EXTH-62. Epigenetic priming with DNMT inhibitors enhances CAR T-Cell efficacy in pediatric brain tumors

Abstract Chimeric antigen receptor (CAR) T-cell therapy has transformed treatment for hematologic malignancies, but its application in pediatric brain tumors remains limited due to poor persistence and T-cell exhaustion. Epigenetic regulation, particularly DNA methylation, plays a key role in modulating T-cell function. We previously demonstrated improved CAR T-cell persistence in immunocompetent models following methylation priming during manufacturing. Here, we investigate the translational relevance of DNA methyltransferase inhibition (DNMTi) using azacytidine (AZA), zebularine, and GSK3685032, added during GMP-compatible manufacturing of B7-H3–targeting human CAR T-cells for pediatric glioma and medulloblastoma. Human T cells from healthy donors were transduced with a B7-H3 CAR or control CAR and treated with low-dose DNMTis early during manufacturing. Functional assessments included repeated antigen stimulation, cytotoxicity assays, flow cytometry for memory and exhaustion markers, cytokine profiling, and in vivo efficacy studies using xenograft models. Outcomes focused on T-cell activation, memory differentiation, persistence, cytokine secretion, and cytotoxic function. DNMTi-treated CAR T cells exhibited enhanced expansion (3–5-fold increase with AZA), greater cytotoxicity, increased cytokine production, and reduced expression of exhaustion markers. Treated cells showed a higher frequency of central memory phenotype and sustained effector function at lower effector-to-target ratios. Zebularine and GSK3685032 yielded similar improvements, with a milder impact on T-cell viability. In vivo, DNMTi-treated CAR T cells achieved comparable tumor control but showed superior persistence, enabling tumor clearance upon rechallenge—an effect not observed with untreated CAR T cells. These effects were consistent across multiple pediatric brain tumor models, including U87 (glioma) and D556 (medulloblastoma). In conclusion, DNMT inhibition during CAR T-cell manufacturing significantly enhances T-cell expansion, function, and persistence while reducing exhaustion. This strategy offers a promising approach to overcome key barriers in solid tumor immunotherapy. Ongoing studies will define the optimal DNMTi regimen and elucidate underlying mechanisms to inform future clinical translation in pediatric brain tumors.