EPHB4-Targeted CAR-T Cells Demonstrate Potent Antitumor Activity in an Orthotopic Tongue PDX Model of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) remains associated with poor outcomes and functional impairment despite multimodal treatment, underscoring the need for effective and less invasive therapeutic strategies. Ephrin type-B receptor 4 (EPHB4) is frequently expressed on OSCC cells and represents a potential target for chimeric antigen receptor (CAR)-T cell therapy. In this study, we evaluated the antitumor activity of EPHB4-targeted CAR-T cells (131CAR) using in vitro cytotoxicity assays against the OSCC cell line HSC-4 and an orthotopic tongue patient-derived xenograft (PDX) model established from an OSCC tumor (OC-15) in NOD scid gamma (NSG) mice. Flow cytometry confirmed EPHB4 expression in HSC-4 cells (~90%) and OC-15-derived tumor cells (~68%). In vitro assays demonstrated effector-to-target ratio–dependent cytotoxicity of 131CAR against HSC-4 cells. In vivo, both intratumoral and intravenous administration of 131CAR significantly reduced tumor burden compared to control groups, with greater tumor reduction observed following intratumoral delivery. Immunohistochemistry revealed infiltration of human CD8⁺ cells and decreased tumor area in treated tumors, and digital pathology analysis corroborated the reduced tumor area. These findings indicate that EPHB4-targeted CAR-T cells exert antitumor activity in an orthotopic OSCC model and suggest that local administration may enhance therapeutic efficacy in anatomically relevant settings.