Establishment of a Highly-Sensitive and Specific anti-EphB2 Monoclonal Antibody Eb₂Mab-12 for Flow Cytometry

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 nervous development through controlling cell migration and axon guidance. EphB2 is overexpressed in tumors, including glioma, breast cancer, hepatocellular carcinoma, and malignant mesothelioma, and it functions as a tumor promoter. Therefore, the development of monoclonal antibodies (mAbs) against EphB2 is essential for tumor diagnosis and therapy for EphB2-positive tumors. In this study, we developed novel mAbs for human EphB2 using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-EphB2 mAbs, Eb2Mab-12 (mouse IgG1, kappa), reacted with EphB2-overexpressed Chinese hamster ovary-K1 (CHO/EphB2) and an endogenously EphB2-expressing cancer cell line (LS174T) by flow cytometry. Using flow cytometry, the dissociation constant (KD) values of Eb2Mab-12 for CHO/EphB2 and LS174T were determined as 1.7 × 10−9 M and 4.4 × 10−10 M, respectively. Furthermore, Eb2Mab-12 did not cross-react with other members of EphA and EphB receptors. These results indicated that Eb2Mab-12 possesses a high affinity and specificity for detecting EphB2 and could apply to tumor therapy.