Abstract Background: Efforts to apply CAR (Chimeric Antigen Receptor) T cell therapy to solid tumors are challenged by antigen heterogeneity, immunosuppressive microenvironments, and insufficient T-cell persistence. EPHA2, a receptor tyrosine kinase, is broadly overexpressed across solid tumors, including osteosarcoma (OS), non-small cell lung cancer (NSCLC), and glioblastoma, and is linked to metastatic progression and poor outcomes. The need is especially urgent in OS, where no new systemic therapy has been approved in over four decades. This study evaluates EPHA2 expression in OS and preclinically validates EPHA2-directed CAR T cells with enhanced tumor clearance and immune activation. Methods: EPHA2 expression was evaluated by immunohistochemistry on an OS patient-derived xenograft (PDX) tissue microarray (n = 55) and assigned H-scores. Fully human EPHA2 antibodies were engineered into 2nd generation CAR constructs containing antibody that recognizes and binds to EPHA2 antibody and costimulatory signaling domain. Cytotoxicity assays were employed to screen EPHA2-directed CAR T cells against EPHA2-high A549 (NSCLC) and 143B (OS) cell lines, enabling identification of the most active construct, EP10. EP10 was then evaluated in immunodeficient MHC-DKO (MHC class I and II double knockout) mice bearing metastatic A549 or 143B xenografts. Tumor burden was quantified by bioluminescence imaging. Event-free survival (EFS) was was assessed by log-rank test with events defined a moribund status. Results: EPHA2 showed broad expression across OS PDX models (range 1.8-265.1; median 99.7), with 88% of models scoring ≥20. EP10 CAR T cells demonstrated potent in vitro cytotoxicity, achieving 75% growth inhibition in A549 and 90% in 143B at a 1:8 effector-to-target ratio, outperforming reference CAR 4H5. EPHA2 surface expression was confirmed by flow cytometry. In A549 xenografts, EP10 significantly reduced tumor burden (p 0.0001) and markedly improved EFS compared to both the non-targeting mCherry (p=4e-04) and 4H5 (p=4e-04). In 143B xenografts, EP10 significantly prolonged EFS compared to the non-targeting mCherry control (p=0.01; EP10 median survival = 53.5 days vs 44 days) and 4H5 (p=0.01; EP10 median survival = 53.5 days vs 49.5 days). Conclusions: EPHA2 is widely expressed in OS and represents a compelling therapeutic target in OS and other solid tumors. We developed a novel EPHA2-targeting CAR T cell demonstrating potent cytotoxicity, tumor control, and survival benefit in OS and NSCLC models. Ongoing armoring efforts aim to enhance T-cell fitness, infiltration, and resistance to antigen-negative escape. Together, these data support ongoing clinical translation efforts of EPHA2-directed CAR T cells. Citation Format: Ali Cihan, Daoqi You, Armaan Siddiquee, Kristina Guillan, Tamar Feinberg, Erin Burns, Jasmine Um, Samantha Brosius, Joan Rou-En Choo, Andrew L. Kung, Anthony F. Daniyan, Filemon S. Dela Cruz, . Development of EPHA2 car t cell therapy for solid tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1540.
Cihan et al. (Fri,) studied this question.