EXTH-88. A novel dual-antigen CAR T cell strategy targeting PDPN and GD2 for the treatment of glioblastoma

Abstract While chimeric antigen receptor (CAR) T cell therapy has achieved encouraging remission rates in hematologic malignancies, effective treatment of glioblastoma (GBM), the most common and most lethal primary malignant brain tumor in adults, has yet to be demonstrated. Especially the heterogeneity and plasticity of the tumor antigen landscape in GBM present substantial challenges to therapeutic efficacy. In this study, we identified Podoplanin (PDPN) and GD2 as the most consistently preserved target antigens through a comprehensive screening campaign involving cell lines, both primary and recurrent GBM samples, and publicly available single-cell RNA sequencing datasets. We developed novel PDPN/GD2-dual-targeting CAR T cell strategies and evaluated their potential to overcome GBM heterogeneity in an extensive preclinical program. This included GBM cell lines (GCLs), patient-derived organoids (PDOs), and orthotopic xenograft models using stereotactic injections. We employed multi-parameter flow cytometry, in-depth cytokine profiling (cytometric bead arrays), proliferation (CFSE assays), and cytotoxicity assays (luciferase-based assays, Caspase immunofluorescence staining, BLI imaging). Our CAR T cells exhibited potent anti-tumor responses against multiple GBM cell lines and PDOs. While monospecific CAR T cells already elicited strong anti-tumor effects in PDO co-cultures, dual-targeting CAR T cells significantly enhanced apoptosis in PDOs and induced favorable cytokine and cytotoxic mediator profiles. In vivo, long-term remission was observed following locoregional administration of PDPN- or GD2-specific CAR T cells into the lateral ventricle of GBM-bearing NSG mice. While heterogeneous orthotopic tumors eventually relapsed in these two groups, dual-targeting therapy led to significantly prolonged overall survival and even achieved cure in the majority of mice. Collectively, our findings suggest that this approach can effectively address GBM heterogeneity and overcome previous limitations of single-antigen CAR T cell therapies.