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PSMA-targeting TGFβ-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: a phase 1 trial

Abstract

Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-β. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-β receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion, >98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-β-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.

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Fig. 1: CART-PSMA-TGFβRDN protocol design and consort diagram.
Fig. 2: Engraftment of CART-PSMA-TGFβRDN cells and cytokine elaboration in peripheral blood.
Fig. 3: Pathological and radiologic evaluations following CART-PSMA-TGFβRDN cell infusion.
Fig. 4: T cell trafficking and DSP of the TME in CRPC biopsies before and after CART-PSMA-TGFβRDN cell infusion.
Fig. 5: Evaluation of clinical responses and other correlatives following adoptive transfer of CAR T cells in a patient with mCRPC.

Data availability

Sequencing data are available at the NCBI Sequence Read Archive under accession no. PRJNA769699. Additional requests for raw and analyzed data and/or materials will be promptly reviewed by the University of Pennsylvania Center for Innovation to determine whether the application is subject to any intellectual property or confidentiality requirements. Patient-related information not included in this report was collected as part of a clinical trial and may be subject to patient confidentiality. Any data and materials that can be shared will be released following execution of a material transfer agreement. CAR-PSMA consists of variable light and heavy chains from the J591 antibody sequence in the patent published on 12 December 2002, titled “Modified antibodies to prostate-specific membrane antigen and uses thereof” (no. WO 02/098897 A2). The variable light and heavy chains were used to construct a single-chain variable fragment fused to 4-1BB and CD3ζ intracellular endodomain sequences listed in the patent published on 8 October 2015, titled “Treatment of cancer using anti-CD19 chimeric antigen receptor” (no. US 2015/0283178 A1). TGFβRDN is comprised of the human TGFβRII sequence with the regions encoding the intracellular kinase domain removed20; the amino acid sequence of the complete CAR-PSMA-TGFβRDN is provided in Supplementary Fig. 7.

References

  1. Lu, X. et al. Effective combinatorial immunotherapy for castration-resistant prostate cancer. Nature 543, 728–732 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Venkatachalam, S., McFarland, T.R., Agarwal, N. & Swami, U. Immune checkpoint inhibitors in prostate cancer. Cancers (Basel) 13, 2187 (2021).

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

    Article  CAS  PubMed  Google Scholar 

  4. Maude, S. L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. New Engl. J. Med. 371, 1507–1517 (2014).

    Article  PubMed  Google Scholar 

  5. Schuster, S. J. et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. New Engl. J. Med. 377, 2545–2554 (2017).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Park, J. H. et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. New Engl. J. Med. 378, 449–459 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Wright, G. L. Jr. et al. Upregulation of prostate-specific membrane antigen after androgen-deprivation therapy. Urology 48, 326–334 (1996).

    Article  PubMed  Google Scholar 

  9. Perner, S. et al. Prostate-specific membrane antigen expression as a predictor of prostate cancer progression. Hum. Pathol. 38, 696–701 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Westdorp, H. et al. Immunotherapy for prostate cancer: lessons from responses to tumor-associated antigens. Front. Immunol. 5, 191 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Wolf, P. et al. Preclinical evaluation of a recombinant anti-prostate specific membrane antigen single-chain immunotoxin against prostate cancer. J. Immunother. 33, 262–271 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Silver, D. A., Pellicer, I., Fair, W. R., Heston, W. D. & Cordon-Cardo, C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin. Cancer Res. 3, 81–85 (1997).

    CAS  PubMed  Google Scholar 

  13. Mhawech-Fauceglia, P. et al. Prostate-specific membrane antigen (PSMA) protein expression in normal and neoplastic tissues and its sensitivity and specificity in prostate adenocarcinoma: an immunohistochemical study using mutiple tumour tissue microarray technique. Histopathology 50, 472–483 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Rubtsov, Y. P. & Rudensky, A. Y. TGFbeta signalling in control of T-cell-mediated self-reactivity. Nat. Rev. Immunol. 7, 443–453 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Timme, T. L. et al. Mesenchymal-epithelial interactions and transforming growth factor-beta expression during mouse prostate morphogenesis. Endocrinology 134, 1039–1045 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Wikstrom, P., Damber, J. & Bergh, A. Role of transforming growth factor-beta1 in prostate cancer. Microsc. Res. Tech. 52, 411–419 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Steiner, M. S. & Barrack, E. R. Transforming growth factor-beta 1 overproduction in prostate cancer: effects on growth in vivo and in vitro. Mol. Endocrinol. 6, 15–25 (1992).

