Establishment of a Novel Anti-EphA4 Monoclonal Antibody, Ea₄Mab-3, for Versatile Applications

Erythropoietin-producing hepatocellular carcinoma (Eph) receptor A4 (EphA4) binds to a broad range of ephrin ligands and is upregulated in various tumors. EphA4 activation plays crucial roles in the maintenance of cancer invasiveness, immune evasion, and stem cell properties. Furthermore, a novel EphA4 ligand, secretory ribonuclease 1, has been identified, and its interaction stimulates EphA4 signaling, leading to the promotion of cancer stem cell properties. Therefore, EphA4 has been considered an attractive target for cancer therapies. Thus, monoclonal antibodies (mAbs) against EphA4 are essential for basic research and mAb-based treatment in the clinic. In this study, we developed a novel anti-human EphA4 mAb, Ea₄Mab-3, using the Cell-Based Immunization and Screening (CBIS) method. Ea₄Mab-3 reacted with EphA4-overexpressed Chinese hamster ovary-K1 (CHO/EphA4) and endogenous EphA4-positive lung squamous cell carcinoma LK-2 in flow cytometry. The binding affinities (apparent dissociation constant K_D values) were determined as 4.5 × 10^-9 M for CHO/EphA4 and 2.5 × 10^-9 M for LK-2. Furthermore, Ea₄Mab-3 did not exhibit cross-reactivity with other Eph receptor-overexpressed CHO-K1 cells. In addition, Ea₄Mab-3 is suitable for immunoblotting and immunohistochemistry. These results indicate that Ea₄Mab-3, established using the CBIS method, facilitates basic studies of EphA4 and is expected to be useful for mAb-based tumor diagnosis and therapy.