Objective: To obtain humanized engineering bifunctional antibody, which has potentialities for clinical application.
Methods: Humanized anti-human hepatocellular carcinoma (HCC) single chain fragment (hscFv25) was linked with human TNF-alpha gene to form anti-HCC bifunctional antibody, then it was subcloned into prokaryotic GST fusion expression vector pGEX 4T-1 and expressed in the host E.coli. Indirect immunofluorescent staining was performed on HCC cell smearing slides in order to evaluate the activity of the purified aim protein, then MTT trial to evaluate the cytotoxicity of hscFv25-TNFalpha to SMMC-7721, finally primary tumor regression trial in nude mice bearing HCC to evaluate the targeting therapeutic value of hscFv25-TNFalpha.
Results: The hscFv25-TNFalpha had the similar specificity to parental antibody HAb25 for SMMC-7721 antigen. One hour predisposed MTT trial in control with parental antibody HAb25 affirmed that hscFv25-TNFalpha was cytotoxic to targeted cell SMMC-7721 with the IC(50) to be 7.1 microg/ml. The cytotoxicity can be inhibited by parental antibody HAb25. This indicated that the cytotoxicity of hscFv25-TNFalpha to targeted cell is antibody-mediated selective cytotoxicity. The tumor regression trial to the 3mm HCC xenografts in nude mice showed that hscFv25-TNFalpha had assured targeting cytotoxicity and the efficiency was nearly up to 3/3 (1/3 complete remission, 2/3 partial remission). The cytotoxicity of the hscFv25-TNFalpha was better than that of TNFalpha, whose efficiency was only 2/3 and without complete remission.
Conclusion: hscFv25-TNFalpha is an anti-HCC bi-functional antibody which has potentialities for clinical application.