Summary/Abstract (PROJECT 1) Chimeric antigen receptor (CAR) T cells directed to the CD19 protein (CART19) have revolutionized the treatment of a variety of B cell malignancies, with complete remission rates as high as 97% in certain types of advanced leukemia. Many of these responses are sustained, but poor CAR T cell expansion and persistence following infusion and antigen-negative escape are common mechanisms of treatment failure. It is critical to investigate factors driving successful CAR T cell function in responding patients. Previous studies have indicated that in lymphoid malignancies, durable remission is associated with activation of specific T cell pathways and only some patients experience therapeutic levels of CAR T cell expansion and antitumor activity. During the previous award period, we successfully developed a comprehensive understanding of the T cell-intrinsic and - extrinsic mechanisms of resistance as well as tumor cell-intrinsic mechanisms that lead to relapse. Here we propose extensive next-generation analyses to understand properties of optimal CAR T cell therapy, elucidate mechanisms of resistance, and develop strategies to overcome resistance and enhance CAR T cell activity to cure more patients with hematologic diseases. We will take advantage of already established successful collaborations with core laboratories possessing expertise in cell manufacturing, gene editing, and state-of-the- art correlative science platforms to explore a number of innovative aims. In Aim 1, we will carry out an extensive analysis of T cell receptor (TCR) rearrangements, vector copy number, T cell subsets analyzed by flow cytometry, serum cytokines and biomarkers (~7,000), and transcriptional landscapes of CAR T cells in blood as well as tumor tissues and formulate multi-omics models linking correlative data and outcome. In Aim 2, we have designed a high-impact clinical trial to knockout CD5, an unconventional negative immune checkpoint molecule and inhibitor of antigen-receptor signaling, with the goal of increasing the therapeutic index of 3-day manufactured CART19 cells for relapsed/refractory B-cell malignancies. Finally, in Aim 3, we will carry out functional analyses of genes implicated in potentiating CAR T cell proliferation, persistence, and antitumor function to develop `best-in-class' products for ALL, CLL, and NHL. Using a structured, multi-pronged strategy, we hope to ameliorate resistance to CAR T cell-based therapies for B-cell malignancies and clinically advance several next-generation synthetic biology tools. This work is expected to open therapeutic horizons in the field of adoptive immunotherapy for cancer and offer new research prospects that could be translated to improving the treatment of other cancers and autoimmune disorders.