Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro


Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs. The approach takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3β. In vitro differentiation of progenitors transduced with a known Tcra gene in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. We purified these cells to generate TCRβ chain libraries pre-enriched for target antigen specificity. Several TCRβ chains paired with a transgenic TCRα chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Agonist signaling drives CD4/CD8 DN TCRαβ+ γδ-like T cell development in vitro.
Figure 2: Ectopic expression of an antigen-specific TCRα chain before β-selection.
Figure 3: Analysis of enhanced-affinity TCRs recovered from the agonist-selected TCRβ library screen.
Figure 4: High-affinity WT1-specific T cells develop in 3D-PYYα retrogenic mice.
Figure 5: Agonist-selection of in vitro-derived human T cells with enhanced affinity for antigen.
Figure 6: Analysis of human enhanced-affinity TCRs.


  1. 1

    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).

    CAS  Article  Google Scholar 

  2. 2

    Porter, D.L., Levine, B.L., Kalos, M., Bagg, A. & June, C.H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N. Engl. J. Med. 365, 725–733 (2011).

    CAS  Article  Google Scholar 

  3. 3

    Kochenderfer, J.N. et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 116, 4099–4102 (2010).

    CAS  Article  Google Scholar 

  4. 4

    Chapuis, A.G. et al. Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients. Sci. Transl. Med. 5, 174ra27 (2013).

    Article  Google Scholar 

  5. 5

    Chapuis, A.G. et al. Transferred melanoma-specific CD8+ T cells persist, mediate tumor regression, and acquire central memory phenotype. Proc. Natl. Acad. Sci. USA 109, 4592–4597 (2012).

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Dudley, M.E. et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J. Clin. Oncol. 26, 5233–5239 (2008).

    CAS  Article  Google Scholar 

  8. 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  Google Scholar 

  9. 9

    Stromnes, I.M. et al. Abrogation of SRC homology region 2 domain-containing phosphatase 1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo. J. Immunol. 189, 1812–1825 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Schmitt, T.M., Ragnarsson, G.B. & Greenberg, P.D. T cell receptor gene therapy for cancer. Hum. Gene Ther. 20, 1240–1248 (2009).

    CAS  Article  Google Scholar 

  11. 11

    Garrido, F., Aptsiauri, N., Doorduijn, E.M., Garcia Lora, A.M. & van Hall, T. The urgent need to recover MHC class I in cancers for effective immunotherapy. Curr. Opin. Immunol. 39, 44–51 (2016).

    CAS  Article  Google Scholar 

  12. 12

    Udyavar, A., Alli, R., Nguyen, P., Baker, L. & Geiger, T.L. Subtle affinity-enhancing mutations in a myelin oligodendrocyte glycoprotein-specific TCR alter specificity and generate new self-reactivity. J. Immunol. 182, 4439–4447 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Zhao, Y. et al. High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines. J. Immunol. 179, 5845–5854 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Richman, S.A. & Kranz, D.M. Display, engineering, and applications of antigen-specific T cell receptors. Biomol. Eng. 24, 361–373 (2007).

    CAS  Article  Google Scholar 

  15. 15

    Wucherpfennig, K.W., Gagnon, E., Call, M.J., Huseby, E.S. & Call, M.E. Structural biology of the T-cell receptor: insights into receptor assembly, ligand recognition, and initiation of signaling. Cold Spring Harb. Perspect. Biol. 2, a005140 (2010).

    Article  Google Scholar 

  16. 16

    Huseby, E.S., Crawford, F., White, J., Marrack, P. & Kappler, J.W. Interface-disrupting amino acids establish specificity between T cell receptors and complexes of major histocompatibility complex and peptide. Nat. Immunol. 7, 1191–1199 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Stadinski, B.D. et al. A role for differential variable gene pairing in creating T cell receptors specific for unique major histocompatibility ligands. Immunity 35, 694–704 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Wang, J.-H. & Reinherz, E.L. The structural basis of αβ T-lineage immune recognition: TCR docking topologies, mechanotransduction, and co-receptor function. Immunol. Rev. 250, 102–119 (2012).

    Article  Google Scholar 

  19. 19

    Cameron, B.J. et al. Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells. Sci. Transl. Med. 5, 197ra103 (2013).

    Article  Google Scholar 

  20. 20

    Linette, G.P. et al. Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood 122, 863–871 (2013).

