We generated a novel HER2 monoclonal antibody (mAb104) which binds to an epitope in domain II of HER2 that is conformationally exposed in tumors in response to HER2 amplification or activation but is not accessible to antibody binding in normal tissues. Consistent with other studies that evaluated antibodies targeting conformationally exposed epitopes, mAb104 lacked in vitro activity however showed potent anti-tumor activity in vivo. The anti-tumor effect in vivo was similar in magnitude to trastuzumab and pertuzumab, whilstand combination with trastuzumab was superior to trastuzumab alone. Immunohistochemistry screening of normal and tumor tissues with mAb104 showed mAb104 did not bind to normal tissues, confirming tumor specificity of mAb104. In vivo biodistribution and imaging data demonstrated specific tumor targeting of mAb104 in HER2 expressing tumors. Confocal microscopy clearly demonstrated internalization of mAb104 into the tumor cells, consistent with mAb104:HER2 trafficking. Mab104 is tumor specific, exhibits potent antitumor activity in HER2-positive models and internalizes into HER2-positive tumor cells. These results demonstrate the potential of mAb104 as a novel HER2 targeting therapy both as a naked antibody for signalling abrogation therapy, and for payload delivery as an antibody-drug conjugate or for beta/alpha particle therapy.