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Microbes & Immunity A novel anti-EphB2 monoclonal antibody for flow cytometry

mAb (C Mab-46) were successfully determined using Funding
48
44
the REMAP method. In addition, the epitopes of anti-
mouse CD39 mAb (C Mab-1) were identified using This research was supported partially by the Japan Agency
for Medical Research and Development (AMED) under
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both REMAP and PAMAP methods. Therefore, further grant numbers: JP24am0521010 (to Y.K.), JP24ama121008
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studies are required to elucidate the epitope and biological (to Y.K.), JP24ama221339 (to Y.K.), JP23am0401013
activities of Eb Mab-12. (to Y.K.), JP24ama221339 (to Y.K.), JP24bm1123027 (to
2
The expression of EphB2 in more than 100 cell lines was Y.K.), and JP24ck0106730 (to Y.K.), and by the Japan Society
investigated using flow cytometry, with LS174T exhibiting for the Promotion of Science (JSPS) Grants-in-Aid for
the highest expression (Figure 3). Since LS174T is a Scientific Research (KAKENHI) grant numbers: 24K11652
transplantable cancer cell line in BALB/c nude mice, the (to H.Satofuka), 22K06995 (to H.Suzuki), 21K20789 (to
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in vivo anti-tumor effects of Eb Mab-12 can be evaluated. T.T.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K).
2
To achieve this, conversion of Eb Mab-12 (mouse IgG )
1
2
to mouse IgG subclass is essential for enhancing effector Conflict of interest
2a
activation ability. Previously, recombinant mAbs were The authors have no conflicts of interest.
produced, converting them into the mouse IgG subclass
2a
from mouse IgG . In addition, defucosylated IgG mAbs Authors contributions
2a
1
were produced using fucosyltransferase 8-deficient CHO- Conceptualization: Mika K. Kaneko, Yukinari Kato
K1 cells to enhance the antibody-dependent cellular Formal analysis: Rena Ubukata
cytotoxicity and in vivo anti-tumor effect in mouse xenograft Investigation: Rena Ubukata, Miu Hirose, Hiroyuki
models. Therefore, a class-switched and defucosylated Satofuka, Tomohiro Tanaka, Hiroyuki Suzuki
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version of Eb Mab-12 could significantly contribute to the Methodology: Mika K. Kaneko
2
treatment of EphB2-positive tumors in pre-clinical studies. Writing – original draft: Rena Ubukata, Hiroyuki Suzuki
The properties of Eb Mabs (Eb Mab-3, 8, and 10), which Writing – review & editing: Hiroyuki Suzuki, Yukinari Kato
2
2
showed cross-reactivity, were also determined (Table 1).
Such mAbs may be useful when targeting multiple EphBs Ethics approval and consent to participate
for mAb-based therapies.
All animal experiments were approved by the Animal Care
Cancer-specific mAbs (CasMabs) have been developed and Use Committee of Tohoku University (Permit number:
against HER2 (H Mab-250), podocalyxin (PcMab-6), 2022MdA-001).
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53
2
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and podoplanin (LpMab-2), demonstrating reactivity
with cancer cells, but not normal cells in flow cytometry. Consent for publication
The strategy used in this study is also applicable for Not applicable.
selecting anti-EphB2 CasMabs from Eb Mabs (Table 1)
2
or from clones derived from remaining positive wells. It Availability of data
has been confirmed that EphB2 is detectable in certain The data presented in this study are available in the article.
normal epithelial cells, prompting the screening of anti-
EphB2 CasMabs. The unique properties of H Mab-250 Further disclosure
2
could facilitate the development of HER2-targeting The paper has been uploaded to a preprint server
chimeric antigen receptor (CAR)-T-cells, which are (DOI: 10.20944/preprints202406.0704.v2).
currently undergoing a clinical phase I study in the
US. Therefore, the development of Eb Mabs for CAR-T References
55
2
applications is deemed necessary for treating EphB2-
positive tumors. 1. Zhu Y, Su SA, Shen J, et al. Recent advances of the Ephrin and
Eph family in cardiovascular development and pathologies.
5. Conclusion iScience. 2024;27(8):110556.
doi: 10.1016/j.isci.2024.110556
An anti-EphB2 mAb, Eb Mab-12 exhibits high affinity and
2
specificity to EphB2, suggesting its potential application in 2. Pasquale EB. Eph receptor signaling complexes in the plasma
tumor diagnosis and therapy. membrane. Trends Biochem Sci. 2024;49(12):1079-1096.
doi: 10.1016/j.tibs.2024.10.002
Acknowledgments
3. Pasquale EB. Eph receptors and ephrins in cancer
None. progression. Nat Rev Cancer. 2024;24(1):5-27.

Volume 2 Issue 3 (2025) 174 doi: 10.36922/mi.5728