Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited therapeutic options. Combining chemotherapy with immune checkpoint inhibitors (ICIs) has improved TNBC treatment, supporting the premise that DNA damage resistance and immune evasion are of importance for TNBC progression. Nonetheless, TNBCs often exhibit intrinsic or acquired resistance to these agents, emphasizing the need for developing new strategies for the treatment of recalcitrant advanced TNBCs. Our work supported by this grant has demonstrated that the transmembrane MUC1-C oncoprotein, which is aberrantly expressed in TNBCs, contributes to the cancer stem cell (CSC) state, DNA damage resistance and immune evasion. MUC1-C consists of a 58 aa extracellular domain and a 72 aa cytoplasmic domain that contribute to TNBC progression. These findings have supported the development of agents that target the MUC1-C extracellular and cytoplasmic domains for the treatment of advanced refractory TNBCs. Funding by this grant supported generation of the MAb 3D1 antibody that targets the MUC1-C extracellular domain at a conserved alpha-3 helix. Our work on MAb 3D1 has resulted in the development of allogeneic anti- MUC1-C CAR T cells with pharma that are now in the clinic for the treatment of TNBCs and other cancers. MAb 3D1 is also being developed as an antibody-drug conjugate (ADC) with the NCI NExT Program for IND- enabling studies and early phase evaluation as an immunotherapy for TNBC patients. We anticipate that, as for other anti-cancer agents, patients with TNBC will develop resistance to these anti-MUC1-C MAb 3D1 CAR T cells and ADCs. To address this potential outcome, our proposed work focuses on the development of novel strategies directed against the MUC1-C extracellular domain at a unique conserved alpha-4 helix. Our objective is to develop new immunotherapeutic agents that circumvent potential resistance mechanisms which emerge against the anti-MUC1-C agents already in the clinic. Our work has also focused on the development of agents that target the MUC1-C cytoplasmic domain, which is activated by chronic inflammation and promotes carcinogenesis. We developed the first-in-class GO- 203 inhibitor that blocks this region and thereby the MUC1-C oncogenic function. The proposed studies will focus on the development of GO-203 and other new agents that target the MUC1-C cytoplasmic domain and are delivered to TNBC CSCs using our recently formulated Quatramer platform. Our rationale is that these anti-MUC1-C agents will, like most therapeutics, be limited by resistance mechanisms and that new approaches will be needed for targeting the MUC1-C extracellular and cytoplasmic domains. The hypothesis is that our new anti-MUC1-C agents will be effective when used alone and in combinations with genotoxic and immune-based therapies, which will be addressed in studies of (i) human TNBC CSCs, (ii) GEMMs, and (iii) TNBC tumor specimens with the objective of advancing TNBC tr...