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.