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

Objectives 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. Methods 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. Results 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. Conclusions 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.