    CAS  PubMed  Google Scholar 

  18. Wikstrom, P., Stattin, P., Franck-Lissbrant, I., Damber, J. E. & Bergh, A. Transforming growth factor beta1 is associated with angiogenesis, metastasis, and poor clinical outcome in prostate cancer. Prostate 37, 19–29 (1998).

    Article  CAS  PubMed  Google Scholar 

  19. Matthews, E. et al. Down-regulation of TGF-beta1 production restores immunogenicity in prostate cancer cells. Br. J. Cancer 83, 519–525 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kloss, C. C. et al. Dominant-negative TGF-beta 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. Tang, N. et al. TGF-beta inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight 5, e133977 (2020).

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Stuber, T. et al. Inhibition of TGF-beta-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer. J. Immunother. Cancer 8, e000676 (2020).

  24. Bendle, G. M., Linnemann, C., Bies, L., Song, J. Y. & Schumacher, T. N. Blockade of TGF-beta signaling greatly enhances the efficacy of TCR gene therapy of cancer. J. Immunol. 191, 3232–3239 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Bollard, C. M. et al. Tumor-specific T-cells engineered to overcome tumor immune evasion induce clinical responses in patients with relapsed Hodgkin lymphoma. J. Clin. Oncol. 36, 1128–1139 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhang, L. et al. Inhibition of TGF-beta signaling in genetically engineered tumor antigen-reactive T cells significantly enhances tumor treatment efficacy. Gene Ther. 20, 575–580 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Ishigame, H., Mosaheb, M. M., Sanjabi, S. & Flavell, R. A. Truncated form of TGF-betaRII, but not its absence, induces memory CD8+ T cell expansion and lymphoproliferative disorder in mice. J. Immunol. 190, 6340–6350 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Li, M. O., Sanjabi, S. & Flavell, R. A. Transforming growth factor-beta controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms. Immunity 25, 455–471 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Gorelik, L. & Flavell, R. A. Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity 12, 171–181 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Chang, S. S. et al. Five different anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-associated neovasculature. Cancer Res. 59, 3192–3198 (1999).

    CAS  PubMed  Google Scholar 

  31. Sweat, S. D., Pacelli, A., Murphy, G. P. & Bostwick, D. G. Prostate-specific membrane antigen expression is greatest in prostate adenocarcinoma and lymph node metastases. Urology 52, 637–640 (1998).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  33. Gattinoni, L., Klebanoff, C. A. & Restifo, N. P. Paths to stemness: building the ultimate antitumour T cell. Nat. Rev. Cancer 12, 671–684 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Basch, E. et al. Development of the National Cancer Institute’s patient-reported outcomes version of the common terminology criteria for adverse events (PRO-CTCAE). J. Natl Cancer Inst. 106, dju244 (2014).

  35. Yuen, K. C. et al. High systemic and tumor-associated IL-8 correlates with reduced clinical benefit of PD-L1 blockade. Nat. Med. 26, 693–698 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Schalper, K. A. et al. Elevated serum interleukin-8 is associated with enhanced intratumor neutrophils and reduced clinical benefit of immune-checkpoint inhibitors. Nat. Med. 26, 688–692 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Scher, H. I. et al. Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone: recommendations of the prostate cancer clinical trials working group. J. Clin. Oncol. 26, 1148–1159 (2008).

    Article  PubMed  Google Scholar 

  38. Eisenhauer, E. A. et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur. J. Cancer 45, 228–247 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Foroozan, M., Roudi, R., Abolhasani, M., Gheytanchi, E. & Mehrazma, M. Clinical significance of endothelial cell marker CD34 and mast cell marker CD117 in prostate adenocarcinoma. Pathol. Res. Pract. 213, 612–618 (2017).

    Article  CAS  PubMed  Google Scholar 

  40. Bettencourt, M. C., Bauer, J. J., Sesterhenn, I. A., Connelly, R. R. & Moul, J. W. CD34 immunohistochemical assessment of angiogenesis as a prognostic marker for prostate cancer recurrence after radical prostatectomy. J. Urol. 160, 459–465 (1998).

    Article  CAS  PubMed  Google Scholar 

  41. Trojan, L. et al. Expression of different vascular endothelial markers in prostate cancer and BPH tissue: an immunohistochemical and clinical evaluation. Anticancer Res. 24, 1651–1656 (2004).

