EXTH-140. EphA3-Directed CAR-T Cell Therapy for Glioblastoma: Dual Targeting of Tumor and Tumor Microenvironment

Abstract BACKGROUND Glioblastoma (GBM) is a universally lethal brain cancer with limited durable responses to immunotherapy, largely due to its immunosuppressive tumor microenvironment (TME). Ephrin receptor A3 (EphA3), a receptor tyrosine kinase minimally expressed in normal tissue, is enriched on GBM cells, glioma stem cells (GSCs), and M2-like tumor-associated macrophages (TAMs). We hypothesize that dual targeting of TAMs and tumor cells by EphA3-CAR-T is a viable treatment strategy. METHODS We developed EphA3-directed second-generation CAR-T cells (EphA3-CAR-T) incorporating the single-chain variable fragment of ifabotuzumab, an anti-EphA3 monoclonal antibody, achieving >90% transduction efficiency. We evaluated antitumor efficacy in vitro; the functional activities of EphA3-CAR-T on GSCs were assessed via selective cytotoxicity, T cell proliferation, degranulation assays, and depletion of M2-like macrophages. In vivo, 1 × 10⁶ EphA3⁺ U251 MG cells and/or 1 × 10⁶ ex vivo–polarized M2-like macrophages were implanted in the flanks of NOD-SCID-γ⁻/⁻ mice. Comparative studies with EGFRvIII-directed CAR-T cells were also performed. RESULTS Immunohistochemistry confirmed EphA3 expression across both tumor and immune compartments in patient-derived specimens. EphA3-CAR-T cells demonstrated potent, selective cytotoxicity (p<0.001), T cell proliferation (p<0.001), and CD107α degranulation (p<0.001) against EphA3⁺ GBM and GSCs, while also reversing TAM-mediated immunosuppression via depletion of ex vivo–polarized M2-like macrophages (p<0.05), compared to other validated CAR-Ts. In vivo, EphA3-CAR-T significantly reduced tumor volume (p<0.001) and improved survival (p<0.01), in the presence of TAMs. In contrast, EGFRvIII CAR-T cells were inactivated under the same TME conditions. EphA3-CAR-T cells remained active in models co-engrafted with macrophages, suggesting preserved function within the immunosuppressive TME. CONCLUSIONS EphA3-CAR-T therapy addresses a major barrier to CAR-T success in GBM: TME-mediated immunosuppression. This tumor- and TME-targeting approach demonstrated robust preclinical activity. These findings support the clinical translation of EphA3-CAR-T cells as a next-generation immunotherapy for GBM.