T-Bet Overexpression Increases 4-1BB-Costimulated CAR T Cell Potency

Data from our lab and others has demonstrated that CAR T cells form a mixed Th1/Th2 population which, in contrast to canonical T cell differentiation, produce both interferon gamma (IFN-γ) and IL-4. Whereas a growing body of evidence has highlighted the importance of IFN-γ in the CAR T cell response, IL-4 can have immune-suppressive functions. IFN-γ from CD4+ CAR T cells (CAR4s), has been shown to promote cytotoxic effector functions of CD8+ CAR T cells (CAR8s), as well as bolster activation of endogenous immune cells including CD8 T cells and NK cells (Boulch et al Sci Immunol. 2021). Given the importance of IFN-γ to effective anti-tumor immune responses, we generated murine CAR T cells that target CD19 with a CD28 costimulatory domain (1928z) and compared them to CAR T cells that overexpress T-bet (T-bet-1928z) in an effort to skew CAR T cells away from IL-4 production and towards IFN-γ production. Upon stimulation of CAR T cells with the murine B-cell acute lymphoblastic leukemia (B-ALL) cell line E2A-PBX, fewer T-bet-1928z CAR4s produced IL-4 compared to 1928z CAR4s. This suggests T-bet overexpression is a viable strategy to reduce Th2-like programming of effector functions in CAR4s. To determine the impact of T-bet overexpression on early CAR T cell efficacy in vivo, we adoptively transferred 1928z or T-bet-1928z CAR T cells into leukemia-bearing C57Bl6/J recipients and analyzed CAR T cells from the bone marrow at peak expansion of CAR T cells as well as post-contraction. Both CARs eliminated CD19+ cells from the bone marrow and mediated long-term survival. T-bet-1928z CAR4s demonstrated increased expansion at Day 4 compared to 1928z CAR4s (p=0.0412), although there was no significant difference in CAR8 numbers at this timepoint. At Day 11 post-CAR, we observed an increase in the number of T-bet-1928z CAR T cells present in the bone marrow (p