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Rewiring T-cell responses to soluble factors with chimeric antigen receptors

Abstract

Chimeric antigen receptor (CAR)-expressing T cells targeting surface-bound tumor antigens have yielded promising clinical outcomes, with two CD19 CAR-T cell therapies recently receiving FDA approval for the treatment of B-cell malignancies. The adoption of CARs for the recognition of soluble ligands, a distinct class of biomarkers in physiology and disease, could considerably broaden the utility of CARs in disease treatment. In this study, we demonstrate that CAR-T cells can be engineered to respond robustly to diverse soluble ligands, including the CD19 ectodomain, GFP variants, and transforming growth factor beta (TGF-β). We additionally show that CAR signaling in response to soluble ligands relies on ligand-mediated CAR dimerization and that CAR responsiveness to soluble ligands can be fine-tuned by adjusting the mechanical coupling between the CAR's ligand-binding and signaling domains. Our results support a role for mechanotransduction in CAR signaling and demonstrate an approach for systematically engineering immune-cell responses to soluble, extracellular ligands.

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Figure 1: GFP-binding CARs can respond to soluble GFP ligands that are capable of dimerizing CARs.
Figure 2: A TGF-β-binding CAR converts soluble TGF-β into a T-cell stimulatory molecule.
Figure 3: The TGF-β CAR converts TGF-β from a T-cell growth suppressant to a T-cell growth stimulant.
Figure 4: TGF-β-mediated CAR dimerization in cis can activate the TGF-β CAR.
Figure 5: TGF-β CARs form actin-dependent clusters.
Figure 6: A mechanotransduction model for TGF-β CAR function and functional tuning.

References

  1. Sadelain, M., Rivière, I. & Riddell, S. Therapeutic T cell engineering. Nature 545, 423–431 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Chang, Z.L. & Chen, Y.Y. CARs: synthetic immunoreceptors for cancer therapy and Beyond. Trends Mol. Med. 23, 430–450 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Brown, C.E. et al. Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N. Engl. J. Med. 375, 2561–2569 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Ali, A. et al. HIV-1-specific chimeric antigen receptors based on broadly neutralizing antibodies. J. Virol. 90, 6999–7006 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Ellebrecht, C.T. et al. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease. Science 353, 179–184 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Hombach, A. et al. An anti-CD30 chimeric receptor that mediates CD3-ζ-independent T-cell activation against Hodgkin's lymphoma cells in the presence of soluble CD30. Cancer Res. 58, 1116–1119 (1998).

    CAS  PubMed  Google Scholar 

  7. Lanitis, E. et al. Redirected antitumor activity of primary human lymphocytes transduced with a fully human anti-mesothelin chimeric receptor. Mol. Ther. 20, 633–643 (2012).

    CAS  PubMed  Google Scholar 

  8. Nolan, K.F. et al. Bypassing immunization: optimized design of “designer T cells” against carcinoembryonic antigen (CEA)-expressing tumors, and lack of suppression by soluble CEA. Clin. Cancer Res. 5, 3928–3941 (1999).

    CAS  PubMed  Google Scholar 

  9. Westwood, J.A. et al. The Lewis-Y carbohydrate antigen is expressed by many human tumors and can serve as a target for genetically redirected T cells despite the presence of soluble antigen in serum. J. Immunother. 32, 292–301 (2009).

    CAS  PubMed  Google Scholar 

  10. Ma, Q., DeMarte, L., Wang, Y., Stanners, C.P. & Junghans, R.P. Carcinoembryonic antigen-immunoglobulin Fc fusion protein (CEA-Fc) for identification and activation of anti-CEA immunoglobulin-T-cell receptor-modified T cells, representative of a new class of Ig fusion proteins. Cancer Gene Ther. 11, 297–306 (2004).

    CAS  PubMed  Google Scholar 

  11. Carpenter, R.O. et al. B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma. Clin. Cancer Res. 19, 2048–2060 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. McGuinness, R.P. et al. Anti-tumor activity of human T cells expressing the CC49-zeta chimeric immune receptor. Hum. Gene Ther. 10, 165–173 (1999).

    CAS  PubMed  Google Scholar 

  13. Chmielewski, M. et al. T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology 143, 1095–1107. e2 (2012).

    CAS  PubMed  Google Scholar 

  14. Irving, B.A. & Weiss, A. The cytoplasmic domain of the T cell receptor ζ chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 64, 891–901 (1991).

    CAS  PubMed  Google Scholar 

  15. Letourneur, F. & Klausner, R.D. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor zeta family proteins. Proc. Natl. Acad. Sci. USA 88, 8905–8909 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Romeo, C. & Seed, B. Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell 64, 1037–1046 (1991).

    CAS  PubMed  Google Scholar 

  17. Leibly, D.J. et al. A suite of engineered GFP molecules for oligomeric scaffolding. Structure 23, 1754–1768 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2010).

