Enhanced Antitumor Effect Via Genetic Modification Of T Cells To Produce Recombinant Single-chain Bifunctional Antibody

A single-chain antibody(sFv) gene comprising the linked heavy and light chain variable regions derived from a monoclonal antibody with high specifity and affinity binding to hepatocellular carcinoma (HCC) was fused inframe with tumour necrosis factor α (TNF- α ) gene. The sFv-TNF- a gene was then cloned into an expression vector pEGFP-N₃ containing the report gene of green fluorescent protein(GFP) and the vector was identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. To investigate whether mammalian cells could produce fusion protein, NIH3T3 cells were transfected with the plasmid pEGFP-sFv-TNF- α using Calcium Phosphate method. Bright green fluorescence in the cytoplasm of the transfected cells was observed under the fluorescence microscope 20 hours after transfection. It suggested that mammalian cells can express antibody-cytokine fusion proteins, which is theoretically necessary for our next step of experiment.In order to test the targeted toxicity effect of this bifunctional antibody both in cell level and in tumour xenograft nude mouse model, the sFv-TNF- α gene was shifted into a retroviral vector pLXSN and the recombinant retroviral vector was then transfected into the PA317 amphotropic packaging cells by calcium phosphate method, followed by 500ug/ml G418 DMEM medium selection for one week, to establish a stable virus producing cell line. The G418-resistant colonies were selected and transfered into six-well dish plate for cells expansion, whose colony forming units(cfu) were determined using NIH3T3 cells as...