Primary targeting of recombinant Fv-immunotoxin hscFv_(25)-mTNFα against hepatocellular carcinoma

摘要

AIM: To obtain human recombinant Fv-immunotoxin hscFv_(25)-mTNFα (mutant human TNFα fused to human scFv_(25)) against hepatocellular carcinoma (HCC). METHODS: Two relevant sites of enzymatic digestion were added to mTNFα by PCR. mTNFα was linked to the 3′ end of hscFv_(25) in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv_(25)-mTNFα was expressed in Escherichia coli and purified from inclusions. After purified by glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot. And then, the purified recombinant Fv-immunotoxin was injected into nude mice with HCC xenografts through their tail veins. mTNFα protein and PBS were used as control at the same time. After treated for two weeks, nude mice were executed. The bulk and weight of tumors were observed. The tumor tissues were stained by immunohistochemical method with TNFα antibody. RESULTS: The expression ratio of recombinant Fv-immunotoxin hscFv_(25)-mTNFα was 12% of bacterial protein. The result of tumor restraining trials of hscFv_(25)-mTNFα showed 2/5 complete remission and 3/5 partial remission. mTNFα restraining trials showed 5/5 partial remission. The therapeutic result of hscFv_(25)-mTNFα was better than that of mTNFα (F=8.70, P<0.05). The hscFV_(25)-mTNFα remedial tumor tissues were positive for TNFα by immunohistochemical staining. The positive granules mainly existed in the cytoplasm of tumor cell. CONCLUSION: Recombinant Fv-immunotoxin hscFv_(25)-mTNFα has better therapeutic effect than mTNFα. It can inhibit the cellular growth of HCC and has some potential of clinical application.