Establishment of a highly sensitive and specific anti-EphB2 monoclonal antibody (Eb₂Mab-12) for flow cytometry

Abstract

Ephrin type-B receptor 2 (EphB2) is a member of the Eph family tyrosine kinase receptors. EphB2 binds to ephrin-B1, ephrin-B2, and ephrin-B3, which are critical regulators of vascular and neural development, influencing cell migration and axon guidance. EphB2 is overexpressed in several tumors, including glioma, breast cancer, hepatocellular carcinoma, and malignant mesothelioma, where it functions as a tumor promoter. Therefore, the development of monoclonal antibodies (mAbs) targeting EphB2 is essential for the diagnosis and treatment of EphB2-positive tumors. In this study, we developed novel mAbs for human EphB2 using the Cell-Based Immunization and Screening method. Among the established anti-EphB2 mAbs, Eb₂Mab-12 (mouse IgG1, kappa) showed reactivity toward EphB2-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB2) and an endogenously EphB2-expressing cancer cell line (LS174T), as confirmed by flow cytometry. The dissociation constant (KD) values of Eb₂Mab-12 for CHO/EphB2 and LS174T were determined to be 1.7 × 10⁻⁹ M and 4.4 × 10⁻¹⁰ M, respectively, using flow cytometry. Furthermore, Eb₂Mab-12 exhibited no cross-reactivity with other members of the EphA and EphB receptors. These results indicate that Eb₂Mab-12 possesses high affinity and specificity in detecting EphB2, suggesting its potential application in tumor therapy.

Keywords

EphB2 / Monoclonal antibody / Cell-Based Immunization and Screening / Flow cytometry