In order to get soluble TNF receptor (sTNFR) II with good neutralizing activity against TNFalpha, we constructed the fusion gene sTNFRII-gAD, which encoded human sTNFR II and the globular domain of adiponectin (gAD), and then expressed it in mammalian cells and analyzed its anti-TNFalpha activity. First, sTNFRII cDNA was obtained by RT-PCR from the total RNA of human peripheral blood lymphocytes, and fused in frame with gAD gene. Then, the fusion gene sTNFRII-gAD was cloned into the expression vector pAAV2neo to result in the plasmid pAAV2neo-sTNFRII-gAD. By immunofluorescent staining with monoclonal antibody either against TNFRII or against adiponectin, we demonstrated that the pAAV2neo-s7NFRII-gAD-transiently-transfected BHK-21S cells were positive. To obtain G418-resistant BHK-21S/pAAV2neo-sTNFRII-gAD cells, we cultured the transfected BHK-21S cells above in 10% FBS containing DMEM media with 800 microg/mL G418 for 15 days, and changed the serum-containing culture media to a serum-free chemically defined media so as to change the cells culturing style from adhesion to suspension. 24 hours later, we harvested the supernatant of the culture for sTNFRII-gAD fusion protein characterization and anti-TNFalpha activity analysis. With monoclonal antibody either against TNFRII or against adiponectin, the Western blotting analysis showed that the sTNFRII-gAD fusion protein was expressed and existed as monomer, trimer and multimer forms in the supernatant. The bioactivity assay demonstrated that the sTNFRII-gAD fusion protein had the ability to neutralize TNFalpha so as to inhibit the cytotoxicity of TNFalpha on L929 cells. Put together, this study has laid the groundwork for large-scale preparation of sTNFRII-gAD fusion protein.
Adiponectin ; biosynthesis ; genetics ; Animals ; Cells, Cultured ; Cricetinae ; Humans ; Protein Structure, Tertiary ; genetics ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Solubility ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

BACKGROUNDChemotherapy is very important in the treatment of advanced non-small cell lung cancer (NSCLC). The aim of this study is to evaluate the efficacy, clinical benefit and toxicity of combined chemotherapy with domestic gemcitabine (ZEFEI) plus carboplatin in the treatment of advanced NSCLC.

METHODSThirty-four previously untreated patients with advanced NSCLC (stage III-IV) received domestic gemcitabine of 1000mg/m² on days 1, 8, and carboplatin of AUC 5 on day 1, with 21 days as a cycle. Each patient received at least three cycles.

RESULTSThe total clinical response rate (complete and partial response) was 44% (15/34). Overall clinical benefit rate was 53% (18/34). The main toxicities were hematological toxicities. The rate of grade III-IV leukopenia and thrombocytopenia was 47% and 24% respectively.

CONCLUSIONSCombined chemotherapy with domestic gemcitabine plus carboplatin is an effective and feasible regimen for advanced NSCLC.