Synthetic Notch CD123 and CLL1 chimeric antigen receptor T-cells (CAR-T) target acute myeloid leukemia and reduce off-tumor effects associated with CLL1 CAR-T cells

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy targeting the C-type lectin-like molecule-1 (CLL1) antigen has emerged as a promising therapeutic approach for acute myeloid leukemia (AML). However, its clinical translation is hindered by on-target, off-tumor toxicity, particularly severe neutropenia caused by CLL1 expression on normal neutrophils. To address this limitation, we constructed a synthetic Notch (SynNotch) dual-antigen AND-gated CAR-T system targeting CD123 and CLL1 to improve tumor specificity and reduce off-tumor toxicity. CD123 and CLL1 expression was first assessed in AML cell lines and primary AML blasts by flow cytometry. The in vitro cytotoxicity, serial killing activity, cytokine secretion, exhaustion-associated protein markers, and transcriptomic profiles of engineered CAR-T cells were then evaluated, and therapeutic efficacy and safety were further examined in AML xenograft models. SynNotch CD123 and CLL1 CAR-T cells showed stronger cytotoxic activity against AML blasts while exhibiting reduced activity toward normal neutrophils. In addition, these cells displayed lower expression of exhaustion-associated genes and markers, together with increased expression of genes related to naive/memory differentiation. In vivo, this CAR-T variant showed improved expansion, longer persistence, and stronger anti-leukemic activity, together with less severe neutropenia. Increased cytokine production in vitro indicated stronger T-cell activation but also suggested the need for careful evaluation of potential cytokine-related toxicity. These findings indicate that SynNotch dual-antigen-gated CAR-T cells may provide a more specific and potentially safer therapeutic strategy for relapsed/refractory AML.