FOXO1 Inhibition Generates Potent Nonactivated CAR T Cells against Solid Tumors

Abstract 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 TAS cells). This approach made T cells directly permissive to lentiviral infection, allowing CAR expression, with enhanced antitumor functions. FOXO1 inhibition in primary T cells (TAS cells) correlated with acquisition of a stem cell memory phenotype, high levels of granzyme B, and increased production of TNFα. TAS cells displayed enhanced proliferative and cytotoxic capacities as well as improved migratory properties. In vivo experiments showed that CAR TAS cells were more efficient at controlling solid tumor growth than classical CAR T cells. The production of CAR TAS from patients’ cells confirmed the feasibility of the protocol in clinic.