[Construction of phage antibody library with predetermined CDR3 gene and screening of humanized Fab of anti-human integrin alphanubeta3 monoclonal antibody].
OBJECTIVE
To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of anti-human integrin alphanubeta(3) 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 alphanubeta(3). 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 2.1x10(6), 2x10(7) colony forming units, respectively. Indirect ELISA and competitive inhibition ELISA analysis demonstrated that three humanized Fab antibody had specific binding activity with human integrin alphanubeta(3). The strongest anti-human integrin alphanubeta(3) reactive D5 clone was sequenced and sequencing analysis showed that the V(kappa) and V(H) were derived from VKIII and VHI, respectively.


CONCLUSIONS
Humanized Fab of anti-human integrin alphanubeta(3) mAb has been successfully obtained by phage display technology which lays the foundation for further clinical research.
Wang
Chen
C
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. wangchen2001@163.ccom
Hou
Li-hua
LH
Du
Gui-xin
GX
Li
Jian-min
JM
Chen
Pu-yan
PY
Tong
Yi-gang
YG
D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D039302:Integrin alphaVbeta3 / Q000276:immunology*; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D039302:Integrin alphaVbeta3; D019151:Peptide Library