Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Co-opting signalling molecules enables logic-gated control of CAR T cells

Nature volume 615, pages 507–516 (2023)Cite this article

Subjects

Abstract

Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3,4,5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Proximal signalling molecules are necessary to propagate CAR T cell activation.
Fig. 2: Proximal signalling molecules are sufficient to propagate CAR T cell activation.
Fig. 3: ZAP-70 CARs bypass upstream signalling elements and can exhibit enhanced anti-tumour activity.
Fig. 4: LAT and SLP-76 CARs bypass upstream signalling elements to function together as a Boolean-logic AND gate.
Fig. 5: Targeted mutations establish LINK CAR specificity.
Fig. 6: LINK CAR mediates tumour eradication while preventing on-target, off-tumour toxicity.

Similar content being viewed by others

Data availability

All new CAR sequences are available in Supplementary Table 1. CAR constructs will be made available through Material Transfer Agreements when possible. The scRNA-seq datasets have been deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through the GEO series accession number GSE216286.

References

  1. Majzner, R. G. & Mackall, C. L. Clinical lessons learned from the first leg of the CAR T cell journey. Nat. Med. 25, 1341–1355 (2019).

    Article  CAS  PubMed  Google Scholar 

  2. June, C. H. & Sadelain, M. Chimeric antigen receptor therapy. N. Engl. J. Med. 379, 64–73 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 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).

    Article  CAS  PubMed  Google Scholar 

  4. Roybal, K. T. et al. Engineering T cells with customized therapeutic response programs using synthetic Notch receptors. Cell 167, 419–432 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Srivastava, S. et al. Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting. Cancer Cell 35, 489–503 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Maldini, C. R., Ellis, G. I. & Riley, J. L. CAR T cells for infection, autoimmunity and allotransplantation. Nat. Rev. Immunol. 18, 605–616 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Amor, C. et al. Senolytic CAR T cells reverse senescence-associated pathologies. Nature 583, 127–132 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Larson, R. C. & Maus, M. V. Recent advances and discoveries in the mechanisms and functions of CAR T cells. Nat. Rev. Cancer 21, 145–161 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Majzner, R. G. et al. Tuning the antigen density requirement for CAR T-cell activity. Cancer Discov. 10, 702–723 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Salter, A. I. et al. Comparative analysis of TCR and CAR signaling informs CAR designs with superior antigen sensitivity and in vivo function. Sci. Signal. 14, eabe2606 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Labanieh, L., Majzner, R. G. & Mackall, C. L. Programming CAR-T cells to kill cancer. Nat. Biomed. Eng. 2, 377–391 (2018).

    Article  CAS  PubMed  Google Scholar 

  13. Rafiq, S., Hackett, C. S. & Brentjens, R. J. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat. Rev. Clin. Oncol. 17, 147–167 (2020).

    Article  PubMed  Google Scholar 

  14. Eshhar, Z., Waks, T., Gross, G. & Schindler, D. G. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc. Natl Acad. Sci. USA 90, 720–724 (1993).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Frank, S. J. et al. Structural mutations of the T cell receptor zeta chain and its role in T cell activation. Science 249, 174–177 (1990).

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Finney, H. M., Lawson, A. D., Bebbington, C. R. & Weir, A. N. Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product. J. Immunol. 161, 2791–2797 (1998).

    Article  CAS  PubMed  Google Scholar 

  19. Pegram, H. J. et al. Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood 119, 4133–4141 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kloss, C. C. et al. Dominant-negative TGF-β receptor enhances PSMA-targeted human CAR T cell proliferation and augments prostate cancer eradication. Mol. Ther. 26, 1855–1866 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lynn, R. C. et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 576, 293–300 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    Article  CAS  PubMed  Google Scholar 

  23. Feucht, J. et al. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nat. Med. 25, 82–88 (2019).

    Article  CAS  PubMed  Google Scholar 

  24. Lamers, C. H. et al. Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity. Mol. Ther. 21, 904–912 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lamers, C. H. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J. Clin. Oncol. 24, e20–e22 (2006).

    Article  PubMed  Google Scholar 

  26. Courtney, A. H., Lo, W. L. & Weiss, A. TCR signaling: mechanisms of initiation and propagation. Trends Biochem. Sci. 43, 108–123 (2018).

    Article  CAS  PubMed  Google Scholar 

  27. Palacios, E. H. & Weiss, A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 23, 7990–8000 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Stein, P. L., Lee, H. M., Rich, S. & Soriano, P. pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells. Cell 70, 741–750 (1992).

