Aim: To obtain human recombinant Fv-immunotoxin hscFv(25)-mTNFalpha (mutant human TNFalpha fused to human scFv(25)) against hepatocellular carcinoma (HCC).
Methods: Two relevant sites of enzymatic digestion were added to mTNFalpha by PCR. MTNFalpha was linked to the 3' end of hscFv(25) in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv(25)-mTNFalpha 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. MTNFalpha 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 TNFalpha antibody.
Results: The expression ratio of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was 12% of bacterial protein. The result of tumor restraining trials of hscFv(25)-mTNFalpha showed 2/5 complete remission and 3/5 partial remission. mTNFalpha restraining trials showed 5/5 partial remission. The therapeutic result of hscFv(25)-mTNFalpha was better than that of mTNFalpha (F=8.70, P<0.05). The hscFv(25)-mTNFalpha remedial tumor tissues were positive for TNFalpha by immunohistochemical staining. The positive granules mainly existed in the cytoplasm of tumor cell.
Conclusion: Recombinant Fv-immunotoxin hscFv(25)-mTNFalpha has better therapeutic effect than mTNFalpha. It can inhibit the cellular growth of HCC and has some potential of clinical application.