The Preparation And Characterization Of An Anti-CD147 Human IgG1

BackgroundCD 147(cluster of differentiation 147)is a transmembrane glycoprotein highly expressed on many types of tumor cells,and it was reported to participate in various diseases development.Research in our laboratory has found that knocking down CD 147 expression significantly enhanced the effects of trastuzumab on HER2-positive breast cancer cells,which suggest CD 147 is a valuable therapeutic target for cancer and autoimmune diseases.At present,mouse monoclonal antibodies against CD 147 have already been used for the targeted therapy of liver cancer.Though human antibodies are superior to murine monoclonal antibodies in vivo therapeutic applications,no relevant reports have been reported yet.ObjectiveA fully human antibody phage display library constructed by our laboratory was used for screening of specific human anti-CD 147-scFvs.A human anti-CD147 IgG1 antibody was then constructed and characterized.MethodsThe anti-CD147-scFv was screened from the phage library by acid elution.After the specificity of the anti-CD 147-scFvs selected were characterized by regular ELISA assay,DNA sequence analysis,cell ELISA and competitive inhibition assay i.There relative affinity was determined via ammonium thiocyanate elution method.Then an eukaryotic expression vector of human anti-CD 147 IgG1 was then constructed via recombinant DNA technology.The fully human anti-CD 147 antibody was expressed in eukaryotic cells,and the antibody was purified from the supernatants by affinity chromatography,and the purity was checked by SDS-PAGE.The binding specificity to human anti-CD147 IgGl was determined by ELISA and Western Blot.Finally,the effect of the antibody on the proliferation of breast cancer cells was analyzed by the CCK-8 method.Results1.After four rounds of enrichment screening,phage antibody was enriched by 750 times;three anti-CD 147-scFv clones(No.13,No.15 and No.168)with different genotype sequences were obtained through regular ELISA assay,sequencing analysis,cell ELISA and competitive inhibition assay identification.Their light and heavy chains of variable regions belong to VλⅠ/VHⅠ,VκⅢ/VHⅢ,and VλⅢ/VHⅢ.Their specificity was further verified after soluble expression of the anti-CD 147-scFvs.The relative affinity indexes(RAI)of the three anti-CD 147-scFvs were 0.2 mol/L,0.35 mol/L and 0.6 mol/L,respectively,measured by ammonium thiocyanate elution method.The relative affinity of 168 was better than those of No.13 and No.15.2.The VH and Vκ/λ of the three anti-CD 147-scFv clones were amplified by PCR and they showed correct band sizes on agarose gel electrophoresis.After further verification of the DNA sequence of three anti-CD 147-scFv clones,IgG1 expression vectors of the fully human anti-CD 147 were constructed successfully.3.The light and heavy chain eukariatic expression vectors were transfected into HEK293F cells in an equal proportion.After 3 days culture in serum-free culture medium,intact human antibody expression in the supernatant was shown by ELISA assay.The antibody was purified from the supernatants on day 7 by affinity chromatography with a purity of>95%as shown by SDS-PAGE.The binding specificity of the antibody to CD-147 antigen was verified by ELISA and Western Blot.4.An inhibitory effect of No.168 human anti-CD147 IgGl on the proliferation of breast cancer cells was demonstrated by CCK-8 method.ConclusionThe specific human anti-CD 147 scFvs have been obtained by screening of a fully human antibody phage display library.After the verification of their specificity,the human anti-CD 147 IgGl has been expressed in eukaryotic cells with the ability to inhibit the growth of breast cancer cells.