    CAS  PubMed  Google Scholar 

  42. Gevaert, T. et al. Comparing the expression profiles of steroid hormone receptors and stromal cell markers in prostate cancer at different Gleason scores. Sci. Rep. 8, 14326 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  43. de Galiza Barbosa, F. et al. Nonprostatic diseases on PSMA PET imaging: a spectrum of benign and malignant findings. Cancer Imaging 20, 23 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Nakamura, R. et al. Expression and regulatory effects on cancer cell behavior of NELL1 and NELL2 in human renal cell carcinoma. Cancer Sci. 106, 656–664 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kiuchi, Z. et al. GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells. FASEB J. 33, 7387–7402 (2019).

    Article  CAS  PubMed  Google Scholar 

  46. Pawlicki, J. M. et al. NPM-ALK-induced reprogramming of mature TCR-stimulated T cells results in dedifferentiation and malignant transformation. Cancer Res. 81, 3241–3254 (2021).

    Article  CAS  PubMed  Google Scholar 

  47. Geyer, M. B. et al. Safety and tolerability of conditioning chemotherapy followed by CD19-targeted CAR T cells for relapsed/refractory CLL. JCI Insight 5, e122627 (2019).

  48. Turtle, C. J. et al. Immunotherapy of non-Hodgkin’s lymphoma with a defined ratio of CD8+ and CD4+ CD19specific chimeric antigen receptor-modified T cells. Sci. Transl. Med. 8, 355ra116 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Hirayama, A. V. et al. The response to lymphodepletion impacts PFS in patients with aggressive non-Hodgkin lymphoma treated with CD19 CAR T cells. Blood 133, 1876–1887 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Fraietta, J. A. et al. Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells. Nature 558, 307–312 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Shah, N. N. et al. Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy. Blood Adv. 3, 2317–2322 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Nobles, C. L. et al. CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration. J. Clin. Invest. 130, 673–685 (2020).

    Article  CAS  PubMed  Google Scholar 

  54. Curtsinger, J. M., Johnson, C. M. & Mescher, M. F. CD8 T cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal 3 cytokine. J. Immunol. 171, 5165–5171 (2003).

    Article  CAS  PubMed  Google Scholar 

  55. Pape, K. A., Khoruts, A., Mondino, A. & Jenkins, M. K. Inflammatory cytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4+ T cells. J. Immunol. 159, 591–598 (1997).

    CAS  PubMed  Google Scholar 

  56. Priceman, S. J. et al. Regional delivery of chimeric antigen receptor-engineered T cells effectively targets HER2(+) breast cancer metastasis to the brain. Clin. Cancer Res. 24, 95–105 (2018).

    Article  CAS  PubMed  Google Scholar 

  57. Mulazzani, M. et al. Long-term in vivo microscopy of CAR T cell dynamics during eradication of CNS lymphoma in mice. Proc. Natl Acad. Sci. USA 116, 24275–24284 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Theruvath, J. et al. Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors. Nat. Med. 26, 712–719 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Brown, C. E. et al. Optimization of IL13Ralpha2-targeted chimeric antigen receptor T cells for improved anti-tumor efficacy against glioblastoma. Mol. Ther. 26, 31–44 (2018).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  62. Vitanza, N. A. et al. Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis. Nat. Med. 27, 1544–1552 (2021).

  63. 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).

  64. Vuk-Pavlovic, S. et al. Immunosuppressive CD14+HLA-DRlow/- monocytes in prostate cancer. Prostate 70, 443–455 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Brusa, D. et al. Circulating immunosuppressive cells of prostate cancer patients before and after radical prostatectomy: profile comparison. Int. J. Urol. 20, 971–978 (2013).

    CAS  PubMed  Google Scholar 

  66. Hossain, D. M. et al. TLR9-targeted STAT3 silencing abrogates immunosuppressive activity of myeloid-derived suppressor cells from prostate cancer patients. Clin. Cancer Res. 21, 3771–3782 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. O’Rourke, D. M. et al. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci. Transl. Med. 9, eaaa0984 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Fraietta, J. A. et al. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia. Blood 127, 1117–1127 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020).

  71. Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Teachey, D. T. et al. Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Cancer Discov. 6, 664–679 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Brady, T. et al. A method to sequence and quantify DNA integration for monitoring outcome in gene therapy. Nucleic Acids Res. 39, e72 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Berry, C., Hannenhalli, S., Leipzig, J. & Bushman, F. D. Selection of target sites for mobile DNA integration in the human genome. PLoS Comput. Biol. 2, e157 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  75. Berry, C. C. et al. Estimating abundances of retroviral insertion sites from DNA fragment length data. Bioinformatics 28, 755–762 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Berry, C. C., Ocwieja, K. E., Malani, N. & Bushman, F. D. Comparing DNA integration site clusters with scan statistics. Bioinformatics 30, 1493–1500 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Scholler, J. et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med. 4, 132ra153 (2012).

