BiTE® Molecule vs. CAR-T targeting FLT3 in AML: How Positive Co-Stimulation Tips the Scale

T-cell-based immunotherapies have revolutionized treatment paradigms in B-cell malignancies, yet their translation to acute myeloid leukemia (AML) has been hindered by a scarcity of tumor-restricted antigens and the risk of on-target off-leukemia toxicity. FLT3 has emerged as a promising therapeutic target with limited expression in healthy hematopoietic tissues. Here, we perform a head-to-head preclinical comparison of a FLT3-directed bispecific T-cell engager (BiTE® molecule) and second-generation FLT3-specific chimeric antigen receptor (CAR) T cells. Both approaches induced potent cytotoxicity against AML cell lines and primary patient-derived blasts while sparing healthy hematopoietic stem and progenitor cells. Despite similar short-term efficacy, prolonged antigen exposure demonstrated progressive functional decline and metabolic exhaustion; however, CAR T cells maintained cytotoxic capacity and proliferative potential over time. In AML xenograft models, CAR T cells achieved superior tumor control, prolonged survival, and greater T-cell infiltration than BiTE® molecule-treated counterparts. Transcriptomic profiling of T cells recovered from bone marrow further revealed a distinct exhaustion-associated gene signature in BiTE®-redirected T cells. Importantly, provision of CD86-mediated co-stimulation significantly enhanced the antitumor activity of BiTE®-redirected T cells in vitro and in vivo. These findings establish FLT3 as a viable and selective immunotherapeutic target in AML and underscore the functional and transcriptional differences between BiTE® molecule-redirected T cells and CAR T cells. Moreover, they reveal the critical role of co-stimulatory signaling in sustaining the efficacy of T-cell-based therapies in vivo, offering a rational path for improving T-cell redirection strategies in myeloid malignancies.