Construction of an immunoconjugate targeting tissue factor and its effect on colorectal cancer cells

AIM: To construct a recombinant eukaryotic expression vector pcDNA3.1(+)-hFVII-LC+hIgG1-Fc, and to produce and purify the immunoconjugate hFVII-LC+hIgG1-Fc protein. METHODS: The target sequences were amplified by RT-PCR from hepatic tissue and lymphocyte RNA, and cloned into eukaryotic expression vector pcDNA3.1(+). After confirmed by restriction endonuclease digestion and DNA sequencing, the recombinant plasmid was transfected into CHO-K1 cells by lipofectamine 2000. The transfectant clones were selected by G418 screening. The positive monoclonals were grown in CHO-K1 serum-free medium Excel 301 and the culture medium was collected. The hFVII-LC+hIgG1-Fc protein was purified by affinity Ni-NTA resin. The immunoconjugate was identified by ELISA with tissue factor (TF) affinity and specificity. Induction of NK cell-mediated antibody-dependent cell cytotoxicity(ADCC) was examined in HT-29 colorectal cancer cell line. RESULTS: Human liver tissue and lymphocytes from Han population were used as template for amplification of hFVII-LC and hIgG1-Fc DNA fragments, which were confirmed by sequencing and were exactly the same as those GenBank reported. The eukaryotic expression vector pcDNA3.1(+)-hFVII-LC+hIgG1-Fc was successfully constructed, and 1.3 mg of hFVII-LC+hIgG1-Fc protein could be prepared from 1 liter of Excel 301 serum-free culture medium through Ni-NTA affinity chromatography. The immunoconjugate was specially bound to TF and induced a significant ADCC response in HT-29 cells. CONCLUSION: The human hFVII-LC+hIgG1-Fc recombinant plasmid and the hFVII-LC+hIgG1-Fc immunoconjugate are obtained, which provide the basis for further study of cancer-targeted therapy.