Preparation And Preliminary Application Of Single-chain Fv Dimers For Hepatocellular Carcinoma

Background Hepatocellular carcinoma(HCC) is the fifth most common cancer worldwide and the second cause of cancer-related death in our country.Traditional therapies,such as resection,chemotherapy and radiotherapy have not satisfactional efficacy.Single chain variable fragments(scFv) can be a good vector of targeted therapy for HCC as a result of their small molecule weight,strong tumor tissue penetration,weak immunogenicity and short half-lives in blood,as well as possessing the same binding ability and specificity as the whole antibody.My work team had obtained a scFv against hepatocellular carcinoma(scFv 4-16,GenBank:DQ640759) through the phage display antibody library technology.After reconstruction of affinity maturation and humanization of scFv,we have attained a scFv with high affinity to HCC and low immunogenicity.However,scFv is monovalent antibody with the shortcoming of low affinity,bad stability,too rapid clearance from the circulation and small-scale preparation in bacterial expression system.This study is to construct the non- covalent single-chain Fv dimers for Hepatocellular carcinoma,express the dimmers in Yeast pichlia pastores,prepare the immunonanoparticles and investigate their effects on HCC cells.It may provide an evidence for hepatocellular carcinoma targeted diagosis and therapy in vovo in the future.Chapter 1 Construction,expression and assay of single-chain Fv dimers for hepatocellular carcinoma in E.coll.Objective:To construct single-chain Fv dimers for hepatocellular carcinoma,express in E.coli and assay the biological activities.Methods:We had reported the construction, screening and humanization of the single-chain Fv for HCC.The linker between VH and VL genes were shortened to 3～5 amino acid residues,cloned into the vector of pCANTABSE.The recombinant vector plasmids were transformed into TG1 cells and sequenced.The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibody.ELISA was used to analyze the binding activity of phage to HCCs.HB2151 were infected with the phage antibodies which had the best binding affinity to hepatocellular carcinoma to express single-chain Fv dimers. Expressed products were identified by SDS-PAGE,West-blotting and ELISA.Results: Three single-chain Fv dimers genes(scFv-3,scFv-4,scFv-5) had been constructed successfully with the binding ability 3.5-6 folds to hepatocellular carcinoma comparing with their parental scFv.The single-chain Fv dimmer(scFv-5,termed BDM3) with the best binding ability was expressed.There was a strip considered to be aim protein in the results of SDS-PAGE and Western blotting.The concentration of BDM3 was only 100μg /L,which was not enough for the following research.Conclusions:The single-chain Fv dimers had been constructed and expressed successfully in E.coli.The expressed products have better antigen binding activity.That was a basic work for the following experiments.Chapter 2 Expression and assay of single-chain Fv dimers for hepatocellular carcinoma in Yeast pichlia pastores.Objective:To express dimers BDM3 in Yeast pichia pastores and assay its biological activities.Methods:We had constructed pGAPZαA-BDM3 and transformed it into DH5α.The recombinant vector plasmids were sequenced and transformed to Yeast pichlia pastores GS115.BDM3 was expressed in Yeast pichlia pastores.Expressed products were identified by SDS-PAGE,Western blotting,size exclusion gel chromatography(SEC),ELISA and immunohistochemistry.Results:SDS-PAGE and Western blotting had shown that the dimers BDM3 was successfully expressed in Yeast pichlia pastores.The SEC results suggested the molecular weight of the expressed products were about 62 kD with 92%dimerization.The concentration of BDM3 was 30mg/L,which was 300 times as many as the products in E.coli.Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv,even after 16 h incubation at 37℃still having 50%binding activity.The purified dimmers were specially binding to tumor antigen of HCC.Conclusions:The functional scFv dimers against hepatocellular carcinoma was achieved,and it was essential to make use of the dimers in hepatocellular carcinoma targeted diagosis and therapy. Chapter 3 Preparation and assay of immunonanoparticles and investigation of their effects on HCC cellsObjective:To obtain and identify the polyelectrolyte nanoparticles,and investigate the effect of BDM3-loading nanoparticles on HCC cells.Methods:By means of IR and TEM,the physicochemical characteristics of the polyelectrolyte nanoparticles were manifested,including the the particle size,morphology and the concentration of loaded protein on the particle size,protein entrapment efficiency of the nanoparticles. Furthermore,we investigated the effect of BDM3-loading nanoparticles on HCC cells. Results:The particle size is ranged from 100nm to 200nm and has a suitable concentration of loaded protein on the particle size with a 53%entrapment efficiency of BDM3.There was a significant inhibition of BDM3-loading nanoparticles on HCC cells growth.Conclusions:A targeted BDM3 nanopaticle has been obtained and it is a basic work for HCC targeted diagosis and therapy in vivo in the future research.