ABSTRACT Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has been successful against some liquid tumors, but has failed to cure solid tumors. A key reason for CAR T cell failure against solid tumors is antigen heterogeneity. However, pre-clinical studies of CAR T cells against solid tumors in animal models show some promise; in a brain tumor mouse model of glioblastoma, CAR T cells recognizing the EGFRvIII tumor-specific antigen are successful in eliminating tumor, but only against homogeneous tumor and only when mice first receive lymphodepletive host conditioning (via total body irradiation) prior to CAR T cell infusion. Although lymphodepletive host conditioning provides immunological space for CAR T cell expansion, it is problematic in the context of heterogeneous solid tumors, as it impairs endogenous host immunity which is critical for targeting alternative antigens found within the solid tumor. For that reason, successful CAR T cell treatment against solid heterogeneous tumors will require innovative methods to improve CAR T cell persistence to eliminate the need for host lymphodepletive conditioning, and allow for preservation of host endogenous immunity. To achieve this, we propose to utilize metabolic reprogramming of EGFRvIII CAR T cells. Many studies over the last decade have now clearly demonstrated a link between T cell differentiation, function, and metabolism. A predominantly oxidative metabolism supports T cell surveillance, survival, and memory, whereas a predominantly glycolytic metabolism supports biosynthesis to promote effector T cell proliferation and function, but is associated with decreased longevity. The objectives of this R21 proposal are to (1) utilize metabolic reprogramming of EGFRvIII CAR T cells to improve CAR T cell persistence in vitro and in vivo, and (2) test the ability of modified EGFRvIII CAR T cells delivered in the absence of lymphodepletive host conditioning to preserve the endogenous immune system and improve heterogeneous tumor killing. We hypothesize that methods that increase oxidative metabolism will improve CAR T cell persistence, eliminating the need for lymphodepletive host conditioning, maintaining host endogenous immunity, and ultimately improving heterogeneous tumor killing. To test our hypothesis, we will perform the following specific aims: 1) Identify genetic and pharmacological strategies to modify EGFRvIII CAR T cells for enhanced metabolic fitness to support persistence; and 2) Test if metabolically fit murine EGFRvIII CAR T cells delivered in the absence of lymphodepletive host conditioning preserve endogenous immunity. If successful, these approaches can be partnered in future studies with strategies to enhance endogenous host immunity against heterogeneous tumors and overcome a hostile immunosuppressive tumor environment. This work, while performed in a brain tumor model, would be relevant for CAR T cell therapy against multiple solid tumors.