IMMU-87. Synergistic TCR’CAR T Cells Enhance Persistence and Cytotoxicity against High-Grade Gliomas

Abstract BACKGROUND Pediatric high-grade gliomas (HGGs) are the most common cancer-related cause of death for children ages 0-19. B7H3-targeting Chimeric Antigen Receptor (CAR) T cells have demonstrated robust preclinical efficacy in models of HGG, yet current clinical trials show limited efficacy. This is partly due to limited CAR T cell cytotoxicity and persistence, driven by suboptimal CAR-mediated T cell activation. TCR’CAR T cells, a combination of TCR- and CAR-based adoptive immunotherapies, may improve T cell cytotoxicity, expansion, and endurance through synergistic signaling. Here, we co-expressed a gp100-specific TCR (a tumor antigen in HGG) with a B7H3 CAR to combine native T cell signaling with CAR signaling, aiming to enhance T cell function and persistence through complementary signaling crosstalk. METHODS T cells expressing the gp100 TCR were transduced with a B7-H3 CAR to generate dual-specific TCR’CAR T cells. Functional analyses included repeated stimulation and MTS cytotoxicity assays against HGG cell lines to assess efficacy and persistence. Cytokine expression was measured by ELISA. Memory phenotyping and exhaustion marker expression were evaluated by flow cytometry. RESULTS Compared to CAR-only T cells, TCR’CAR T cells demonstrated significantly enhanced persistence (up to 15 stimulations, median 7 vs. 3 stimulations), proliferation (maximum fold change of ~8,000 vs. 4.4), and sequential killing capacity. While initial cytotoxicity was comparable, TCR’CAR T cells maintained superior cytotoxicity at lower effector-to-target ratios with up to a 34.3-fold and 5.8-fold increase in IL-2 and IFNγ production, respectively, compared to CAR-only T cells during repeated stimulations. Early mechanistic results suggest that TCR’CAR T cells undergo changes in memory T cell phenotypes and exhaustion-related pathways during repeated stimulation. CONCLUSION These findings support integrating TCR and CAR platforms to enhance T cell function and persistence. Future studies will evaluate in vivo efficacy and underlying molecular mechanisms, aiming to improve CAR T cell therapy for HGG.