The EphA3 Antibody Enhances CD47 Antibody-Mediated Phagocytosis by Repolarizing M2-Like Tumor-Associated Macrophages in a Preclinical Model of Recurrent Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is characterized by a highly immunosuppressive tumor microenvironment (TME), with tumor-associated macrophages (TAMs) playing a central role in resistance to therapy. The immune checkpoint molecule CD47, known for its "don't eat me" signal, and EphA3, a receptor tyrosine kinase, are both upregulated in radiation-resistant HNSCC. However, their cooperative role in regulating TAMs and therapeutic resistance remains poorly understood. We established a radiation-resistant HNSCC model (MOC2R) using repeated irradiation and confirmed increased expression of CD47 and EphA3 in these cells. The effects of single and dual blockade of CD47 and EphA3 were evaluated through in vitro phagocytosis assays, Western blot, co-immunoprecipitation, and in vivo tumor models. Transcriptomic analysis and immunofluorescence staining were performed to elucidate molecular pathways involved in TAM modulation. We demonstrated in three independent experiments (n = 3) with statistical significance (P < 0.05). CD47 and EphA3 were upregulated in radioresistant HNSCC and recurrent tumors. Dual blockade synergistically enhanced macrophage-mediated phagocytosis (P < 0.01) and significantly reduced clonogenic tumor cell survival compared to single blockade (P < 0.0001). EphA3 inhibition reprogrammed tumor-associated macrophages toward an M1-like phenotype, with increased expression of pro-inflammatory markers and decreased M2-associated markers. In vivo, combination therapy significantly reduced tumor volume and weight and increased M1 macrophage infiltration, while reducing M2 macrophages. Mechanistically, EphA3 regulated macrophage polarization via the β-catenin/FOSL2/ARID5A axis, enhancing the efficacy of CD47 blockade. Our findings demonstrate that EphA3, in cooperation with CD47, promotes an immunosuppressive TME by modulating β-catenin/FOSL2/ARID5A signaling in TAMs. Dual targeting of EphA3 and CD47 represents a promising strategy to reprogram macrophages and enhance anti-tumor immunity in radiation-resistant HNSCC.