Epidermal Growth Factor Receptor VⅢ Antibody Development And Application In Hepatocellular Carcinoma Therapy

EGFRvⅢhave only been observed in cancer but not normal tissues which make it an ideal target for cancer therapy.Antibody therapy has been developed to be potential option for treating cancer.In this study, we developed a monoclonal antibody 12H23 that can specifically recognize EGFRvⅢand also overexpressing EGFR.Additionally,we found EGFRvⅢhave played an important role in HCC tumorigenesis and 5-FU resistance. Hence we tried to use the anti-EGFRvⅢmAb 12H23 for therapy of HCC.The results shown that mAb 12H23 can inhibit EGFRvⅢpositive Huh-7 growth in vivo,and enhance therapeutic effect of 5-FU,which make it a promising agent of HCC therapy.Thereafter,we succefully made the chimeric antibody C12 derived from mAb 12H23.The CHO cell line stably expressing chimeric antibody C12 with a yield of 20 pg/cell/day.Moreover,mAb C12 remains most of the binding affinity and specificity of mAb 12H23.Thus,mAb C12 is promising for the preclinical study.SectionⅠEGFRvⅢtargeting antibody development【Objective】To obtain candidate anti-EGFRvⅢantibodies.【Methods】Recombinant EGFRvⅢand NIH-3T3-EGFRvⅢcell line were used for immunization of BLAB/c mice.Anti-EGFRvⅢantibodies were obtained by fusion of spleen B cell with SP2/0 cell and following hybridoma cloning. Western blot,immuno-staining and FACS were applied to identify antibody specificity.In vivo fluorescent imaging system was used to monitor the in vivo distribution of cyanin 5.5 labeled mAb 12H23.Antibody affinity was measured by SPR analysis while antibody subtype and epitope were identified by Elisa analysis.【Results】An antibody clone 12H23 was obtained by hybridoma screening.In FACS assay,the antibody has strong binding to U87-EGFRvⅢ(stably expressing EGFRvⅢ) cells,a relative week binding to A431 cell(overexpressing EGFR),while has no obviously binding to Wt U87 cell.Results from the in vivo fluorescent imaging clearly showed the accumulation of mAb 12H23 to U87-EGFRvⅢxenograft but not U87 xenograft.Further study showed mAb 12H23 is an IgGl subtype, and its binding epitope is located in CC16 peptide(287aa-302aa of EGFR). SPR analysis also showed that 12H23 bind the epitope at a dissociation constant Kd=1.73×10^-10M.【Conclusion】We have successfully obtained a candidate antibody targeting EGFRvⅢ.SectionⅡEpidermal Growth Factor Receptor vⅢenhance the tumorigenicity and 5-FU resistance in Human Hepatocellular Carcinoma【Objective】To determine the biological significance of EGFRvⅢin HCC including tumor progression and chemotherapy resistance.【Methods】To delineate biological characteristic of EGFRvⅢin human HCC cells, different HCC cell lines were evaluated for EGFRvⅢexpression,cell growth and drug resistance.At the same time,EGFRvⅢexpression cassette was introduced by lentivirus vector into representative Huh-7 cell line. The EGFRvⅢ-expressing transfectant was compared with its parent cell for cell proliferation and 5-FU induced apoptosis resistance.【Results】Expression of EGFRvⅢin Huh-7 cells produced constitutively activated EGFRvⅢreceptors.And the EGFRvⅢtransfectants exhibited an about 1.5 fold in cell proliferation in vitro and 2.7 fold in vivo.Huh7-EGFRvⅢcells also showed about 3 fold increase of migration when quantified by transwell migration assay.In addition,Huh7-EGFRvⅢshowed significantly contribution to 5-FU resistance in vitro and in vivo.【Conclusion】EGFRvⅢnot only plays a pivotal role in tumorigenicity of human hepatocellular carcinoma but also contribute to the 5-FU resistance of HCC.SectionⅢAn anti-EGFRvⅢantibody 12H23 as an effective therapeutic agent solely or combining with 5-FU in Human Hepatocellular Carcinoma【Objective】To examine the therapeutic effect of anti-EGFRvⅢantibody 12H23 solely or combining with 5-FU on Human Hepatocellular Carcinoma therapy.【Methods】MTT assay were performed of evaluate effect of mAb 12H23 or combination with 5-FU on HCC cells and HCC tumor xenograft therapy with mAb 12H23 or combination with 5-FU also been performed for in vivo effects. The enzymes correlated with 5-FU(TS,DPD,OPRT) and transcription factor E2F-1 in the cells treating with the indicated drugs also were examined.【Results】mAb 12H23 can strongly inhibit the growth of Huh7-EGFRvⅢxenograft in vivo.Additionally,12H23 and 5-FU have synergetic growth inhibition effect on the Huh7-EGFRvⅢboth in vitro and in vivo.mab 12H23 treatment on Huh7-EGFRvⅢcan downregulate the expression of E2F-1,TS and DPD while upregulate that of OPRT.【Conclusion】Antibody 12H23 have potential to be used as therapeutics solely or combination with 5-FU for the treatment of EGFRvⅢ-positive HCC.mAb 12H23 treatment can downregulate the expression of E2F-1,TS and DPD while upregulate that of OPRT.SectionⅣEngineering and characterization of chimeric monoclonal antibody(C12) for EGFRvⅢtargeting therapy 【Objective】To produce chimeric mAb derived from 12H23.【Methods】Genes encoding variable fragments were amplified from 12H23 hybridoma,and cloned into expression vector carrying constant region of human heavy chain and light chain.After transfection,CHO-DG44 cell line stably expressing the chimeric antibody named as C12 was selected with methotrexate(MTX).Thereafter,immunostaining and ELISA were applied to analyze the bingding characteristics of the chimeric antibody C12.【Results】The vector encoding the chimeric antibody C12 was successfully constructed and introduced into CHO cells.CHO cells can stably express C12 with a yield of 20 pg/cell/day.Additionally,C12 showed almost the same binding affinity and specificity as its parent antibody 12H23.【Conclusion】The chimeric monoclonal antibody C12 derived from 12H23 was successfully generated.C12 remains most of the binding affinity and specificity of 12H23.