Abstract B021: Epigenetic reprogramming with DNA methylation inhibitors during CAR T cell manufacturing enhances anti-tumor activity, persistence, and cytotoxicity against solid and brain tumors

Abstract Pediatric brain tumors are the most common and deadliest childhood cancers, emphasizing the urgent need for improved treatments. Chimeric Antigen Receptor (CAR) T-cell therapy offers a promising, targeted approach but is limited by epigenetic challenges, particularly T-cell exhaustion. DNA methylation, a key regulatory mechanism, contributes to these challenges by suppressing CAR T-cell function. This study investigates enhancing CAR T-cell anti-tumor activity in solid and brain tumors through targeted manipulation of the DNA methylome using DNA methyltransferase inhibitors (DNMTis) during manufacturing. We hypothesize that altering the DNA methylome in CAR T-cells will enhance immune activation and anti-tumor efficacy. We optimized a transduction protocol that includes early treatment with DNMTis (including 5-Azacytidine and GSK-3484862) during the manufacturing process of murine and human CAR T-cells targeting B7-H3. Standard in vitro assays were conducted to assess whether DNMTi-treated B7-H3 CAR T-cells showed improved effector functions, including differentiation, cytokine response, persistence, and cytotoxicity. Results showed that incorporating DNMT inhibition during CAR T-cell manufacturing significantly enhanced proliferation and persistence, with improved sequential killing capacity in vitro. DNMTi-treated CAR T-cells expanded up to 55-fold more upon repeated stimulation with B7-H3-positive tumor cells and showed sustained cytokine secretion across stimulations. Treated CAR T-cells also maintained high cytotoxicity at lower effector-to-target ratios through 12 repeated stimulations, while untreated cells lost efficacy over time. Mechanistic insights indicated an enriched memory T-cell phenotype and epigenetic remodeling of exhaustion markers in DNMTi-treated CAR T-cells. In vivo studies in glioma and pediatric brain tumor models are ongoing, and preliminary data from ovarian cancer models show reduced disease burden and ascites in DNMTi-enhanced CAR T-treated groups. These findings highlight DNMT inhibition as a strategy to overcome epigenetic limitations in CAR T-cell therapy, providing a potential pathway to more effective treatments for glioma and other solid tumors through methylome-targeted reprogramming. Further research will validate these findings and assess their clinical potential. Citation Format: Elton L VanNoy, Zhongzhen Yi, Abigail Lee, Lynne El Ghorayeb, Katherine Chiappinelli, Dalia Haydar. Epigenetic reprogramming with DNA methylation inhibitors during CAR T cell manufacturing enhances anti-tumor activity, persistence, and cytotoxicity against solid and brain tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B021.