Metabolic reprogramming by ex vivo glutamine inhibition endows CAR-T cells with less-differentiated phenotype and...

The inadequate in vivo persistence of chimeric antigen receptor (CAR)-modified T cells has been shown to lead to poor therapeutic efficacy and disease recurrence. In vivo persistence is associated with the differentiation subsets infused, with less differentiated T_N or T_CM conferring superior renewal capacity and antitumor immunity compared to T_EM or T_EFF. However, ex vivo expanded CAR-T cells exhibit phenotypic heterogeneity with majority of T_EM or T_EFF subsets and very low populations of T_N and T_CM. The transition of differentiation subsets is closely correlated with T cell metabolism fitness. Effector T cell differentiation from T_N or T_CM requires glutamine uptake and metabolism. Using a CD19-specific CAR, we demonstrated that glutamine inhibition by adding the glutamine antagonist 6-Diazo-5-oxo-l-norleucine (DON) into the culture endows CAR-T cells with enhanced mitochondrial OXPHOS utilizing fatty acids and reduced glycolytic activity, and retains more T_N or T_CM subsets. DON- pretreated CAR-T cells exhibited stronger cytotoxic lysis in vitro and more robust elimination of tumor burdens in vivo. This study suggests that glutamine inhibition ex vivo would be a potential approach for modulating metabolism and differentiation state to improve the efficacy of CAR-T cell therapy.