    Article  CAS  PubMed  Google Scholar 

  29. Yamakami, K., Akao, S., Wakabayashi, K., Tadakuma, T. & Yoshizawa, N. Mice lacking protein tyrosine kinase fyn develop a T helper-type 1 response and resist Leishmania major infection. Environ. Health Prev. Med. 6, 132–135 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ledbetter, J. A. et al. CD28 ligation in T-cell activation: evidence for two signal transduction pathways. Blood 75, 1531–1539 (1990).

    Article  CAS  PubMed  Google Scholar 

  31. Zhao, Q., Williams, B. L., Abraham, R. T. & Weiss, A. Interdomain B in ZAP-70 regulates but is not required for ZAP-70 signaling function in lymphocytes. Mol. Cell. Biol. 19, 948–956 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Long, A. H. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med. 21, 581–590 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Majzner, R. G. et al. CAR T cells targeting B7-H3, a pan-cancer antigen, demonstrate potent preclinical activity against pediatric solid tumors and brain tumors. Clin. Cancer Res. 25, 2560–2574 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Maher, J., Brentjens, R. J., Gunset, G., Riviere, I. & Sadelain, M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ/CD28 receptor. Nat. Biotechnol. 20, 70–75 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Sugawara, T. et al. An improved retroviral gene transfer technique demonstrates inhibition of CD4−CD8− thymocyte development by kinase-inactive ZAP-70. J. Immunol. 161, 2888–2894 (1998).

    Article  CAS  PubMed  Google Scholar 

  36. Au-Yeung, B. B., Shah, N. H., Shen, L. & Weiss, A. ZAP-70 in signaling, biology, and disease. Annu. Rev. Immunol. 36, 127–156 (2018).

    Article  CAS  PubMed  Google Scholar 

  37. Paz, P. E. et al. Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells. Biochem. J 356, 461–471 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhang, W. et al. Association of Grb2, Gads, and phospholipase C-γ1 with phosphorylated LAT tyrosine residues. effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling. J. Biol. Chem. 275, 23355–23361 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Balagopalan, L., Coussens, N. P., Sherman, E., Samelson, L. E. & Sommers, C. L. The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harb. Perspect. Biol. 2, a005512 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Motto, D. G., Ross, S. E., Wu, J., Hendricks-Taylor, L. R. & Koretzky, G. A. Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production. J. Exp. Med. 183, 1937–1943 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P. & Samelson, L. E. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92, 83–92 (1998).

    Article  CAS  PubMed  Google Scholar 

  42. Labanieh, L. et al. Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell 185, 1745–1763 (2022).

    Article  CAS  PubMed  Google Scholar 

  43. Maude, S. L. et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N. Engl. J. Med. 378, 439–448 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Neelapu, S. S. et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N. Engl. J. Med. 377, 2531–2544 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Berdeja, J. G. et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study. Lancet 398, 314–324 (2021).

    Article  CAS  PubMed  Google Scholar 

  46. Wagner, J., Wickman, E., DeRenzo, C. & Gottschalk, S. CAR T cell therapy for solid tumors: bright future or dark reality? Mol. Ther. 28, 2320–2339 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Fuca, G., Reppel, L., Landoni, E., Savoldo, B. & Dotti, G. Enhancing chimeric antigen receptor T-cell efficacy in solid tumors. Clin. Cancer Res. 26, 2444–2451 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Hegde, M. et al. Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma. Nat. Commun. 11, 3549 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  49. Majzner, R. G. et al. GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature 603, 934–941 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  50. Lajoie, M. J. et al. Designed protein logic to target cells with precise combinations of surface antigens. Science 369, 1637–1643 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  51. Cho, J. H., Collins, J. J. & Wong, W. W. Universal chimeric antigen receptors for multiplexed and logical control of T cell responses. Cell 173, 1426–1438 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. MacKay, M. et al. The therapeutic landscape for cells engineered with chimeric antigen receptors. Nat. Biotechnol. 38, 233–244 (2020).

    Article  CAS  PubMed  Google Scholar 

  53. Jing, Y. et al. Expression of chimeric antigen receptor therapy targets detected by single-cell sequencing of normal cells may contribute to off-tumor toxicity. Cancer Cell 39, 1558–1559 (2021).

    Article  CAS  PubMed  Google Scholar 

  54. Parker, K. R. et al. Single-cell analyses identify brain mural cells expressing CD19 as potential off-tumor targets for CAR-T immunotherapies. Cell 183, 126–142 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kolanus, W., Romeo, C. & Seed, B. T cell activation by clustered tyrosine kinases. Cell 74, 171–183 (1993).

    Article  CAS  PubMed  Google Scholar 

  56. Fitzer-Attas, C. J., Schindler, D. G., Waks, T. & Eshhar, Z. Harnessing Syk family tyrosine kinases as signaling domains for chimeric single chain of the variable domain receptors: optimal design for T cell activation. J. Immunol. 160, 145–154 (1998).

