Abstract 4801: T-bet overexpression promotes CAR T cell potency and responsiveness to low antigen density leukemia

Abstract Data from our lab and others have demonstrated that CAR T cells, contrary to canonical T cell subsets, form populations that appear to be a mixed Th1/Th2 phenotype and produce both IFNγ and IL-4. IL-4 has been shown to have immune-suppressive functions, whereas CAR T cell-derived IFNγ has been shown to promote anti-tumor responses by bolstering CAR8 cytotoxicity and promoting the activation and function of endogenous immune cells. We sought to diminish the Th2-like phenotype and boost CAR T cell potency by overexpressing T-bet, a transcription factor that is essential for Th1 differentiation of CD4 T cells and promotes effector functions in CD8 T cells. We tested overexpression of T-bet in 2nd generation CD19 CAR T cells (1928z and 19BBz) and found that T-bet reduced production of IL-4, demonstrating successful redirection to a Th1/Tc1 fate. Production of IFNγ, TNFα, and IL-2 were enhanced with T-bet overexpression, especially against leukemia cells expressing low levels of CD19 (CD19lo). T-bet overexpression also improved polyfunctional cytokine secretion from these CAR T cells. Furthermore, 1928z and 19BBz CAR T cells showed increased effector functions, and degranulated and proliferated at higher rates against CD19lo leukemia when T-bet was overexpressed. To determine the impact of T-bet overexpression on CAR T cell efficacy in vivo, we adoptively transferred CAR+ T cells co-transduced to express a 1928z or 19BBz CAR with T-bet-EGFR or an EGFR only control into NSG mice engrafted with WT NALM6 leukemia expressing high levels of CD19. T-bet overexpression negatively impacted leukemia burden and survival of mice treated with 1928z CAR T cells. However, T-bet overexpression had no significant impact on the in vivo efficacy of 19BBz CAR T cells against WT leukemia. Since CD28-containing CAR T cells are known to signal with a higher intensity than 4-1BB-containing CAR T cells, we hypothesized that T-bet overexpression may be beneficial only in the context of suboptimal antigen receptor signaling. To this end, we tested the impact of T-bet overexpression on CAR T cell responses against CD19lo leukemia, as downstream CAR signaling is reduced against low-antigen target cells. We found that T-bet overexpression did not significantly affect the leukemia clearance or survival of mice engrafted with CD19lo leukemia treated with 1928z CAR T cells. However, T-bet overexpression enhanced the leukemia clearance and survival of mice engrafted with CD19lo leukemia treated with 19BBz CAR T cells. Together, this data supports a model in which the impact of T-bet overexpression on CAR T cell efficacy is dependent on the CAR signal intensity, with T-bet enhancing responses of CAR T cells receiving a low-intensity signal. Thus, we demonstrate T-bet overexpression as a viable therapeutic strategy to enhance 4-1BB- co-stimulated CAR T cell responses to low-density antigens, overcoming a well-known obstacle limiting CAR T cell efficacy. Citation Format: Jennifer M. Cimons, Kole R. Degolier, Samuel D. Burciaga, M. Eric Kohler, Terry J. Fry. T-bet overexpression promotes CAR T cell potency and responsiveness to low antigen density leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4801.