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Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry

文献类型： 外文期刊

作者： Xu, Liming 1 ; Zhang, Yu 2 ; Wang, Qiuying 3 ; Zhao, Jingzhuang 1 ; Liu, Miao 1 ; Guo, Mo 3 ; Jiang, Yuanyuan 4 ; Cao, Ho 1 ;

作者机构： 1.CAFS, Heilongiang River Fishery Res Inst, Harbin 150070, Peoples R China

2.CAAS, Harbin Vet Res Inst, State Key Lab Vet Biotechnol, Div Avian Infect Dis, Harbin 150001, Peoples R China

3.Northeast Agr Univ, Biopharmaceut Labs, Harbin 150030, Peoples R China

4.Harbin Pharmaceut Grp Bioengn Co, Harbin 150025, Peoples R China

5.HeiLongJiang BaYi Agr Univ, Coll Biol Sci & Technol, Daqing 163319, Peoples R China

关键词： Bacteria display technology;Bi-specific antibody;Collagen induced arthritis;Flow cytometry;Rheumatoid arthritis

期刊名称：INTERNATIONAL IMMUNOPHARMACOLOGY （ 影响因子：4.932； 五年影响因子：4.624 ）

ISSN： 1567-5769

年卷期： 2015 年 24 卷 2 期

页码：

收录情况： SCI

摘要： Using conventional approaches, the antigen-binding affinity of a novel format of bi-specific antibody (BsAb) cannot be determined until purified BsAb is obtained. Here, we show that new lipoprotein A (NlpA)-based bacteria display technology, combined with flow cytometry (FCM), can be used to detect antigen-binding affinity of BsAbs, in the absence of expression and purification work. Two formats of BsAb,-scFv2-CH/CL and Diabody-CH/CL, specific for human interleukin 113 (hIL-1 beta) and human interleukin 17A (hIL-17A), were constructed and displayed in Escherichia coli using NlpA-based bacteria display technology. Conversion of these cells to spheroplasts, and their incubation with fluorescently conjugated antigens resulted in the selective labeling of spheroplasts expressing BsAb; enabling their antigen-binding affinity to be analyzed with FCM. The association and dissociation of BsAbs for binding to hIL-1 beta and hIL-17A were analyzed using FCM-based assays. The results showed that antigen-binding affinity of Diabody-CH/CL was significantly higher than that of scFv2-CH/CL. To confirm these results of FCM-based assays, BsAbs were expressed, purified and subjected to relative affinity measurements, in vitro and in vivo bioactivity analysis. The results showed that Diabody-CH/CL had greater relative affinities for both antigens, resulting in better blocking bioactivities on cellular level and effects on alleviating joint inflammation, and cartilage destruction and bone damage in collagen induced arthritis (CIA) mice model. These results indicate that BsAbs with good antigen-binding affinity can be identified by FCM-based assays without expression and purification work, and the indentified BsAb can serve as a lead compound for further drug development. (C) 2014 Elsevier B.V. All rights reserved.

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