Creation of functional muteins using phage libraries for pharmacoproteomic-based drug discovery and development of DDS

Abstract

Tumor necrosis factor-alpha (TNF-α) has been expected to be a promising new antitumor agent, but toxic side effects by the systemic administration of TNF-α limit its clinical application. In this study, we attempted to improve the therapeutic potency of TNF-α by using our protein-drug innovation systems. Among phage libraries displaying various mutant TNF-αs, we isolated some lysine-deficient super mutant TNF-αs, typified by mTNF-α-K90R, with higher TNF-receptor affinities and stronger bioactivity in vitro, in spite of the importance of lysine residues for trimer formation and receptor binding. mTNF-α-K90R showed more than 10 times stronger in vivo antitumor effects and 1.3 times less toxicity than wild-type TNF-α (wTNF-α). Site-specifically mono-PEGylated mTNF-α-K90R (sp-PEG-mTNF-α-K90R) at N-terminus showed higher in vitro bioactivity than unmodified wTNF-α, whereas randomly mono-PEGylated wTNF-α at a lysine residue (ran-PEG-wTNF-α) had less than 6% of the bioactivity of wTNF-α. The antitumor therapeutic window of sp-PEG-mTNF-α-K90R was extended by about 5 times, 60 times and 18 times compared with those of mTNF-α-K90R, wTNF-α and ran-PEG-wTNF-α, respectively. sp-PEG-mTNF-α-K90R may, thus, be a potential systemic antitumor therapeutic agent. These data suggested that our fusion protein-drug innovation system composed of a creation system of functional mutant proteins based on phage display technique and a site-specific PEGylation system may open up a new avenue to the optimal protein therapy. © 2004 The Pharmaceutical Society of Japan.