To explore and design a novel bi-specific chimeric antigen receptor (CAR) structure. To obtain the corresponding CAR-T cells and verify killing effects on tumor cells in vitro and in vivo.Five kinds of bi-specific CAR structures including humanized CD19 scFv and CD79b scFv, CD8 hingeTM-4-1BB-CD3ζ and/or CD3ε chain intracellular regions were constructed and prepared. CAR-19-79b cells were obtained. Five kinds of CAR-T cells were co-incubated with the 3M-CD19-CD79b-Luc target cells. Luciferase assay and ELISA were used to detecte the killing ability of these five groups of CAR-T cells and the secretion of cytokines and compared. The optimal structure of CAR-T cells was used to treat the leukemia mouse model constructed by Daudi-Luc cells. And the treatment efficacy was evaluated. At the same time, other targets were used in this structure. With the same methods, the stability and effectiveness of the structure were verified.CAR-19-79b-T cells were cultured for 7 days, the expression rates of CAR-19 and CAR-79b were 21.6%-36.3% and 21.7%-37.8%, respectively. The killing rates of 5 kinds of CAR-19-79b-T cells prepared by T cells from 3 healthy donors on 3M-CD19-CD79b-Luc cells were significantly higher than those of the T cell control group at the effect-target ratio of 10∶1. Among them, the killing rates of CAR-19-79b-T cells with No. III and No. IV structures were the strongest. After co-incubation with 3M-CD19-CD79b-Luc target cells, the amount of IFN-γ and TNF-α secreted by CAR-T cells with CAR IV and CARV structures was the lowest. And there was no significance between the two groups (A novel bi-specific CAR structures was successfully designed, which could efficiently kill the corresponding tumor cells and secrete less cytokines (such as TNF-α, IFN-γ). Moreover, it shows obvious therapeutic effect on Daudi lymphoma mouse model. The bi-specific CAR structure shows good killing specificity and safety.特殊结构双靶点CAR-T细胞对肿瘤细胞的杀伤作用.探索并设计一种新颖的双靶点嵌合抗原受体(chimeric antigen receptor,CAR)结构,获得相应CAR-T细胞并验证其对肿瘤细胞的体内外杀伤效果.构建并制备5种包含人源化CD19 scFv和CD79b scFv、CD8 hingeTM-4-1BB-CD3ζ和/或CD3ε链胞内区的双靶点CAR-T细胞,荧光素酶法和ELISA法检测CAR-T细胞对3M-CD19-CD79b-Luc靶细胞的杀伤能力和细胞因子的分泌,选取最优结构CAR-T细胞治疗Daudi-Luc细胞构建的白血病小鼠模型并评估其疗效,同时使用其他靶点代入优选结构,相同方法验证该结构的稳定性及有效性.CAR-19-79b-T细胞培养7 d后,CAR-19表达率为21.6%-36.3%,CAR-79b表达率为21.7%-37.8%。CAR-19-79b-T细胞在10∶1效靶比时,对3M-CD19-CD79b-Luc细胞的杀伤率均显著高于T细胞对照组,其中CAR Ⅲ、CAR Ⅳ号结构细胞杀伤能力最强,均显著高于T细胞组(成功设计了一种新颖的抗双靶点CAR-T细胞,能够高效杀伤相应的肿瘤细胞,同时分泌较少的细胞因子(如TNF-α,IFN-γ),而且对Daudi淋巴瘤小鼠动物模型有显著的治疗效果,该双靶点CAR结构表现出良好的杀伤特异性和安全性.