    CAS  Article  Google Scholar 

  21. 21

    von Boehmer, H. et al. Pleiotropic changes controlled by the pre-T-cell receptor. Curr. Opin. Immunol. 11, 135–142 (1999).

    CAS  Article  Google Scholar 

  22. 22

    Pennington, D.J., Silva-Santos, B. & Hayday, A.C. Gammadelta T cell development--having the strength to get there. Curr. Opin. Immunol. 17, 108–115 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Terrence, K., Pavlovich, C., Matechak, E. & Fowlkes, B. Premature expression of T cell receptor (TCR)αβ suppresses TCRγδ gene rearrangement but permits development of γδ lineage T cells. J. Exp. Med. 192, 537–548 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Egawa, T., Kreslavsky, T., Littman, D. & von Boehmer, H. Lineage diversion of T cell receptor transgenic thymocytes revealed by lineage fate mapping. PLoS One 3, 1512 (2008).

    Article  Google Scholar 

  25. 25

    Baldwin, T.A., Sandau, M.M., Jameson, S.C. & Hogquist, K.A. The timing of TCR alpha expression critically influences T cell development and selection. J. Exp. Med. 202, 111–121 (2005).

    CAS  Article  Google Scholar 

  26. 26

    von Boehmer, H., Kirberg, J. & Rocha, B. An unusual lineage of α/β T cells that contains autoreactive cells. J. Exp. Med. 174, 1001–1008 (1991).

    CAS  Article  Google Scholar 

  27. 27

    Schmitt, T.M. & Zúñiga-Pflücker, J.C. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756 (2002).

    CAS  Article  Google Scholar 

  28. 28

    Schmitt, T.M. & Zúñiga-Pflücker, J.C. T-cell development, doing it in a dish. Immunol. Rev. 209, 95–102 (2006).

    Article  Google Scholar 

  29. 29

    Schmitt, T.M. et al. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5, 410–417 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Hogquist, K.A., Jameson, S.C. & Bevan, M.J. Strong agonist ligands for the T cell receptor do not mediate positive selection of functional CD8+ T cells. Immunity 3, 79–86 (1995).

    CAS  Article  Google Scholar 

  31. 31

    Page, D.M., Kane, L.P., Allison, J.P. & Hedrick, S.M. Two signals are required for negative selection of CD4+CD8+ thymocytes. J. Immunol. 151, 1868–1880 (1993).

    CAS  PubMed  Google Scholar 

  32. 32

    Schmitt, T.M. et al. Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection. Blood 122, 348–356 (2013).

    CAS  Article  Google Scholar 

  33. 33

    Chervin, A.S., Aggen, D.H., Raseman, J.M. & Kranz, D.M. Engineering higher affinity T cell receptors using a T cell display system. J. Immunol. Methods 339, 175–184 (2008).

    CAS  Article  Google Scholar 

  34. 34

    Buckler, A.J., Pelletier, J., Haber, D.A., Glaser, T. & Housman, D.E. Isolation, characterization, and expression of the murine Wilms' tumor gene (WT1) during kidney development. Mol. Cell. Biol. 11, 1707–1712 (1991).

    CAS  Article  Google Scholar 

  35. 35

    Scharnhorst, V., van der Eb, A.J. & Jochemsen, A.G. WT1 proteins: functions in growth and differentiation. Gene 273, 141–161 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Holst, J., Vignali, K.M., Burton, A.R. & Vignali, D.A. Rapid analysis of T-cell selection in vivo using T cell–receptor retrogenic mice. Nat. Methods 3, 191–197 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Smith, T.R.F., Verdeil, G., Marquardt, K. & Sherman, L.A. Contribution of TCR signaling strength to CD8+ T cell peripheral tolerance mechanisms. J. Immunol. 193, 3409–3416 (2014).

    CAS  Article  Google Scholar 

  38. 38

    Robins, H.S. et al. Comprehensive assessment of T-cell receptor β-chain diversity in αβ T cells. Blood 114, 4099–4107 (2009).

    CAS  Article  Google Scholar 

  39. 39

    Snauwaert, S. et al. In vitro generation of mature, naive antigen-specific CD8+ T cells with a single T-cell receptor by agonist selection. Leukemia 28, 830–841 (2013).

