Project Summary/Abstract CD19-targeted chimeric antigen receptor (CAR) T cell therapy has shown remarkable treatment effects in B-cell malignancies, but many patients suffer from limited response and CD19-negative tumor relapse. Recently, we demonstrated that next-generation “armored” CAR T cells constitutively expressing the immune-stimulatory molecule CD40 ligand (CD40L) exhibited superior antitumor efficacy in preclinical studies but have not yet been fully explored in the context of CAR antigen-negative tumor relapse. There remains an urgent need for the non-invasive in vivo tracking of transfused T cells to determine their biodistribution, expansion, and functionality and to increase the CAR T cells’ killing capacity in case of imminent treatment failure. To overcome these limitations, we began developing methods for monitoring the in vivo kinetics of CAR T cells based on the concept of immune cell radiohapten capture. We demonstrated for the first time that T cells can be successfully transduced with a DOTA-antibody reporter, the DAbR1, enabling their in vivo tracking via PET and SPECT. In the current proposal, we build on this work and optimize our approach for translation of immune cell radiohapten capture from animals to patients, based on greatly improved components of 1) radiohapten capture reporter scFv C825 with picomolar binding affinity, and 2) optimized radiohaptens, the next- generation Proteus-DOTA (Pr) series suitable for imaging and targeted alpha therapy (TAT). The primary objectives of this study are to develop a clinically applicable PET imaging strategy of CAR T cell trafficking in B- cell malignancies and further study the effect of CD40L on counteracting the immune inhibition in syngeneic models of B-cell malignancies. The secondary objectives are to deliver TAT to enhance T cells’ killing capacity in cases of imminent treatment failure and improve the potency of CAR T cell therapies. We project to achieve our aims by generating second-generation and armored CD 19 CAR T cells expressing cell-surface anchored scFv C825. Syngeneic and immunodeficient xenograft murine models of CD19+ B cell malignancies including antigen-loss variants will be employed. After successful transduction, we will assess in vitro functionality of the CAR and the reporter, as well as radiation toxicity, followed by in vivo functionality, imaging sensitivity, and biodistribution, and develop an armored CAR T cell-based theranostic approach with the novel pair [86Y]YPr/[225Ac]AcPr. Finally, as a prerequisite to clinical translation of this novel platform, we will conduct studies using human second-generation and armored CAR T cells in clinically relevant xenograft models. The availability of such a single platform would provide crucial information for safer, more effective clinical trials. The aims thus have immediate translational relevance for our current clinical CD19 CAR T studies and other planned CAR T cell therapy trials.