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227 Targeting TGFβ pathway to enhance CAR-T therapy for glioblastoma
  1. Darya Alizadeh,
  2. Robyn Nee,
  3. Zhiqiang Wang,
  4. Madeleine Maker,
  5. Franklin Chen,
  6. Jonathan Hibbard and
  7. Christine Brown
  1. City of Hope, Duarte, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Chimeric antigen receptor-(CAR) T cell therapy is seeing encouraging progress in solid tumors, however, factors such as the suppressive tumor microenvironment (TME) hinder its effectiveness.1 We recently completed a phase I trial of IL13Rα2-targeted CAR-T cells for recurrent glioblastomas (GBM) and other high-grade gliomas, the largest clinical study completed to date (NCT02208362).2–5 Samples from this trial were evaluated for TGFβ, an immunosuppressive cytokine highly upregulated in GBM.

Methods TGFβ levels were measured using ELISA. Syngeneic mouse studies were used to evaluate the impact of TGFβ blockade on CAR-T therapy. Flow cytometry was used to characterize the TME and CRISPR/Cas technology was utilized to knockout TGFβR2.

Results We measured TGFβ levels in 781 patient samples collected from the tumor site (tumor fluid; TF) or cerebral spinal fluid (CSF) before and after CAR-T administration. Compared to CSF, TF exhibited a higher concentration of TGFβ indicating a more immunosuppressive environment in tumor bed. We also found that post CAR-T therapy TGFβ levels decreased in the TF, but not the CSF. Furthermore, higher TGFβ levels in TF correlated with poor prognosis and lack of response to CAR-T therapy. These results indicate that targeting the TGFβ pathway may enhance CAR-T therapy against GBM. Using our syngeneic glioma model, we evaluated the importance of inhibiting the TGFβ pathway. We demonstrated that pretreatment with a TGFβR1 inhibitor (LY3200882) significantly augmented the efficacy of CAR-T therapy and improved overall survival of mice bearing large established tumors. TME characterization revealed upregulation of a migratory phenotype for both CAR and endogenous T cells. These results indicate that targeting TGFβ in the TME augments CAR-T cell function by potentially enhancing both endogenous and CAR T cell migration and trafficking into the tumor. Next, we assessed the impact of blocking TGFβ-signaling on human CAR-T cells. Targeting the TGFβ pathway (using TGFβR1 inhibitor) enhanced CAR-T cell antitumor activity and proliferation following multiple tumor-rechallenges in vitro. Gene expression analyses revealed upregulation of genes associated with cytotoxic and memory-stem-like phenotypes and down-regulation of genes associated with exhaustion and inhibitory pathways. Based on these results, we next evaluated TGFβ-resistant CAR-T cells in vivo and demonstrated that blocking TGFβ-signaling through TGFβR2 knockout augmented the efficacy of CAR-T cells in a large immunosuppressive GBM tumor model in syngeneic mice.

Conclusions Collectively, our results indicate that inhibiting the TGFβ pathway either in TME or CAR T cells is essential for enhancing CAR-T cell efficacy in GBM.

Acknowledgements This work was supported in part by Mustang Bio., Inc, Cancer Center Support Grant P30 CA33572, and The Kenneth T. and Eileen L. Norris Foundation. We also thank Lilly for providing the TGFbR1 inhibitor (LY3200882) for our studies.


  1. Yap T, et al. First-In-Human Phase I Study of a Next-Generation, Oral, TGFb Receptor 1 Inhibitor, LY3200882, in Patients with Advanced Cancer. Clin Cancer Res 2021;27(24):6666–76

  2. Brown CE, et al. Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy. N Engl J Med, 2016;375(26):2561–9.

  3. Alizadeh D, et al. IFNg Is Critical for CAR T Cell-Mediated Myeloid Activation and Induction of Endogenous Immunity. Cancer Discov. 2021;11(9):2248–65.

  4. Tang N, et al. TGF-beta inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight 2020;5(4).

  5. Han J, et al. TGF-beta signaling and its targeting for glioma treatment. Am J Cancer Res, 2015;5(3):945–55.

Ethics Approval All studies related to analyzing patient samples or healthy donors were obtained with informed written consent (whenever necessary) after protocols were approved by the City of Hope IRB and IND committee. All animal experiments were performed using protocols approved by the City of Hope IACUC.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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