HER3 (ErbB3) is a member of the human epidermal growth factor receptor (HER) tyrosine kinase family that includes EGFR and HER2, two well-known targets for oncology therapy. Kinase signaling of the HER family is initiated by ligand binding that induces receptor homodimerization or heterodimerization and transphosphorylation of the intracellular domain. In this regards, HER3 is unique among the HER kinase family members. Despite being able to bind ligand, HER3 does not possess a catalytically active kinase domain. To turn on the signaling pathway, HER3 needs to form a heterodimer with another HER family member. However, this does not make HER3 inferior with respect to HER pathway signaling, since dimerization of HER3 with HER2 elicits one of the most powerful oncogenic signals. Similar to EGFR and HER2, HER3 expression and activity have been linked to the pathogenesis of various cancers including breast, ovarian and melanoma, and has been associated with resistance to several cancer therapies. The significance of HER3 in cancer pathogenesis prompted us to develop HER3 antagonistic antibodies. A large number of HER3 antibodies were generated by immunizing mice with HER3 expressing cell lines, and screened for the ability to bind human and monkey HER3. Additionally, since HER3 and activated HER3 (phospho-HER3) were detected in the majority of primary as well as metastatic breast cancer tissues by immunohistochemistry staining, antibodies were screened for their ability to inhibit the HER3 ligand-induced proliferation as well as the basal proliferation of breast cancer cell lines. Three HER3 antibodies, HER3-3, HER3-8 and HER3-10, were exceptionally potent in inhibiting the basal proliferation of four breast cancer cell lines that constitutively express phospho-HER3, where other HER3 antibodies (i.e. U1-59 and MM-121) had little or no activity. In SKBR3 cells, these HER3 antibodies inhibited cell proliferation, comparable to the activity of trastuzumab although the HER3 expression level was thirty-fold lower than the HER2 level. All three HER3 antibodies recognized overlapping epitopes. However, only HER3-8 and HER3-10 antibodies strongly competed for the binding of HER3 ligand and inhibited HER2-HER3 dimerization. These HER3 antibodies were also very potent in inhibiting ligand-induced HER3 signaling and tumor cell proliferation. Because of its best overall activities, HER3-8 was humanized by variable domain resurfacing technology. Humanized HER3-8 retained all of the biological properties and activities of muHER3-8. In conclusion, we have generated a novel humanized HER3 antibody, huHER3-8 that is not only active in inhibiting exogenous ligand-induced HER3 signaling and tumor cell growth, but also effective in inhibiting the basal proliferation of HER3-positive tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4564. doi:10.1158/1538-7445.AM2011-4564