Systemic pre-conditioning favors effector over exhausted CD8 T-cell subsets following Sup2-IL33 armored CAR T-cell therapy

Background: Systemic pre-conditioning prior to chimeric antigen receptor (CAR) T-cell infusion enhances engraftment and increases infiltration and activity of adoptively transferred cells within the tumor. Despite promising responses in some patients, additional strategies are needed to enhance overall efficacy of CAR T cells in patients with solid tumors. Armoring CAR T cells to express immunomodulatory cytokines can enhance treatment efficacy. In this study, we assess how systemic pre-conditioning impacts CAR T cells alone and in combination with interleukin (IL)-2 superkine (Sup2) and IL-33 cytokine armoring in the context of B16F10 melanoma. Methods: Local and systemic pre-conditioning regimens were assessed in combination with TRP1-specific CAR T cells with or without Sup2 and IL-33 armoring in immunocompetent mice with B16F10 melanoma. CAR T-cell expansion kinetics and phenotype were evaluated with luminescence imaging, flow cytometry, and single-cell RNA sequencing. Results: Systemically preconditioned mice treated with Sup2-IL33 CAR T cells elicited greater tumor control and extended overall survival compared with non-conditioned or locally preconditioned mice. Depletion of Sup2-IL33 CAR T cells in preconditioned mice resulted in loss of durable tumor control. While total body irradiation (TBI) increased unarmored CAR T-cell engraftment, Sup2-IL33 armoring increased tumor infiltration of effector cells over TOX+exhausted cells. RNA velocity trajectory analysis predicted divergent differentiation of tumor-infiltrating progenitor cells into either effector or exhausted T cells. Conclusions: Systemic preconditioning increased the expansion and persistence of transferred CAR T cells.TBI preconditioning in combination with cytokine armoring promoted the emergence of diverse effector phenotypes that correlated with enhanced efficacy of Sup2-IL33 CAR T cells in the context of B16F10 melanoma. Durable responsiveness and extended survival following combined therapy required continued CAR T-cell persistence despite the ability of Sup2-IL33 armoring to induce endogenous tumor immunity.