Rejuvenating Effector/Exhausted CAR T Cells to Stem Cell Memory–Like CAR T Cells By Resting Them in the Presence of CXCL12 and the NOTCH Ligand

T cells with a stem cell memory (T(SCM)) phenotype provide long-term and potent antitumor effects for T-cell transfer therapies. Although various methods for the induction of T(SCM)-like cells in vitro have been reported, few methods generate T(SCM)-like cells from effector/exhausted T cells. We have reported that coculture with the Notch ligand–expressing OP9 stromal cells induces T(SCM)-like (iT(SCM)) cells. Here, we established a feeder-free culture system to improve iT(SCM) cell generation from expanded chimeric antigen receptor (CAR)-expressing T cells; culturing CAR T cells in the presence of IL7, CXCL12, IGF-I, and the Notch ligand, hDLL1. Feeder-free CAR-iT(SCM) cells showed the expression of cell surface markers and genes similar to that of OP9-hDLL1 feeder cell–induced CAR-iT(SCM) cells, including the elevated expression of SCM-associated genes, TCF7, LEF1, and BCL6, and reduced expression of exhaustion-associated genes like LAG3, TOX, and NR4A1. Feeder-free CAR-iT(SCM) cells showed higher proliferative capacity depending on oxidative phosphorylation and exhibited higher IL2 production and stronger antitumor activity in vivo than feeder cell–induced CAR-iT(SCM) cells. Our feeder-free culture system represents a way to rejuvenate effector/exhausted CAR T cells to SCM-like CAR T cells. SIGNIFICANCE: Resting CAR T cells with our defined factors reprograms exhausted state to SCM-like state and enables development of improved CAR T-cell therapy.