FOXO1 Inhibition Generates Potent Nonactivated CAR T Cells against Solid Tumors

Chimeric antigen receptor (CAR) T cells have shown promising results in the treatment of B-cell malignancies. Despite the successes, challenges remain. One of them directly involves the CAR T-cell manufacturing process and especially the ex vivo activation phase. While this is required to allow infection and expansion, ex vivo activation dampens the antitumor potential of CAR T cells. Optimizing the nature of the T cells harboring the CAR is a strategy to address this obstacle and has the potential to improve CAR T-cell therapy, including for solid tumors. Here, we describe a protocol to create CAR T cells without ex vivo preactivation by inhibiting the transcription factor FOXO1 (CAR T^AS cells). This approach made T cells directly permissive to lentiviral infection, allowing CAR expression, with enhanced antitumor functions. FOXO1 inhibition in primary T cells (T^AS cells) correlated with acquisition of a stem cell memory phenotype, high levels of granzyme B, and increased production of TNFa. T^AS cells displayed enhanced proliferative and cytotoxic capacities as well as improved migratory properties. In vivo experiments showed that CAR T^AS cells were more efficient at controlling solid tumor growth than classical CAR T cells. The production of CAR T^AS from patients’ cells confirmed the feasibility of the protocol in clinic.