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 performed a head-to-head preclinical comparison of an FMS-like tyrosine kinase 3 (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 cells but spared healthy hematopoietic stem and progenitor cells in vitro. 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 the bone marrow further revealed a distinct exhaustion-associated gene signature in samples from mice that had been treated with the FLT3 BiTE molecule. Importantly, provision of CD86-mediated costimulation enhanced 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 a critical role for costimulatory signaling in sustaining the efficacy of T-cell-based therapies in vivo, offering a rationale for improving T cell-redirection strategies in myeloid malignancies.
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