interactions. J. Mol. Recognit. 16, 324–332 (2003).

    CAS  Article  Google Scholar 

  24. Cohen, C.J. et al. Direct phenotypic analysis of human MHC class i antigen presentation: visualization, quantitation, and in situ detection of human viral epitopes using peptide-specific, MHC-restricted human recombinant antibodies. J. Immunol. 170, 4349–4361 (2003).

    CAS  Article  Google Scholar 

  25. Biddison, W.E. et al. Tax and M1 peptide/HLA-A2-specific Fabs and T cell receptors recognize nonidentical structural features on peptide/HLA-A2 complexes. J. Immunol. 171, 3064–3074 (2003).

    CAS  Article  Google Scholar 

  26. Held, G. et al. Dissecting cytotoxic T cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments. Eur. J. Immunol. 34, 2919 (2004).

    CAS  Article  Google Scholar 

  27. Anderton, S.M., Radu, C.G., Lowrey, P.A., Ward, E.S. & Wraith, D.C. Negative selection during the peripheral immune response to antigen. J. Exp. Med. 193, 1–11 (2001).

    CAS  Article  Google Scholar 

  28. Garboczi, D.N., Hung, D.T. & Wiley, D.C. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA 89, 3429–3433 (1992).

    CAS  Article  Google Scholar 

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Acknowledgements

We would like to thank the following for supplying plasmids containing wild-type TCR genes: W.E. Biddison for A6, V. Cerundolo for 1G4 and MM15, E. Gostick for ILAK, S. Burrows for LC13, G.F. Gao for JM22, H. Gaston for AH1.23, S. Gadola for CD1d and Paul Bowness for GRb. We would like to thank M. Sami, P. Todorov and A. Johnson for assistance with protein purification, Martin Green, R. Ashfield and N. Lissin for helpful discussions and critical reading of the manuscript, B. Laugel and A.K. Sewell for additionally sharing data before publication and D. Sutton for assistance in preparing figures.

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Correspondence to Jonathan M Boulter.

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Competing interests

Authors with affiliation to Avidex Ltd. are or have been employees of Avidex. J.M.B. and B.K.J. are also minor shareholders. Avidex Ltd. is developing high affinity TCRs as therapeutic targeting agents. We do not believe that this has in any way influenced our presentation or interpretation of the data presented in this manuscript.

Supplementary information

Supplementary Fig. 1

Global fit analysis of 1G4c113 binding (PDF 86 kb)

Supplementary Fig. 2

Competition analysis of 1G4c113 binding (PDF 199 kb)

Supplementary Table 1

Peptide-HLA complexes used for testing A6c134 specificity. Peptide-HLA complexes used for testing 1G4c113 specificity. (PDF 50 kb)

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Li, Y., Moysey, R., Molloy, P. et al. Directed evolution of human T-cell receptors with picomolar affinities by phage display. Nat Biotechnol 23, 349–354 (2005). https://doi.org/10.1038/nbt1070

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