    CAS  PubMed  Google Scholar 

  19. Tang, J.C.Y. et al. A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation. Cell 154, 928–939 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Mack, M., Riethmüller, G. & Kufer, P. A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity. Proc. Natl. Acad. Sci. USA 92, 7021–7025 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Urbanska, K. et al. A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor. Cancer Res. 72, 1844–1852 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Tamada, K. et al. Redirecting gene-modified T cells toward various cancer types using tagged antibodies. Clin. Cancer Res. 18, 6436–6445 (2012).

    CAS  PubMed  Google Scholar 

  23. Rabinovich, G.A., Gabrilovich, D. & Sotomayor, E.M. Immunosuppressive strategies that are mediated by tumor cells. Annu. Rev. Immunol. 25, 267–296 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Flavell, R.A., Sanjabi, S., Wrzesinski, S.H. & Licona-Limón, P. The polarization of immune cells in the tumour environment by TGF-β. Nat. Rev. Immunol. 10, 554–567 (2010).

    CAS  PubMed  Google Scholar 

  25. Koehler, H., Kofler, D., Hombach, A. & Abken, H. CD28 costimulation overcomes transforming growth factor-beta-mediated repression of proliferation of redirected human CD4+ and CD8+ T cells in an antitumor cell attack. Cancer Res. 67, 2265–2273 (2007).

    CAS  PubMed  Google Scholar 

  26. Bunnell, S.C. et al. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Campi, G., Varma, R. & Dustin, M.L. Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling. J. Exp. Med. 202, 1031–1036 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Kim, S.T. et al. The alphabeta T cell receptor is an anisotropic mechanosensor. J. Biol. Chem. 284, 31028–31037 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Li, Y.-C. et al. Cutting edge: mechanical forces acting on T cells immobilized via the TCR complex can trigger TCR signaling. J. Immunol. 184, 5959–5963 (2010).

    CAS  PubMed  Google Scholar 

  30. Liu, B., Chen, W., Evavold, B.D. & Zhu, C. Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling. Cell 157, 357–368 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang, Y. et al. A flow cytometry method to quantitate internalized immunotoxins shows that taxol synergistically increases cellular immunotoxins uptake. Cancer Res. 70, 1082–1089 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Hargreaves, P.G. & Al-Shamkhani, A. Soluble CD30 binds to CD153 with high affinity and blocks transmembrane signaling by CD30. Eur. J. Immunol. 32, 163–173 (2002).

    CAS  PubMed  Google Scholar 

  33. Schwesinger, F. et al. Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates. Proc. Natl. Acad. Sci. USA 97, 9972–9977 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Hu, K.H. & Butte, M.J. T cell activation requires force generation. J. Cell Biol. 213, 535–542 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhang, H., Cordoba, S.-P., Dushek, O. & van der Merwe, P.A. Basic residues in the T-cell receptor ζ cytoplasmic domain mediate membrane association and modulate signaling. Proc. Natl. Acad. Sci. USA 108, 19323–19328 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Dobbins, J. et al. Binding of the cytoplasmic domain of CD28 to the plasma membrane inhibits Lck recruitment and signaling. Sci. Signal. 9, ra75 (2016).

    PubMed  PubMed Central  Google Scholar 

  37. van der Merwe, P.A. & Dushek, O. Mechanisms for T cell receptor triggering. Nat. Rev. Immunol. 11, 47–55 (2011).

    CAS  PubMed  Google Scholar 

  38. Yokosuka, T. et al. Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C θ translocation. Immunity 29, 589–601 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Sadelain, M. Chimeric antigen receptors: driving immunology towards synthetic biology. Curr. Opin. Immunol. 41, 68–76 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kalos, M. & June, C.H. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39, 49–60 (2013).

    CAS  PubMed  Google Scholar 

  41. Matsuda, M., Koga, M., Nishida, E. & Ebisuya, M. Synthetic signal propagation through direct cell-cell interaction. Sci. Signal. 5, ra31 (2012).

    PubMed  Google Scholar 

  42. Gordon, W.R. et al. Mechanical allostery: evidence for a force requirement in the proteolytic activation of Notch. Dev. Cell 33, 729–736 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Morsut, L. et al. Engineering customized cell sensing and response behaviors using synthetic Notch receptors. Cell 164, 780–791 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Schwarz, K.A., Daringer, N.M., Dolberg, T.B. & Leonard, J.N. Rewiring human cellular input-output using modular extracellular sensors. Nat. Chem. Biol. 13, 202–209 (2017).

    CAS  PubMed  Google Scholar 

  45. Hanash, S.M., Pitteri, S.J. & Faca, V.M. Mining the plasma proteome for cancer biomarkers. Nature 452, 571–579 (2008).

