用抗原表位定向选择方法人源化抗TNF-α抗体Fab段.docVIP

???? 用抗原表位定向选择方法人源化?抗TNF-α抗体Fab段 ???? ????【摘要】　目的　用抗原表位定向选择(epitope guided selection)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。结论　 抗原表位定向选择方法提供了一种有效的鼠单抗人源化路线。　　【主题词】　噬菌体基因库　抗原决定簇　选择(遗传学)　肿瘤坏死因子 Humanization of anti-TNF-α murine Fab with the method of epitope guided selection　Wang Zhuozhi*, Wang Yan, Li Jing, et al.* Navy General Hospital, Beijing 100037　　【Abstract】　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.　　【Subject words】　Phage antibody librar