Construction & Seclection Of Na(?)ve Phage Display Antibody Library And Characterization Of Anti-DR5 Monoclonal Antibodies

Since the mid-1990s, antibodies have emerged as important new drug class. As therapeutics, human antibody can overcome a number of drawbacks of murine monoclonal antibody, including human anti-mouse antibody reaction (HAMA), short circulating time in vivo, unable to induce ADCC and CDC.Antibody phage display technology is one of the most powerful tools for production human antibodies. Library size is a major determinant in successful selection against any potential antigen and it also correlates with the affinity of the isolated antibodies. Therefore, using modern molecular biology methods, we constructed a large na?ve human ScFv antibody phage library based on Cre/Loxp recombination system. Subsequently, the specific anti-DR5 antibodies derived from the library were expressed in prokaryotic and eukaryotic systems, and their characters were analyzed. In detail, the research results were shown as following:Construction and selection of the na?ve phage display antibody libraryDiverse VL and VH genes were amplified by RT-PCR from lymphocytes collected from adult peripheral blood and newborn cord blood (>180). The V genes were spliced to form ScFv by overlap PCR and cloned into the vector pDF. After transferring phagemid into XL1-BLUE, the primary library was generated. By infecting the phagemid into Cre+ bacteria BS1365 at high multiplicity of infection (MOI), the VH genes were recombined to create a very large phage antibody library. Finally, the secondary repertoire was obtained by infecting XL1-BLUE at low MOI. The results showed that the variable genes were amplified effectively, the size of primary library was 3.6×10⁷, and the final repertoire was calculated to have a diversity of 1.8×10¹¹. Based on Cre/Loxp system, a very large phage antibody library was constructed successfully. To validate the library, five different antigens (i.e. B Lymphocytes stimulator (Blys), death receptor 5 (DR5), tumor necrosis factors-α(TNF-α), Interleukin 6 (IL-6) and Ricin) were chosen and screened respectively. After four round panning, the specific human ScFvs against all the 5 tested antigens were obtained. Furthermore, the detailed sequence analysis results demonstrated that the acquired variable genes were included various subfamily, which suggested the phage antibody library possessed good diversity and could be used for preparation human antibody potentially.Expression and characterization of the specific anti-DR5 antibodiesTo reduce the expense and the cycle of experiment, using bioinformatics theory and computer-guide molecular modeling method, the anti-DR5 mABs derived form library were evaluated theoretically based on multi-sequence alignments and conformational stability of the antibody Fvs. Considering the special binding experimental results, the three factors (i.e. conformational stability, binding activity and occurrence frequency) were chosen as theoretical screening method and the four antibodies (i.e. C11, B2, A2 and A8) were selected to further experiment.The four recombination ScFv antibody genes were inserted into pET28a vector and expressed in BL21 strain. The results showed that the ScFvs were expressed effectively. Furthermore, Using ELISA and Western Blot methods, the expressed ScFvs in supernatant after ultrasonic, could recognize the purified DR5 antigen. The result showed that the ScFv mAbs derived from library possessed the ability to recognized and bind DR5 antigen.To express whole antibody, the VH & VL variable genes of the four antibodies were respectively subcloned into pCMV-Express, a eukaryotic express vector, and transiently transfected into 293T cell. The Western blot experiments showed that the whole antibody molecules were expressed in supernatant, and could be recognized specifically by goat-anti-human IgG-HRP. Moreover, the expressed antibodies could specially bind the DR5 antigen transfected in NC membrane. The competition ELISA experiment revealed that the antibodies could be competed in concentration dependence with the natural ligand TRAIL.