This is a title in sentence case:synnotch CD123 and CLL1 CAR-T can specifically target AML to avoid the off-target effects

Abstract Objective Chimeric antigen receptor T-cell (CAR-T) therapy targeting CLL1 has emerged as a promising immunotherapeutic approach for acute myeloid leukemia (AML), demonstrating potential to improve patient prognosis. However, the expression of CLL1 on neutrophils leads to the simultaneous eradication of both tumor cells and neutrophils by CAR-T cells, resulting in severe granulocytopenia and subsequent infections. SynNotch CAR-T cells, which integrate synthetic Notch receptors with CAR-T technology, represent an innovative immunotherapy strategy that leverages the strengths of both components to enhance the treatment of hematologic malignancies. Methods We first evaluated the expression levels of CD123 and CLL1 in AML cell lines and primary blasts using flow cytometry. Subsequently, we engineered SynNotch CD123 and CLL1 CAR-T cells and assessed their cytotoxic activity in vitro against AML cell lines and primary blasts. Additionally, we examined the cytotoxicity of SynNotch CAR-T cells against normal neutrophils. Metabolomics sequencing analysis was used to evaluate the expression of exhaustion-associated genes, as well as naïve and memory differentiation genes, in CLL1 CAR-T cells and SynNotch CD123 and CLL1 CAR-T cells. Finally, we validated the therapeutic efficacy and safety of SynNotch CD123 and CLL1 CAR-T cells in vivo using a relapsed AML xenograft model. Results SynNotch CD123 and CLL1 CAR-T cells exhibited significantly enhanced cytotoxicity against AML cell lines and primary blasts compared to single CLL1 CAR-T cells. These cells demonstrated robust recognition and elimination of AML cells expressing multiple antigens, resulting in significant anti-tumor activity and survival benefits in vitro. Metabolomics sequencing analysis revealed that SynNotch CAR-T cells had lower exhaustion-associated genes LTA, BATF3, GZMB, LAG3, JUNB and higher naïve and memory differentiation genes IL7R, LEF1, CD44, KLF3 compared to CLL1 CAR-T cells. Moreover, SynNotch CD123 and CLL1 CAR-T cells significantly prolonged the survival of xenograft models while reducing the occurrence rate of granulocytopenia compared to the group treated with CLL1 CAR-T cells. ConclusionThis study demonstrates the promising therapeutic potential of SynNotch-engineered CAR-T cells in AML. We developed a CD123-targeting SynNotch receptor by fusing an anti-CD123 single-chain variable fragment (scFv) to the intracellular domain of the Notch core, coupled with the Gal4VP64 transcription factor to drive CLL1-specific CAR expression. This innovative approach enables precise tumor targeting while significantly reducing off-target effects, resulting in enhanced T cell persistence and superior therapeutic efficacy. In our forthcoming clinical trials, we aim to evaluate the safety and clinical effectiveness of this SynNotch CD123-CLL1 CAR-T cell therapy in patients with refractory or relapsed AML. These trials will assess whether this targeted immunotherapy can improve clinical outcomes and potentially transform the treatment paradigm for high-risk AML patients.