Anti-hepatoma Fully Human Single-chain Fv And Adriamycin Immunoconjugate Preparation And Its Antitumor Effect

Objectives:To construct anti-hepatoma ScFv-SA3protein, prokaryotic expression vector pET21a (+) was used. Oxidized Dextran T10(Dex-T10) was used as intermediate bridge for drug Adriamycin (ADM) and antibody ScFv-SA3. The conjugate or mixture of drug and antibody were compared to the free drug for their effect in vitro and in vivo. Conjugate was tested for its capacity of suppression cells proliferation and tumor development.Methods:To get the antibody ScFv-SA3, prokaryotic expression vector pET21a (+) and PCR were used. Protein expression was done using E.coli BL21(DE3) previously transformed with above recombinant pET21a (+) plasmid, and then induced by IPTG Protein were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).The soluble protein was purified by His-Tag Ni-Agarose under the natural condition for western blot analysis and ELISA. The specifically bonding to HepG2cells was tested by Immunocytochemistry. Oxidized Dextran T10(Dex-T10) was used as intermediate carriers for conjugates drug ADM and ScFv-SA3antibody.The activity of ScFv-SA3in conjugate was tested by indirect ELISA. Its antitumor effect was tested by MTT, plate colony formation assay as well as xenograft model of hepatocellular carcinoma in nude mice.Results:The prokaryotic expression plasmid pET21a (+)-SA3was successfully constructed and ScFv-SA3was expressed in E.coli BL21(DE3). We got the purified ScFv-SA3of40KD and Western blot show that ScFv-SA3was expressed correctly. The activity of ScFv-SA3was verified by ELISA and Immunocytochemistry; it can bond to the HepG2cells. Immunoconjugate ADM-Dex-ScFv-SA3was prepared and the mole ratio of ADM to antibody was1:14.21,. In vitro experiments revealed that ADM-Dex-ScFv-SA3inhibited the HepG2cells proliferation and colony formation ability of the cells, toxicity to non-target cells is less than the free ADM. In vivo, ADM-Dex-ScFv-SA3has effect on suppression of tumor growth of tumor-bearing mice and the median survival time of mice in conjugate group is longer than the others.Conclusions:1. The prokaryotic expression plasmid pET21a (+)-SA3was successfully constructed and natural fully human single-chain Fv ScFv-SA3was expressed.2. Immunoconjugate ADM-Dex-ScFv-SA3was prepared.3. ADM-Dex-ScFv-SA3inhibited the HepG2cells proliferation in vitro; In vivo, tumor growth of tumor-bearing mice was suppressed and the median survival time of mice was prolonged.