AccScience Publishing / MI / Online First / DOI: 10.36922/mi.5728
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Establishment of a highly sensitive and specific anti-EphB2 monoclonal antibody (Eb2Mab-12) for flow cytometry

Rena Ubukata1 ,  Hiroyuki Suzuki1 ,  Miu Hirose1 ,  Hiroyuki Satofuka1 ,  Tomohiro Tanaka1 ,  Mika K. Kaneko1 ,  Yukinari Kato1*
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1 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
MI, 5728 https://doi.org/10.36922/mi.5728
Submitted: 30 October 2024 | Revised: 21 December 2024 | Accepted: 3 January 2025 | Published: 16 January 2025
© 2025 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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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
Funding
This research was supported partially by the Japan Agency for Medical Research and Development (AMED) under grant numbers: JP24am0521010 (to Y.K.), JP24ama121008 (to Y.K.), JP24ama221339 (to Y.K.), JP23am0401013 (to Y.K.), JP24ama221339 (to Y.K.), JP24bm1123027 (to Y.K.), and JP24ck0106730 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant numbers: 24K11652 (to H.Satofuka), 22K06995 (to H.Suzuki), 21K20789 (to T.T.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K).
Conflict of interest
The authors have no conflicts of interest.
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