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Abstract
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.
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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