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| 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 |
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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-Mab1# and Mab2# 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-Mab1# and Mab2# 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 Mab1# 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 Mab1# 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.
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Received: 12 January 2015
Published: 25 March 2015
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