靶向抗PD-1/CD19双特异性抗体的表达与活性鉴定

Expression and activity identification of bispecific antibody targeting PD-1/CD19

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0.306μg/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/CD19BsAb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(Bs Ab)targeting PD-1 and CD19 and evaluate its activity.Methods:With p CAR1 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 Bs Ab was purified by Affinity chromatography and then identified by SDS-PAGE and WB.The blocking activity of Bs Ab 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 p CAR1-19 X3 was successfully constructed,and anti-PD-1/CD19 Bs Ab was successfully expressed in CHO-S cells,named p CAR1-19 X3-TY.p CAR1-19 X3-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 0.306μg/ml.ADCC results showed that p CAR1-19 X3-TY could mediate the cytotoxicity of PBMC against Raji cells,and the curve showed a linear upward trend;when the effect/target ratio was 50∶1,the target cell lysis rate of pCAR1-19 X3-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.pCAR1-19 X3-TY has the potential application value in the treatment of B-cell malignant tumor.