EXTH-101. CAR T-cells targeted to B7-H3 are effective against ependymomas but show limited response in the immune competent setting

Targeted treatments are desperately needed for ependymomas (EPN). Chimeric antigen receptor (CAR) T-cells have the potential to transform patient outcomes. However, little is known regarding the antitumor efficacy of CAR T-cells against EPNs. We found that B7-H3 is highly and consistently expressed on EPNs, making it a promising CAR T-cell target. The goal of this project is to develop a safe, effective B7-H3-targeted CAR T-cell immunotherapy for EPN. We generated second-generation mouse and human B7-H3-CAR T-cells and examined their anti-EPN functionality in vitro and in vivo. Human B7-H3.CAR T-cells demonstrated in vitro antitumor activity against six EPN cell lines. In repeated-stimulation assays, CAR T-cells remained functional for 7–10 rounds of killing and expanded up to 325,000-fold. Multiplex cytokine analysis revealed sustained Th1 response and a significant increase in Th2 response from the 1st to the 4th stimulation. We found that EPN cells constitutively secrete high levels of C-C motif chemokine ligand 2 (CCL2), which increased significantly (up to 100,000-fold) in co-culture with CAR T-cells. In vivo, 11/17 human B7-H3.CAR-treated mice durably cleared xenograft EPNs, while the remaining animals were treatment refractory. In a fully syngeneic EPN model, despite robust in vitro efficacy, murine B7-H3.CAR T-cells showed in vivo efficacy in only 1/9 mice, which eventually relapsed 76 days after CAR T-cell treatment. IHC analysis of tumors at endpoint revealed robust infiltration of macrophages and microglia within tumors, regardless of treatment. This observation is consistent with CCL2’s role as a major chemoattractant of monocytes and macrophages. Together, our study establishes clinically relevant models to study CAR T-cell therapy resistance in EPN, and preliminary mechanistic insight into CAR T-cell persistence and modulation through CCL2 signaling. Additionally, our work is the first to evaluate CAR T-cell efficacy against EPNs in the syngeneic setting.