重组抗人上皮黏附因子人源化单克隆抗体的瞬时表达及体外活性检测

Transient Expression and in vitro Activity Test of Recombinant Humanized Anti-EpCAM Monoclonal Antibody

人上皮黏附因子(EpCAM)在多种上皮相关癌症表面过表达,介导肿瘤的增殖、分化、转移及信号传导等多种活动,是一种有潜力的癌症治疗靶点。本研究通过构建哺乳动物细胞表达载体,优化转染条件,使用瞬时基因表达技术在HEK293E细胞中表达了重组抗EpCAM人源化IgG1型单克隆抗体(rhEpCAM-mAb)。抗体经纯化后,采用乳酸脱氢酶释放法初步鉴定了其介导的抗体依赖细胞毒作用。结果表明,rhEpCAM-mAb在HEK293E细胞中以DNA∶PEI为1∶5,轻链∶重链为1∶3的最优条件进行瞬时表达时,抗体表达量可达46.05 mg/L,抗体对EpCAM的亲和力为5.56×10-12 mol/L,它对SK-OV-3、SW480和OVCAR-3细胞抗体依赖的细胞毒作用的IC50分别为12.36、5.43、102.90 ng/ml。本研究利用瞬时基因表达技术能够快速获得重组单克隆抗体,并对其生物活性进行研究,为生物技术药物开发及生产提供理论支持。

Epithelial cell adhesion molecule(EpCAM)is a potential cancer treatment target,which can be overexpressed on the surface of a variety of epithelial cancers,and participate in tumor proliferation,differentiation,metastasis,signal transmission and other activities.In this study,the recombinant anti-EpCAM humanized IgG1 monoclonal antibody(rhEpCAM-mAb)was expressed in HEK293E cells by constructing mammalian cell expression vectors and optimizing transfection conditions using transient gene expression technology.After the antibody was purified,the lactate dehydrogenase release method was used to preliminarily identify its antibody-dependent cytotoxicity.The results showed that when rhEpCAM-mAb was transiently expressed in HEK293E cells under the optimal conditions of DNA∶PEI with 1∶5 and light chain∶heavy chain with 1∶3,the antibody expression amount could reach 46.05 mg/Land the affinity for EpCAM is 5.56×10-12 mol/L.The IC50s for antibody-dependent cytotoxicity on SK-OV-3,SW480 and OVCAR-3 cells were 12.36,5.43,102.90 ng/ml,respectively.This study used transient gene expression technology to obtain recombinant monoclonal antibodies quickly and studied their biological activities,which could provide theoretical support for biotechnology drug development and production.