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

Guanjie Li1 ,  Tomohiro Tanaka1 ,  Mika K. Kaneko1 ,  Hiroyuki Suzuki1* ,  Yukinari Kato1*
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1 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
MI, 025300066 https://doi.org/10.36922/MI025300066
Received: 25 July 2025 | Revised: 5 September 2025 | Accepted: 9 October 2025 | Published online: 8 December 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

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 (KD 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
Funding
This research was supported in part by the Japan Agency for Medical Research and Development under grant numbers JP25am0521010 (to Y.K.), JP25ama121008 (to Y.K.), P25ama221153 (to Y.K.), JP25ama221339 (to Y.K.), and JP25bm1123027 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) under grant numbers 24K18268 (to T.T.) and 25K10553 (to Y.K.).
Conflict of interest
The authors declare that they have no competing interests.
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Microbes & Immunity, Electronic ISSN: 3029-2883 Print ISSN: 3041-0886, Published by AccScience Publishing
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