Co-Expression of IL-2 Enhances the Efficacy of FLT3-CAR-γδT Cells in Acute Myeloid Leukemia

Background: B-cell malignancies have been effectively treated using chimeric antigen receptor-T (CAR-T) treatment employing traditional αβT cells. However, because of several obstacles, application in acute myeloid leukemia (AML) is still restricted. A safer "off-the-shelf" alternative can be supplied by CAR-γδT cells, which have major histocompatibility complex (MHC)-independent tumor identification capabilities and a decreased risk of graft versus host disease (GvHD). This study aimed to develop FLT3-targeted CAR-γδT cells that co-express cytokines (IL-2 or IL-7) to increase their anti-AML persistence and therapeutic efficacy. Methods: FLT3-CAR-γδT cells, FLT3-IL2-CAR-γδT cells, and FLT3-IL7-CAR-γδT cells were constructed. Their antitumor potency was comprehensively assessed through cytotoxicity assays, cytokine release, and persistence evaluation in vitro (using AML cell lines and primary AML cells) and in vivo (via mouse model). Results: Superior cytotoxicity against AML cell lines (OCI-AML3, MOLM-13, THP-1, and MV4-11) was demonstrated by FLT3-IL2-CAR-γδT cells, which also released higher levels of granzyme B, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). FLT3-IL2-CAR-γδT cells exhibited cytotoxicity in some primary AML cells in vitro. During the antigen-repeated stimulation assay, FLT3-IL2-CAR-γδT cells preserved the stem cell-like memory T (T_SCM) cell subsets, sustained cytokine release, and maintained excellent viability. FLT3-IL2-CAR-γδT cells considerably slowed the development of AML in vivo and extended the existence (>68 days) of mice. Conclusions: FLT3-IL2-CAR-γδT cells exhibit potent and durable anti-AML activity, providing a novel strategy for clinical AML immunotherapy.