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c-Kit signaling potentiates CAR T cell efficacy in solid tumors by CD28- and IL-2-independent co-stimulation

Nature Cancer volume 4, pages 1001–1015 (2023)Cite this article

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Abstract

The limited efficacy of chimeric antigen receptor (CAR) T cell therapy for solid tumors necessitates engineering strategies that promote functional persistence in an immunosuppressive environment. Herein, we use c-Kit signaling, a physiological pathway associated with stemness in hematopoietic progenitor cells (T cells lose expression of c-Kit during differentiation). CAR T cells with intracellular expression, but no cell-surface receptor expression, of the c-Kit D816V mutation (KITv) have upregulated STAT phosphorylation, antigen activation-dependent proliferation and CD28- and interleukin-2-independent and interferon-γ-mediated co-stimulation, augmenting the cytotoxicity of first-generation CAR T cells. This translates to enhanced survival, including in transforming growth factor-β-rich and low-antigen-expressing solid tumor models. KITv CAR T cells have equivalent or better in vivo efficacy than second-generation CAR T cells and are susceptible to tyrosine kinase inhibitors (safety switch). When combined with CD28 co-stimulation, KITv co-stimulation functions as a third signal, enhancing efficacy and providing a potent approach to treat solid tumors.

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Fig. 1: KITv CAR T cells demonstrate constitutive STAT phosphorylation and higher antigen-specific target cell lysis in vitro.
Fig. 2: KITv CAR T cells show potent antitumor efficacy in mesothelioma and lung cancer models in vivo.
Fig. 3: KITv co-stimulation enhances the antitumor activity of CAR T cells in a TGFβ-rich tumor microenvironment.
Fig. 4: KITv potentiates the antitumor efficacy of CAR T cells against low-antigen (MSLN+) lung cancer targets.
Fig. 5: KITv potentiates the antitumor efficacy of CAR T cells against low-antigen (MSLN+) mesothelioma cancer targets without cytotoxicity against mesothelial cells.
Fig. 6: KITv enhances the antitumor efficacy of PSMA-targeting CAR T cells.
Fig. 7: M28z-KITv CAR T cells are susceptible to tyrosine kinase inhibitors in vivo.

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Data availability

RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE229026. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

No custom code was generated in the course of this study.

References

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

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Cappell, K. M. & Kochenderfer, J. N. A comparison of chimeric antigen receptors containing CD28 versus 4-1BB costimulatory domains. Nat. Rev. Clin. Oncol. 18, 715–727 (2021).

    CAS  PubMed  Google Scholar 

  3. Grosser, R., Cherkassky, L., Chintala, N. & Adusumilli, P. S. Combination immunotherapy with CAR T cells and checkpoint blockade for the treatment of solid tumors. Cancer Cell 36, 471–482 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Kosti, P., Maher, J. & Arnold, J. N. Perspectives on chimeric antigen receptor T-cell immunotherapy for solid tumors. Front. Immunol. 9, 1104 (2018).

    PubMed  PubMed Central  Google Scholar 

  5. Guedan, S. et al. Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability. J. Clin. Invest. 130, 3087–3097 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Wijewarnasuriya, D., Bebernitz, C., Lopez, A. V., Rafiq, S. & Brentjens, R. J. Excessive costimulation leads to dysfunction of adoptively transferred T cells. Cancer Immunol. Res. 8, 732–742 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Drakes, D. J. et al. Optimization of T-cell receptor-modified T cells for cancer therapy. Cancer Immunol. Res. 8, 743–755 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Zuccolotto, G. et al. PSMA-specific CAR-engineered T cells for prostate cancer: CD28 outperforms combined CD28-4-1BB “super-stimulation”. Front. Oncol. 11, 708073 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Fraietta, J. A. et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat. Med. 24, 563–571 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Shum, T. et al. Constitutive signaling from an engineered IL7 receptor promotes durable tumor elimination by tumor-redirected T cells. Cancer Discov. 7, 1238–1247 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang, Y. et al. An IL-4/21 inverted cytokine receptor improving CAR-T cell potency in immunosuppressive solid-tumor microenvironment. Front. Immunol. 10, 1691 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Luo, H. et al. Coexpression of IL7 and CCL21 increases efficacy of CAR-T cells in solid tumors without requiring preconditioned lymphodepletion. Clin. Cancer Res. 26, 5494–5505 (2020).

