EXTH-03. CAR T-CELLS TARGETED TO B7-H3 OR HER2 FUNCTION INDEPENDENTLY OF ANTIGEN DENSITY IN XENOGRAFT MODELS OF EPENDYMOMA

Abstract BACKGROUND Targeted treatment options are desperately needed for ependymomas (EPN). Chimeric antigen receptor (CAR) T-cells have immense potential to positively transform treatment outcomes; however, little is known regarding antitumor efficacy of CAR T-cells against EPNs. Specifically, antigen density has emerged as a major influencer of CAR T-cell potency. However, the specific high and low antigen density thresholds governing CAR T-cell efficacy for EPN are unknown. The goal of this project is to determine the role of antigen density in CAR T-cell potency against EPN. METHODS Using quantitative flow cytometry, we determined antigen density of B7-H3 and HER2 (the two CAR targets currently in pediatric EPN clinical trials), on EPN patient derived cell lines (1425, ST1, CPITT, ST2, EPI, L46SJ). We generated B7-H3 and HER2-CAR T-cells with CD28z signaling domain and compared long-term cytolytic activity, expansion, and cytokine production against EPNs. RESULTS We found that all EPN cell lines express remarkably high yet variable density of B7-H3 (62,700 – 335,440 molecules/ cell). In contrast, surface density of HER2 was consistently and significantly lower (860 – 24,488 molecules/ cell). In repeated-stimulation assays, B7-H3-CAR T-cells killed 7-10 times and expanded up to 325,360-fold when cultured against EPN. However, we observed no significant difference in total expansion or correlation with antigen density against EPNs. Surprisingly, expansion of HER2-CAR T-cells against EPNs was drastically higher and greatly exceeded that of B7-H3-CAR-T cells against the same EPN cells despite much lower antigen density. CONCLUSIONS Our work shows that both B7-H3 and HER2 are good targets against EPN, despite drastic differences in antigen density. Results suggest that lower antigen density of HER2 may be superior for CAR T-cell activity. Future work will validate whether HER-CAR T-cells have superior antitumor efficacy in vivo to further delineate role of antigen density thresholds in CAR T-cell potency.