Preparation Of Anti-human CD86-scFv And Study Of Its Biological Characteristics

CD86, also called B7-2, mainly expressed in antigen-presenting cells, is one of theimportant costimulatory molecular of B7family. The ligand of CD86is CD28, whencombination with CD28can support a second set of signals for T cell activation andproliferation of T cells. CD86consists of306amino acids, belonging to type Itransmembrane glycoprotein.Single chain variable fragment (scFv) is a novel recombinant antibody, which includes thevariable heavy (VH) and variable light (VL) domain of an antibody. The VH domain is linked to theVL domain by a flexible polypeptide linker. The advantage of this kind of antibody is that it hassmaller size, reduced immunogenicity, a shorter half life time and improved penetration into a solidtumor. On the basis of obtained murine hybridoma cell1D1secreting anti-human CD86monoclonalantibody and human-mouse chimeric antibody against-human CD86, this research established theexpression plasmid of anti-human CD86-scFv and transfected into CHO cells. The cell line whichcan stably and constantly secret anti-human CD86-scFv has been obtained.Part I Construction and expression of anti-human CD86-scFvObjective: To construct and express the single chain variable fragment (scFv)of monoclonal antibody against human CD86, then select the cell line which canstably and constantly secret anti-human CD86-scFv.Methods: The VH and VL genes were amplified by RT-PCR from murinehybridoma cell line1D1which produced anti-human CD86monoclonal antibody(mAb). The CD86-scFv gene was constructed and integrated into eukaryotic expressingvector to construct pIRES2-EGFP/scFv. Then the plasmid was transfected into CHOcells with LipofectamineTM2000and efficiently expressed after G418selection. Tothe cell line expressing anti-human CD86-scFv stably by flow cytometry.Results: The CD86-scFv gene consist of780bp and include genes encoding thesignal peptide and linker gene. One stable expressing CD86-scFv cell line was obtained.The positive rate of culture supernatant and L929-CD86cells was64.8%. Conclusion: Anti-human CD86-scFv has been successfully expressed in CHOcells (named SA-IV), and it provides the material basis for the future study of thebiology activity of the antibody.Part II Analysis the biological characteristics of anti-humanCD86-scFvObjective: Prepare anti-human CD86-scFv and analysis its biologicalcharacteristics.Methods: Eukarya expression cell line SA-IV had been cultured and culturesupernatant was collected. The CD86-scFv was obtained through IMAC affinitychromatography. The experiment evaluated the potency and analyzed the identificationof CD86-scFv to membrane CD86. Then, the identification of CD86-scFv binding tomembrane CD86was performed through competitive inhibition assay. The inhibitoryeffect of anti-human CD86-scFv on proliferation of Raji cells in vitro was detected byMTT. Raji cells were incubated with CD86-scFv, and the cells were transplanted intoBALB/c nude mice, then the inhibition tumorigenesis of Raji was observed in vivo.Results: About4.2milligram CD86-scFv were purified from culture supernatantand its positive binding rate with L929-CD86cells, Raji cell and Daudi cells is67.0%、72.3%and80.5%, respectively. Moreover, CD86-scFv could inhibit the growth of Rajicells and the inhibition rate was28.3%. In addition, the apoptosis rate of Raji cells was11.2%. Raji cells were incubated with CD86-scFv, and then cells were transplanted intoBALB/c nude mice. Compared with the control group, experimental group micetumor onset delayed for6-8days. The average volume of the tumor was smaller thancontrol group.Conclusion: Anti-human CD86-scFv can combine with corresponding antigen.CD86-scFv can inhibit the growth of tumor cells both in vitro and in vivo.