EPHB4-Targeted CAR-T Cells Demonstrate 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 the main in vivo experiment, intratumoral administration of 131CAR suppressed tumor growth and significantly reduced the cytokeratin-defined carcinoma area compared with control groups. Digital pathology further showed a reduced proportion of EPHB4-positive area within the residual carcinoma and increased accumulation of human CD8+ cells in 131CAR-treated tumors. These findings indicate that EPHB4-targeted CAR-T cells exert antitumor activity in an orthotopic OSCC model and support further preclinical evaluation of this therapeutic approach in anatomically relevant settings.