    Article  CAS  PubMed  Google Scholar 

  57. Deindl, S. et al. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70. Cell 129, 735–746 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Lee, K. M. et al. Molecular basis of T cell inactivation by CTLA-4. Science 282, 2263–2266 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  59. Fry, T. J. et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat. Med. 24, 20–28 (2018).

    Article  CAS  PubMed  Google Scholar 

  60. Walker, A. J. et al. Tumor antigen and receptor densities regulate efficacy of a chimeric antigen receptor targeting anaplastic lymphoma kinase. Mol. Ther. 25, 2189–2201 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Jena, B. et al. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials. PLoS ONE 8, e57838 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  62. Roederer, M., Nozzi, J. L. & Nason, M. C. SPICE: exploration and analysis of post-cytometric complex multivariate datasets. Cytometry A 79, 167–174 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Lareau, C. A. et al. Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling. Nat. Biotechnol. 39, 451–461 (2021).

    Article  CAS  PubMed  Google Scholar 

  64. Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was funded in part by the NIH Director’s New Innovator Award (DP2 CA272092 to R.G.M.), the Parker Institute for Cancer Immunotherapy (R.G.M., C.L.M., A.T.S., E.W.W. and C.L.) and a Lloyd J. Old STAR Award from the Cancer Research Insitute (A.T.S.). E.W.W. is supported by a Bridge Fellow Award from the Parker Institute for Cancer Immunotherapy. C.L. is supported by a Stanford Science Fellowship, a Parker Scholarship and NHGRI K99HG012579. We thank H. Chang for insightful comments, and SciStories (S. Knemeyer, V. Yeung and S. M. Suh) for providing the schematics (Figs. 1, 2, 4 and 5 and Extended Data Figs. 1, 6–8 and 10) and consulting on figure design.

Author information

Authors and Affiliations

  1. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Aidan M. Tousley, Maria Caterina Rotiroti, Lea Wenting Rysavy, Won-Ju Kim, Guillermo Nicolas Dalton, Crystal L. Mackall & Robbie G. Majzner

  2. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Louai Labanieh

  3. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

    Louai Labanieh, Caleb Lareau, Evan W. Weber, Yajie Yin, Ansuman T. Satpathy, Crystal L. Mackall & Robbie G. Majzner

  4. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Caleb Lareau, Yajie Yin & Ansuman T. Satpathy

  5. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA

    Caleb Lareau, Elena Sotillo, Skyler P. Rietberg, Yajie Yin, Dorota Klysz, Peng Xu, Ansuman T. Satpathy, Crystal L. Mackall & Robbie G. Majzner

  6. Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Evan W. Weber

  7. Department of Chemical Engineering, Stanford University, Stanford, CA, USA

    Eva L. de la Serna & Alexander R. Dunn

  8. Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA

    Alexander R. Dunn

  9. Biophysics Program, Stanford University, Stanford, CA, USA

    Alexander R. Dunn

  10. Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Crystal L. Mackall

Authors
  1. Aidan M. Tousley
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Caterina Rotiroti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Louai Labanieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lea Wenting Rysavy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Won-Ju Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Caleb Lareau
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Elena Sotillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Evan W. Weber
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Skyler P. Rietberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Guillermo Nicolas Dalton
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yajie Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Dorota Klysz
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Peng Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Eva L. de la Serna
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Alexander R. Dunn
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Ansuman T. Satpathy
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Crystal L. Mackall
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Robbie G. Majzner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.M.T. conceptualized the work, cloned constructs, designed and performed experiments, analysed data and wrote the manuscript. M.C.R. conceptualized the work, designed and performed experiments and analysed data. L.L. conceptualized the work, cloned constructs and designed experiments. L.W.R. cloned constructs, performed experiments and analysed data. W.-J.K. designed and performed experiments and analysed data. C.L. and Y.Y. performed and analysed scRNA-seq experiments. E.S. designed experiments and performed molecular analyses. E.W.W. designed experiments and analysed scRNA-seq data. S.P.R. cloned constructs, designed and performed experiments and analysed data. G.N.D. performed in vivo experiments. D.K. designed experiments. P.X. performed in vivo experiments. E.L.d.l.S. performed experiments. A.T.S., C.L.M. and A.R.D. supervised and/or funded some elements of the work. R.G.M. conceptualized, funded and supervised the work, designed experiments and wrote the manuscript. All authors contributed to the editing of the manuscript.

Corresponding author

Correspondence to Robbie G. Majzner.

