AMPK agonism optimizes the in vivo activation and antileukemic efficacy of chimeric antigen receptor T cells.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have achieved remarkable clinical success. However, up to 50% of patients with CAR T-cell treated leukemia relapse and long-term survivor data indicate that CAR T cell persistence is key to enforcing extended, 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 CAR T cells capable of increased in vivo function and enhanced leukemia clearance. METHODS: CAR T cells were generated from healthy human donor T cells via lentiviral transduction, followed by exposure to either Compound 991 or dimethyl sulfoxide (DMSO) for 96 hours. 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 1 week later with 991- versus DMSO-expanded CAR T cells. Leukemia burden and antileukemia efficacy were 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. Mechanistically, 991 treatment mimicked nutrient starvation, with increased autophagy and generation of mitochondrially protective metabolites. Importantly, receipt of 991-exposed CAR Ts 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 for in vivo administration, but sustained activation and differentiation often negatively impact in vivo persistence and function. Here, we demonstrate that promoting AMPK activity during in vitro CAR T expansion metabolically reprograms cells without limiting T cell yield, increases early activation 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 CAR T construct, or genetically altering the cells. Together, 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.