Page 176 - MI-2-3

Microbes & Immunity

COMMUNICATION
Establishment of a highly sensitive and specific

anti-EphB2 monoclonal antibody (Eb Mab-12)
2
for flow cytometry

Rena Ubukata, Hiroyuki Suzuki , Miu Hirose, Hiroyuki Satofuka,
Tomohiro Tanaka, Mika K. Kaneko , and Yukinari Kato*
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Miyagi, Japan

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
*Corresponding author:
Yukinari Kato EphB2 using the Cell-Based Immunization and Screening method. Among the
(yukinari.kato.e6@tohoku.ac.jp) established anti-EphB2 mAbs, Eb Mab-12 (mouse IgG , kappa) showed reactivity
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2
Citation: Ubukata R, Suzuki H, toward EphB2-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB2) and an
Hirose M, et al. Establishment endogenously EphB2-expressing cancer cell line (LS174T), as confirmed by flow
of a highly sensitive and specific cytometry. The dissociation constant (K ) values of Eb Mab-12 for CHO/EphB2 and
anti-EphB2 monoclonal antibody D 2
−10
−9
(Eb2Mab-12) for flow cytometry. LS174T were determined to be 1.7 × 10 M and 4.4 × 10 M, respectively, using
Microbes & Immunity. flow cytometry. Furthermore, Eb Mab-12 exhibited no cross-reactivity with other
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2025;2(3):168-177. members of the EphA and EphB receptors. These results indicate that Eb Mab-12
doi: 10.36922/mi.5728 2
possesses high affinity and specificity in detecting EphB2, suggesting its potential
Received: October 30, 2024 application in tumor therapy.
1st revised: December 6, 2024
2nd revised: December 21, 2024 Keywords: EphB2; Monoclonal antibody; Cell-Based Immunization and Screening; Flow
Accepted: January 3, 2025 cytometry
Published online: January 16,
2025
Copyright: © 2025 Author(s). 1. Introduction
This is an Open-Access article
distributed under the terms of the The mammalian ephrin and Eph system comprises eight cell surface ephrin ligands (five
Creative Commons Attribution ephrin-As and three ephrin-Bs) and 14 receptor tyrosine kinases (nine EphA and five
License, permitting distribution,
1-6
and reproduction in any medium, EphB receptors). The interaction between Eph receptors and ephrins occurs through
provided the original work is dimerization or oligomerization, which leads to the tyrosine phosphorylation of Eph
properly cited. receptor and ephrin-B. The phosphorylated tyrosine recruits cytoplasmic effectors
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Publisher’s Note: AccScience containing Src-homology 2 domains, phosphotyrosine-binding domains, and PDZ
Publishing remains neutral with domains. Consequently, the Eph receptor-ephrin complexes activate bidirectional
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regard to jurisdictional claims in
published maps and institutional signaling – forward signaling from the Eph receptors and reverse signaling from the
affiliations. ephrins – which is essential for intercellular communication among similar or different
Volume 2 Issue 3 (2025) 168 doi: 10.36922/mi.5728