Ethics declarations

Competing interests

A.M.T., R.G.M., M.C.R., L.L. and C.L.M. are inventors on a pending patent application (PCT/US2022/017544, applicant: The Board of Trustees of the Leland Stanford Junior University) for the novel CARs described in this manuscript. R.G.M. and C.L.M. are co-founders of and hold equity in Link Cell Therapies. R.G.M., C.L.M. and L.L. are co-founders of and hold equity in Cargo Therapeutics (formerly Syncopation Life Sciences). C.L.M. is a co-founder of and holds equity in Lyell Immunopharma. R.G.M., L.L. and E.W.W. are consultants for and hold equity in Lyell Immunopharma. S.P.R. is a former employee of and holds equity in Lyell Immunopharma. R.G.M. is a consultant for NKarta, Arovella Pharmaceuticals, Innervate Radiopharmaceuticals, GammaDelta Therapeutics, Aptorum Group, Zai Labs, Immunai, Gadeta, FATE Therapeutics (DSMB) and Waypoint Bio. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Allogene Therapeutics and Merck Research Laboratories. C.L. is a consultant for Cartography Biosciences. A.M.T. and M.C.R. are consultants for and hold equity in Link Cell Therapies. A.M.T. is a consultant for and holds equity in Cargo Therapeutics (formerly Syncopation Life Sciences). E.W.W. is a consultant for and holds equity in VISTAN Health and consults for Umoja Biopharma. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature thanks Joseph Fraietta and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Required proximal signalling molecules for CD19-4-1BBζ and CD19-CD28ζ CAR signalling.

a, Schematic illustrating the TCR signalling pathway wherein LCK phosphorylates ITAM motifs in CD3ζ, creating a binding site for ZAP-70. ZAP-70 is then activated and phosphorylates adaptor proteins LAT and SLP-76. LAT and SLP-76 then form a scaffold for the recruitment of PLCγ1 and other downstream effector molecules that initiate T cell activation. b, Flow cytometric data exhibiting CD19-4-1BBζ CAR expression in unedited and edited T cells prior to stimulation. c, Quantification of TNF+IL-2+ and CD107a+IFNγ+ populations in edited CD19-4-1BBζ CAR T cells as shown in Fig. 1d,e. Baseline measurements from the unstimulated controls were subtracted from stimulated conditions. Shown are mean values ± s.d. of three experimental replicates. Data is representative of 3 independent experiments with different blood donors. Groups were compared using one-way ANOVA with correction for multiple comparisons (p < 2 x 10−16 for no guide vs LCK KO, no guide vs ZAP-70 KO, no guide vs LAT KO, and no guide vs SLP-76 KO). d, Flow cytometric data exhibiting CD19-CD28ζ CAR expression in unedited and edited T cells prior to stimulation. e, Flow cytometric plots demonstrating knockout efficiencies for proximal signalling molecules in CAR T cells shown in d. f,g, After CRISPR/Cas9-mediated knockout of the indicated proximal signalling molecules, CD19-CD28ζ CAR T cells were stimulated with Nalm6 tumour cells. Shown is flow cytometric data of TNF x IL-2 (f) and CD107a x IFNγ (g) in knockout populations designated in e. h, Quantification of TNF+IL-2+ and CD107a+IFNγ+ populations in edited CD19-CD28ζ CAR T cells as shown in f,g. Baseline measurements from the unstimulated controls were subtracted from stimulated conditions. Shown are mean values ± s.d. of three experimental replicates. The experiment shown in f-h was performed one time. Groups were compared using one-way ANOVA with correction for multiple comparisons (p < 2 x 10−16 for no guide vs LCK KO, no guide vs ZAP-70 KO, no guide vs LAT KO, and no guide vs SLP-76 KO). i, Schematic illustrating the protein domains of ZAP-70; dashed lines indicate the Kinase + Interdomain B (KIDB) region contained in the ZAP-70KIDB CAR. j, Flow cytometric data exhibiting expression of CD19-ZAP-70, CD19-ZAP-70Kinase, and CD19-ZAP-70KIDB CARs. k, Flow cytometric data exhibiting expression of HER2-targeting proximal signalling CARs.

Extended Data Fig. 2 ZAP-70 CAR T cells show potent in vitro and in vivo activity.

