Preliminary Study On The Construction, Expression And Function Of Genetically Engineered Anti-CD3×CD87 Bispecific Antibody

Background:Bispecific antibodies(BsAbs)have been considered as a promising immunotherapy strategy against tumors.In the treatment of cancer,BsAbs are constitutive of the variable light(VL)and heavy chains(VH)of two monoclonal antibodies,which recognize two different antigens present on T cells and tumor surface.CD3 is an antigen that is expressed on the surface of T cells and the CD3 complex has the potential to trigger T cells.Therefore anti-CD3 bispecific antibodies are extensively studied.Studies have shown that the expression level of CD87 is related to the degree of tumor malignancy.Compared with normal tissues,CD87 is over-expressed in human malignant tumor tissues.Thus we choose CD87 as another target for single chain bispecific antibody(scBsAb).Method:First,the heavy and light chains of the anti-human CD3 monoclonal antibody were cross-linked with the heavy and light chains of the anti-human CD87 monoclonal antibody by SnapGene Viewer software.The whole sequence of scBsAb was obtained by gene synthesis.The prokaryotic expression vector of pET24a/scBsAb was constructed and transformed into competent cells of E.coil.BL-21(DE3)by genetic engineering technique.After induction with IPTG,the bacteria were ultrasonicated,and scBsAb in the form of inclusion bodies were harvested by centrifugation.The scBsAb was further purified by affinity chromatography under denaturing conditions and refolded by dilution.The SDS-PAGE and Western Blotting were used to detect and identify the scBsAb protein.Second,the antigens binding activity of scBsAb was examined by flow cytometry.Furthermore,scBsAb-mediated cytotoxic activity was determined.In the killing process of scBsAb-mediated peripheral blood mononuclear cells(PBMCs)targeting CD87-positive tumor cells,level of IFN-y was determined by commercial ELISA kit.Results:Our results showed that scBsAb with high purity was obtained.The scBsAb could both specifically bind to CD87 and CD3.The scBsAb-mediated killing of PBMCs against CD87+PC-3 cells was related to the concentration of used scBsAb and the ratio of effector to target(E:T).When E:T was 10:1 and the scBsAb concentration was 100 ?g/mL,the killing rate was 40.86%;whereas when the E:T ratio was 40:1 and scBsAb concentration was 100 ?g/mL,the killing rate was 60.9%.In addition,ELISA assay showed that IFN-y level in the co-culture system was significantly increased during the killing process.Our data indicate that anti-CD3xanti-CD87 scBsAb could mediate PBMCs to kill CD87+tumor cells in vitro.Conclusion:In this study,we used Escherichia coli expression system to obtain anti-CD3×anti-CD87 scBsAb,and its biological activity was studied.We found that scBsAb was able to specifically bind to CD3 and CD87.In vitro studies have shown that scBsAb mediates the cytotoxicity of PBMCs in targeting CD87+tumors.This has laid the foundation for further in vivo experiments and the clinical application of anti-CD3×anti-CD87 scBsAb.