Treatment options for patients with refractory castrate resistant prostate cancer (CRCP) and the highly aggressive form of neuroendocrine prostate cancer (NEPC) are limited by a lack of actionable molecular targets. Immunotherapeutic agents, such as immune checkpoint inhibitors (ICIs), have also been largely ineffective against refractory CRPC and NEPC. Mucin 1 (MUC1) is aberrantly overexpressed in CRPC and is associated with poor progression-free and overall survival. MUC1 consists of an extracellular N-terminal mucin subunit (MUC1-N) that is shed from the cell surface, and an oncogenic non-shed transmembrane C-terminal subunit (MUC1-C). Recent findings have identified MUC1-C as a master effector of lineage plasticity driving progression of CRPC to NEPC and as a potential druggable target for eliminating CRPC/NEPC cancer stem cells (CSCs). We have generated a novel monoclonal antibody (MAb), designated 3D1, against the alpha-3 helix in MUC1-C extracellular domain. MAb 3D1 has been humanized and conjugated to MMAE as an antibody-drug conjugate (ADC). Based on findings that the huMAb-3D1-MMAE ADC is highly effective against MUC1-C- expressing cancer cells, the NCI NExT Program is supporting IND-enabling studies for their potential clinical development. To date, trials with ICIs have had limited success in the treatment of patients with PC, emphasizing the need for developing novel approaches for the immunotherapy of this disease. Our hypothesis is that huMAb- 3D1-MMAE ADCs will be effective against MUC1-expressing PC CSCs and that eliminating these tumor cells could reverse, in part, the immune suppressive effects of MUC1-C-expressing CSCs on the tumor microenvironment. The objective of the proposed work is to assess the effectiveness of the huMAb-3D1-MMAE ADC against in vitro and in vivo models of CRPC/NEPC when used alone and in combination with ICIs. If our proposed studies are successful in validating MUC1-C as a target for CRPC/NEPC, the huMAb-3D1-MMAE ADC would be advanced for conducting the early phase trials in patients with refractory disease. In summary, the innovative aspects of our proposed research are that we have (i) identified MUC1- C as a target of importance for CRPC/NEPC CSCs, and (ii) generated an ADC against MUC1-C for the potential treatment of patients with refractory CRPC/NEPC.