    CAS  PubMed  Google Scholar 

  13. Ma, X. et al. Interleukin-23 engineering improves CAR T cell function in solid tumors. Nat. Biotechnol. 38, 448–459 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Hawkins, E. R., D’Souza, R. R. & Klampatsa, A. Armored CAR T-cells: the next chapter in T-cell cancer immunotherapy. Biologics 15, 95–105 (2021).

    PubMed  PubMed Central  Google Scholar 

  15. Kagoya, Y. et al. A novel chimeric antigen receptor containing a JAK–STAT signaling domain mediates superior antitumor effects. Nat. Med. 24, 352–359 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Rojas-Sutterlin, S., Lecuyer, E. & Hoang, T. Kit and Scl regulation of hematopoietic stem cells. Curr. Opin. Hematol. 21, 256–264 (2014).

    CAS  PubMed  Google Scholar 

  17. Ali, S. Role of c-Kit/SCF in cause and treatment of gastrointestinal stromal tumors (GIST). Gene 401, 38–45 (2007).

    CAS  PubMed  Google Scholar 

  18. Lennartsson, J. & Rönnstrand, L. Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol. Rev. 92, 1619–1649 (2012).

    CAS  PubMed  Google Scholar 

  19. Ceredig, R. & Rolink, T. A positive look at double-negative thymocytes. Nat. Rev. Immunol. 2, 888–897 (2002).

    CAS  PubMed  Google Scholar 

  20. Bluman, E. M. et al. The c-Kit ligand potentiates the allogeneic mixed lymphocyte reaction. Blood 88, 3887–3893 (1996).

    CAS  PubMed  Google Scholar 

  21. Frumento, G. et al. CD117 (c-Kit) is expressed during CD8+ T cell priming and stratifies sensitivity to apoptosis according to strength of TCR engagement. Front. Immunol. 10, 468 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Falchi, L. & Verstovsek, S. Kit mutations: new insights and diagnostic value. Immunol. Allergy Clin. North Am. 38, 411–428 (2018).

    PubMed  Google Scholar 

  23. Raghav, P. K., Singh, A. K. & Gangenahalli, G. A change in structural integrity of c-Kit mutant D816V causes constitutive signaling. Mutat. Res. 808, 28–38 (2018).

    CAS  PubMed  Google Scholar 

  24. Hirota, S. et al. Gain-of-function mutations of c-Kit in human gastrointestinal stromal tumors. Science 279, 577–580 (1998).

    CAS  PubMed  Google Scholar 

  25. Longley, B. J. et al. Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm. Nat. Genet. 12, 312–314 (1996).

    CAS  PubMed  Google Scholar 

  26. Beghini, A. et al. c-Kit mutations in core binding factor leukemias. Blood 95, 726–727 (2000).

    CAS  PubMed  Google Scholar 

  27. Jawhar, M. et al. Molecular profiling of myeloid progenitor cells in multi-mutated advanced systemic mastocytosis identifies KIT D816V as a distinct and late event. Leukemia 29, 1115–1122 (2015).

    CAS  PubMed  Google Scholar 

  28. Abbaspour Babaei, M., Kamalidehghan, B., Saleem, M., Huri, H. Z. & Ahmadipour, F. Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells. Drug Des. Devel. Ther. 10, 2443–2459 (2016).