a, Representative flow cytometric plots of CAR, LAG-3, TIM-3, and PD-1 expression for T cells bearing CD19 or HER2-specific CARs containing 4-1BBζ or ZAP-70KIDB endodomains (day 10 after T cell activation). Representative of three experiments with different blood donors. b, IFNγ secretion (as measured by ELISA) by GD2-4-1BBζ and GD2-ZAP-70KIDB CAR T cells following 24-h culture in the absence of target cells. Shown are mean values ± s.d. of three experimental replicates. Representative of three experiments with different blood donors. Groups were compared with the unpaired t-test (two-tailed). c, IL-2 secretion by B7-H3 or GD2-specific CAR T cells containing ZAP-70KIDB or 4-1BBζ endodomains following co-culture with Nalm6 cells expressing B7-H3/GD2 or 143B osteosarcoma cells. Shown are mean values ± s.d. of three experimental replicates. Representative of four independent experiments with different blood donors. Groups were compared with the unpaired t-test (two-tailed). d, Tumour cell killing of GFP+ human neuroblastoma CHLA-255 cells co-cultured with B7-H3 or GD2-specific CAR T cells containing ZAP-70KIDB or 4-1BBζ endodomains at a 1:1 ratio of T cells to tumour cells. Shown are mean values ± s.d. of three experimental replicates. Representative of four independent experiments with different blood donors. e, Percent CAR+ T cells recovered from the bone marrow of CHLA-255-bearing mice on days 7 and 14 after treatment with 1 x 107 B7-H3-4-1BBζ or B7-H3-ZAP-70KIDB CAR T cells 7 days after tumour inoculation. Shown are mean values ± s.e.m. for n = 5 mice (B7-H3-ZAP-70KIDB) or n = 4 mice (B7-H3-4-1BBζ) per group per time point. Experiment was performed once at two time points. Groups were compared with the Mann–Whitney test (two-tailed). f, Absolute number of CAR T cells recovered from the spleens of CHLA-255-bearing mice on day 28 after treatment with 1 x 107 B7-H3-4-1BBζ or B7-H3-ZAP-70KIDB CAR T cells. Shown are mean values ± s.e.m. for n = 4 mice. Experiment was performed once. Groups were compared with the Mann–Whitney test (two-tailed). g,h, NSG mice bearing CHLA-255-luciferase xenografts were treated intravenously with 3 x 106 B7-H3-ZAP-70KIDB CAR T cells on day 7 post CHLA-255 engraftment, then rechallenged with B7-H3+Nalm6-luciferase or WT B7-H3-Nalm6-luciferase leukaemia lines on day 30 post initial tumour engraftment (dashed vertical line). g, Quantification of tumour progression for each individual mouse as measured by flux values acquired by BLI. h, Survival curves for mice bearing tumours shown in g. Experiment was performed once with n = 5 mice per group. Tumour growth in g was compared between groups using repeated measures one-way ANOVA with correction for multiple comparisons and survival in h was compared with the log-rank test. i, Flow cytometric plots of CAR expression on B7-H3-CD28ζ, B7-H3-CD28ζ(1XX), B7-H3-ζ(1XX), or B7-H3-ZAP-70KIDB CAR T cells (day 10 after T cell activation). j, NSG mice bearing 143B osteosarcoma xenografts were treated intravenously with 1 x 107 B7-H3-CD28ζ, B7-H3-CD28ζ(1XX), B7-H3-ζ(1XX), or B7-H3-ZAP-70KIDB CAR T cells on day 13 post tumour inoculation. Tumours were measured at least twice weekly, and tumour area was calculated as the product of the tumour width and depth. Experiment was performed one time with n = 5 mice per group (n = 6 mice for B7-H3-CD28ζ, n = 4 mice for B7-H3-ζ(1XX)). Tumour growth curves were compared with repeated measures one-way ANOVA with correction for multiple comparisons.

Extended Data Fig. 3 ZAP-70KIDB CAR T cells show distinct transcriptomic profiles.

a, Uniform manifold approximation and projection (UMAP) embedding visualization showing overlaid B7-H3-4-1BBζ versus B7-H3-ZAP-70KIDB CAR T cells ± stimulation with B7-H3+Nalm6. b, UMAP embedding visualization showing an overlay annotating the three CAR T cell donors. c, Numbers of differentially expressed genes in B7-H3-4-1BBζ versus B7-H3-ZAP-70KIDB CAR T cells ± stimulation with B7-H3+Nalm6. Differentially expressed genes were defined by a Benjamini–Hochberg adjusted p-value < 0.01 and abs(logFC) > 0.5 from Seurat’s FindMarkers function (two-sided Wilcoxon signed rank test). d, Violin plots characterizing single-cell gene expression modules for T cell exhaustion (left panel; n = 61 genes) and T cell memory signature (right panel; n = 69 genes) in unstimulated B7-H3-4-1BBζ versus B7-H3-ZAP-70KIDB CAR T cells. Module scores were computed using the FindModuleScores function in Seurat. Comparisons were performed with the Mann–Whitney (two-tailed) test. e, UMAP embedding analysis showing KLF2 expression in overlaid B7-H3-4-1BBζ versus B7-H3-ZAP-70KIDB CAR T cells ± stimulation with B7-H3+Nalm6. f, Heat map exhibiting differentially expressed subsets of genes associated with T cell effector, exhaustion, function, and memory signalling pathways in B7-H3-4-1BBζ versus B7-H3-ZAP-70KIDB CAR T cells ± stimulation with B7-H3+Nalm6.

