用抗原表位定向选择方法人源化抗TNF-α抗体Fab段

Humanization of anti TNF α murine Fab with the method of epitope guided selection

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.