PEGylated recombinant human tumor necrosis factor alpha: preparation and anti-tumor potency

AIM: To assess the merits of polyethylene glycol-modified recombinant human tumor necrosis factor alpha (PEG-rHuTNF-alpha). METHODS: The rHuTNF-alpha was modified with N-succinimidyl succinnate monomethoxy polyethylene glycol (SS-PEG) of three different molecular weights. The PEG-rHuTNF-a was separa ted into fractions of various molecular weights by gel filtration chromatog raphy. In vitro activities of various fractions were determined with L929 c ell assay and in vivo antitumor potencies of main fractions were studied wi th respect to necrosis of S-180 solid tumor. RESULTS: The rHuTNF-alpha coul d be modified using SS-PEG under mild conditions. The main fraction of PEG( 5000-)rHuTNF-alpha contained four PEG molecules, and PEG(12000-)rHuTNF-alph a and PEG(20000-)rHuTNF-alpha contained two PEG molecules, respectively. Th ere was a higher activity when rHuTNF-alpha was coupled to less numbers' of the same molecular weight PEG molecules. When PEG-rHuTNF-alpha was of the same molecular weight, rHuTNF-alpha modified with bigger molecular weight P EG molecules had a higher activity. PEG-rHuTNF-alpha was resistant to prote olysis, and over 70 % activity remained after 8 h, but the activity of rHuT NF-a was time-dependently diminished by incubation with bovine trypsin. PEG (5000-)HuTNF-alpha (1500 IU per mouse) had a similar anti-tumor potency com pared with rHuTNF-alpha (3000 IU per mouse). PEG(12000-)rHuTNF-alpha (1500 IU per mouse) had an increased anti-tumor potency compared with rHuTNF-alph a (3000 IU per mouse). In particular, PEG(20000-)rHuTNF-alpha at a dose of 1500 IU per mouse had a higher anti-tumor potency than rHuTNF-alpha at a do se of 6000 IU per mouse. CONCLUSION: PEG-modified rHuTNF-alpha could be mor e suitable for therapeutic use.