    PubMed  PubMed Central  Google Scholar 

  29. Omori, I. et al. D816V mutation in the KIT gene activation loop has greater cell-proliferative and anti-apoptotic ability than N822K mutation in core-binding factor acute myeloid leukemia. Exp. Hematol. 52, 56–64 (2017).

    CAS  PubMed  Google Scholar 

  30. Orfao, A., Garcia-Montero, A. C., Sanchez, L. & Escribano, L., REMA. Recent advances in the understanding of mastocytosis: the role of KIT mutations. Br. J. Haematol. 138, 12–30 (2007).

  31. Xiang, Z., Kreisel, F., Cain, J., Colson, A. & Tomasson, M. H. Neoplasia driven by mutant c-KIT is mediated by intracellular, not plasma membrane, receptor signaling. Mol. Cell. Biol. 27, 267–282 (2007).

    CAS  PubMed  Google Scholar 

  32. Chaix, A. et al. Mechanisms of STAT protein activation by oncogenic KIT mutants in neoplastic mast cells. J. Biol. Chem. 286, 5956–5966 (2011).

    CAS  PubMed  Google Scholar 

  33. Harir, N. et al. Oncogenic Kit controls neoplastic mast cell growth through a STAT5/PI3-kinase signaling cascade. Blood 112, 2463–2473 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Larrue, C. et al. Oncogenic KIT mutations induce STAT3-dependent autophagy to support cell proliferation in acute myeloid leukemia. Oncogenesis 8, 39 (2019).

    PubMed  PubMed Central  Google Scholar 

  35. Wang, H. et al. The proto-oncogene c-Kit inhibits tumor growth by behaving as a dependence receptor. Mol. Cell 72, 413–425 (2018).

    CAS  PubMed  Google Scholar 

  36. Adusumilli, P. S. et al. Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity. Sci. Transl. Med. 6, 261ra151 (2014).

    PubMed  PubMed Central  Google Scholar 

  37. Cherkassky, L. et al. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. J. Clin. Invest. 126, 3130–3144 (2016).

    PubMed  PubMed Central  Google Scholar 

  38. Adusumilli, P. S. et al. A phase I trial of regional mesothelin-targeted CAR T-cell therapy in patients with malignant pleural disease, in combination with the anti-PD-1 agent pembrolizumab. Cancer Discov. 11, 2748–2763 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Ghosn, M. et al. Image-guided interventional radiological delivery of chimeric antigen receptor (CAR) T cells for pleural malignancies in a phase I/II clinical trial. Lung Cancer 165, 1–9 (2022).

    CAS  PubMed  Google Scholar 

  40. Cherkassky, L., Hou, Z., Amador-Molina, A. & Adusumilli, P. S. Regional CAR T cell therapy: an ignition key for systemic immunity in solid tumors. Cancer Cell 40, 569–574 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Nicolet, B. P. et al. CD29 identifies IFN-γ-producing human CD8. Proc. Natl Acad. Sci. USA 117, 6686–6696 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Bhat, P., Leggatt, G., Waterhouse, N. & Frazer, I. H. Interferon-γ derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity. Cell Death Dis. 8, e2836 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Smith, K. A. Interleukin-2: inception, impact, and implications. Science 240, 1169–1176 (1988).

    CAS  PubMed  Google Scholar 

  44. Linnekin, D. Early signaling pathways activated by c-Kit in hematopoietic cells. Int. J. Biochem. Cell Biol. 31, 1053–1074 (1999).

    CAS  PubMed  Google Scholar 

  45. Xu, Y. et al. Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15. Blood 123, 3750–3759 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med. 17, 1290–1297 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Ulloa, L., Doody, J. & Massagué, J. Inhibition of transforming growth factor-β/SMAD signalling by the interferon-γ/STAT pathway. Nature 397, 710–713 (1999).

    CAS  PubMed  Google Scholar 

  48. Kuga, H. et al. Interferon-γ suppresses transforming growth factor-β-induced invasion of gastric carcinoma cells through cross-talk of Smad pathway in a three-dimensional culture model. Oncogene 22, 7838–7847 (2003).

