表达全人源抗人IgE单抗的细胞株构建及筛选

doi:10.13523/j.cb.20150310

中国生物工程杂志

2015, Vol. 35

Issue (3): 66-74 DOI: 10.13523/j.cb.20150310

技术与方法

表达全人源抗人IgE单抗的细胞株构建及筛选

张银川¹, 刘萌萌¹, 张雅婷¹, 桂芳¹, 张爱华², 闭兰¹, 潘勇兵¹

1. 武汉生物制品研究所有限责任公司抗体研究室武汉 430207;
2. 中国生物技术股份有限公司科研与国际合作部北京 100029

Construction and Screening of Recombinant Cell Line Expressing Fully-human mAbs against Human IgE

ZHANG Yin-chuan¹, LIU Meng-meng¹, ZHANG Ya-ting¹, GUI Fang¹, ZHANG Ai-hua², BI Lan¹, PAN Yong-bin¹

1. Laboratory of Antibody Drugs, Wuhan Institute of Biological Products Co., Ltd. Wuhan 430207, China;
2. Department of Research and International Cooperation, China National Biotec Group, Beijing 100029, China

全文: PDF(1991 KB) HTML

摘要：

目的:构建及筛选高效表达原创性全人源抗人IgE单克隆抗体的重组工程细胞株.方法:将采用核糖体展示技术筛选到的原创性全人源抗人IgE单链抗体(scFv)基因改构设计为IgG1κ型全长抗体,构建重组真核表达质粒并电转染CHO-S细胞,Dot-blot法选取多株高表达克隆进行40ml摇瓶批次培养,再据细胞生长特征及抗体表达量选取高表达克隆进行40ml摇瓶及3L摇瓶流加培养研究,选取候选细胞株并对改构前后抗体的生物学活性进行比较研究.结果:成功构建了pMH3-H、pMH3-L、pCApuro-H、pCApuro-L四种重组真核表达质粒并成功共转染CHO-S细胞.完成了4次电转染8轮细胞克隆筛选,获得两株表达量较高的候选克隆Mab和Mab,在3L摇瓶流加培养中抗体表达量分别达到470mg/L及499mg/L.生物膜光干涉技术(Bio-Layer Interferometry,BLI)亲和力结果显示Mab^1#及Mab^2#两株单抗亲和力均达到nmol/L级(10^-9),与现有唯一上市的抗人IgE单抗药物奥马珠单抗(Omalizumab)的亲和力相当.选取Mab^1#全长抗体与其改构前的母本单链抗体的表面等离子共振技术(surface plasmon resonance,SPR)中和活性比较结果显示Mab^1#抑制hIgE与FcεRI结合的EC50为3nmol/L,EC90为9nmol/L,较改造前亲和力提高了4.3倍,中和活性(EC50)提高了23.7倍,中和活性(EC90)提高了41.3倍.结论:成功将表达原创性全人源抗人IgE的单链抗体(约25kDa)改造为亲和力及中和活性均大幅提升的全长抗体(约150kDa),获得2个候选细胞株.

关键词： 人免疫球蛋白E; 全人源; 单克隆抗体; 细胞株; 哮喘

Abstract:

Objective: Construction and screening the recombinant cell line that is highly expressed of original fully-human anti-human IgE monoclonal antibodies. Methods: The screened originally fully-human anti-human IgE single-chain antibody (scFv) by the ribosome display technology is re-constructed to full length IgG1κ antibody. Construct the recombinant eukaryotic expression vectors and electric-transfect CHO-S cells. Select multiple highly expressed clones for the 40ml flask batch culture by the Dot-blot method. Then according to the growth characteristics of the cells and the antibody expression quantity select highly expressed clones for the 40ml flask and 3L flask fed-batch culture study. The antibody biological activity before and after the reconstruction of the candidate cell lines are compared. Results: Four kinds of eukaryotic expression plasmids-pMH3-H, pMH3-L, pCApuro-H, pCApuro-L are successfully constructed and co-transfected CHO-S cells. Complete four times of electric transfection and eight rounds cell clones screens and obtain two highly expressed candidate clones-Mab^1# and Mab^2# whose antibody expression quantity reaches 470mg/L and 499mg/L in 3L flask fed-batch culture. The affinity result by the Bio-Layer Interferometry (BLI) technology shows that the affinity of two monoclonal antibody-Mab^1# and Mab^2# reaches nM (10^-9) level and are comparable with the only existing anti-human IgE monoclonal antibody drug Omalizumab on the market. Compare the activity of Mab^1# strain full-length antibody with its parent single-chain antibody by the surface plasmon resonance (SPR). The result shows the level of EC50 which inhibits the binding of hIgE and FcεRI in Mab^1# is 3nM, with affinity increases 4.3 times, the neutral activity of EC50 increases 23.7 times and the neutral activity of EC90 increases 41.3 times. Conclusion: The single-chain antibody (about 25kDa) of originally expressed fully-human anti-human IgE is successfully transformed to full length antibody (about 150kDa) whose affinity and neutral activity level are significantly increased and obtain two candidate cell lines.

Key words: Human IgE Fully human Monoclonal antibody Cell line Asthma

收稿日期: 2015-01-12 出版日期: 2015-03-25

ZTFLH:

Q813

基金资助:

国家科技重大专项资助项目(2011ZX09506-005)

通讯作者: 潘勇兵 E-mail: yongbingpan@163.com

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引用本文:

张银川, 刘萌萌, 张雅婷, 桂芳, 张爱华, 闭兰, 潘勇兵. 表达全人源抗人IgE单抗的细胞株构建及筛选[J]. 中国生物工程杂志, 2015, 35(3): 66-74.

ZHANG Yin-chuan, LIU Meng-meng, ZHANG Ya-ting, GUI Fang, ZHANG Ai-hua, BI Lan, PAN Yong-bin. Construction and Screening of Recombinant Cell Line Expressing Fully-human mAbs against Human IgE. China Biotechnology, 2015, 35(3): 66-74.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150310 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I3/66

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