Project Abstract HER2 overexpression is a powerful prognostic marker in node-positive patients with breast cancer. Immunohistochemical (IHC) staining is the most widely used approach in the clinic for scoring HER2 status as positive (3+), equivocal (2+), and negative (0 or 1+). Breast tumors with low levels of HER2 (1+ or 2+) are not considered as positive for HER2 overexpression and such tumors do not well respond to HER2-targeted agents anti-HER2 antibody (trastuzumab) and ado-trastuzumab emtansine (T-DM1). Due to their genetic diversity and clinical heterogeneity, the therapeutic challenges for HER2-low breast cancer are the paucity of actionable targets and lack of targeted therapies. We found that heterozygous deletion of chromosome 17p (17p loss) is the most prevalent event in breast cancer (56%) as well as in TNBC (53%). Within the 17p deletion region is the tumor suppressor TP53 (encoding p53), whose deletion or mutation is known as a primary tumorigenic driver. Our recent study identified POLR2A in the TP53-neighboring region as a collateral vulnerability target in the TNBC tumors with 17p loss, suggesting that inhibition of POLR2A may be a precision therapy approach. To accelerate the translational development of our important finding, we are developing antibody-drug conjugates (ADC) with α-amanitin (POLR2A inhibitor) as a warhead. This approach inhibits the specific uptake of α-amanitin into hepatocytes and increases tumor-specific targeting using tumor-specific monoclonal antibodies. In this project, we will first develop α-amanitin-conjugated trastuzumab (T-Ama) and test their efficacy in treating 17ploss TNBC tumors with low levels of HER2. Second, we will determine how the 17p loss leads to reduced T cell infiltration and cytotoxicity, thereby leads to immune evasion of the TNBC tumor. To determine the underlying molecular mechanism, we will examine how the 17p loss negatively impacts tumor antigen presentation and tumor immune response in TNBC. Finally, we will determine the efficacy of T-Ama as a single agent or in combination with immune checkpoint blockade in treating TNBC with 17p loss.