Preparation Of Anti-human CD80- ScFv And Diabody And Study Of Their Biological Function

CD80,also called B7-1,is one important costimulatory molecule of B7 family.Human CD80 molecule,mainly expressed in APC such as dendritic cells, mononuclear phagocytes and B cells,is a 44-54kDaⅠtype transmember protein, whose encoding genes are directed at human chromosome 3q13.3-q21, containing 1491bp. CD80 ligand is CD28 and CTLA-4.While combination with CD28 can support a second set of signals for T cell activation and promote the proliferation and differiation of T cells,bind with CTLA-4 strongly diminishs T cells response but maintains T cell homeostasis.If T cells activation lacks the second signals,there is a serious problem that antigen peptide-MHC complexes can not activate specific T cells effectively,but conversely make T cells anergy or tolerance.Now it was demonstrated that CD80 is not merely the critically regulatory molecule in the course of T cell activiation,but involved in many diseases.Single-chain variable fragment(ScFv) is a small recombinant antibody fragment, containing the complete antigen binding site, which includes the variable heavy (VH) and variable light (VL) domains of an antibody. The VH domain is linked to a VL domain by an introduced ?exible polypeptide linker,whose length is commonly 10-25 amino acids.The common peptide linker is (Gly4Ser)3.Diabody,a dimer of ScFv,is formed by shorting the polypeptide linker between VH and VL to 3-10 amino acids,which can promote pair between intermolecules. Objective: To construct and express the single chain variable fragment (ScFv) of monoclonal antibody against human CD80 and study its biological functions.Methods: The VH and VL genes were cloned by RT-PCR from a murine hybridoma cell line 4E5,which produces monoclonal antibody (McAb) aginst human CD80 antign.Splicing by overlapping extension PCR (SOE-PCR) was used to splice the VH and VL genes to construct L-VH-Linker-VL CD80-ScFv genes, which were cloned into the eukaryotic expressing vector pIRES2-EGFP. CHO cells were transfected by pIRES2-EGFP/ScFv plasmids through the liposome-mediated method and selected by G418.Anti-human CD80-ScFv was purified from culture supernatant.Then the experiments evaluated the potency and analyzed the identification of ScFv to membrane CD80;competitive inhibition experiment was applied to analyze the binding ability of anti-human CD80-ScFv with the corresponding antigen. The inhibitory effect of anti-human CD80-ScFv on in vitro proliferation of Raji cells and the effect of ScFv on costimulatory signals mediated by CD80 were detected by MTT methods.Results: The ScFv genes consist of 828bp and include genes encoding the signal peptide and linker. The highly secreting and stable cells were harvested. About 15.12 milligram ScFv antibodies, whose relative potency is 2.0μg/5×105cells,were purified from one liter culture supernatantat,and its positive binding rate with Raji and Daudi is 96.6% and 95.0% respectively and cannot bind with Jurkat cells.The competitive inhibition rate of anti-human CD80-ScFv against murine parent antibody 4E5 is 98.67%,and anti-human CD80-ScFv could inhibit growth of Raji cells in vitro.In addition to them ,it can also make the proliferation rate of PBMC decline by 43.48% via blocking costimulatory signals mediated by CD80.Conclusion: Anti-human CD80-ScFv has been successfully expressed in CHO cells(named SA-Ⅱ) and could identify membrane CD80 molecule in the cell surface and mediate the related biological functions. Its preparation possess an important value on fundamental and clinical research of CD80 and related molecules.To construct and express anti-human CD80 bivalent dimer(diabody) and study its biological functions.Methods: VH and VL genes were cloned by PCR,and then cloned into the eukaryotic expressing vector pIRES2-EGFP to construct recombinant pIRES2- EGFP/ diabody. CHO cells was transfected by above plasmids through the liposome-mediated methods and selected by G418 to harvest stably highly secreting cell lines.Anti-human CD80-diabody was purified from culture supernatant by Ni catridges.Reducing and non-reducing SDS-PAGE was used to analyze its bivalent.Next the potency of diabody was detected by FCM.Then the experiments analyzed the identification of diabody to membrane CD80 and competitive inhibition against parent antibody 4E5 via immunofluorescence and FCM ; The effect of anti-human CD80-diabody on proliferation of Raji cells which express CD80 higtly and naturely,was determined by MTT methods and we also researched blockade of diabody on CD80-mediated costimulatory signal simultaneously.Results: It was demonstrated that pIRES2-EGFP/diabody was constructed successfully,concluding 798bp.One cell lines named SA-Ⅲ,which can secret the diabody stably were generated.About 15 milligram diabody were purified from one liter culture supernatantat.Mr of the dimer was about 53kDa(anti-human CD80-ScFv Mr is about 27kDa).And the potency of anti-human CD80-diabody is 1.5μg/5×105 cells in comparation with murine parent antibody 4E5,and its positive binding rate with L929-CD80,Raji and Daudi cells is 96.5%,98.1% and 93% respectively,but diabody don't bind with Jurkat cells.The diadoby not only can inhibit combination of 4E5 with CD80 antigen and its inhibition rate is 97.11%,but also inhibit in vitro proliferation of Raji cells and corresponding rate is 31.27%.Finally the antibody can also block costimulatory signal.Compared with ScFv,the affinity of diabody has improved obviously.Conclusion: Anti-human CD80-diabody secreting cell SA-Ⅲwas generated and the diabody have good biological function. The affinity of diabody improved clearly.All those supported a base for future study of CD80.