SUMMARY Our overarching goal is to develop a novel theranostic prostate-cancer-specific CAR-T cell paradigm that will allow for successful targeting of antigen-heterogeneous neuroendocrine prostate cancer (NEPC) under image guidance. Our proposal and experiments are based on the observation that NEPC develops in most cases through increasing de-differentiation from the clinical state of castrate-resistant disease; along this trajectory, DLL3 expression increases and PSMA expression decreases on tumor cells. Hence, targeting DLL3 as well as PSMA with a bi-specific immunotherapeutic is a logical choice when developing a novel targeted therapeutic approach for this disease state. Our approach takes advantage of existing institutional expertise in developing CAR-T-cell-based therapeutic strategies following very encouraging clinical outcomes in hematologic malignancies and in mesothelioma. Here, we aim to extend this approach to metastatic NEPC. We realize that translation of the clinical CAR-T cell success in hematologic malignancies to metastatic solid tumors must overcome several obstacles. It necessitates systemic delivery of CAR-T cells, and any therapeutic success will require the delivery of CAR-T cells to metastatic sites in sufficient quantity, their survival and potent effector function at these sites, and the prevention of tumor immune escape due to plasticity and heterogeneity of targeted surface antigens. Therefore, we have designed a series of experiments to address these challenges in logical sequence. Our research team combines expertise in CAR-T cell design, development of T-cell tracking techniques, translation of novel imaging probes in prostate cancer, and clinical trial design. We will conduct a series of pre-clinical studies in order to refine the technology and prepare for translation in patients with NEPC. We will begin by evaluating the therapeutic efficacy of DLL3-specific CAR-T cells in NEPC models and determining the lower DLL3-expression threshold on cancer cells for CAR-T tumoricidal effect. Next, we will develop DLL3/PSMA-bi-specific CAR-T cells and evaluate their ability to eradicate tumors with heterogeneous DLL3 and PSMA expression using real-time multi-reporter bioluminescent imaging. To facilitate clinical translation of this approach, we will verify that these bi-specific CAR-T cells can be tracked by PET imaging with the clinically approved 18F-MFBG radiotracer, and their efficacy predicted and measured using the clinically established DLL3- and PSMA-specific radiotracers 89Zr-DFO-SC16 and 68Ga-PSMA, respectively. In summary, this proposal brings together translatable CAR-based therapeutic and imaging components in genetically engineered T-cells, allowing for non-invasive monitoring of CAR-T cell trafficking, tumor targeting, and tumor response. Of note, the theranostic paradigms that will be developed and validated in this proposal can also be translated and used to address comparable clinical questions in other...