EXTH-02. MODIFYING CAR T CELL EPIGENETIC PROGRAMS TO IMPROVE THEIR PERSISTENCE AGAINST GROUP 3 MEDULLOBLASTOMAS

Abstract BACKGROUND Safe, effective therapies are desperately needed to improve outcomes for patients with medulloblastoma (MB), a malignant pediatric brain cancer. Chimeric antigen receptor (CAR)-T cells are a promising therapeutic option as they target T-cell cytotoxicity exclusively to tumor associated antigens. B7 homolog 3 (B7-H3) is an ideal target for CAR-T cell therapy as all MB subtypes express B7-H3 but healthy tissues do not. However, a major roadblock to tumor clearance and control is the inability of CAR-T cells to persist, suggesting that improving persistence will improve therapeutic efficacy. The goal of this study was to compare head-to-head the impact of knocking out TET2 and DNMT3A negative epigenetic regulators of CAR-T cell persistence against group 3 MB (G3MB), the most malignant MB subtype. METHODS We generated B7-H3 CAR-T cells with CD28ζ signaling domain and performed CRISPR/Cas9 knockout (KO) of TET2, DNMT3A, and AAVS1 as a control. We compared resulting CAR-T cell persistence via repeat stimulation assays in vitro against G3MB cell lines with varying B7-H3 antigen density (D341low, HDMB03mid, and D425high). We further compared KO CAR-T cells in vivo against D341low, HDMB03mid, and D425high orthotopic xenografts. RESULTS Our data shows that DNMT3A KO, but not TET2 KO, consistently improves the persistence and expansion of CAR-T cells against G3MB in vitro. In vivo, KO of epigenetic regulators, improves CAR-T cell therapy against highly aggressive D341low, HDMB03mid, and D425high but is insufficient to elicit curative responses. CONCLUSIONS Our study demonstrates that genetic KO of negative epigenetic regulators of CAR-T cell persistence can improve anti-tumor function against G3MB. We conclude that for the B7-H3.CD28ζ CAR, DNMT3A KO is superior for improving persistence over TET2 KO, but that DNMT3A KO is insufficient in vivo.