    Article  Google Scholar 

  78. Medvec, A. R. et al. Improved expansion and in vivo function of patient T cells by a serum-free medium. Mol. Ther. Methods Clin. Dev. 8, 65–74 (2018).

    Article  CAS  PubMed  Google Scholar 

  79. Merritt, C. R. et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat. Biotechnol. 38, 586–599 (2020).

    Article  CAS  PubMed  Google Scholar 

  80. He, Z. & Zhou, J. Empirical evaluation of a new method for calculating signal-to-noise ratio for microarray data analysis. Appl. Environ. Microbiol. 74, 2957 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32, 2847–2849 (2016).

    Article  CAS  PubMed  Google Scholar 

  82. Bates, D., Bolker, M. M., Walker, B. & Fitting, S. Linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015).

    Article  Google Scholar 

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Acknowledgements

We thank the patients and their families for participation in this clinical trial, which was sponsored by the University of Pennsylvania. We acknowledge the Human Immunology Core at the University of Pennsylvania for providing leukocytes; the Hospital of the University of Pennsylvania Apheresis Unit for PBMC collections; and the Data Safety Monitoring Board for data analysis. We also thank the contributors who supported development and execution of the clinical trial: the Clinical Cell and Vaccine Production Facility, the Translational and Correlative Studies Laboratory and the Product Development Lab from the University of Pennsylvania Center for Cellular Immunotherapies. We thank D. Maseda for the kind gift of Y664F NPMALK-transformed T cells and lentiviral constructs encoding NPM-ALK fusion kinases. This work was supported by a Prostate Cancer Foundation Challenge Award (N.B.H. and C.H.J.), Tmunity Therapeutics, Inc., the George Weiss Funding Group, an Alliance for Cancer Gene Therapy Investigator Award in Cell and Gene Therapy for Cancer (J.A.F. and N.B.H.), a Prostate Cancer Foundation Young Investigator Award (V.N.), U54 CA244711-01 (C.H.J. and J.A.F.), R01 CA241762-03 (F.D.B. and J.A.F.) and ACC P30 Core Grant no. P30 CA016520-42 (S.F.L., W.-T.H., J.A.F. and N.H.).

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V.N., J.S.B.-R., I.-Y.J., S.F.L., A.J.R., M.M.D., W.-T.H., P.L., E.L.C., G.P., N.V., A.C., M.M., R.A.S., M.D.F., A.M., J.G., L.L., K.D., S.E.C., T.D.H., J.X., M.Gohil, T.H.B., S.S.Y., V.E.G., I.K., F.C., L.T., K.T., C.L.N., H.R., F.D.B., C.H.J., J.A.F. and N.B.H. participated in the design, execution and/or interpretation of the reported experiments or results. V.N., J.S.B.-R., I.-Y, J., A.J.R., M.M.D., W.-T.H., P.L., S.L.M., S.E.C., T.D.H., V.E.G., I.K., F.C., L.T., P.C.C.T.P.I., E.O.H., D.L.S., F.D.B., J.A.F. and N.B.H. participated in the acquisition or analysis of data. V.N., J.S.B., C.H.J., J.A.F. and N.B.H. wrote the paper, with all authors contributing to writing and providing feedback. C.H.J., J.A.F. and N.B.H. supervised all aspects of the research.

Corresponding authors

Correspondence to Carl H. June, Joseph A. Fraietta or Naomi B. Haas.

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Competing interests

Patents, royalties, other intellectual property: S.F.L., M.M.D., D.L.S., C.H.J. and J.A.F. have filed patent applications in the field of T cell therapy for cancer and have received royalties. C.H.J. and A.C. are cofounders of Tmunity Therapeutics. M.M.D. has received research funding from Tmunity Therapeutics and serves on the Scientific Advisory Board for Cellares Corporation. S.F.L. has served as a consultant for Novartis Pharmaceuticals, Kite Pharma and Wugen, and receives clinical trial funding from Novartis Pharmaceuticals. J.A.F. is a member of the Scientific Advisory Boards of Cartography Bio. and Shennon Biotechnologies Inc. The remaining authors declare no competing interests.

