A genetically engineered single-chain FV/TNF molecule possesses the anti-tumor immunoreactivity of FV as well as the cytotoxic activity of tumor necrosis factor.
Recombinant DNA techniques were used to clone, construct and express the fused gene FV-TNF in E. coli under control of the strong T7 bacteriophage promoter in the expression vector pT7-7-FV-TNF. The fusion protein FV/TNF in inclusion bodies from the bacteria homogenate was solubilized in the denaturing solution containing 6 mol/l guanidine and 0.3 mol/l DTT and refolded in refolding buffer containing 8 mmol/l GSSG. The FV/TNF was purified by ion exchange chromatography. The yield of FV/TNF was estimated at 10 mg/l. The purified FV/TNF displayed a single band of 42 kD under reducing conditions, whereas it showed three forms including its monomer (40/42 kD), its dimer (84 kD) and its trimer (126 kD) under non-reducing conditions. Our data showed that this fusion protein retained its bifunctional activities well, namely the anti-TAG72 immunoreactivity of the FV portion and the cytotoxic activity of the TNF moiety. Therefore, the FV/TNF fusion protein may prove useful in targeting the biological effect of TNF to tumor cells as well as in stimulating the immune destruction of tumor cells.
Yang
J
J
Saskatoon Cancer Center, University of Saskatchewan, Canada.
Moyana
T
T
Xiang
J
J
D000230:Adenocarcinoma / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D003110:Colonic Neoplasms / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000276:immunology
D018033:Antibodies, Bispecific; D000951:Antigens, Neoplasm; D004274:DNA, Recombinant; D006023:Glycoproteins; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C055966:immunoglobulin Fv; C047052:tumor-associated antigen 72