ABSTRACT: BACKGROUND: Chimeric antigen receptor (CAR) T cells have achieved remarkable clinical success. However, up to 50% of CAR T cell-treated leukemia patients relapse and long-term survivor data indicate that CART cell persistence is key to enforcing relapse-free survival. Unfortunately, ex vivo expansion protocols often drive metabolic and functional exhaustion, reducing in vivo efficacy. Preclinical models have demonstrated that redirecting metabolism can improve in vivo T cell function. Here, we hypothesized that exposure to an agonist targeting AMP-activated protein kinase (AMPK) would create CARTs capable of increased in vivo function and enhanced leukemia clearance. METHODS: CART cells were generated from healthy human donor T cells via lentiviral transduction, followed by exposure to either Compound 991 or DMSO for 96 hours, followed by a 48-hour washout. During and after agonist treatment, T cells were harvested for metabolic and functional assessments. To test in vivo efficacy, immunodeficient mice were injected with luciferase+ NALM6 leukemia cells, and one week later with 991- or DMSO-expanded CARTs. Leukemia burden and anti-leukemia efficacy was assessed via radiance imaging and overall survival. RESULTS: Compound 991 treatment activated AMPK without limiting cellular expansion, and increased both mitochondrial density and handling of reactive oxygen species (ROS). Mechanistically, 991 treatment mimicked nutrient starvation, with increased autophagic flux and generation of mitochondrially-protective metabolites in treated cells. Importantly, receipt of 991-exposed CARTs significantly improved in vivo leukemia clearance and prolonged recipient survival, likely as a result of elevated activation and increased CD4+ T cell yields at early times post-injection. DISCUSSION: Ex vivo expansion is necessary to generate sufficient cell numbers, but sustained activation and differentiation often negatively impact in vivo persistence and function. Here, we demonstrate that promoting AMPK activity during in vitro CART expansion metabolically reprograms cells without limiting T cell yield, increases early activation marker expression following in vivo transfer, and ultimately enhances anti-leukemia efficacy. Importantly, Compound 991 treatment achieves these results without further modifying the expansion media, changing the CART construct, or genetically altering the cells. Altogether, these data highlight AMPK agonism as a potent and readily translatable approach to improve the metabolic profile and in vivo efficacy of adoptively transferred T cells.
