Modified CD15/CD16-CLL1 inhibitory CAR-T cells for mitigating granulocytopenia toxicities in the treatment of acute myeloid leukemia.

CLL1 Chimeric antigen receptor T-cell (CAR-T) therapy, as a promising immunotherapeutic approach, has demonstrated its potential to enhance the prognosis of patients diagnosed with acute myeloid leukemia (AML). However, due to the overexpression of CLL1 on neutrophils, CAR-T cells not only eliminated tumor cells but also eradicated neutrophils simultaneously, resulting in severe granulocytopenia and subsequent infections. Considering the distinct expression levels of CD15/CD16 on neutrophils and AML blasts, we have devised novel modified CD15 /CD16-CLL1 iCAR structures incorporating diverse inhibitory elements. Through extensive screening of structural optimization, we have successfully identified CD16-CLL1 iCAR-T cells that combine PD1 and 2B4 blockade, as well as a single VHH fragment replacing the entire CD16 scFv recognition domain. These modified cells demonstrate enhanced cytotoxicity against blasts while minimizing neutrophil elimination. Furthermore, their functionality has been effectively validated through both in vitro and in vivo experiments. In conclusion, we have successfully engineered innovative CD16-CLL1 iCAR-T cells, which preserves the cytotoxicity against tumor cells while preventing elimination of neutrophils, thereby significantly reducing the incidence of granulocytopenia during CAR-T therapy. Furthermore, our future objectives encompass the meticulous validation of both the efficacy and safety profile of this groundbreaking CAR-T therapy in clinical trials, as well as a comprehensive assessment of its potential to enhance the prognosis of patients diagnosed with AML.