Functionalization of tumor necrosis factor-alpha using phage display technique and PEGylation improves its antitumor therapeutic window.

PURPOSE In this study, the optimization of antitumor therapy with tumor necrosis factor-alpha (TNF-alpha) was attempted. EXPERIMENTAL DESIGN Using the phage display technique, we created a lysine-deficient mutant TNF-alpha (mTNF-K90R). This mutant had higher affinities to both TNF receptors, despite reports that certain lysine residues play important roles in trimer formation and receptor binding. RESULTS The mTNF-K90R showed an in vivo therapeutic window that was 13-fold higher than that of the wild-type TNF-alpha (wTNF-alpha). This was due to the synergistic effect of its 6-fold stronger in vitro bioactivity and its 2-fold longer plasma half-life derived from its surface negative potential. The reason why the mTNF-K90R showed a higher bioactivity was understood by a molecular modeling analysis of the complex between the wTNF-alpha and TNF receptor-I. The mTNF-K90R, which was site-specifically mono-PEGylated at the NH2 terminus (sp-PEG-mTNF-K90R), had a higher in vitro bioactivity and considerably longer plasma half-life than the wTNF-alpha, whereas the randomly mono-PEGylated wTNF-alpha had 6% of the bioactivity of the wTNF-alpha. With regard to effectiveness and safety, the in vivo antitumor therapeutic window of the sp-PEG-mTNF-K90R was 60-fold wider than that of the wTNF-alpha. CONCLUSIONS These results indicated that this functionalized TNF-alpha may be useful not only as an antitumor agent but also as a selective enhancer of vascular permeability in tumors for improving antitumor chemotherapy.