药学学报, 2014, 49(12): 1665-1673
李路军, 杨艳群, 胡雪平, 谢冕, 刘梦元. 抗TNF-α/抗ED-B双特异抗体的构建与功能分析[J]. 药学学报, 2014, 49(12): 1665-1673.
LI Lu-jun, YANG Yan-qun, HU Xue-ping, XIE Mian, LIU Meng-yuan. Construction and functional analysis of a bispecific antibody that targets TNF-α and ED-B[J]. Acta Pharmaceutica Sinica, 2014, 49(12): 1665-1673.

李路军1,2,3, 杨艳群1,3, 胡雪平1,3, 谢冕1,3, 刘梦元1,3
1. 暨南大学第二临床医学院/深圳市老年医学研究所, 广东 深圳 518020;
2. 湖北大学生命科学学院感染与免疫研究中心, 湖北 武汉 430062;
3. 湖北绿色生物资源转换协同创新中心, 湖北 武汉 430062
为了赋予TNF-α抗体对炎症组织的特异性, 构建了抗TNF-α/抗纤维连接蛋白额外域B (ED-B) 的基因工程双特异抗体BsDb.BsDb在大肠杆菌中获得了高效表达, 表达产物经鉴定、纯化和复性制备后, 进行了生物学活性和药动学分析.BsDb可同时结合重组人TNF-α和ED-B, 并中和TNF-α生理作用.在胶原诱导的小鼠关节炎模型中, BsDb快速从血浆和正常组织清除, 但能选择性的积累和保留于炎症关节.说明BsDb具有炎症组织的特异性和正常组织的低毒性, 在类风湿关节炎和其他自身免疫性疾病的治疗上具有较大潜力, 为其临床前期研究奠定了基础.
关键词:    双特异抗体      类风湿关节炎      纤维连接蛋白      肿瘤坏死因子α      额外域B     
Construction and functional analysis of a bispecific antibody that targets TNF-α and ED-B
1. The Second Clinical Medical College of Jinan University, Shenzhen Institute of Geriatrics, Shenzhen 518020, China;
2. Center for Infection and Immunity Research, College of Life Sciences, Hubei University, Wuhan 430062, China;
3. Hubei Collaborative Innovation Center for Green Transformation of Bioresources, Wuhan 430062, China
In order to enhance the specificity of TNF-α monoclonal antibody to inflamed site, a bispecific antibody BsDb that targets TNF-α and the extra-domain B (ED-B) of fibronectin (FN) was constructed by covalently linking the anti-TNF-α single chain Fv antibody (TNF-scFv) and the anti-ED-B scFv L19 via a flexible peptide linker deriving from human serum albumin (HSA). ED-B is an antigen specifically expressed at the inflamed site. BsDb is expressed in E. coli, identified by immunoblot, and purified with affinity chromatography. This was followed by further examination of its bioactivities and pharmacokinetics. We demonstrated that BsDb retained the immunoreactivity of its original antibodies as it could simultaneously bind to TNF-α and ED-B and neutralize the biological action of TNF-α. In the collagen-induced arthritis mice model, BsDb selectively accumulate in the inflamed joint with a maximal uptake of (12.2 ± 1.50) % ID/g in a single inflamed paw and retain in the inflamed paw for at least 72 h. In contrast, BsDb showed a short serum half-life of (0.50 ± 0.05) h and a rapid clearance from normal tissues. The findings reported herein indicate that BsDb has good specificity to the inflamed site and low toxicity to normal tissues. BsDb is therefore likely to have greater clinical applications in the treatment of rheumatoid arthritis and other autoimmune diseases. This laid a stable basis for its preclinical study.
Key words:    bispecific antibody    rheumatoid arthritis    fibronectin    tumor necrosis factor α    extro-domain B   
收稿日期: 2014-06-24
基金项目: 国家自然科学基金资助项目(30973669).
通讯作者: 刘梦元
Email: liumengy@tom.com
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谢冕  在本刊中的所有文章
刘梦元  在本刊中的所有文章

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