    Article  Google Scholar 

  40. 40

    Van Coppernolle, S. et al. Functionally mature CD4 and CD8 TCRαβ cells are generated in OP9-DL1 cultures from human CD34+ hematopoietic cells. J. Immunol. 183, 4859–4870 (2009).

    CAS  Article  Google Scholar 

  41. 41

    Liu, B. et al. 2D TCR-pMHC-CD8 kinetics determines T-cell responses in a self-antigen-specific TCR system. Eur. J. Immunol. 44, 239–250 (2014).

    CAS  Article  Google Scholar 

  42. 42

    Huang, J. et al. The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness. Nature 464, 932–936 (2010).

    CAS  Article  Google Scholar 

  43. 43

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

    CAS  Article  Google Scholar 

  44. 44

    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 

  45. 45

    Jones, L.L., Colf, L.A., Stone, J.D., Garcia, K.C. & Kranz, D.M. Distinct CDR3 conformations in TCRs determine the level of cross-reactivity for diverse antigens, but not the docking orientation. J. Immunol. 181, 6255–6264 (2008).

    CAS  Article  Google Scholar 

  46. 46

    Hunder, N.N. et al. Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N. Engl. J. Med. 358, 2698–2703 (2008).

    CAS  Article  Google Scholar 

  47. 47

    Tran, E. et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344, 641–645 (2014).

    CAS  Article  Google Scholar 

  48. 48

    Schmitt, T.M., Stromnes, I.M., Chapuis, A.G. & Greenberg, P.D. New strategies in engineering T-cell receptor gene-modified t cells to more effectively target malignancies. Clin. Cancer Res. 21, 5191–5197 (2015).

    CAS  Article  Google Scholar 

  49. 49

    Stone, J.D. & Kranz, D.M. Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies. Front. Immunol. 4, 244 (2013).

    CAS  Article  Google Scholar 

  50. 50

    Mehrotra, S. et al. A coreceptor-independent transgenic human TCR mediates anti-tumor and anti-self immunity in mice. J. Immunol. 189, 1627–1638 (2012).

    CAS  Article  Google Scholar 

  51. 51

    Fujiwara, H. et al. Antileukemia multifunctionality of CD4+ T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer. Leukemia 29, 2393–2401 (2015).

    CAS  Article  Google Scholar 

  52. 52

    Brochet, X., Lefranc, M.-P. & Giudicelli, V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res. 36, W503–W508 (2008).

    CAS  Article  Google Scholar 

  53. 53

    Giudicelli, V., Brochet, X. & Lefranc, M.-P. IMGT/V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences. Cold Spring Harb. Protoc. 2011, pdb.prot5633 (2011).

    PubMed  Google Scholar 

  54. 54

    Letourneur, F. & Malissen, B. Derivation of a T cell hybridoma variant deprived of functional T cell receptor α and β chain transcripts reveals a nonfunctional α-mRNA of BW5147 origin. Eur.J.Immunol. 19, 2269–2274 (1989).

    CAS  Article  Google Scholar 

Download references


This work was supported by the US National Institutes of Health (NIH) (P01 CA18029-40 and R01 CA033084-32, P.D.G.; NIH CA178844, D.M.K.) and the Guillot Family Fund. T.M.S. is supported by the Jose Carreras International Leukemia Foundation 2013 E.D. Thomas Post-Doctoral Fellowship. We would also like to thank C. Delaney for providing cord blood samples, L. Badenhorst for technical assistance, S. Tan for help with in vivo experiments, and N. Duerkopp for administrative assistance.

Author information




T.M.S. conceptualized, designed and performed the research, interpreted results and wrote the manuscript; D.H.A. contributed to experimental design and developed the 3D-PYY TCR; K.I.-T. performed and optimized experiments; S.O. contributed to experimental design; D.M.K. designed experiments and interpreted results and P.D.G. designed the experiments, assisted in writing the manuscript and supervised the study.

Corresponding author

Correspondence to Philip D Greenberg.

Ethics declarations

Competing interests

P.D.G. is a co-founder of Juno therapeutics, which is developing T-cell-based immunotherapies including TCR gene therapy. P.D.G. and T.M.S. are co-inventors on a patent application covering the technology described here.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schmitt, T., Aggen, D., Ishida-Tsubota, K. et al. Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro. Nat Biotechnol 35, 1188–1195 (2017).

Download citation

Further reading


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