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248 SMART CAR-T cells resist tumor immunosuppressive microenvironment with enhanced efficacy against solid tumors
  1. Qi Dong1,
  2. Wenjie Yin1,
  3. Manli Yin1,
  4. Tao Wang1,
  5. Di Wu1,
  6. Xinxin Wang2,
  7. William Cao1 and
  8. Lianjun Shen3
  1. 1Gracell Biotechnologies Co., Ltd., Shanghai, China
  2. 2Gracell Biotechnologies Co., Ltd., San Diego, CA, USA
  3. 3Gracell Biotechnologies, Shrewbury, MA, 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 Transforming growth factor-β (TGF-β) is a major mediator of T cell suppression in the tumor microenvironment (TME). It has been shown that co-expression of a dominant-negative TGF-β receptor 2 (dnTGFBR2) in chimeric antigen receptor T (CAR-T) cells increased proliferation of lymphocytes, enhanced cytokine secretion, and maintained long-term efficacy in vivo. To combat immunosuppressive TME and further improve the persistence and efficacy of CAR-T cells against solid tumors, we designed CAR-T with a novel module (Suppressive Molecule Activated and Rejuvenated T cells; or, ‘SMART’) that combines the ectodomain of dnTGFBR2 and an intracellular cytokine receptor signaling domain to convert the suppressive signal by TGFBR2 into a supportive signal in such CAR-T cells (termed ‘SMART CAR-T cells’).

Methods The in vitro tumoricidal capacities of SMART CAR-T cells specific to human mesothelin (MSLN) or claudin 18.2 (CLDN18.2) were tested in short-term tumor killing assays and repeated tumor challenge assays in the presence or absence of exogenous of TGF-β1, 2, 3. Cell apoptosis and exhaustion were monitored by flow cytometry at various time points. Human cell line-derived xenograft models in severe immunodeficient mice were utilized to study the in vivo anti-tumor efficacy and preclinical safety profiles of SMART CAR-T cells.

Results SMART CAR-T cells and their conventional counterparts displayed comparable efficacy in short-term cytotoxicity assays against multiple tumor cell lines in vitro; however, upon repeated stimulation with these cells, SMART CAR-T cells showed more potent and longer-lasting tumor-specific lysis than the conventional CAR-T. In addition, SMART CAR-T cells were more resistant to cell death. In multiple xenograft mouse models, SMART CAR-T cells exhibited stronger and more durable tumoricidal activities (figure 1). In tumor re-challenge studies, SMART CAR-T cells were highly efficacious in tumor eradication even > 100 days after initial CAR-T dosing. SMART CAR-T cells recovered from mouse bone marrow and spleens 5 months after injection were still able to lyse tumor cells and proliferate upon stimulation, further demonstrating the durability of SMART CAR-T cells in vivo. Moreover, in mice with a high tumor burden (>1000 mm3), SMART CAR-T cells showed more significant suppression of tumor growth when compared with conventional CAR-T cell controls.

Conclusions SMART CAR-T cells comprising of dnTGFBR2 and a cytokine receptor signaling domain resisted the immunosuppressive TME and maintained long-term proliferation and cytotoxicity both in vitro and in vivo. The enhanced preclinical efficacy and safety profile of SMART CAR-T cells warrants further study in clinical trials.

Abstract 248 Figure 1

SMART CAR-T cells specific to MSLN mounted deeper and longer-lasting tumoricidal responses than conventional CAR-T cells in TGF-β-replete preclinical animal models. (A) Schematic mechanism of action of SMART CAR-T cells (B-C) SMART CAR-T cells were able to suppress high tumor burden (B) or prevent the regrowth of rechallenged tumor xenografts (C).

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