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Eph receptor tyrosine kinases, particularly EphB4, exhibit diverse roles in cancer biology, acting as either tumor promoters or suppressors depending on the cellular environment and ligand engagement. EphB4 is frequently overexpressed in breast cancer and contributes to dysregulated signaling and tumor progression through the abnormal interaction with its ligand Ephrin\u2010B2. We herein developed an improved anti\u2010EphB4 monoclonal antibody, Eb\n                    <jats:sub>4<\/jats:sub>\n                    Mab\u20107\u2010mG\n                    <jats:sub>2a<\/jats:sub>\n                    , which can be characterized as a subclass\u2010switched IgG\n                    <jats:sub>2a<\/jats:sub>\n                    variant designed to enhance immune effector function, specifically antibody\u2010dependent cellular cytotoxicity (ADCC) and complement\u2010dependent cytotoxicity (CDC). Our findings showed that Eb\n                    <jats:sub>4<\/jats:sub>\n                    Mab\u20107\u2010mG\n                    <jats:sub>2a<\/jats:sub>\n                    effectively blocked Ephrin\u2010B2\u2010induced ERK phosphorylation and proliferation in EphB4\u2010positive MCF\u20107 breast cancer cells but had no effect on EphB4\u2010knockout (KO) MCF\u20107 cells. Flow cytometry confirmed high\u2010affinity binding between Eb\n                    <jats:sub>4<\/jats:sub>\n                    Mab\u20107\u2010mG\n                    <jats:sub>2a<\/jats:sub>\n                    and EphB4\u2010expressing cells, whereas in\u00a0vitro assays demonstrated potent and selective ADCC and CDC activities against EphB4\u2010positive tumor cells. In\u00a0vivo experiments showed that Eb\n                    <jats:sub>4<\/jats:sub>\n                    Mab\u20107\u2010mG\n                    <jats:sub>2a<\/jats:sub>\n                    significantly suppressed xenograft\u00a0growth in models bearing EphB4\u2010overexpressing CHO\u2010K1 and EphB4\u2010positive MCF\u20107, but showed no therapeutic effect in EphB4\u2010negative CHO\u2010K1 and EphB4\u2010KO MCF\u20107 xenografts. Immunohistochemical analysis revealed reduced Ki\u201067 proliferation indices in treated tumors, supporting the antiproliferative effects of the developed antibody. Overall, these findings demonstrate that Eb\n                    <jats:sub>4<\/jats:sub>\n                    Mab\u20107\u2010mG\n                    <jats:sub>2a<\/jats:sub>\n                    exerts dual\u2010action antitumor activity through ligand blockade and immune effector engagement. Further evaluations in other EphB4\u2010overexpressing cancers and in combination with immune checkpoint inhibitors are warranted. Humanization and tumor\u2010selective engineering may enhance its clinical potential for precision oncology.\n                  <\/jats:p>","DOI":"10.1111\/cas.70198","type":"journal-article","created":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T03:29:29Z","timestamp":1759202969000},"page":"3417-3430","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Design and Evaluation of\n                    <scp>\n                      Eb\n                      <sub>4<\/sub>\n                      Mab\n                    <\/scp>\n                    \u20107\u2010\n                    <scp>\n                      mG\n                      <sub>2a<\/sub>\n                    <\/scp>\n                    : A Dual\u2010Action Anti\u2010\n                    <scp>EphB4<\/scp>\n                    Monoclonal Antibody for Targeted Breast Cancer Therapy"],"prefix":"10.1111","volume":"116","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9039-4474","authenticated-orcid":false,"given":"Tomokazu","family":"Ohishi","sequence":"first","affiliation":[{"name":"Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research Foundation  Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2646-5132","authenticated-orcid":false,"given":"Hiroyuki","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Department of Antibody Drug Development, Tohoku University Graduate School of Medicine  Sendai Miyagi Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mika K.","family":"Kaneko","sequence":"additional","affiliation":[{"name":"Department of Antibody Drug Development, Tohoku University Graduate School of Medicine  Sendai Miyagi 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