EXTH-139. Identification of clinically actionable drugs that enhance CAR-T cell expansion and cytotoxicity against DMG

Despite decades of research, there have been no advancements in treatment options for children diagnosed with diffuse midline glioma (DMG). The emergence of cell-based immunotherapies, specifically the use of chimeric antigen receptor (CAR) T cells has shown some initial promise in pediatric brain tumors. However, there are both tumor and CAR T cell intrinsic biological limitations that hinder their overall efficacy. Therefore, we sought to identify compounds that could be used in combination with CAR T cells to overcome their shortcomings as a monotherapy. To conduct a targeted screen of selected compounds, we first labeled DMG tumor cells with Renilla luciferase and B7-H3 CAR T cells with firefly luciferase. We determined that an effector-to-target (E: T) ratio of 1:16 provided the most consistent tumor cell killing at both 24 and 72 hours. We prioritized 32 compounds for screening based on FDA approval, blood-brain barrier permeability, and clinical relevance to brain tumors. Staurosporine and DMSO were used as positive and negative controls for cell death, respectively, while Dasatinib served as a T cell-specific negative control. Tumor and CAR T cell dynamics were then assessed using cell-specific luminescent substrates in response to each compound. From our targeted screen, we identified several compounds that enhanced CAR T cell expansion, promoted tumor cell killing, or did both (20 out of 32). Notably, our screen recovered previously reported hits such as Lenalidomide and Birinipant, supporting the validity of our approach. We are currently evaluating the top three compounds that enhance both CAR T cell expansion and tumor cell killing in vitro and in vivo. Collectively, we have optimized a method to perform screens involving multiple variables which will support the ongoing efforts to investigate combinatorial treatment strategies for CAR T cells against DMGs.