ID #549 TCR-CAR T Cells as a Novel Approach for Pediatric High-Grade Glioma Therapy

Abstract Background CAR T cell therapy for pediatric high-grade gliomas (HGGs) is limited by poor persistence and suboptimal cytotoxicity. Co-expressing a tumor-specific TCR with a CAR (TCR-CAR) may enhance T cell activation, stabilize immune synapses, and improve effector function. We investigated whether gp100-specific TCR co-expression with a B7H3 CAR improves T cell performance for pediatric HGG therapy. Methods TCR-CAR T cells were generated by transducing gp100 TCR-expressing T cells with a B7H3 CAR. In vitro functional analyses included repeated stimulation, MTS cytotoxicity assays, cytokine production, and memory/exhaustion phenotyping by flow cytometry. Immunocompetent murine models were established with intracranial HGG implantation followed by T cell treatment. Clinical translation was considered using standard clinical-grade T cell manufacturing protocols. Results TCR-CAR T cells demonstrated superior in vitro persistence (median 7.5 vs. 3 stimulations), proliferation (fold change ∼8,000 vs. ∼42), and sequential killing compared to CAR-only T cells. While initial cytotoxicity was similar, TCR-CAR T cells maintained enhanced cytotoxicity with higher IL-2 and IFNγ production over repeated stimulations. Phenotyping data suggests shifts in memory subsets and exhaustion pathways over time. In vivo, TCR-CAR–treated mice showed reduced survival compared to CAR-only–treated mice, potentially due to overactivation or tumor microenvironment-mediated trapping. Conclusion Dual TCR-CAR strategies enhance T cell function, persistence, and cytokine production in vitro, supporting integration of TCR and CAR platforms for pediatric HGG therapy. The in vivo discrepancy highlights potential challenges with overactivation or tumor trapping, underscoring the need for mechanistic studies. These findings support continued development and clinical-grade manufacturing toward safe and effective translation for children with HGG.