Abstract 3489: Functional enhancement of hypoxia-activated chimeric antigen receptor T-cells by a chimeric switch receptor recognizing TGF-β

Abstract Background: We have previously demonstrated that hypoxia-activated chimeric antigen receptor (CAR) T-cells could execute robust anti-tumor efficacy with controllable on-target, off-tumor (OTOT) toxicities in the clinical setting. However, some normal tissues or organs, such as bone marrow, rectum, stomach, gallbladder, and epidermis, also exhibit hypoxic microenvironment, which might lead to potential side effects or constraints for the clinical use of hypoxia-responsive CAR T-cells. TGF-β is a significant immunosuppressive factor that impedes immune response in the tumor microenvironment, while its expression in the hypoxic normal tissues is limited. We optimized the construct by introducing an AND-gated circuit complementing the hypoxia-driven conditional activation to reduce OTOT toxicities further. Methods: The AND-gated CAR, BTRPTB001L, consisted of a hypoxia-activated CAR and a constitutively expressing TGF-β chimeric switch receptor (CSR). T-cells isolated from human peripheral blood mononuclear cells were stimulated with anti-CD3/CD28 beads, lentivirally transduced, and expanded ex vivo. CAR/CSR expression was assessed by flow cytometry. IncuCyte was used to analyze the in-vitro cytotoxicity in the presence of TGF-β. The TGF-β sponging capability was assessed by ELISA. Anti-tumor efficacy was evaluated in subcutaneously inoculated xenograft mouse models. Safety evaluation was performed using the AND-gated CAR T-cells targeting the murine orthologous antigen (BTRPTB001L’) in immunocompromised mice. Results: In BTRPTB001L, the fold change of CAR expression level in 1% O2 compared to 21% O2 was 6.33 (P < 0.05), while the CSR expression remained unaffected under either condition. BTRPTB001L showed significantly reduced cytotoxicity under normoxic conditions compared to second-generation (2nd-gen) hypoxia-activated CAR T-cells by co-culturing with target cells. Interestingly, in the presence of TGF-β and under hypoxic conditions, BTRPTB001L showed enhanced cytotoxicity, indicating that the CSR could reverse the inhibitory signal and be specifically activated by the soluble TGF-β in the tumor microenvironment. Notably, the CSR could also sponge TGF-β in the culture supernatant. The improved anti-tumor efficacy of BTRPTB001L was also demonstrated in a xenograft mouse model of pancreatic cancer. Moreover, while the constitutively expressing 2nd-gen CAR recognizing the murine orthologous TAA caused a lethal weight loss phenotype in mice, the BTRPTB001L’-treated group showed no prominent adverse events. Conclusion: BTRPTB001L demonstrated robust anti-tumor potential and improved safety profile compared to hypoxia-responsive 2nd-gen CAR T-cell, supporting its further clinical development. Citation Format: Zhihong Huang, Deping Han, Jean-Paul Thiery, Xi Zhang. Functional enhancement of hypoxia-activated chimeric antigen receptor T-cells by a chimeric switch receptor recognizing TGF-β [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3489.