Objective To construct a novel dual-specific antibody targeting human CD 123(CD 123 DuAb)and study its effects in acute myeloid leukemia(AML).Methods Based on the variable region of the CD 123 monoclonal antibody independently developed at our institution,the CD 123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein.Through a series of in vitro experiments,its activation and proliferation effect on T cells,as well as the effect of promoting T-cell killing of AML cells,were verified.Results ① A novel CD 123 DuAb plasmid targeting CD 123 was successfully constructed and expressed in the Expi-CHO eukaryotic system.②The CD 123 DuAb could bind both CD3 on T cells and CD 123 on CD 123+tumor cells.③When T cells were co-cultured with MV4-11 cells with addition of the CD 123 DuAb at a concentration of 1 nmol/L,the positive expression rates of CD69 and CD25 on T cells were 68.0%and 44.3%,respectively,which were significantly higher than those of the control group(P<0.05).④Co-culture with CD 123 DuAb at 1 nmol/L promoted T-cell proliferation,and the absolute T-cell count increased from 5× 105/ml to 3.2× 106/ml on day 9,and CFSE fluorescence intensity decreased significantly.(5)With the increase in CD123 DuAb concentration in the culture system,T-cell exhaustion and apoptosis increased.When the CD 123 DuAb was added at a concentration of 1 nmol/L to the culture system,the proportion of CD8+PD-1+LAG-3+T cells was 10.90%,and the proportion of propidium iodide(PI)-Annexin V+T cells and PI+Annexin V+T cells was 18.27%and 11.43%,respectively,which were significantly higher than those in the control group(P<0.05).⑥ The CD123 DuAb significantly activated T cells,and the activation intensity was positively correlated with its concentration.The expression rate of CD107a on T cells reached 16.05%with 1 nmol/L CD123 DuAb,which was significantly higher than that of the control group(P<0.05).⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L,and the concentration of IFN-γ and TNF-a in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml,respectively,which was significantly higher than that of the control group(P<0.05).⑧When CD 123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD 123+tumor cells,the killing intensity of T cells significantly increased,and the residual rates of CD123+MV4-11 cells,CD123+Molm13 cells,and CD123+THP-1 cells were 7.4%,6.7%,and 14.6%on day 3,respectively,which were significantly lower than those in the control group(P<0.05).Conclusion In this study,a novel CD 123 DuAb was constructed and expressed.In vitro experiments verified that the DuAb binds to CD123+tumor cells and T cells simultaneously,promotes T-cell activation and proliferation,and facilitates their anti-leukemia effect,which provides a basis for further clinical research.