EXTH-56. Optimizing CAR T cell therapy for pediatric glioma: Enhancing persistence and efficacy with DNA methylation inhibition during manufacturing

Chimeric antigen receptor (CAR) T cell therapy has revolutionized hematological cancer treatment but faces significant challenges in targeting solid tumors. A key barrier is the epigenetic regulation of T cell exhaustion, which limits therapeutic efficacy. Chronic antigen stimulation leads to increased DNA methylation, restricting effector gene expression essential for CAR T cell persistence and function. This study investigates the use of DNA methyltransferase inhibitors (DNMTi) during CAR T cell manufacturing to reprogram the DNA methylome and improve anti-glioma activity. We hypothesize that DNMTi treatment can enhance the immune activation and antitumor function of engineered adoptive T cells. We optimized a transduction protocol that incorporates adding DNMTi early during the manufacturing process of murine CAR T cells targeting B7-H3. Subsequently, standard in-vitro assays were conducted, and results show that integrating DNMTi during CAR T-cell manufacturing extends their persistence and anti-glioma activity in-vitro. In repeated-stimulation assays, DNMTi-treated CAR T-cells exhibited significantly higher fold-expansion and longer persistence compared to those without DNMTi exposure. Additionally, the treated CAR T cells showed increased cytotoxicity over time, with improved effector function resulting in enhanced tumor cell clearance up to the 12th repeat stimulation. This was associated with remarkably enhanced secretion of immune-stimulatory cytokines that persisted across repetitive stimulations. These findings are further supported by in-vivo studies in immunocompetent, glioma-bearing mice, where treatment with DNMTi-exposed CAR T cells resulted in enhanced survival. Mechanistic studies are ongoing to identify differentially methylated regions (DMRs) and uncover transcriptional programs modulated by DNMTi treatment. These findings offer a promising strategy to overcome CAR T cell exhaustion and enhance the efficacy of adoptive cellular therapies in glioma. The use of DNMTis represents a clinically accessible and easily integrable approach, supporting the translational potential of this strategy. More broadly, these results highlight the value of epigenetic modulation in improving treatment outcomes for solid and brain tumors, warranting further investigation.