摘要
目的用抗原表位定向选择(epitopeguidedselection)方法,将鼠源性抗TNF-α单抗Fab段改造成完全人源性Fab。方法首先用鼠单抗Fd段基因与人的κ链基因库相互配对,构建成人-鼠杂合噬菌体抗体库,筛选可与TNF-α结合的克隆,所获得的人κ链基因再与人Fd基因库组合,建立人源噬菌体抗体库,再进行筛选;类似地以鼠κ链基因和人Fd库组合构建杂合抗体库,筛选人Fd基因,再和筛到的人κ链配对,选择与TNF-α特异结合的克隆。结果以鼠抗体Fd段定向选择人κ链获得能与TNF-α特异结合的杂合抗体Fab片段,再以这些人κ链定向选择人Fd段获得能与TNF-α结合的完全人源性的Fab,但这些Fab除与TNF-α反应外,与一些无关蛋白有交叉反应。以鼠单抗κ链定向选择人Fd段获得能与TNF-α特异结合的杂合Fab,其中一个克隆显示很强的结合活性,以该克隆的Fd与筛到的人κ链配对,得到了能与TNF-α特异结合的人源性Fab片段。
Objective Humanizing murine anti TNF α antibody Fab fragment with the method of epitope guided selection. Methods Firstly, the Fd gene of a murine anti TNF α antibody was paired with a repertoire of human κ chain, and displayed on the filamentous phage forming a hybrid phage antibody library. After four round panning against TNF α, the hybrid antibody fragments containing human κ chain were obtained. The selected human κ chain genes were in turn paired with a repertoire of human Fd fragments forming a human phage antibody library, and the phages were selected against TNF α again. The other way, human Fd gene was first selected by pairing human Fd repertoire with mouse anti TNF α κ chain. Then the human Fd gene was paired with selected human κ chains, and their binding activity to TNF was determined. Results Human Fabs obtained by first selecting human κ chains using mouse Fd chain as template showed nonspecific binding to some unrelated proteins. When human Fd chain was selected first and paired with selected human κ chain, the resulted human Fab bound to TNF α specifically, and their human origin was proved. Conclusion The epitope guided selection is thougt to be a valid method for humanizing mouse antibodies.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1998年第6期490-494,共5页
Chinese Journal of Microbiology and Immunology
基金
国家科委863项目
关键词
噬菌体
基因库
抗原决定簇
选择
肿瘤坏死因子
Phage antibody library Epitope guided selection Tumor necrosis factor