兼有无岩藻糖基化和抗凋亡体外培养特性的CHO抗体生产细胞系的建立

Monoclonal antibodies (mAbs), the most important kind of therapeutics for a variety of human diseases, have shown magnificent economical and social value. CHO cell line-the most preferred host for production of mAbs-is still unideal due to its relatively low in vivo efficacy resulting from high fucosylation and decreased producitivity resulting from cell apoptosis. In this study, CHO will be improved in its productivity for antibodies and in vitro culture characteristics by fucose removal and increased anti-apoptosis. The study will be accomplished with tremendous dependence on the innovative TALEN technology. We will design TALENs targeting to special DNA sites within CHO FUT8 gene and donors for homologous recombination containing anti-apoptotic E1B-19K sequence. The TALEN pair will be expressed in a biotin-based regulatory expression system. FUT8 gene will be knocked-out simultaneously with the site-specific integration of E1B-19K. Finally, Rituxan (mAb for CD20) will be expressed in the resultant CHO cell line to validate its charateristics of nonfucosylated antibody production and improved in vitro anti-apoptosis.

单克隆抗体是目前针对多种人类疾病最为重要的治疗制剂，具有巨大的经济效益和社会价值。CHO细胞是单抗药物生产最常用的宿主细胞系，但CHO细胞仍存在着高度岩藻糖化导致单抗体内效能较低和细胞凋亡等因素导致单抗表达水平较低等问题。本研究拟通过去除核心岩藻糖基化以增强抗体体内效能，通过增强细胞抗凋亡能力以改善CHO细胞体外培养效率，以期获得抗体生产能力和体外培养性状显著改善的CHO细胞系。本研究采用新兴的人工核酸内切酶TALEN技术，以CHO为研究对象，设计针对CHO细胞岩藻糖转移酶（FUT8）基因内特异靶位点的TALEN分子和含抗凋亡基因E1B-19K的同源重组供体,并应用基于生物素的可调控型基因表达系统表达TALEN分子,在定点整合E1B-19K基因的同时实现FUT8基因的敲除。将由此获得的细胞系用于表达Rituxan（CD20单抗），以验证该细胞系生产无岩藻糖基化抗体和体外培养抗凋亡的能力。