    CAS  PubMed  Google Scholar 

  49. Koh, J. et al. Regulatory (FoxP3+) T cells and TGF-β predict the response to anti-PD-1 immunotherapy in patients with non-small cell lung cancer. Sci. Rep. 10, 18994 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Hasegawa, Y. et al. Transforming growth factor-β1 level correlates with angiogenesis, tumor progression, and prognosis in patients with nonsmall cell lung carcinoma. Cancer 91, 964–971 (2001).

    CAS  PubMed  Google Scholar 

  51. Jakubowska, K., Naumnik, W., Niklinska, W. & Chyczewska, E. Clinical significance of HMGB-1 and TGF-β level in serum and BALF of advanced non-small cell lung cancer. Adv. Exp. Med. Biol. 852, 49–58 (2015).

    CAS  PubMed  Google Scholar 

  52. Urso, L. et al. Detection of circulating immunosuppressive cytokines in malignant pleural mesothelioma patients for prognostic stratification. Cytokine 146, 155622 (2021).

    CAS  PubMed  Google Scholar 

  53. Stockhammer, P. et al. Detection of TGF-β in pleural effusions for diagnosis and prognostic stratification of malignant pleural mesothelioma. Lung Cancer 139, 124–132 (2020).

    PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Feng, Y. et al. A novel human monoclonal antibody that binds with high affinity to mesothelin-expressing cells and kills them by antibody-dependent cell-mediated cytotoxicity. Mol. Cancer Ther. 8, 1113–1118 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Chen, J. et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530–534 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Tripathi, S. K. et al. Genome-wide analysis of STAT3-mediated transcription during early human TH17 cell differentiation. Cell Rep. 19, 1888–1901 (2017).

    CAS  PubMed  Google Scholar 

  59. Seo, H. et al. BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells. Nat. Immunol. 22, 983–995 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Fucà, 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).

    PubMed  PubMed Central  Google Scholar 

  61. Foster, B. M., Zaidi, D., Young, T. R., Mobley, M. E. & Kerr, B. A. CD117/c-Kit in cancer stem cell-mediated progression and therapeutic resistance. Biomedicines 6, 31 (2018).

    PubMed  PubMed Central  Google Scholar 

  62. Pistillo, M. P. et al. IFN-γ upregulates membranous and soluble PD-L1 in mesothelioma cells: potential implications for the clinical response to PD-1/PD-L1 blockade. Cell. Mol. Immunol. 17, 410–411 (2020).

    CAS  PubMed  Google Scholar 

  63. Chen, N., Li, X., Chintala, N. K., Tano, Z. E. & Adusumilli, P. S. Driving CARs on the uneven road of antigen heterogeneity in solid tumors. Curr. Opin. Immunol. 51, 103–110 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Morello, A., Sadelain, M. & Adusumilli, P. S. Mesothelin-targeted CARs: driving T cells to solid tumors. Cancer Discov. 6, 133–146 (2016).

    CAS  PubMed  Google Scholar 

  65. Ferrao, P., Gonda, T. J. & Ashman, L. K. Expression of constitutively activated human c-Kit in Myb transformed early myeloid cells leads to factor independence, histiocytic differentiation, and tumorigenicity. Blood 90, 4539–4552 (1997).

    CAS  PubMed  Google Scholar 

  66. Sepulveda, H., Cerwenka, A., Morgan, T. & Dutton, R. W. CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation. J. Immunol. 163, 1133–1142 (1999).

    CAS  PubMed  Google Scholar 

  67. Matos, M. E. et al. Expression of a functional c-Kit receptor on a subset of natural killer cells. J. Exp. Med. 178, 1079–1084 (1993).

