ABSTRACT Adoptive T cell therapy (ACT) is a promising approach for treating patients with advanced malignancies. Advances in molecular biology and genetic engineering have led to the design and use of modified T cells that can recognize tumors to achieve significant tumor control upon ACT to patients. These T cells are either transduced with tumor antigen reactive T cell receptors (TCR), or chimeric antigen receptors (CARs). Yet, elimination of established tumors has not been effectively achieved, typically due to loss of T-cell effector function or failure of long-term survival. Most ACT trials use rapidly expanded T cells that are terminally differentiated and exhibit an effector memory (Tem) phenotype. These Tem phenotypes bearing cells are more susceptible to the adverse effects of the tumor microenvironment (TME). Therefore, the development of novel mechanism based therapeutic strategies that can reprogram anti-tumor T cells to maintain their metabolic fitness and effector function in a tumor microenvironment is urgently needed to enhance the therapeutic value of ACT. Our Phase I clinical trial in this proposal builds on strong in vivo pre-clinical tumor control data obtained using novel ex vivo programming conditions that merge robust phenotypes of both Th1 and Th17 cells to generate a hybrid Th1/17 cell. We have recently established that programming conditions that bring together ‘anti-tumor effector function’ of Th1 cells and ‘stemness’ of Th17 cells lead to a superior hybrid Th1/17 (and Tc/17) cell exhibiting long-term tumor control. CD19 CAR T-cell therapy (CD19-CTCT) represents an enormous scientific and clinical breakthrough for patients with CD19-positive non-Hodgkin lymphoma (NHL), however, major issues still remain regarding toxicity and with the majority of patients succumbing to disease relapse. Thus, we hypothesize that ex vivo expansion and programming of CD19-CAR-Ts to metabolically enhanced hybrid T1/17 (Th1/17 and Tc1/17) phenotype will lead to robust anti-tumor control even with fewer adoptively transferred cells. Additionally, we have used cytoplasmically truncated CD34 tag (CD34t) into the CAR T cell construct to enable a more purified CAR T-cell product via CD34 selection, which aims to improve CAR T-cell antigen reactivity, persistence, and reduces off-target toxicities. Following specific aims are proposed to establish and develop our approach for commercialization: Specific Aim 1: Define a safe dose of meCD19- CD34t-CAR-T cells while evaluating efficacy for R/R B-cell NHL and CLL/SLL. Specific Aim 2: Establish pre- and post-infusion molecular signature and its correlation to anti-tumor response. We believe that this proposal will help adopt the novel ex vivo programming conditions for generating robust anti-tumor CAR-Ts that could be used in future in clinical trials to target other malignancies.