Development of an anti-human EphA2 monoclonal antibody Ea₂Mab-7 for multiple applications

Ephrin type A receptor 2 (EphA2) binds to membrane-bound ligands, ephrin A1, A2, and A5, eliciting bidirectional signaling. This signaling regulates many physiological processes, such as tissue development, homeostasis, and regeneration. The dysregulation of the EphA2-ephrins axis contributes to various diseases, including cancers. The high expression of EphA2 is observed in various cancers, which promotes cancer malignancy, whereas its levels are relatively low in most normal adult tissues. Therefore, EphA2 is a promising target for cancer therapy. We developed anti-human EphA2 monoclonal antibodies in this study using the Cell-Based Immunization and Screening method. Among them, a clone Ea₂Mab-7 (IgG₁, κ) exhibited a high affinity and sensitivity in flow cytometry. The dissociation constant values of Ea₂Mab-7 for CHO/EphA2 and MDA-MB-231 cells were determined as 6.2 ± 1.3 × 10^-9 M and 1.6 ± 0.4 × 10^-9 M, respectively. Furthermore, Ea₂Mab-7 can detect endogenous EphA2 in Western blot and immunohistochemistry. Therefore, the Ea₂Mab-7 is highly versatile for basic research and is expected to contribute to clinical applications, such as antibody therapy and tumor diagnosis.