Extended Data Fig. 4 ZAP-70KIDB CAR T cells exhibit phosphorylation of key signalling molecules with reduced baseline activation.

a,b, GD2-4-1BBζ, GD2-ZAP-70KIDB, GD2-CD28ζ and CD19-4-1BBζ CAR T cells were stimulated for 5, 15 or 45 min with anti-idiotype antibody-coated beads at a 4:1 bead to T cell ratio. a, Phospho-PLCg1S1248, total PLCg1, phospho-SLP-76S376, total SLP-76, phospho-AKT1/2/3S473, total AKT1/2/3, phospho-ERK1/2T202 Y204, total ERK1/2, phospho-NF-κBS536, total NF-κB, and phospho-c-JUNS73, were measured by western blot. b, Densitometric quantifications of phosphorylation levels observed in a, normalized to the unstimulated conditions. Intensities of phospho-protein bands were normalized to that of each correspondent total protein. Fold change in band intensity was calculated relative to unstimulated sample. Data in a and b is representative of two experiments with different blood donors. Uncropped data with molecular weight markers is displayed in Supplementary Fig. 2.

Extended Data Fig. 5 ZAP-70KIDB CARs depend on ZAP-70 kinase activity on LAT and SLP-76.

a, Flow cytometric data exhibiting surface CAR and intracellular CD247/CD3ζ expression of B7-H3-ZAP-70KIDB (± CD247 CRISPR–Cas9 knockout) (left panel), and IL-2 secretion (right panel) by B7-H3-ZAP-70KIDB (± CD247 knockout) CAR T cells following co-culture with B7-H3+Nalm6 cells or anti-CD3/anti-CD28 beads. Shown are mean values ± s.d. of three experimental replicates. Performed one time. b, Flow cytometric data exhibiting surface CAR and CD3 expression of B7-H3-ZAP-70KIDB (± TRAC knockout) (left panel), and IL-2 secretion (right panel) by B7-H3-ZAP-70KIDB (± TRAC knockout) CAR T cells following co-culture with B7-H3+Nalm6 cells or anti-CD3/anti-CD28 beads. Shown are mean values ± s.d. of three experimental replicates. Representative of two experiments with different blood donors. c, Flow cytometric data exhibiting the expression of B7-H3-ZAP-70KIDB CARs ± D461N mutation. d, Flow cytometric data exhibiting B7-H3-ZAP-70KIDB CAR expression in unedited and edited T cells prior to stimulation. e, Flow cytometric plots demonstrating knockout efficiencies for proximal signalling molecules in CAR T cells shown in d. f, Quantification of TNF+IL-2+ and CD107a+IFNγ+ populations as shown in Fig. 3h,i. Baseline measurements from the unstimulated controls were subtracted from the stimulated conditions. Shown are mean values ± s.d. of three experimental replicates. Representative of three experiments performed with two different blood donors. Groups were compared via one-way ANOVA with correction for multiple comparisons.

Extended Data Fig. 6 LAT and SLP-76 CARs jointly mediate T cell activation.

a, Flow cytometric data exhibiting CAR expression of co-transduced CD19 and HER2 proximal signalling CAR (LCK, FYN, LAT, and SLP-76) combinations. b, IL-2 secretion (as measured by ELISA) by T cells shown in a following co-culture with CD19+HER2+Nalm6 cells. Shown are mean values ± s.d. of three experimental replicates. Representative of four experiments performed with three different blood donors. Statistical comparisons performed with one-way ANOVA with correction for multiple comparisons (p < 2 x 10−16 for all comparisons to CD19-LAT + HER2-SLP-76). c, Flow cytometric expression of LAT and SLP-76 CARs on T cells utilized in Fig. 4f,g. d, Schematic illustrating incorporation of a dual Cysteine-to-Alanine (2CA) mutation in the CD28 hinge–TM domain of the LAT CAR component of the LINK system. e, Flow cytometric data exhibiting  LINK CAR (±2CA mutation) expression. f,g, IL-2 secretion (f) and tumour cell killing (g) by LINK CAR T cells (±2CA mutation) following co-culture with indicated cell lines. Shown are mean values ± s.d. of three experimental replicates. Representative of eight independent experiments performed with five different blood donors. Comparisons in f performed with unpaired t-test (two-tailed). h, Flow cytometric data exhibiting LINK CAR (CD19-28TM-LAT + HER2-8TM-SLP-76) expression in unedited and edited T cells before stimulation with CD19+HER2+ Nalm6. i, Flow cytometric plots demonstrating knockout efficiencies for proximal signalling molecules in CAR T cells utilized in Fig. 4h,i. j, Quantification of TNF+IL-2+ and CD107a+IFNγ+ populations as shown in Fig. 4h,i. Baseline measurements from the unstimulated controls were subtracted from the stimulated conditions. Shown are mean values ± s.d. of three experimental replicates. Comparisons were performed via one-way ANOVA with correction for multiple comparisons. Representative of two independent experiments with different blood donors. k, Flow cytometric expression of LINK CAR (±LAT Y132F).

