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

Nature Medicine volume 28, pages 724–734 (2022)Cite this article

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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.

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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.

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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.).

Author information

Author notes

  1. These authors contributed equally: In-Young Jung, Simon F. Lacey, Andrew J. Rech, Megan M. Davis.

  2. These authors jointly supervised this work: Carl H. June, Joseph A. Fraeitta, Naomi B. Haas.

Authors and Affiliations

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

    Vivek Narayan, Neha Vapiwala, Elizabeth O. Hexner & Naomi B. Haas

  2. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Vivek Narayan, In-Young Jung, Andrew J. Rech, Priti Lal, Erica L. Carpenter, Neha Vapiwala, Ronnie A. Sebro, Michael D. Farwell, Thomas H. Buckingham, Stephanie S. Yee, Kyle Tien, Elizabeth O. Hexner, Carl H. June, Joseph A. Fraietta & Naomi B. Haas

  3. Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Julie S. Barber-Rotenberg, In-Young Jung, Simon F. Lacey, Andrew J. Rech, Megan M. Davis, Gabriela Plesa, Anne Chew, Michael Moniak, Amy Marshall, Joan Gilmore, Lester Lledo, Karen Dengel, Jun Xu, Mercy Gohil, Vanessa E. Gonzalez, Irina Kulikovskaya, Fang Chen, Lifeng Tian, Whitney Gladney, Diane Frazee, Mary Truran, Elizabeth Veloso, Holly McConville, Jonathan Aguedelo, Samantha Hower, Sophia Ngo, Julie Jadlowsky, J. Joseph Melenhorst, Minnal Gupta, Farzana Nazimuddin, Chelsie Bartoszek, Natalka Koterba, Rachael Reynolds, Farris Ellington, Christopher C. Kloss, Jihyun Lee, Yangbing Zhao, John Scholler, James L. Riley, Bruce L. Levine, Elizabeth O. Hexner, Donald L. Siegel, Carl H. June & Joseph A. Fraietta

  4. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    In-Young Jung, Christopher L. Nobles, Hayley E. Raymond, Aoife Roche, John Everett, James L. Riley, Frederic D. Bushman & Joseph A. Fraietta

  5. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Simon F. Lacey, Andrew J. Rech, Priti Lal, J. Joseph Melenhorst, Bruce L. Levine, Donald L. Siegel, Carl H. June & Joseph A. Fraietta

  6. Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA

    Andrew J. Rech, Erica L. Carpenter, Thomas H. Buckingham, Stephanie S. Yee, Kyle Tien, Carl H. June & Joseph A. Fraietta

  7. Department of Biostatistics, Epidemiology and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Wei-Ting Hwang

  8. Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Shannon L. Maude

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

    Shannon L. Maude

  10. Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ronnie A. Sebro & Michael D. Farwell

  11. NanoString Technologies, Inc., Seattle, WA, USA

    Sarah E. Church, Tyler D. Hether, Christina Bailey, Andrew White, Briana Hudson & Pearl Chang

  12. Cellular Immunotherapy Program, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA, USA

    Marcela V. Maus

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  1. Vivek Narayan
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  2. Julie S. Barber-Rotenberg
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Consortia

Prostate Cancer Cellular Therapy Program Investigators

  • Diane Frazee
  • , Mary Truran
  • , Elizabeth Veloso
  • , Holly McConville
  • , Jonathan Aguedelo
  • , Samantha Hower
  • , Sophia Ngo
  • , Julie Jadlowsky
  • , J. Joseph Melenhorst
  • , Aoife Roche
  • , John Everett
  • , Minnal Gupta
  • , Farzana Nazimuddin
  • , Chelsie Bartoszek
  • , Natalka Koterba
  • , Rachael Reynolds
  • , Farris Ellington
  • , Christopher C. Kloss
  • , Jihyun Lee
  • , Yangbing Zhao
  • , John Scholler
  • , James L. Riley
  • , Christina Bailey
  • , Andrew White
  • , Briana Hudson
  • , Pearl Chang
  • , Marcela V. Maus
  •  & Bruce L. Levine

Contributions

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.

Ethics declarations

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.

Peer review

Peer review information

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.

Additional information

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

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
Full size table
Extended Data Table 2 Individual patient SAEs following CART-PSMA-TGFβRDN cell infusion
Full size table

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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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