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
Authors: VanNoy Elton L , Yi Zhongzhen , Lee Abigail , El Ghorayeb Lynne , Chiappinelli Katherine , Haydar Dalia 
Published: February 2025

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.