期刊
PROTEIN ENGINEERING DESIGN & SELECTION
卷 23, 期 8, 页码 667-677出版社
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzq034
关键词
antibody engineering; conformational isomer; isomerization; single-chain diabody; V-H-V-L interface
Thrombopoietin receptor agonist humanized VB22B single-chain diabody (hVB22B (scFv)(2)) was found to be expressed as a mixture of two conformational isomers, a single-chain diabody form and a bivalent scFv form, which had different V-H/V-L (variable region of the heavy chain/light chain) association patterns. The single-chain diabody form showed significantly higher biological activity than the bivalent scFv form and, when incubated at elevated temperatures, exhibited novel isomerization to the inactive bivalent scFv form. Therefore, therapeutic development of hVB22B (scFv)(2) would require separation of the purified single-chain diabody form from the mixture of the two conformational isomers and also inhibition of isomerization into an inactive bivalent scFv form during storage. Novel V-H/V-L interface engineering in hVB22 (scFv)(2), in which hydrogen bonding between H39 and L38 was substituted with electrostatic interaction to enhance the desired V-H/V-L association and inhibit the undesired V-H/V-L association, enabled selective expression of the desired conformational isomer without any reduction in biological activity or thermal stability. Moreover, V-H/V-L interface-engineered hVB22 (scFv)(2) was completely resistant to isomerization. Because the hydrogen bonding interaction between H39 and L38 and the surrounding residues are highly conserved in human antibody sequences, V-H/V-L interface engineering could be generally applied to various (scFv)(2) molecules for selective expression and inhibition of the isomerization of conformational isomers.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据