Project Summary/Abstract Chimeric antigen receptor (CAR) T cell therapy redirects T cells to activate and subsequently kill antigen- expressing cancer cells. This is achieved by coupling a cancer antigen-specific extracellular single-chain variable IgG fragment (scFv) to intracytoplasmic, endogenous T cell activation signaling domains. CAR T cell therapy has shown promise for treating hematopoietic malignancies; however, relapse of antigen-negative tumors remains a significant source of failure for these patients. Further, little success has been seen in treating solid tumors with immunosuppressive microenvironments. Combination therapy with CAR T cells and checkpoint blockade is a possible approach to overcome these obstacles. Checkpoint blockade therapy antagonizes the signaling pathways that suppress the immune system. Current checkpoint blockade strategies have focused on altering T cell-tumor interactions, but recent studies also show promise in abrogating innate immune checkpoints, specifically the CD47-SIRPα signaling axis. This pathway, known as the “do not eat me” signal, prevents both antibody mediated macrophage phagocytosis and active cross priming of T cells by dendritic cells, and is thus involved in suppressing both innate and adaptive immune processes. Cancer cells have co-opted this pathway to evade immune attack. However, early stage clinical trials of anti-CD47 agents show systemic toxicities of anemia and thrombocytopenia. Our long-term goal is to engineer a more potent CAR T cell that can overcome antigen loss relapse and the immunosuppressive tumor microenvironment. To accomplish this, we propose to investigate the combination of CAR T cell therapy with intrinsic SIRPα protein secretion to activate antibody therapy and antigen presentation, as this combination should potently engage both innate and adaptive immunity to lead to a more complete antitumor response. We have already engineered human CD19 CAR T cells to secrete a small molecule, high affinity, SIRPα mimic, CV1. These CV1-secreting CAR T cells, named OrexiCAR T cells, retain their cytotoxic function and the cell-secreted CV1 can potentiate mAb therapy. In addition, we have shown that cancer antigen stimulation of the OrexiCAR T cells in vitro leads to a large increase in secreted CV1. Here, we propose to study OrexiCAR T cells in a fully immunocompetent, syngeneic setting to determine which mechanisms contribute to their potency. We believe the proposed research will allow a better understanding of OrexiCAR T cell efficacy and its applicability to the clinic. The Aims are: 1) To construct mouse CD19 OrexiCAR vectors, transduce into primary mouse cells, and validate functions of CAR and CV1 in vitro and 2) To evaluate the anti-tumor effect of mOrexiCAR T cells in an immunocompetent, syngeneic mouse model and to discover and describe the immunologic mechanism