Construction of phage antibody library with predetermined CDR3 gene and screening of humanized Fab of antiª²human integrin ¦Á¦Í¦Â3 monoclonal antibody

    WANG Chen1, 2, HOU Liª²hua2, DU Guiª²xin2, LI Jianª²min2, CHEN Puª²yan1, TONG Yiª²gang2*

    1College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095; Beijing Institute of Microbiology & Epidemiology, Beijing 100071, China

    [Abstract] AIM: To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of antiª²human integrin ¦Á¦Í¦Â3 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 ¦Á¦Í¦Â3. 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¡Á106, 2¡Á107 colony forming units, respectively. Indirect ELISA and competitive inhibition ELISA analysis demonstrated that three humanized Fab antibody had specific binding activity with human integrin ¦Á¦Í¦Â3. The strongest antiª²human integrin ¦Á¦Í¦Â3 reactive D5 clone was sequenced and sequencing analysis showed that the V¦Ê and VH were derived from VKIII and VHI, respectively. CONCLUSION: Humanized Fab of antiª²human integrin ¦Á¦Í¦Â3 mAb has been successfully obtained by phage display technology which lays the foundation for further clinical research. ......