Prostate cancer is the most frequently diagnosed cancer for men in the United States and VA cancer patients and is the second largest cause of cancer-related death in men. Advanced prostate cancer, which develops in 10-20% of all prostate cancer patients, is typically bone-metastatic and resistant to androgen deprivation therapy. The relative 5-year survival rate for patients with advanced prostate cancer is 28% and the median overall survival ranges between 9-13 months. Therefore, novel treatment options are needed for patients with advanced prostate cancer. Adoptive transfer of T lymphocytes engineered to express chimeric antigen receptors (CARs) is a novel therapeutic modality that has generated unprecedented responses in patients with highly refractory CD19' B cell leukemia and lymphoma and BCMA' multiple myeloma and holds promise as a potential treatment for prostate cancer. In a recent phase I trial hailed as a clinical milestone in prostate cancer, 13 patients were treated with autologous PSMA-specific CAR T cells armored with a dominant-negative TGF-β receptor. Four patients achieved a >/=30% reduction in PSA and the failure of the CAR T cell therapy was accompanied by an adaptive response in the tumor microenvironment (TME) that led to upregulation of multiple localized inhibitory molecules. The central hypothesis of this proposal is that adaptive resistance utilizes multiple mechanisms to escape surveillance and attack from the immune system; therefore, immune cells engineered with multipronged approaches to target the tumor, the TME, and overcome adaptive resistance mechanisms will prevent CART cell failure and tumor escape. In prior studies, CAR T cell therapies targeting glycoforms of mucin1 and fibronectin post-translationally modified with the Tn antigen, a truncated O-glycan, demonstrated tumor-specific activity against prostate cancer in xenograft models and a reduction in tumor metastases. These studies identified loss of interferon-gamma signaling as an adaptive resistance mechanism that desensitizes tumor cells to CART cell-mediated cytotoxicity and agonists of TLR signaling that induced an interferon-like gene signature synergized with CAR T cells to overcome this resistance mechanism. The rationale for this research is that identification of counter resistance mechanisms that maintain CAR T cell activity in the context of adaptive resistance by prostate tumors. The central hypothesis of this proposal will be tested by pursuing three independent specific aims: 1) to design dual-targeting CAR T cells and strategies to modulate target antigen expression to prevent tumor escape; 2) to identify mechanisms of CART cell and TLR agonist synergy in prostate tumors deficient in interferon-gamma signaling; 3) to enhance CART cell resistance to immune checkpoint and immunosuppressive cytokines. This research will enhance the depth of understanding on the mechanisms of tumor escape through adaptive resistance as well as the breadth of im...