Abstract 4278: Regulatory T cell inspired engineering of CAR-T cells enhances anti-tumor efficacy in solid tumors

Abstract Chimeric antigen receptor (CAR)-T cells have revolutionized treatment for hematologic malignancies. Yet, their efficacy against solid tumors remains limited due to the immunosuppressive tumor microenvironment (TME), characterized by nutrient deprivation, hypoxia, and lactate accumulation. Regulatory T cells (Tregs) thrive under such conditions due to expression of the transcription factor FOXP3, which enhances their intratumoral fitness. Leveraging this biology, we engineered effector CAR-T cells to overexpress FOXP3 and evaluated their therapeutic potential in melanoma. In vitro, CAR-T cells targeting the melanoma-associated antigen TRP1 efficiently killed B16F10 melanoma cells, with FOXP3 overexpression modestly reducing cytotoxicity. We next tested CD4+, CD8+, or total CAR-T cell subsets in immunocompetent C57BL/6 mice bearing B16F10 melanomas. In vivo NanoLuc luciferase imaging revealed superior persistence of FOXP3-expressing CAR-T cells compared to control CAR-T cells. Strikingly, while conventional CAR-T cells exhibited minimal tumor control, FOXP3 overexpression produced divergent outcomes: CD4+ or CD8+ CAR-FOXP3-T cells alone unexpectedly accelerated melanoma growth, whereas total CAR-FOXP3-T cells significantly delayed tumor progression. Mechanistic analyses revealed that FOXP3 overexpression did not alter the extent of CAR-induced T cell activation but did protect CAR-T cells from activation-induced cell death. Further characterization revealed that total CAR-FOXP3-T cells were predominantly CD8+, leading us to hypothesize that supplemental CD4+ CAR-T cells could enhance anti-tumor activity. Indeed, co-administration of CD4+ CAR-T cells and total CAR-FOXP3-T cells resulted in sustained control of melanoma growth in vivo, indicating a synergistic interaction between the two cell populations. Our findings provide proof-of-concept for novel CAR-T cell therapy design principles to overcome barriers in solid tumor immunotherapy and inform the development of next-generation cellular therapies. Funding: This work was supported by Swim Across America Grant 23-1579 to LMRF and an MUSC Specialized Center of Research Excellence (SCORE) 5U54DA016511-18 Pilot Project Award to AC. This study was supported in part by the Flow Cytometry and Cell Sorting Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313). Citation Format: Avik Chattopadhyay, Erin O’Connor, Leonardo M. Ferreira, . Regulatory T cell inspired engineering of CAR-T cells enhances anti-tumor efficacy in solid tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4278.