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High-throughput identification of antigen-specific TCRs by TCR gene capture

Abstract

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen–reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

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Figure 1: Rapid assembly of a CG antigen–specific TCR library by TCR gene capture.
Figure 2: TCR gene capture allows TCR identification in bulk antigen-specific T cell populations obtained from clinical material by frequency-based matching.
Figure 3: Identification of NY-ESO-1–specific TCRs in antigen-specific T cell populations from mice with a diverse human TCR repertoire.
Figure 4: Unbiased identification of tumor-reactive TCRs within oligoclonal intratumoral T cell populations.

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References

  1. Restifo, N.P., Dudley, M.E. & Rosenberg, S.A. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12, 269–281 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dudley, M.E. et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298, 850–854 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Besser, M.J. et al. Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clin. Cancer Res. 16, 2646–2655 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Schumacher, T.N. T-cell-receptor gene therapy. Nat. Rev. Immunol. 2, 512–519 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Linnemann, C., Schumacher, T.N. & Bendle, G.M. T-cell receptor gene therapy: critical parameters for clinical success. J. Invest. Dermatol. 131, 1806–1816 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Morgan, R.A. et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126–129 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Johnson, L.A. et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114, 535–546 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Robbins, P.F. et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J. Clin. Oncol. 29, 917–924 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Simpson, A.J., Caballero, O.L., Jungbluth, A., Chen, Y.T. & Old, L.J. Cancer/testis antigens, gametogenesis and cancer. Nat. Rev. Cancer 5, 615–625 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Scanlan, M.J., Simpson, A.J. & Old, L.J. The cancer/testis genes: review, standardization, and commentary. Cancer Immun. 4, 1 (2004).

    PubMed  Google Scholar 

  11. Reddy, S.T. et al. Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nat. Biotechnol. 28, 965–969 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Kwakkenbos, M.J. et al. Generation of stable monoclonal antibody-producing B cell receptor–positive human memory B cells by genetic programming. Nat. Med. 16, 123–128 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Wrammert, J. et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667–671 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Heemskerk, M.H. et al. Efficiency of T cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex. Blood 109, 235–243 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Mamedov, I.Z. et al. Quantitative tracking of T cell clones after haematopoietic stem cell transplantation. EMBO Mol. Med. 3, 201–207 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bolotin, D.A. et al. Next generation sequencing for TCR repertoire profiling: Platform-specific features and correction algorithms. Eur. J. Immunol. 42, 3073–3083 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Andersen, R.S. et al. Dissection of T cell antigen specificity in human melanoma. Cancer Res. 72, 1642–1650 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Kvistborg, P. et al. TIL therapy broadens the tumor-reactive CD8+ T cell compartment in melanoma patients. OncoImmunology 1, 409–418 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Derbinski, J. & Kyewski, B. How thymic antigen presenting cells sample the body's self-antigens. Curr. Opin. Immunol. 22, 592–600 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Sadovnikova, E. & Stauss, H.J. Peptide-specific cytotoxic T lymphocytes restricted by nonself major histocompatibility complex class I molecules: reagents for tumor immunotherapy. Proc. Natl. Acad. Sci. USA 93, 13114–13118 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Amir, A.L. et al. PRAME-specific allo-HLA-restricted T cells with potent antitumor reactivity useful for therapeutic T-cell receptor gene transfer. Clin. Cancer Res. 17, 5615–5625 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Li, Y. et al. Directed evolution of human T-cell receptors with picomolar affinities by phage display. Nat. Biotechnol. 23, 349–354 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Li, L.P. et al. Transgenic mice with a diverse human T cell antigen receptor repertoire. Nat. Med. 16, 1029–1034 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Stanislawski, T. et al. Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer. Nat. Immunol. 2, 962–970 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Wilde, S. et al. Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity. Blood 114, 2131–2139 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Traggiai, E. et al. Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 304, 104–107 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Gimeno, R. et al. Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2−/− γc−/− mice: functional inactivation of p53 in developing T cells. Blood 104, 3886–3893 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Hombrink, P. et al. High-throughput identification of potential minor histocompatibility antigens by MHC tetramer-based screening: feasibility and limitations. PLoS ONE 6, e22523 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jorritsma, A. et al. Selecting highly affine and well-expressed TCRs for gene therapy of melanoma. Blood 110, 3564–3572 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Hodis, E. et al. A landscape of driver mutations in melanoma. Cell 150, 251–263 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pleasance, E.D. et al. A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature 463, 184–190 (2010).

    Article  CAS  PubMed  Google Scholar 

  32. Wang, G.C., Dash, P., McCullers, J.A., Doherty, P.C. & Thomas, P.G. T cell receptor αβ diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection. Sci. Transl. Med. 4, 128ra142 (2012).

    Google Scholar 

  33. Seitz, S. et al. Reconstitution of paired T cell receptor α- and β-chains from microdissected single cells of human inflammatory tissues. Proc. Natl. Acad. Sci. USA 103, 12057–12062 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Matsushita, H. et al. Cancer exome analysis reveals a T-cell–dependent mechanism of cancer immunoediting. Nature 482, 400–404 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Castle, J.C. et al. Exploiting the mutanome for tumor vaccination. Cancer Res. 72, 1081–1091 (2012).

    Article  CAS  PubMed  Google Scholar 

  36. van Rooij, N. et al. Tumor exome analysis reveals neo-antigen–specific T cell reactivity in an ipilimumab-responsive melanoma. J. Clin. Oncol. 10.1200/JCO.2012.47.7521 (16 September 2013).

