[Construction and selection of human Fab antibody phage display library of extracellular domain of HER 2]

To construct the fully humanized anti-extracellular domain (ECD) of HER2 Fab fragment phage library, select antibodies against HER2 ECD specifically and identify its characteristics.

Peripheral blood monouclear cells (PBMCs) of breast cancer patients with HER2-overexpressing were immunized in vitro with purification protein of recombinant HER2 ECD and were then transformed by Epstein-Barr virus (EBV). After total RNA was extracted, the heavy chain Fd and k/λ light chain were amplified by RT-PCR. Following restrictive digestion with Sac I/Xba I and Xho I/Spe I, the light chain k/λ genes and heavy chain genes Fd were inserted into the phagemid vector pComb3 successively and then electroporated into E.coil XL1-Blue.The humanized Fab phage antibody library against HER2 ECD was constructed by infection of helper phage VCSM13.The libraries were enrich after panned three cycles by purification protein of recombinant HER2 ECD.Then random clones were tested by ELISA to select the positive ones, which were furher identified their antigen binding acticities by Western blot, and the strongest binding to HER2 ECD clone was sequenced.

The Fab phage antibody library with 2.5 x 10(7) volume was constructed and four positive clones which specifically recognized the HER2 ECD were isolated and further demonstrated by Western blot. Sequence analysis of the positivest clone showed that the variable heavy domains(VH) and variable light domains(VL) were highly homologous with the human embryonal Ig heavy chain V region sequences and kappa light chain sequences, respectively.

A fully humanized Fab phage antibody library is successfully constructed and specific antibodies against HER2 ECD are obtained, which provides an experimental foundation for new humanized anti-HER2 ECD monoclonal antibodies.