Design and Evaluation of <scp> Eb ₄ Mab </scp> ‐7‐ <scp> mG _2a </scp> : A Dual‐Action Anti‐ <scp>EphB4</scp> Monoclonal Antibody for Targeted Breast Cancer Therapy

Breast cancer remains a leading cause of cancer mortality worldwide, underscoring the urgent need for novel and effective therapeutic strategies. 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-B2. We herein developed an improved anti-EphB4 monoclonal antibody, Eb₄Mab-7-mG_2a, which can be characterized as a subclass-switched IgG_2a variant designed to enhance immune effector function, specifically antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Our findings showed that Eb₄Mab-7-mG_2a effectively blocked Ephrin-B2-induced ERK phosphorylation and proliferation in EphB4-positive MCF-7 breast cancer cells but had no effect on EphB4-knockout (KO) MCF-7 cells. Flow cytometry confirmed high-affinity binding between Eb₄Mab-7-mG_2a and EphB4-expressing cells, whereas in vitro assays demonstrated potent and selective ADCC and CDC activities against EphB4-positive tumor cells. In vivo experiments showed that Eb₄Mab-7-mG_2a significantly suppressed xenograft growth in models bearing EphB4-overexpressing CHO-K1 and EphB4-positive MCF-7, but showed no therapeutic effect in EphB4-negative CHO-K1 and EphB4-KO MCF-7 xenografts. Immunohistochemical analysis revealed reduced Ki-67 proliferation indices in treated tumors, supporting the antiproliferative effects of the developed antibody. Overall, these findings demonstrate that Eb₄Mab-7-mG_2a exerts dual-action antitumor activity through ligand blockade and immune effector engagement. Further evaluations in other EphB4-overexpressing cancers and in combination with immune checkpoint inhibitors are warranted. Humanization and tumor-selective engineering may enhance its clinical potential for precision oncology.