Tandem bispecific CD123/CLL‐1 CAR‐T cells exhibit specific cytolytic effector functions against human acute myeloid leukaemia

The treatment of refractory and recurrent acute myeloid leukaemia (AML) is still a challenge with poor response rates and short survival times. In an attempt to solve this problem, we constructed a tandem bispecific chimeric antigen receptor (CAR) targeting CD123 and C-type lectin-like molecule 1 (CLL-1), two different AML antigens, and verified its cytotoxic effects in vitro.We established and cultured K562 cell lines expressing both CD123 and CLL1 antigens. Single-target CAR-T cells specific to CD123 and CLL1 were engineered, alongside tandem CD123/CLL1 bispecific CAR-T cells. Flow cytometry was used to determine cell phenotypes, transfection efficiencies, cytokine release, and CAR-T-cell proliferation, and an lactate dehydrogenase assay was used to detect the cytotoxicity of CD123/CLL-1 bispecific tandem CAR-T cells in vitro.Two types of tandem CAR-T cells exhibited significant killing effects on CLL-1 + CD123+ leukaemia cell lines and primary AML tumour cells. The killing efficiency of tandem CAR-T cells in the case of single antigen expression is comparable to that of single target CAR-T cells. When faced with dual target tumour cells, dual target CAR-T cells significantly surpass single target CAR-T cells. CD123/CLL-1 CAR-T cells in tandem targeted and killed CD123- and CLL-1-positive leukaemia cell lines and released a large number of cytokines.CD123/CLL-1 CAR-T cells in tandem can simultaneously target CD123 and CLL-1 on AML cells, demonstrating a significant ability to kill single antigens and multi-target tumour cells. This suggests that CD123/CLL-1 CAR-T cells exhibit significant advantages in the expression of multiple antigens in a wide range of target cells, which may help overcome the challenges posed by tumour heterogeneity and evasion mechanisms.