PROJECT SUMMARY/ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 21-100. Novel therapies for pediatric osteosarcoma (OS) are urgently needed. Patients with recurrent and metastatic disease have dismal prognoses, with less than 20% surviving beyond three years. As intensification of conventional chemotherapy is unlikely to yield further benefit according to expert consensus, alternative therapeutic classes such as immunotherapies must be developed. The long-term goal of this research is to develop an effective immunotherapy for OS. Immunotherapy with chimeric antigen receptor (CAR) T cells has shown great preclinical promise in solid tumor treatment. However, clinical efficacy has been limited, in part due to ineffective homing of CAR T cells to tumor sites. Our preliminary studies show that OS tumors secrete the chemokine CXCL16, but that less than 5% of activated T cells express the cognate receptor CXCR6, indicating a ligand/receptor mismatch that adversely affects CAR T cell efficacy. We therefore hypothesize that expressing CXCR6 in CAR T cells will enhance their ability to home to OS tumor sites and result in enhanced cytotoxicity. Our preliminary data also shows that OS tumors express very little of the chemokine XCL1, which functions to recruit dendritic cells for antigen uptake and presentation and to attract endogenous immune cells to tumor sites. We therefore further hypothesize that the antitumor activity of CAR T cells can be augmented by expression of XCL1, thereby attracting additional immune cells into the tumor at the time of CAR-mediated killing and priming the endogenous immune system for antitumor response. We will evaluate these hypotheses using CAR T cells against the tumor-associated antigen B7H3, which is highly expressed on OS tumors. In Aim 1, we will determine the in vitro and in vivo efficacy, including functional characteristics, homing, and cytotoxicity, of CXCR6-modified B7H3.CAR T cells in an immunocompetent model of OS. In Aim 2, we will evaluate XCL1-expressing B7H3.CAR T cell performance in our immunocompetent murine model, specifically investigating endogenous immune cell recruitment in response to vXCL1.mB7H3.CAR T cell treatment. In Aim 3, we will combine these strategies to seek a truly synergistic CAR T cell therapy in which mCXCR6.B7H3.CAR T cells are engineered to specifically deliver vXCL1 to tumor sites and express vXCL1 during CAR-mediated killing. We will also further characterize the response of the endogenous immune system to XCL1 expression at tumor sites and assess whether mice who have received XCL1-expressing CAR T cells are able to reject subsequent OS tumor challenges through endogenous immune activity post-CAR T cell treatment. At the end of this work, we expect to have developed an effective and novel CAR T cell therapy approach for OS that synergistically combines enhanced chemokine-mediated homing and endogenous immune s...