While immunotherapies have made strides in the treatment of other cancers, castrate resistant prostate cancer (CRPC) remains largely unresponsive, underscoring the need for novel approaches. One such approach relies on the administration of autologous T cells genetically modified to express a chimeric antigen receptor (CAR) that recognize specific tumor-associated antigens. Prostate Stem Cell Antigen (PSCA) is widely expressed in prostate cancer and we have previously developed a CAR targeting PSCA that has potent in vivo efficacy. To target bone metastatic CRPC (mCRPC), we propose to manipulate a specific subset of T cells, called γδ that can be driven to the skeleton via systemic treatment with bisphosphonates such as zoledronate (ZOL) that is clinically used to limit cancer-induced bone disease in men with bone mCRPC. Importantly, ZOL treatment induces accumulation of phosphoantigens in tumor cells, which are detected by γδ T cells. Our preliminary findings show that ZOL can enhance γδ T-cells' homing to bone where they can prevent cancer growth via CAR and via endogenous T-cell receptor (TCR) recognition. γδ CAR-T treatment, in presence or absence of ZOL, can mitigate cancer-induced bone deterioration. Moreover, we found that soluble factors secreted by bone marrow derived mesenchymal stromal cells (MSC) can increase the cytotoxic potential of γδ CART cells. Finally, we found that the choice of CAR structural and costimulatory moieties affects the phenotype and fuction αβ and γδ T cells differentially, requiring the design of CARs optimized for γδ T cells. Based on these preliminary findings we hypothesize that the homing and cytotoxic activity of γδ CAR-T cells for the treatment of bone metastatic CRPC can be greatly enhanced through genetic, pharmacological, and microenvironmental approaches. We will test our hypothesis by; 1) Defining the optimum γδ CAR-T design that will significantly enhance CRPC cytotoxicity. We will test the biological implications of choosing alternative CAR transmembrane and costimulatory domains, with a specific focus on their ability to modulate the expression of cytokine receptors. We will also dissect the specific signaling pathways that can govern γδ CAR- T cell persistence. Finally, we will identify the molecular signaling pathways triggered by CARs with different costimulatory domains. 2) Determining if ZOL can drive γδ CAR-T recruitment and anti-bone mCRPC activity in vivo. We will use xenograft and PDX models of bone mCRPC to characterize the bioavailability and therapeutic efficacy of γδ CAR-T + ZOL; and an immunocompetent model to map sites of phosphoantigen accumulation. 3) Dissecting the reciprocal effects of γδ CAR-T on the bone mCRPC microenvironment. We will analyze the impact of γδ CAR-T + ZOL treatment on the structure and physiology of the bone, and the effects of MSC on the performance of γδ CAR-T cells in vivo. Based on the anticipated results, characterizing the specific properties of the bone/t...