HG-108MULTISPECIFIC CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELLS OVERCOME INTER-PATIENT TUMOR HETEROGENEITY AND EXHIBIT ENHANCED ANTITUMOR FUNCTIONALITY IN THE TREATMENT OF GLIOBLASTOMA

Abstract

Background: Glioblastomas have poor outcomes in children. HER2-specific CAR T-cells kill autologous glioblastoma, but recurrences occur due to antigen escape variants tumor cells that maintain positivity for two other glioma antigens, IL13Ra2 and EphA2. We hypothesized that co-targeting these escape variants will enhance the anti-glioma activity of CAR T-cells. Purpose: The intent of this project is to develop trispecific-specific CAR T-cell (triCAR T-cells) for the adoptive immunotherapy of glioblastoma that will overcome inter-and intra-patient heterogeneity. DESIGN/Methods: We generated and tested the effector functions of patients' triCART-cellsagainst autologous glioblastoma cells derived from surgical excision samples. Results: Primary glioblastoma excision samples exhibited varied expression of HER2, IL13Ra2 and EphA2. We generated T-cells trispecific for HER2, IL13Ra2 and EphA2 (triCAR T-cells) by integrating a tricistronic transgene encoding the 3 respective CAR molecules into the T cell genome. Flow cytometry indicated proportionate stable expression of individual CAR molecules on the surface of T-cells. These T-cells show distinct specificity for each of the three glioma antigens evidenced byactivation,proliferation,andcytolyticfunction.Additionally,weusedconfocal microscopy to examine the T-cell/tumor immunological synapse and discovered microtubuleorganizingcomplexessignificantlyclosertothe synapse,indicatinga bettercytolytic potential of triCART-cells. In vitro and invivo testing in an autologous setup demonstrated enhanced antitumor activity of triCAR T-cells. Conclusion: The heterogeneity of glioblastoma justifies targeting multiple glioma antigens simultaneously. We have generated and tested a tri-specific T cell product that exhibits an enhanced anti-glioma efficacy.