    CAS  PubMed  Google Scholar 

  46. Hu, S., Loo, J.A. & Wong, D.T. Human body fluid proteome analysis. Proteomics 6, 6326–6353 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Fedorov, V.D., Themeli, M. & Sadelain, M. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Sci. Transl. Med. 5, 215ra172 (2013).

    PubMed  PubMed Central  Google Scholar 

  48. Kloss, C.C., Condomines, M., Cartellieri, M., Bachmann, M. & Sadelain, M. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat. Biotechnol. 31, 71–75 (2013).

    CAS  PubMed  Google Scholar 

  49. Grada, Z. et al. TanCAR: a novel bispecific chimeric antigen receptor for cancer immunotherapy. Mol. Ther. Nucleic Acids 2, e105 (2013).

    PubMed  PubMed Central  Google Scholar 

  50. Zah, E., Lin, M.-Y., Silva-Benedict, A., Jensen, M.C. & Chen, Y.Y. T Cells expressing CD19/CD20 bispecific chimeric antigen receptors prevent antigen escape by malignant B cells. Cancer Immunol. Res. 4, 498–508 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Jonnalagadda, M. et al. Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy. Mol. Ther. 23, 757–768 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Nguyen, P., Moisini, I. & Geiger, T.L. Identification of a murine CD28 dileucine motif that suppresses single-chain chimeric T-cell receptor expression and function. Blood 102, 4320–4325 (2003).

    CAS  PubMed  Google Scholar 

  53. Moeller, M. et al. A functional role for CD28 costimulation in tumor recognition by single-chain receptor-modified T cells. Cancer Gene Ther. 11, 371–379 (2004).

    CAS  PubMed  Google Scholar 

  54. Zhao, Y. et al. A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity. J. Immunol. 183, 5563–5574 (2009).

    CAS  PubMed  Google Scholar 

  55. Gressner, A.M., Weiskirchen, R., Breitkopf, K. & Dooley, S. Roles of TGF-beta in hepatic fibrosis. Front. Biosci. 7, d793–d807 (2002).

    CAS  PubMed  Google Scholar 

  56. Junker, U. et al. Transforming growth factor beta 1 is significantly elevated in plasma of patients suffering from renal cell carcinoma. Cytokine 8, 794–798 (1996).

    CAS  PubMed  Google Scholar 

  57. Yu, Q. & Stamenkovic, I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-β and promotes tumor invasion and angiogenesis. Genes Dev. 14, 163–176 (2000).

    PubMed  PubMed Central  Google Scholar 

  58. Zhang, Q. et al. Adoptive transfer of tumor-reactive transforming growth factor-beta-insensitive CD8+ T cells: eradication of autologous mouse prostate cancer. Cancer Res. 65, 1761–1769 (2005).

    CAS  PubMed  Google Scholar 

  59. Foster, A.E. et al. Antitumor activity of EBV-specific T lymphocytes transduced with a dominant negative TGF-beta receptor. J. Immunother. 31, 500–505 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. El Hentati, F.-Z., Gruy, F., Iobagiu, C. & Lambert, C. Variability of CD3 membrane expression and T cell activation capacity. Cytometry B Clin. Cytom. 78, 105–114 (2010).

    PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Institutes of Health (DP5OD012133, grant to Y.Y.C.; F30CA183528, fellowship to Z.L.C.). We thank M. Jensen (Seattle Children's Research Institute), S. Forman (City of Hope National Medical Center), D. Kohn (University of California, Los Angeles), X. Lin (University of Texas MD Anderson Cancer Center), A. Weiss (University of California, San Francisco), and T. Yeates (University of California, Los Angeles) for materials used in this work. We also thank Y. Choi, H. Ho, and R. Smolkin for assistance and support in the lab.

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Contributions

Z.L.C. and Y.Y.C. designed the project, participated in data analysis throughout, and wrote the manuscript. Z.L.C., M.H.L., and Y.Y.C. edited and revised the manuscript. Z.L.C. developed the TGF-β CAR system and performed and analyzed microscopy, western blot, and computational modeling experiments. Z.L.C., M.H.L., and U.T. performed and analyzed TGF-β CAR flow cytometry, cytokine production, and cell expansion experiments. X.C. performed the CD19 CAR experiments. Z.L.C. and N.J.B. developed and tested the GFP CAR system.

Corresponding author

Correspondence to Yvonne Y Chen.

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Z.L.C. and Y.Y.C. declare competing financial interests in the form of a pending patent application whose value may be affected by the publication of this manuscript.

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Supplementary Data Set 1

Lack of bystander cell lysis by TGF-β CAR-T cells (XLSX 38 kb)

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Chang, Z., Lorenzini, M., Chen, X. et al. Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nat Chem Biol 14, 317–324 (2018). https://doi.org/10.1038/nchembio.2565

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