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Nature Medicine thanks James Gulley, Julie Graff and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Knockout of the endogenous TGFβRII in CART-PSMA cells enhances in vivo prostate tumor control independently of T cell proliferative capacity, early memory differentiation or inhibitory phenotype.

(a) Schema of CAR T cell transfer into prostate tumor (luciferase-expressing PC3 cell)-engrafted mice. (b) Longitudinal bioluminescent tumor burden of PBS or CAR T cell treated mice (n = 6). Error bars depict s.e.m. P values were calculated using a two-tailed t-test between the two CAR T cell treated groups at day 52 post-tumor injection. (c) Violin plots showing the absolute counts (d) memory and (e) inhibitory phenotypes of CART-PSMA-AAVS1KO or CART-PSMA-TGFβRKO cells in the peripheral blood of mice at the peak of T cell expansion (day 48). Thick dashed lines indicate the median and thin dotted lines show the first and third quartiles. P values were determined with a two-tailed t-test.

Extended Data Fig. 2 Transgenic expression of TGFβRDN significantly increases the proliferative capacity, but not the effector function of CART-PSMA cells compared to knockout of the endogenous TGFβRII.

(a) Efficiency of CRISPR/Cas9-mediated knockout (KO) of the endogenous TGFβRII (TGFβRKO) in CART-PSMA cells derived from n = 4 different subjects, as determined by Sanger sequencing and TIDE analysis. Editing efficiency is presented relative to AAVS1 knockout in donor-matched CART-PSMA cells. Error bars depict the SEM. (b) Representative flow cytometry showing levels of pSMAD2/3 in CART-PSMA cells with knockout of AAVS1, TGFβRII or co-expression of TGFβRDN that were unstimulated or stimulated with recombinant human TGFβ (representative of 3 independent experiments). (c) Expansion capacity of CART-PSMA-TGFβRDN versus CART-PSMA-TGFβRKO cells following serial re-stimulation (indicated by black arrows) with TGFβ-expressing irradiated PC3 prostate tumor cells. Proliferation is presented as a change in fold expansion over the longitudinal growth of stimulated CART-PSMA-AAVS1KO cells. Cells were manufactured from 4 different subjects, with pooled data from 3 independent experiments. Error bars depict the s.e.m. P values were calculated using a two-tailed t-test. (d) Killing kinetics of CART-PSMA-TGFβRDN, CART-PSMA-TGFβRKO and CART-PSMA-AAVS1KO cells co-cultured with PC3 tumor targets. CAR T cells directed against CD19 (irrelevant CAR) served as a negative control. Data are representative of 3 individual experiments performed with engineered T cells from 3 independent subjects. Error bars indicate the s.e.m. (e) Cytokine production from CART-PSMA-TGFβRKO and CART-PSMA-TGFβRDN compared to control CART-PSMA cells following stimulation with PC3 cells. Each data point represents a CAR T cell sample derived from an independent donor. Error bars depict the s.e.m. P values were calculated with a two-tailed t-test.

Extended Data Fig. 3 Characterization of baseline apheresis products and preinfusion TGFβRDN expressing PSMA-directed CAR T cells (CART-PSMA-TGFβRDN).

(a) Frequencies of apheresed CD45+, CD45+CD3+, CD45+CD3+CD4+, CD45+CD3+CD8+ cells and CD28+ T cells were assessed by flow cytometry. (b) Proportions of various CD3+CD8+ T cell subsets at the time of apheresis are shown: naive-like, CD27+CD45RO-; central memory, CD27+CD45RO+; effector memory, CD27-CD45RO+; effector, CD27-CD45RO-. (c) Percentages of FoxP3+CD25+ regulatory T cells in apheresis material. (d) CD4:CD8 cell ratio in the pre-infusion CAR T cell product is depicted. (e) Fold expansion of CAR T cell infusion product over 9-days of clinical manufacturing is shown. (f) Frequencies of expanded patient CD3+CD45+ T cells expressing the anti-PSMA CAR and TGFβRDN are plotted. Individual data points for each patient and means (denoted by a black horizontal line) are shown in panels a-f. (g) Expression of a TGFβRDN on manufactured PSMA-targeted CAR T cells prevents TGFβ signaling through SMAD2/3 phosphorylation. Individual data points for each patient and means are shown in all panels. IL-2 denotes patient products manufactured in the presence of this cytokine; IL-7/15 indicates CAR T cell manufacturing using these cytokines. Thick dashed lines in violin plots depict the median and thin dotted lines indicate the first and third quartiles. P values were determined with a two-tailed Student’s t-test for paired samples.

