Zhang Guoquan, Liu Jie, Fan Wanting, Chen Qianyi, Shi Bin
The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedical Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei. China.
Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong. China.
Curr Pharm Biotechnol. 2017;18(4):351-357. doi: 10.2174/1389201018666170320110545.
Recombinant monoclonal antibodies (mAbs) are useful in research, diagnosis, and therapy. The increased demands of recombinant mAbs require efficient production systems. A variety of expression vectors have been developed for stable or transient production of mAbs in mammalian cells. Although a few commercial expression systems of mAbs can be listed, the high expense often impedes academic research.
In this study, we described the development of a transient mammalian system based on a bicistronic vector, which contained an internal ribosome entry site (IRES) and enhancer elements to express the IgG1 light chain (LC) and heavy chain (HC) in one transcript. Optimization of all components of the expression system, including gene transfer methods and regulatory elements, yielded maximal expression levels in serum-free 293 suspension cells (Freestyle 293-F).
This method enabled consistent production of the anti-programmed cell death protein 1 (PD-1) mAb up to 300 mg/L in less than one week under transiently transfected conditions. Furthermore, purified anti-PD-1 IgGs showed specific affinity to the target antigen human PD-1 and PHA-stimulated human T cells.
The simplicity of the procedure made it suitable for the fast and high-yield production of IgG antibodies in small scales, which expedited the screening of a large number of recombinant candidates.
重组单克隆抗体（mAb）在研究、诊断和治疗中具有重要作用。对重组单克隆抗体需求的增加需要高效的生产系统。已开发出多种表达载体用于在哺乳动物细胞中稳定或瞬时生产单克隆抗体。虽然可以列出一些商业化的单克隆抗体表达系统，但高昂的成本常常阻碍学术研究。
在本研究中，我们描述了一种基于双顺反子载体的瞬时哺乳动物系统的开发，该载体包含一个内部核糖体进入位点（IRES）和增强子元件，以便在一个转录本中表达IgG1轻链（LC）和重链（HC）。对表达系统的所有组件进行优化，包括基因转移方法和调控元件，在无血清293悬浮细胞（Freestyle 293-F）中获得了最大表达水平。
该方法能够在瞬时转染条件下，在不到一周的时间内，以高达300 mg/L的产量持续生产抗程序性细胞死亡蛋白1（PD-1）单克隆抗体。此外，纯化的抗PD-1 IgG对靶抗原人PD-1和PHA刺激的人T细胞显示出特异性亲和力。
该方法操作简单，适合小规模快速高产生产IgG抗体，加快了大量重组候选抗体的筛选。