Extended Data Fig. 7 Disrupting GADS interactions eliminates LINK CAR leakiness.

a, Schematic illustrating LINK CAR bearing both the Cysteine-to-Alanine (2CA) mutations and GADS-binding site deletions/truncations (ΔGADS). b, Flow cytometric expression of LINK CARs (±2CA, ±ΔGADS) on T cells utilized in Fig. 5b–d. c, Quantification of CD107a+ and CD69+ on indicated LINK CAR T cells following co-culture with indicated cell lines as shown in Fig. 5d. Representative of five independent experiments with four different blood donors. Shown are mean values ± s.d. of three experimental replicates. Comparisons were performed with one-way ANOVA with correction for multiple comparisons. d, Flow cytometric expression of LINK CAR T cells bearing either Y171F/Y191F point mutations (2YF) or deletion/truncation of the GADS-binding regions (ΔGADS). e, IL-2 secretion (as measured by ELISA) by indicated LINK CAR T cells following co-culture with indicated cell lines shown in Fig. 4c. Representative of two independent experiments with different blood donors. Shown are mean values ± s.d. of three experimental replicates. Note that data for CD19-28TM-LAT + HER2-8TM-SLP-76 and CD19-28TM-LATΔGADS + HER2-8TM-SLP-76ΔGADS conditions are identical to Fig. 5b. Comparisons were performed via one-way ANOVA with correction for multiple comparisons. f, Tumour cell killing of Nalm6 by CD19-CD28ζ, CD19-4-1BBζ, LINK (CD19-28TM-LAT + CD19-8TM-SLP-76), or LINK2CA+ΔGADS (CD19-28TM2CA-LATΔGADS + CD19-8TM-SLP-76ΔGADS) CAR T cells at a 1:1 ratio of T cells to tumour cells. Shown are mean values ± s.d. of three experimental replicates. Representative of three independent experiments with different blood donors.

Extended Data Fig. 8 Testing ROR1-targeting CARs in a model of on-target, off-tumour toxicity.

a, Schematic illustrating on-target, off-tumour toxicity in the lungs of tumour-bearing mice treated with ROR1 targeted CAR T cells. b, Flow cytometry plots exhibiting detection of ROR1-CD28ζ CAR on T cells with both recombinant human and mouse ROR1. c, IL-2 secretion (as measured by ELISA) by ROR1-CD28ζ CAR T cells after 24-h incubation with plate-bound human or mouse ROR1 protein at the indicated concentrations. Shown are mean values ± s.d. of three experimental replicates. Representative of three independent experiments with different blood donors. d, ROR1 and CD19 expression on single/double antigen positive Nalm6 lines used for experiments. e, Flow cytometric expression of ROR1-CD28ζ and indicated ROR1-and-CD19-targeted LINK CARs on T cells. f, Tumour cell killing of cell lines shown in d when co-cultured with the indicated CAR T cells at a 2:1 ratio of T cells to tumour cells. Shown are mean values ± s.d. of three experimental replicates. Representative of four independent experiments performed with two different blood donors. g,h, NSG mice bearing ROR1+CD19+Nalm6-luciferase were treated with 3 x 106 CD19-CD28ζ, CD19-4-1BBζ, or indicated LINK CAR T cells 3 days after tumour inoculation. g, Quantification of tumour progression for each individual mouse as measured by flux values acquired by BLI, normalized to tumour-free mice. h, Survival curves for mice bearing tumours shown in g. Performed once with n = 5 mice per group. Comparisons performed by the log-rank test. i–k, NSG mice bearing ROR1+CD19+Nalm6-luciferase were treated with 8 x 106 of the indicated CAR+ T cells three days after tumour inoculation. i, Absolute number of CAR+ T cells recovered from spleens on day 28 post treatment. j, Representative flow cytometric plots and k, quantification of CAR+ T central memory cells (TCM, CD45RA−CD62L+), T stem cell memory cells (TSCM, CD45RA+CD62L+), T effector memory cells (TEM, CD45RA−CD62L−), and T effector memory CD45RA+ cells (TEMRA, CD45RA+CD62L−) recovered from the spleens on day 28 post treatment. Data shown in i,k are mean values ± s.e.m. Performed one time with n = 5 mice (CD19-4-1BBζ and LINKΔGADS) or n = 4 mice (CD19-CD28ζ and LINK2CA+ΔGADS) per group. Comparisons made with the Mann–Whitney test (two-tailed).

