(P 237) DEVELOPMENT OF EPHA2 CAR T CELL THERAPY FOR OSTEOSARCOMA

Objective: Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents, with poor outcomes in metastatic or relapsed cases. EPHA2, a receptor tyrosine kinase minimally expressed in normal tissues but overexpressed in OS, non-small cell lung cancer (NSCLC), and glioblastoma, is associated with aggressive disease. Application of CAR T cell therapy in solid tumors has been hampered by antigen heterogeneity and an immunosuppressive tumor microenvironment. This study aims to preclinically validate EPHA2-targeted CAR T cells with improved tumor clearance and immune activation.

Methods: 24 fully human EPHA2-specific antibodies were selected based on strong binding to soluble and cell-surface EPHA2 (EC50 < 25 nM by ELISA). Second-generation CAR T cells were engineered and screened in vitro using luciferase-based cytotoxicity assays and live-cell imaging against OS (143B, SAOS2) and high EPHA2-expressing A549 NSCLC cell lines. The top 3 candidates were evaluated in vivo in metastatic 143B and A549 xenograft models in MHC DKO mice. Tumor burden by bioluminescence imaging and time to disease progression were assessed and compared across treatment groups.

Results: In cytotoxicity assays, 75% growth inhibition in A549 and 90% in 143B was seen with the lead CAR T construct (EP10) compared to 41% and 53% with a contemporaneous construct, 4H5, respectively (1:8 Effector:Target ratio). Significant reduction in tumor burden with EP10 was also observed in A549 (p < 0.0001, 2-way ANOVA) and 143B xenografts (p < 0.005) compared to 4H5 and a non-targeting control (p < 0.0001 in A549 and 143B). Significant progression free survival was also observed in EP10 treated A549 model (vs 4H5, p< 0.0005, median survival (MS) 115 days; vs control, p< 0.0001, MS 105 days) and aggressive 143B model (MS 54 days, vs 4H5, p< 0.01, MS 49 days; vs control, p< 0.005, MS 44 days) compared to 4H5 and control.

Conclusion: We describe initial development of a novel EPHA2-targeted CAR T cells with superior cytotoxicity, tumor control, and survival benefit over a contemporaneous product in OS and NSCLC models. Construct optimization with cytokine armoring is ongoing. Results from these studies will provide preclinical support for development of an investigator-initiated clinical trial in relapsed/metastatic OS.