Ea₅Mab-7, a novel anti-EphA5 monoclonal antibody for flow cytometric and immunohistochemical applications

Abstract

Erythropoietin-producing hepatocellular (Eph) receptor A5 (EphA5) binds to ephrin ligands and initiates intracellular signaling through its tyrosine kinase activity. EphA5 is upregulated or mutated in various tumors, and these alterations are associated with tumor progression and sensitivity to immunotherapy, respectively. Therefore, EphA5 has been proposed as an attractive target for tumor diagnosis and therapy. A monoclonal antibody (mAb) against EphA5 is essential for basic research and for the development of mAb-based tumor therapies. In this study, we developed a novel anti-human EphA5 mAb, Ea₅Mab-7, using the Cell-Based Immunization and Screening (CBIS) approach. Ea₅Mab-7 reacted with EphA5-overexpressed Chinese hamster ovary-K1 (CHO/EphA5), EphA5-overexpressed glioblastoma LN229 (LN229/EphA5), and Saos-2 osteosarcoma cells endogenously expressing EphA5, as assessed using flow cytometry. The binding affinities (K_D values) were determined to be 3.2 × 10⁻⁹ M for CHO/EphA5, 3.0 × 10⁻⁹ M for LN229/EphA5, and 1.3 × 10⁻⁹ M for Saos-2. Furthermore, Ea₅Mab-7 did not exhibit cross-reactivity with other ephrin receptor-overexpressed CHO-K1 cells. In addition, Ea₅Mab-7 was suitable for immunohistochemistry. These results indicate that Ea₅Mab-7, established using the CBIS method, facilitates basic studies of EphA5 and is a potential candidate for mAb-based tumor diagnosis and therapy.

Keywords

EphA5 / Monoclonal antibody / Cell-Based Immunization and Screening / Flow cytometry / Immunohistochemistry

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