Extended Data Fig. 9 Both the LAT and SLP-76 components of LINK CAR protect normal tissues expressing shared antigens.

a, Flow cytometric expression of ROR1-and-CD19-targeted LINK CARs on T cells. b-d, NSG mice bearing ROR1+CD19+Nalm6-luciferase were treated with 8 x 106 of the indicated LINK CAR T cells with the SLP-76 CAR bearing specificity for ROR1 on D+2 following tumour inoculation. b, Weights for individual mice over time plotted as a percentage of the weight on day 0. c, Quantification of tumour progression for each individual mouse as measured by flux values acquired by BLI. (d) Survival curves for mice bearing tumours shown in b,c. Comparisons were performed with the log-rank test. b–d was performed once with n = 5 mice per group. e–h, To assess on-target, off-tumour toxicity against normal B cells, NSG mice that had been intravenously injected with 2 x 106 human B cells on D-7 and ROR1+CD19+Nalm6-luciferase cells on D-3 were infused with 8 x 106 CD19-CD28ζ, LINK2CA+ΔGADS, or control (anti-CD19 scFv tethered to a CD8 hinge–TM with no signalling domain) CAR T cells from the same blood donor on D0. e, Weights for individual mice over time plotted as a percentage of the weight on day 0. f, Quantification of tumour progression for each individual mouse as measured by flux values acquired by BLI, normalized to tumour-free mice. g, Survival curves for mice bearing tumours shown in e-f. Comparisons performed by the log-rank test. h, Absolute number of CD20+ B cells recovered from the spleens of mice depicted in e-g on day 9 following treatment. Shown are mean values ± s.e.m. for n = 3 mice per group. Comparisons performed with the Mann–Whitney test (two-tailed). Data is representative of three different blood donors.

Extended Data Fig. 10 LINK CAR outperforms both the SynNotch and the SPLIT CAR system.

a, Schematic illustrating the potential for on-target, off-tumour toxicity for SynNotch CAR T cells. b, Flow cytometric expression of CD19-CD28ζ, ROR1-CD28ζ, ROR1-and-CD19-targeted LINK2CA+ΔGADS, SPLIT (ROR1-8TM-ζ + CD19-28TM-CD28), and SynNotch (CD19-SynNotch → ROR1-CD28ζ) CARs on T cells day 10 after activation. c, Inducible ROR1-CD28ζ CAR expression (detected with recombinant ROR1 protein) following stimulation through the CD19-SynNotch receptor after a 24-h co-culture with CD19+Nalm6. d, Killing of bystander ROR1+CD19− Nalm6-GFP either in the presence of ROR1−CD19− Nalm6-TdTomato (top panel) or ROR1+CD19+ Nalm6-TdTomato (bottom panel) by CD19-CD28ζ, LINK, or SynNotch CAR T cells on day 10 post T cell activation. Shown are mean values ± s.e.m. from four independent experiments with different blood donors. e–g, NSG mice bearing ROR1+CD19+Nalm6-luciferase were treated with 8 x 106 of the indicated CAR+ T cells three days after tumour inoculation. e, Weights for individual mice over time plotted as a percentage of the weight on day 0. f, Quantification of tumour progression for each individual mouse as measured by flux values acquired by BLI. g, Survival curves for mice bearing tumours shown in e,f. Comparisons performed with the log-rank test. Note: e–g is a repeat with a different blood donor of the experiment shown in Fig. 6d–f but without the CD19-CD28ζ group. n = 5 mice per group (n = 4 mice for Mock).

Supplementary information

Supplementary Figure 1

Gating strategies for flow cytometric data. Representative flow cytometry gating strategies for indicated experiments appearing in the main figures and extended data figures. Also included are unstimulated controls used for gating positive TNF x IL-2 and CD107a x IFNγ populations in proximal signalling molecule CRISPR knockout experiments.

Reporting Summary

Peer Review File

Supplementary Figure 2

Raw data for western blots. Raw, full scanned western blot data with molecular weight markers and loading controls, corresponding to the data shown in Extended Data Figure 4a-b. Some samples that were unrelated to this manuscript were run on the same gels and are indicated by a transparent text box.

Supplementary Table 1

CAR sequences. Amino acid sequences for CAR constructs, including scFvs, hinge–TM domains, costimulatory domains, and signalling domains. Includes sequences of novel proximal signalling CARs such as ZAP-70KIDB and LINK LAT/SLP-76 components (with all described mutations and truncations).

Supplementary Table 2

Source Data for in vivo experiments. Source data for all in vivo graphs and quantifications appearing in the Main Figures and Extended Data Figures.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tousley, A.M., Rotiroti, M.C., Labanieh, L. et al. Co-opting signalling molecules enables logic-gated control of CAR T cells. Nature 615, 507–516 (2023). https://doi.org/10.1038/s41586-023-05778-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-023-05778-2

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research