通过将 F10 抗体的高可变区嫁接到人纤维连接蛋白结构域支架上来构建 TNF 结合蛋白。

Construction of TNF-binding proteins by grafting hypervariable regions of F10 antibody on human fibronectin domain scaffold.

机构信息

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

出版信息

Biochemistry (Mosc). 2012 Jan;77(1):62-70. doi: 10.1134/S0006297912010075.

DOI:10.1134/S0006297912010075

Abstract

Tumor necrosis factor (TNF) plays a key role in the pathogenesis of various diseases. To study the possibility of constructing TNF-binding proteins by grafting hypervariable regions of immunoglobulins (CDR), we have replaced amino acid sequences of loops from the tenth type III domain of human fibronectin ((10)Fn3) by amino acid sequences of CDR from the light and heavy chains of the anti-TNF antibody F10. The assessment of TNF-binding properties of the resulting proteins by ELISA has revealed the highest activity of Hd3 containing sequences CDR-H1 and CDR-H2 of the antibody F10 and of Hd2 containing sequences CDR-H1 and CDR-H3. The proteins constructed by us on the fibronectin domain scaffold specifically bound TNF during Western blotting and also weakened its cytotoxic effect on L929 line cells. The highest neutralizing activity was demonstrated by the proteins Hd2 and Hd3, which induced, respectively, 10- and 50-fold increase in the EC(50) of TNF.

摘要

肿瘤坏死因子（TNF）在各种疾病的发病机制中起着关键作用。为了研究通过移植免疫球蛋白的高变区（CDR）构建 TNF 结合蛋白的可能性，我们用抗 TNF 抗体 F10 的轻链和重链的 CDR 替换了人纤维连接蛋白第 10 型 III 结构域（(10)Fn3）中的环的氨基酸序列。通过 ELISA 评估所得蛋白的 TNF 结合特性，结果显示，含有抗体 F10 的 CDR-H1 和 CDR-H2 的 Hd3 以及含有 CDR-H1 和 CDR-H3 的 Hd2 序列的蛋白活性最高。我们在纤维连接蛋白结构域支架上构建的蛋白在 Western blot 中特异性结合 TNF，并且还减弱了 TNF 对 L929 细胞系的细胞毒性作用。具有最高中和活性的是 Hd2 和 Hd3 蛋白，它们分别使 TNF 的 EC(50)增加了 10 倍和 50 倍。