Simple SummaryHuman epidermal growth factor receptor 2 (HER2) is a protein present in increased amounts in breast cancer compared with healthy breast tissue. Anti-cancer drugs directed at HER2 can therefore kill cancer cells while reducing harmful effects to adjacent normal cells that do not have HER2. HER2-positive breast cancers often spread to bone as the disease progresses, resulting in incurable bone metastases, bone fractures and breaks, and severe pain for the patient. We have developed HER2-TTC (HER2-targeted thorium-227 conjugate), an intravenously injected cancer therapy that delivers lethal radiation to HER2-positive cancer cells. We tested HER2-TTC in cell culture and mouse models, including those that mimic HER2-positive human breast cancer metastases in bone. In the in vivo models, HER2-TTC treatment killed breast cancer cells and prevented cancer-induced abnormal changes in bone. These results suggest that HER2-TTC treatment could be beneficial to patients with HER2-positive breast cancer with bone metastases.AbstractHuman epidermal growth factor receptor 2 (HER2) is overexpressed in 15–30% of breast cancers but has low expression in normal tissue, making it attractive for targeted alpha therapy (TAT). HER2-positive breast cancer typically metastasizes to bone, resulting in incurable disease and significant morbidity and mortality. Therefore, new strategies for HER2-targeting therapy are needed. Here, we present the preclinical in vitro and in vivo characterization of the HER2-targeted thorium-227 conjugate (HER2-TTC) TAT in various HER2-positive cancer models. In vitro, HER2-TTC showed potent cytotoxicity in various HER2-expressing cancer cell lines and increased DNA double strand break formation and the induction of cell cycle arrest in BT-474 cells. In vivo, HER2-TTC demonstrated dose-dependent antitumor efficacy in subcutaneous xenograft models. Notably, HER2-TTC also inhibited intratibial tumor growth and tumor-induced abnormal bone formation in an intratibial BT-474 mouse model that mimics breast cancer metastasized to bone. Furthermore, a match in HER2 expression levels between primary breast tumor and matched bone metastases samples from breast cancer patients was observed. These results demonstrate proof-of-concept for TAT in the treatment of patients with HER2-positive breast cancer, including cases where the tumor has metastasized to bone.