    CAS  PubMed  Google Scholar 

  68. Poli, A. et al. CD56bright natural killer (NK) cells: an important NK cell subset. Immunology 126, 458–465 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Gong, M. C. et al. Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen. Neoplasia 1, 123–127 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Krutzik, P. O. & Nolan, G. P. Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55, 61–70 (2003).

    PubMed  Google Scholar 

  71. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  Google Scholar 

  72. Engström, P. G. et al. Systematic evaluation of spliced alignment programs for RNA-seq data. Nat. Methods 10, 1185–1191 (2013).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S. Koop and D. Sewell of the Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, for excellent editorial assistance. Work from the Adusumilli laboratory is supported by grants from the National Institutes of Health (grant numbers P30 CA008748, R01 CA236615-01, R01 CA235667 and U01 CA214195), the US Department of Defense (grant numbers CA170630, CA180889 and CA200437), the Baker Street Foundation, the Batishwa Fellowship, the Comedy versus Cancer Foundation, the Cycle for Survival Fund, the Dallepezze Foundation, the Derfner Foundation, the Esophageal Cancer Education Fund, the Geoffrey Beene Foundation, the Memorial Sloan Kettering Technology Development Fund, the Miner Fund for Mesothelioma Research, Mr. William H. Goodwin and Alice Goodwin, the Commonwealth Foundation for Cancer Research and the Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center. The Adusumilli laboratory also received research support from ATARA Biotherapeutics. The research support sources did not have any role in study design, collection, analysis and interpretation of data, writing of the article or the decision to submit the article for publication.

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Authors and Affiliations

  1. Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Yuquan Xiong, Meriem Taleb, Kyohei Misawa, Zhaohua Hou, Srijita Banerjee, Alfredo Amador-Molina, David R. Jones, Navin K. Chintala & Prasad S. Adusumilli

  2. Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Prasad S. Adusumilli

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Contributions

P.S.A. is responsible for the original concept of the study. P.S.A. and Y.X. designed and guided the studies. Y.X., M.T., K.M., S.B., N.K.C., Z.H., A.A.-M. and P.S.A. contributed to the study methodology and investigations. Y.X., M.T., N.K.C. and P.S.A. interpreted results and performed study analysis. Y.X. and P.S.A. wrote the original draft of the manuscript. Y.X., M.T., N.K.C. and P.S.A. performed scientific editing and revision. Y.X., M.T., K.M., S.B., N.K.C., Z.H., A.A.-M., D.R.J. and P.S.A. reviewed the manuscript draft. P.S.A. acquired funding.

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Correspondence to Prasad S. Adusumilli.

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Competing interests

Y.X. and P.S.A. have a pending patent application on the KITv mutation as a co-stimulatory domain to use in T cells. P.S.A. declares research funding from ATARA Biotherapeutics and Novocure; is a scientific advisory board member and consultant for Adjuvant Genomics, ATARA Biotherapeutics, Abound Bio, Bio4t2, Carisma Therapeutics, Imugene, ImmPACT Bio, Johnson & Johnson, Link Immunotherapeutics, Orion Pharma, Outpace Bio, Pluri-biotech, Putnam Associates and Verismo Therapeutics; has patents, royalties and intellectual property on MSLN-targeted CAR and other T cell therapies licensed to ATARA Biotherapeutics; has issued a patent method for the detection of cancer cells using virus; and has pending patent applications on PD-1 dominant-negative receptors, a wireless pulse-oximetry device and an ex vivo malignant pleural effusion culture system. All other authors declare no competing interests. Memorial Sloan Kettering Cancer Center has licensed intellectual property related to MSLN-targeted CARs and T cell therapies to ATARA Biotherapeutics and has associated financial interests.

