Inhibition of HER2 signaling and breast cancer cell growth with a novel antibody targeting HER2 ECD III/IV.

Human epidermal growth factor receptor 2 (HER2), a ligand-independent tyrosine kinase receptor belonging to the EGFR family, serves as a key oncogenic driver in breast, gastric, and several other solid tumors. Although anti-HER2 therapies have substantially improved survival outcomes-particularly in breast and gastric cancers-treatment resistance and cancer recurrence remain major clinical challenges. Thus, developing novel antibodies exhibiting complementary effects to the current anti-HER2 therapies could provide additional therapeutic benefits. Here, we describe two novel HER2-targeting monoclonal antibodies, m66 (murine-derived) and r40 (rabbit-derived), which inhibit breast cancer cell proliferation in vitro. Of the two, antibody r40 exhibits stronger suppression of the PI3K/AKT and MAPK signaling pathways compared to m66. Moreover, the addition of r40 to the combination of trastuzumab and pertuzumab leads to a significantly enhanced inhibitory effect. We also determined the cryo-EM structures of the HER2-m66-trastuzumab ternary complex and the HER2-r40-trastuzumab-pertuzumab tetrameric complex, at overall resolutions of 3.2 Å and 3.1 Å, respectively. Structural analyses reveal that m66 recognizes an epitope overlapping with that of pertuzumab, whereas r40 binds within the HER2 ECD III/IV-a region distinct from the binding sites of both trastuzumab and pertuzumab. These findings identify r40 as a promising therapeutic candidate for use in combination with trastuzumab and pertuzumab in the treatment of HER2-positive breast cancer.