目的：设计并制备一种分别靶向B细胞表面抗原CD19和CD22的CAR-T细胞，检测其对肿瘤细胞的体内外杀伤效果。方法：将含有人源化 CD19 ScFv的二代CAR分子和带有CD3ε链作为共刺激结构域的CD22 ScFv CAR分子以P2A自剪切肽连接，序列连接于慢病毒载体pLTR-CMV-MCS中，以HEK-293T细胞包装相应的慢病毒载体，感染健康志愿者提供的T细胞制备CAR-19-22-T细胞，同时以相同二代结构分别构建单靶向CAR-T细胞作为参照。构建表达荧光素酶、CD19和/或CD22的前列腺癌3M细胞（靶细胞）。将各种CAR-T细胞与靶细胞共同培养，采用荧光素酶化学发光法和ELISA法检测其对靶细胞的杀伤能力和细胞因子的分泌水平。通过尾静脉注射Raji-Luc细胞构建NOD-SCID免疫缺陷小鼠白血病模型，分别注射各组CAR-T细胞进行治疗并评估其疗效。结果：培养7 d的CAR-19-22-T细胞的CAR-19表达率为13.7%，CAR-22表达率为14.3%。CAR-19-22-T细胞在10∶1效靶比时，对3M-CD19-Luc、3M-CD22-Luc和3M-CD19-CD22-Luc细胞的杀伤率均显著高于T细胞（[ 78.1±14.4）% vs（11.1±4.3）%、（46.7±10.7）% vs（12.4±2.7）%、（90.5±4.3）%vs（14.3±3.7）%，均P<0.01]；与3M-CD19-Luc、3M-CD22-Luc、3M-CD19-CD22-Luc靶细胞共培养后，CAR-19-22-T 细胞 IFN-γ、TNF-α 和 IL-2 水平均显著低于 CAR-19-T 和 CAR-22-T 细胞（P<0.05 或P<0.01）。CAR-19-22-T细胞对移植 Raji-Luc细胞模型小鼠治疗效果明显，其生存期显著长于T细胞组（P<0.01），与CAR-19-T组和CAR-22-T组荷瘤小鼠比较差异均无统计学意义（均P>0.05）。结论：成功设计并制备了一种双靶点CAR-19-22-T细胞，其能够有效杀伤表达CD19和/或CD22抗原的肿瘤细胞，对Raji-Luc细胞的白血病模型小鼠有显著的治疗效果。

Objective: To design and prepare a bi-specific chimeric antigen receptor (CAR)-T cell targeting both CD19 and CD22 antigens on the surface of B lymphocytes and to detect its tumor-killing effects in vitro and in vivo. Methods: Second-generation CAR molecules containing CD19 ScFv (human originated) and CD22 ScFv (CD3ε chain as costimulatory domain) were connected with P2A self-cleaving peptide. The synthesized fragment was packaged into lentivival vector pLTR-CMV-MCS. The lentiviruses were packaged with HEK-293T cells and infected with healthy human T cells to prepare CAR-19-22-T cells. Single targeted second-generation CAR-T cells were constructed with the same CD19 ScFv CAR molecules and CD22 ScFv CAR molecules respectively. Prostate cancer 3M cells expressing luciferase,CD19 and/or CD22 were constructed as target cells. Various CAR-T cells were co-cultured with target cells. Luciferase and ELISA methods were used to detect the target-cell killing ability of each group of CAR-T cells and their cytokine secretion. Mouse leukemia models were constructed by injecting Raji-Luc cells through the tail veins of the mice. Different groups of CAR-T cells were then injected respectively for treatment and the treatment efficacy was evaluated. Results: For CAR-19-22-T cells cultured for 7 days, the expression rates of CAR-19 and CAR-22 were 13.7% and 14.3% respectively.The killing rates of 3M-CD19-Luc, 3M-CD22-Luc and 3M-CD19-CD22-Luc cells by CAR-19-22-T cells at 10∶1 efficiency target ratio were significantly higher than those of T cells [(78.1±14.4)% vs (11.1±4.3)%, (46.7± 10.7)% vs (12.4±2.7)%, (90.5±4.3)% vs (14.3±3.7)%, all P<0.01]. CAR-19-22-T cells co-cultured with 3M-CD19-Luc, 3M-CD22-Luc or 3M-CD19-CD22-Luc target cells secreted significantly lower amounts of IFN-γ, TNF-α and IL-2 than those for CAR-19-T or CAR-22-T (P<0.05 or P<0.01). CAR-19-22-T cells had significantly better treatment efficacy on mice infected with Raji-Luc cells. The survival time of these mice was significantly longer than that of the mice in the T-cell group (P<0.01) and compared with those of the tumor-bearing mice in the CAR-19-T group and CAR-22-T group, was not statistically significant (all P>0.05). Conclusion: Bi-specific CAR-19-22-T cells were successfully designed and prepared, which could effectively kill tumor cells expressing CD19 and/or CD22 antigens and have a significant treatment effect on mouse leukemia model of Raji-Luc cells.

湖南省自然科学基金资助项目（No.2020JJ4403）；湖南省社会发展重点研发项目（No.2019SK2091）