Off-the-shelf CD117 CAR-iNKT cells as a safe and effective allogeneic immunotherapy targeting AML and its immunosuppressive microenvironment

Abstract Acute myeloid leukemia (AML) is a highly aggressive hematologic malignancy with a five-year overall survival rate of approximately 30%. AML disrupts normal hematopoiesis and impairs immune response, resulting in a profoundly immunosuppressive tumor microenvironment (TME) that contributes to rapid disease progression and high relapse rates. While chimeric antigen receptor (CAR) T cell therapy has shown promise in B cell malignancies, its efficacy in AML has been limited due to antigen heterogeneity, immune evasion, and the risks of rejection and graft-versus-host disease (GVHD) associated with allogeneic cells. Invariant natural killer T (iNKT) cells are a rare population of innate-like T cells that recognize glycolipids presented by CD1d, which is frequently expressed on AML cells and tumor-associated macrophages. iNKT cells mediate antitumor activity both directly, via CD1d recognition, and indirectly, through immune modulation, such as cross-priming CD8⁺ dendritic cells. Importantly, iNKT cells do not induce GVHD and have been shown to protect against it in preclinical models from studies in our group and others. Moreover, CAR-engineered iNKT cells have demonstrated potent antitumor effects in xenograft models. These unique properties highlight the potential of iNKT cells for allogeneic cell therapy. In this study, we developed allogeneic CD117-targeted CAR-iNKT cells as a potential off-the-shelf immunotherapy for AML. Using immunocompetent C57BL/6 mouse models of spontaneous MLL-AF9 and MLL-ENL AML, we evaluated both the cytotoxic and immunomodulatory functions of iNKT cells in a clinically relevant setting. CD117 CAR-T and CAR-iNKT cells derived from FVB mice exhibited significantly enhanced cytotoxicity in vitro compared to untransduced controls. Notably, our findings demonstrated that CAR-iNKT cells specifically targeted and eliminated CD117high AML cells. In vivo, adoptive transfer of CD117 CAR-T and CAR-iNKT cells modestly extended survival in aggressive AML models (AF9 alone, median 23 days; allogenic T, median 26 days; allogenic CART, median 32.5 days; allogenic iNKT, median 27 days; allogenic CAR iNKT, median 30.5 days; allogenic CART vs T, p = 0.001; allogenic CAR-iNKT vs iNKT, p = 0.0145). We observed functional exhaustion in CAR-iNKT cells following extensive in vitro engineering. Incorporation of murine IL-15 into the CAR construct significantly mitigated exhaustion and improved both persistence and antitumor efficacy in vivo (AF9 alone, median 31.5 days; allogenic CAR iNKT, median 44 days; allogenic CAR iNKT-IL15, median 85 days; allogenic CAR-iNKT vs CAR-iNKT, p = 0.0029). Furthermore, CAR-iNKT cells were capable of eliminating CD1d⁺ tumor-associated macrophages in the spleen and bone marrow, thereby contributing to the reversal of TME-mediated immunosuppression. Together, these findings support CD117 CAR-iNKT cells as a promising and safe allogeneic immunotherapy for AML.