Screening Human Single Chain Fv Antibodies Against Hepatocellular Carcinoma And Determining Their Affinity

Hepatocellular carcinoma (HCC) is a common disease in certain areas of the world, including subSaharan Africa, southeast Asia, Oceania and China and is responsible for at least 1.3 million deaths every year. It is clear that new treatment modalities must be developed and gene therapy is a leading candidate. A major challenge for non-viral gene delivery systems is the efficient targeting of these genes to the tumor tissue. One strategy to provide non-viral vectors with affinity for certain tissues or organs is the construction of complexes of DNA with ligands for specific receptors such as the asialoglycoprotein receptor, whose expression is limited to hepatocytes and HCC cells. Many gene-transfer methods have been developed that adopt the asialoglycoprotein receptor-mediated endocytosis pathways for the delivery of DNA into liver, such as asialofetuin, galactosylated liposomes, and galactosylated cationic liposomes for drug or gene delivery to the liver, especially to HCC. But this gene transfer efficiency is not sufficient partly due to the size of the ligand. The idea of reducing the size of the ligand ("minimal ligands") has already been exploited for the development of this gene transfer system.The specificity of the receptor for D-galactose or D-galactoseamine is accomplished by specific hydrogen bonding of the 3 and 4-hydroxyl groups with the carbohydrate recognition domain. The crystal structure of H1 subunit showed that three calcium ions form an integral part of the structure, which indicates its calcium-dependence for the carbohydrate-recognition. A minimal 600 bp proximal region of the major subunit of the mouse ASGPR exhibits hepatic-specific promoter activity in HepG2 cells. A functional mimic of the CRD has been developed by modification of the domain amino acid residues. The modified CRD displayed 40-fold preferential binding to N-acetylgalactosamine compared with galactose, making it a good functional mimic for ASGPR.The use of a single chain antibody domain for H1 subunit recognition allows the construction of smaller DNA carriers and could reduce the immunogenicity of the final protein-DNA complex in in vivo applicationsIn this study we described the use of a recombinant fusion protein ASGPR containing the H1 subunit of human asialoglycoprotein receptor to screen a phage-display library. Using capture phage ELISA to select anti-ASGPR scFv antibody by screening human single fold scFv library and compare with indirect phage ELISA. The selected phages were induced to express scFvs and determined affinity.Objective: To identify the scFv antibody fragments specific for asialoglycoprotein receptor by biopanning based on recombinant proteins rCRDH1 from a large human naive scFv phage display library. Using capture phage ELISA to select anti-H1 scFv antibody by screening human single fold scFv library and compare with indirect phage ELISA. Through the soluble expression and purification of human single chain Fv antibody against human hepatic asialoglycoprotein receptor and to determining the affinity constant of the purified W1 scFv to fine a potential way suitable for the gene targeting therapy of HCC in vivo.Methods: According to the gene sequence of H1, the primers were designed. The full length cDNA encoding H1 amplified from pEA1 by PCR were subcloned into an preukaryotic expression vector (pET-32c). A single colony of E. coli BL21 containing the plasmid H1/pET-32c was inoculated LB broth, then diluted 1/100 into 1000ml LB broth and induced with 1mmol/L IPTG. The recombinant H1 was purified with Ni²⁺ chelating HiTrap HP column. Recombinant H1 was used to immune the rabbit for preparing polyclonal antibody.The amplified H1 cDNA from H1/pET3b was subcloned into prokaryotic vector pET-32c. Expression was induced by IPTG. The recombinant CRDH1 was purified with Ni2+ chelating HiTrap HP column. After four rounds of biopanning by capture phage ELISA with recombinant H1 and Trx-His-s tag, respectively, the specific anti-H1 phage clones were transfected into E.coli HB₂₁₅₁, and induced for secreted expression of scFv antibody with IPTG. The positive colonies were sequenced, expressing,purified and identified using Western Blot.The specific anti-ASGPR phage clone W1 was transfected into E.coli HB2151. The single colony was chosen to be inoculated into 2×TY medium and shaken (250 r/min) overnight at 37℃. After 1 in 100 dilution in 2×TY medium and induced for secreted expression, scFv W1 was induced at different concentrations (0.25, .5 and 1.0 mmol/L IPTG) overnight at 37℃, 25℃ and 20℃, respectively. The supernatant was precipitated with saturated ammonium sulfate and its sediment was analyzed by SDS-PAGE. In addition, the sediment was resuspended in 30mL PBS and dialyzed against PBS overnight at 4℃. The expressed scFv W1 was purified by Ni2⁺ chelating HiTrap HP column and the purity of the purified scFv W1 was identified by SDS-PAGE. Then affinity constant of scFv W1 was determined by noncompetitive enzyme immunoassay.Results: The recombinant H1 protein about 50.3kDa was expressed in E.coli as inclusion body; rASGPR was prepared with Ni²⁺ column purification; The polyclonal antibody was also successfully prepared by rASGPR and was proved by the methods of Western blot analysis of 6his-H1 and GST-H1 recombinant protein.The recombinant ASGPR protein about 35 000 was expressed in inclusion bodies in E.coli, rASGPR was prepared with Ni2+ column purification. Forty-four positive colonies were obtained after four rounds of biopanning, there were only one colony can also react with Trx/His tag, 9 of the rest 45 colonies showed differences in nucleotide sequence which induced with IPTG to produce soluble scFv antibody. Purified scFvs can also only reacted with rASGPR. so using this methods, the false positive rate is only 2%, otherwise, using indirect phage ELISA, this figure is 58%.After induced with 0.5mmol/L IPTG overnight at 25℃, the amount of expressed scFv W1 increased grently and its Mr was about 28 000, existed in culture supernant in soluble form. The purity of scFv W1 by nickel-agarose column was above 95% and its yield was about 0.8mg/L. The affinity constant of the purified scFv W1 was confirmed to be (2.31±0.36) ×10^-7M.Conclusions: High-level expression of ASGPR was achieved in E. coli, ASGPR expression can now be monitored in vitro and in vivo gene transfer studies with the the polyclonal antibody.Capture phage ELISA can be used to exclude false positives caused by Trx/His tag of the recombinant protein which coated as antigen to screen a phage library. Using this methods, total 9 anti-ASGPR specific scFv antibodies have successfully and quickly screened out.The E.coli HB2151 infected with phage W1 clones may express soluble scFv W1 with low affinity, which has potential applications to gene therapy of hepatocellular carcinoma .