目的：重组表达和纯化靶向抗PD-1/CD19双特异性抗体（bispecific antibody, BsAb）并验证其活性。方法：以pCAR1为载体，利用分子克隆技术构建抗PD-1/CD19 BsAb真核表达载体，通过PEI试剂转染哺乳动物细胞株CHO-S瞬时表达抗体。利用亲和层析法对BsAb进行纯化，用SDS-PAGE和WB实验进行BsAb蛋白鉴定。用荧光素酶报告基因检测BsAb对PD-1/PD-L1的体外阻断活性，乳酸脱氢酶细胞毒实验检测BsAb依赖的PBMC介导细胞毒性（ADCC）活性。结果：成功构建双质粒真核表达载体pCAR1-19X3，BsAb在CHO-S细胞中成功表达，命名为pCAR1-19X3-TY。pCAR1-19X3-TY在体外能够有效地阻断PD-1与其配体PD-L1的结合，其量效曲线的EC50为0.306 μg/ml。ADCC结果显示，pCAR1-19X3-TY能介导PBMC对Raji细胞产生细胞毒性，曲线呈现直线上升的趋势；当效靶比为50∶1时，pCAR1-19X3-TY的杀伤率为（38.9±0.3）%，与阳性处理组的杀伤率（46.7±4.9）%的差异比较无统计学意义（P>0.05），明显高于阴性对照组（1.2±0.1）%杀伤率（P<0.05）。结论：重组表达的靶向抗PD-1/CD19 BsAb能有效阻断PD-1和PD-L1的结合、激活PBMC介导的Raji细胞毒性作用，具有开发用于治疗B细胞恶性肿瘤的潜力。

Objective: To construct and purify the recombinant bispecific antibody (BsAb) targeting PD-1 and CD19 and evaluate its activity.Methods: With pCAR1 plasmid as the vector, the eukaryotic expression vector of anti-PD-1/CD19 BsAb was constructed by molecular cloning technology, and then transfected into mammalian cell line CHO-S by PEI reagent for transiently expressing antibody. The BsAb was purified by Affinity chromatography and then identified by SDS-PAGE and WB. The blocking activity of BsAb on PD-1/PD-L1 in vitro was detected by Luciferase reporter gene assay. The activity of antibody (BsAb)-dependent cell (PBMC)-mediated cytotoxicity (ADCC) in vitro was evaluated by lactate dehydrogenase (LDH) cytotoxicity assay. Results: The double plasmid eukaryotic expression vector pCAR1-19X3 was successfully constructed, and anti-PD-1/CD19BsAb was successfully expressed in CHO-S cells, named pCAR1-19X3-TY. pCAR1-19X3-TY could effectively block the binding of PD-1 to its ligand PD-L1 in vitro, and the EC50 based on the dose-response curve was 0.306 μg/ml.ADCC results showed that pCAR1-19X3-TY could mediate the cytotoxicity of PBMC against Raji cells, and the curve showed a linear upwardtrend; when the effect/target ratio was 50∶1, the target cell lysis rate of pCAR1-19X3-TY was (38.9±0.3)%, which was not significantly different from that of the positive treatment group (46.7±4.9)% (P>0.05), but significantly higher than that of the negative control group (1.2±0.1)% (P<0.05). Conclusion: The recombinant anti-PD-1/CD19 BsAb can effectively block the binding of PD-1 and PD-L1 and activate PBMC mediated cytotoxicity against Raji cells. pCAR1-19X3-TY has the potential application value in the treatment of B-cell malignant tumor.

天津市重点研发计划科技支撑重点项目（No.18YFZC‐SY01340）