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Extended data

Extended Data Fig. 1 Mz-KITv and M28z-KITv CAR T cells show relatively lower levels of PD1 expression.

a, Proportion of PD1+ cells in CD4 and CD8 CAR T cells after stimulation with antigen-expressing (MSLN+) target cells (n = 5 biological replicates; two-tailed paired t test **p = 0.0018, M28z vs. Mz-KITv; ***p = 0.0009, M28z vs. M28z-KITv in CD4 T cells; and **p = 0,0013, M28z vs. M28z-KITv; **p = 0.0075, M28z vs. Mz-KITv for CD8 T cells). b, Proportion and kinetics of PD1+ cells in CD4 and CD8 CAR T cells upon stimulation with MSLN+ target cells every four days, as indicated by arrows. All data shown are mean ± standard deviation and are representative of three independently repeated experiments with at least three independent donors.

Source data

Extended Data Fig. 2 Long-term functional persistence of M28z-KITv CAR T cells.

Mice were initially established with A549-M lung cancer metastatic tumor and treated with a single dose (1 × 105) of M28z-KITv CAR T cells. After tumor eradication for 122 days, confirmed by multiple tumor bioluminescence imaging (BLI), mice were rechallenged with intraperitoneal (i.p) injection of 1 × 106 A549-M cells on day (d) 123 after initial CAR T-cell treatment, and tumor burden was monitored with BLI (n = 4 mice).

Source data

Extended Data Fig. 3 KITv CAR T cells have molecular expression profiles associated with potent cytotoxic activity.

Mesothelin (MSLN)–targeted M28z and M28z-KITv CD8+ CAR T cells alone (unstimulated) or cocultured (stimulated) with MSLN-expressing 3T3-M cells for 24 hours (M28z Sti and M28z-KITv Sti) were collected, and their gene expression profiles were analyzed by RNA-seq analysis. a, Heat maps of the top 50 differentially expressed genes between unstimulated M28z and M28z-KITv CAR T cells. b, Individual gene expression of AP-1-bZip and IRF gene family members from the bulk RNA-seq analysis in unstimulated M28z (blue) and M28z-KITv (red) CAR T cells. Data are mean ± standard deviation from three samples across three different donors (*p < 0.05; **p < 0.01; ***p < 0.001) determined by multiple t tests. c, Gene-set enrichment analysis, comparing the gene expression of M28z-KITv and M28Z CAR T cells for JAK-STAT3 and IL2-STAT5 gene sets. d, Heat maps of the top 50 differentially expressed genes between M28z Sti and M28z-KITv Sti CAR T cells. e, Gene-set enrichment analysis for the expression profiles of stimulated M28z-KITv Sti compared with M28z Sti CAR T cells for IFN-α and IFN-γ gene sets. Nominal p values and false-discovery rate q values are indicated.

Source data

Extended Data Fig. 4 Dasatinib inhibits CAR T-cell cytotoxicity without affecting viability in vitro.

a, M28z-KITv CAR T cells were cultured in the presence or absence of dasatinib for 72 h. Target-cell viability was monitored by flow cytometry. The experiment was performed twice, and a representative example is shown. b, Luciferase-based cytotoxicity assay assessing M28z KITv CAR T-cell killing at 18 h when cocultured with ffLuc-expressing, mesothelin-positive target cells in the presence or absence of dasatinib (n = 3 technical replicates). The experiment was performed three times, and a representative example is shown.

Source data

Extended Data Fig. 5 Representative gating strategy used for flow cytometry analysis.

a, Tumor cells gating strategy corresponding to data shown in Figs. 1d, 2e, 4b, and h. b, CAR T cells in vitro gating strategy corresponding to data shown in Fig. 1e, h, and j. c, IFNγ KO CAR T cells in vitro gating strategy corresponding to data shown in Fig. 3c. d, CAR T cells in vivo gating strategy corresponding to data shown in Fig. 6d.

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Xiong, Y., Taleb, M., Misawa, K. et al. c-Kit signaling potentiates CAR T cell efficacy in solid tumors by CD28- and IL-2-independent co-stimulation. Nat Cancer 4, 1001–1015 (2023). https://doi.org/10.1038/s43018-023-00573-4

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