  37. de Witte, M.A. et al. An inducible caspase 9 safety switch can halt cell therapy-induced autoimmune disease. J. Immunol. 180, 6365–6373 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Kieback, E., Charo, J., Sommermeyer, D., Blankenstein, T. & Uckert, W. A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer. Proc. Natl. Acad. Sci. USA 105, 623–628 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Kalos, M. et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced Leukemia. Sci. Transl. Med. 3, 95ra73 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kebriaei, P. et al. Infusing CD19-directed T cells to augment disease control in patients undergoing autologous hematopoietic stem-cell transplantation for advanced B-lymphoid malignancies. Hum. Gene Ther. 23, 444–450 (2012).

    Article  CAS  PubMed  Google Scholar 

  41. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Friedman, K.M. et al. Tumor-specific CD4+ melanoma tumor-infiltrating lymphocytes. J. Immunother. 35, 400–408 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. van Lent, A.U. et al. In vivo modulation of gene expression by lentiviral transduction in ″human immune system″ Rag2−/− γc−/− mice. Methods Mol. Biol. 595, 87–115 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. Legrand, N. et al. Functional CD47/signal regulatory protein α (SIRPα) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo. Proc. Natl. Acad. Sci. USA 108, 13224–13229 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Yssel, H., De Vries, J.E., Koken, M., Van Blitterswijk, W. & Spits, H. Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones. J. Immunol. Methods 72, 219–227 (1984).

    Article  CAS  PubMed  Google Scholar 

  48. Spits, H. & Yssel, H. Cloning of human T and natural killer cells. Methods 9, 416–421 (1996).

    Article  CAS  PubMed  Google Scholar 

  49. Schiavetti, F., Thonnard, J., Colau, D., Boon, T. & Coulie, P.G. A human endogenous retroviral sequence encoding an antigen recognized on melanoma by cytolytic T lymphocytes. Cancer Res. 62, 5510–5516 (2002).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to A. Pfauth, F. van Diepen and B. Hooibrink for assistance with flow cytometry and to M. Nagasawa, T. Heidebrecht, E. Siteur-van Rijnstra, K. Weijer, M. Böhne, M. van der Maas, W. van de Kasteele and T. de Jong for technical assistance. The Bloemenhove Clinic (Heemstede, The Netherlands) provided fetal tissues. We thank A. Kaiser, S. Naik and J. Rohr for critical discussions. C.L. is a fellow in the PhD Fellowship Program of Boehringer Ingelheim Fonds – Foundation for Basic Research in Biomedicine. M.A.T., I.Z.M., D.A.B. and D.M.C. are supported by the Molecular and Cell Biology program of the Russian Academy of Sciences RFBR 12-04-33139 and 12-04-00229-a. G.M.B. is the recipient of a Leukaemia and Lymphoma Research Bennett Senior Non-clinical Fellowship (12004). T.B. is supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich TR36. This work was supported by grants from the Dutch Cancer Society (NKI 2009-4282 to T.N.M.S., J.B.A.G.H. and G.M.B. and NKI 2006-3530 to T.N.M.S. and H.S.) and the Danish Council for Strategic Research (09-065152 to S.R.H. and T.N.M.S.).

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Authors

Contributions

C.L. designed, performed, analyzed and interpreted all experiments and wrote the paper. B.H. designed, performed, analyzed and interpreted experiments for the analysis of bulk antigen-specific T cell responses and T cell subsets in TILs and helped in designing, performing, analyzing and interpreting CG-TCR validation experiments. P.K. identified CG antigen–specific T cell responses and helped in designing, performing, analyzing and interpreting CG-TCR validation experiments. R.J.C.K. and A.V. performed bioinformatic analysis to identify chromosomal rearrangements. X.C. designed, performed, analyzed and interpreted experiments in AaBbDII mice. R.S., N.L. and R.G.-E. designed, performed, analyzed and interpreted experiments with HIS mice. L.J. and P.H. designed, performed, analyzed and interpreted experiments to raise allogeneic TCRs against CD79b. C.J.S. identified CG antigen–specific T cell responses in TILs, contributed to RNA-bait library design and performed initial validations. K.B. and S.M. assisted in TCR validation experiments. M.N. and R.M.K. performed TCR gene capture and Illumina sequencing and analyzed and interpreted data. C.U.B. and J.B.A.G.H. provided patient material. M.A.T., I.Z.M., D.A.B. and D.M.C. developed the CDR3 identification algorithm, performed bioinformatic analysis to identify CDR3s and analyzed and interpreted data. H.S. designed and interpreted experiments in HIS mice. S.R.H. identified CG antigen–specific T cell responses and provided patient material. M.H.M.H. designed and interpreted experiments to raise allogeneic TCRs against CD79b. T.B. designed and interpreted experiments in AaBbDII mice. G.M.B. co-supervised the project, designed, performed, analyzed and interpreted all experiments and wrote the paper. T.N.M.S. developed the concept of TCR gene capture, supervised the project, designed and interpreted all experiments and wrote the paper.

Corresponding authors

Correspondence to Gavin M Bendle or Ton N M Schumacher.

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Linnemann, C., Heemskerk, B., Kvistborg, P. et al. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med 19, 1534–1541 (2013). https://doi.org/10.1038/nm.3359

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