构建预设CDR3基因噬菌体抗体库筛选抗人整合素ανβ3mAb人源化.docVIP

构建预设CDR3基因噬菌体抗体库筛选抗人整合素ανβ3mAb人源化 　 作者：王臣 侯利华 杜桂鑫 李建明 陈溥言 童贻刚 【关键词】 噬菌体抗体库;,,整合素ανβ3;,人源化Fab 　　［Abstract］ AIM: To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of antihuman integrin ανβ3 monoclonal antibody (mAb) by epitope guided selection. METHODS: LCDR3 gene of mAb E10 was inserted into human light chain variable region gene library. Hybrid phage antibody library was constructed by cloning E10 chimeric Fd gene and human light chain variable region gene into pComb3. Humanized light chain gene was obtained by screening against human integrin ανβ3. Likewise, humanized Fab were gained by panning human phage antibody library, which was constructed by cloning humanized light chain gene and human heavy chain Fd gene with E10 HCDR3 into pComb3. RESULTS: Three humanized Fab clones was obtained by screening hybrid phage antibody library and human phage antibody library, which contained 2.1×106, 2×107 colony forming units, respectively. Indirect ELISA and competitive inhibition ELISA analysis demonstrated that three humanized Fab antibody had specific binding activity with human integrin ανβ3. The strongest antihuman integrin ανβ3 reactive D5 clone was sequenced and sequencing analysis showed that the Vκ and VH were derived from VKIII and VHI, respectively. CONCLUSION: Humanized Fab of antihuman integrin ανβ3 mAb has been successfully obtained by phage display technology which lays the foundation for further clinical research. 　　［Keywords］phage antibody library; integrin ανβ3; humanized Fab 　　［摘 要］ 目的: 构建预设CDR3基因的噬菌体抗体库, 通过抗原表位导向选择方法筛选抗人整合素ανβ3单克隆抗体(mAb)人源化Fab。方法: 将鼠mAb LCDR3重组到人轻链可变区文库中并与人/鼠嵌合重链Fd基因配对构建杂合噬菌体抗体库, 用固相人整合素ανβ3抗原筛选人源化轻链基因。再用所获人轻链基因与移植有鼠mAb HCDR3的人重链Fd基因配对构建人源噬菌体抗体库, 筛选人源化Fab。结果: 分别构建了库容为2.1×106和2×107的杂合噬菌体抗体库和人源噬菌体抗体库, 筛选到3株人源Fab克隆。经间接ELISA及竞争抑制ELISA证实, 能特异结合整合素ανβ3抗原, 其中人源D5株Fab克隆的基因序列表明, 人轻链可变区基因属VKIII亚群, 人重链可变区基因属VH1亚群。结论: 利用噬菌体抗体库技术, 成功地进行了鼠抗人整合素ανβ3 mAb E10人源化的改造, 为进一步临床应用奠定了基础。 　　［关键词］噬菌体抗体库; 整合素ανβ3; 人源化Fab 　　鼠源单