Humanization of mouse anti-human bladder cancer McAb by CDR3 directed phage antibody library
Objective To humanize an murine anti-human bladder cancer monoclonal antibody BDI. Methods Using overlap PCR and DNA recombination technology, the murine V H and Vκ CDR3 of BDI were spliced into human diverse Fd and κ genes and cloned into a phage antibody expression vector pAL, in which the Fd is flanked by two non-homologous loxp sites, forming a CDR3-incorporated human phage antibody library. Through cre-loxp mediated recombination, the Fd and κ chains at different vectors were shuffled and a large human antibody library was constructed. Panning of the phage antibody library against bladder cancer EJ cells were conducted to select humanized BDI antibodies. The antigen binding activity was determined by ELISA. Results A primary BDI CDR3-incorporated human phage antibody library 2×10 7 of in size was constructed, and a large antibody library of 1×10 10 in size was obtained usingcre-loxp recombination system. Human anti-bladder cancer antibodies were successfully cloned by panning of the large phage antibody library against bladder cancer cells. Binding competition studies demonstrated that they bound the same epitope of the Ag as the parental Ab. Conclusion Using CDR3 directed phage antibody library technique, human counterpart of murine McAb could be selected without the parental murine V region as a template.