Extended Data Fig. 4 Longitudinal cytokine, chemokine and growth factor profiles in the peripheral blood of mCRPC patients treated with CART-PSMA-TGFβRDN cells.

Fold changes in serum cytokine, chemokine and growth factor levels from baseline (preCAR T cell infusion) to each time point postCART-PSMA-TGFβRDN cell administration were measured in patients by multiplex analysis and are depicted as line graphs.

Extended Data Fig. 5 Antitumor responses and clinical outcomes in subjects infused with CART-PSMA-TGFβRDN cells.

(a) Spider plot showing longitudinal serum PSA changes in patients treated with CART-PSMA-TGFβRDN cells. (b) Overall survival (OS) and (c) progression-free survival (PFS) graphed as Kaplan-Meier estimates for all patients. The x-axis is shown in months. Tick marks indicate each censored subject (that is, patients who are alive at the data cutoff point).

Extended Data Fig. 6 Analysis of CAR lentiviral integration sites in mCRPC and advanced leukemia patients.

(a) The word clouds illustrate CAR-PSMA-TGFβRDN lentiviral integration sites near genes of the most abundant clones from each Patient 9 sample, where the numeric ranges represent the upper and lower clonal abundances. (b) The relative abundances of cell clones are summarized as stacked bar plots. The different bars in each panel denote the major cell clones, as marked by integration sites where the x-axis indicates timepoints and the y-axis is scaled by the proportion of total cells sampled. The top 10 most abundant clones have been named by the nearest gene while the remaining sites are grouped as low abundance. The total number of unique sites are listed above each plot. (c) This panel displays the frequency of NELL2- and GLCCI1-disrupted clones observed at each timepoint across advanced leukemia patients treated with CD19 CAR T cells. The size of the points indicates the number of clones observed at the same timepoint and sharing the same abundance. (d) The distribution of integrated pro-vectors across NELL2 and GLCCI1. Each row of lines and boxes indicates a different splice variant of the transcription unit (5 for NELL2 and 1 for GLCCI1). The points indicate the observed integrated pro-vectors. The color of the points indicates the orientation of the integrated element. Points were displaced vertically for aesthetics, as the vertical distances between points hold no value.

Extended Data Fig. 7 CRISPR/Cas9-mediated mutagenesis of NELL2 and GLCCI1 does not alter the proliferative capacity of CART-PSMA-TGFβRDN cells.

(a) Efficiency of CRISPR/Cas9-induced mutagenesis of NELL2 and GLCCI1 in CART-PSMA-TGFβRDN cells from n = 4 different individuals, as assessed by Sanger sequencing and TIDE analysis. Knockout (KO) effectiveness is shown relative to AAVS1 in subject-matched CART-PSMA-TGFβRDN cells. (b) In vitro proliferative capacity of CRISPR/Cas9-edited CART-PSMA-TGFβRDN cells (n = 4 independent donor samples) following serial restimulation (indicated by black arrows) with irradiated PC3 prostate tumor cells. Error bars indicate the s.e.m. (c) Antigen-dependent fold expansion of AAVS1 and GLCCI1 KO CART-PSMA-TGFβRDN cells in the presence or absence of dexamethasone (DEX; E-4M). Box plots show minimum, lower quartile, median, upper quartile and maximum (n = 6 biologically independent samples).

Extended Data Fig. 8 In vitro assessment of Patient 9 CART-PSMA-TGFβRDN cell transformation.

(a) Assessment of proliferation and (b) viability of Patient 9 CART-PSMA-TGFβRDN cells (derived from the CAR T cell infusion product and day 28 postinfusion PBMC) under cytokine- and stimulation-free conditions. T cells from an unrelated donor transformed with an NPM-ALK fusion kinase were cultured in parallel as a control. (c) Absolute cell counts and (d) viability measurements of the same day 28 cells from Patient 9 above that were stimulated in the presence of anti-CD3/CD28 agonistic antibodies and IL-2 (untransduced = not transduced with NPM-ALK). The patient’s cells were transduced with a lentivirus encoding NPM-ALK and cultured separately as a control for transformation.

Extended Data Table 1 Study-related SAEs following CART-PSMA-TGFβRDN cell infusion
Extended Data Table 2 Individual patient SAEs following CART-PSMA-TGFβRDN cell infusion

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Narayan, V., Barber-Rotenberg, J.S., Jung, IY. et al. PSMA-targeting TGFβ-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: a phase 1 trial. Nat Med 28, 724–734 (2022). https://doi.org/10.1038/s41591-022-01726-1

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