[Creation of functional muteins using phage libraries for pharmacoproteomic-based drug discovery and development of DDS].
Tumor necrosis factor-alpha (TNF-alpha) has been expected to be a promising new antitumor agent, but toxic side effects by the systemic administration of TNF-alpha limit its clinical application. In this study, we attempted to improve the therapeutic potency of TNF-alpha by using our protein-drug innovation systems. Among phage libraries displaying various mutant TNF-alphas, we isolated some lysine-deficient super mutant TNF-alphas, typified by mTNF-alpha-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-alpha-K90R showed more than 10 times stronger in vivo antitumor effects and 1.3 times less toxicity than wild-type TNF-alpha (wTNF-alpha). Site-specifically mono-PEGylated mTNF-alpha-K90R (sp-PEG-mTNF-alpha-K90R) at N-terminus showed higher in vitro bioactivity than unmodified wTNF-alpha, whereas randomly mono-PEGylated wTNF-alpha at a lysine residue (ran-PEG-wTNF-alpha) had less than 6% of the bioactivity of wTNF-alpha. The antitumor therapeutic window of sp-PEG-mTNF-alpha-K90R was extended by about 5 times, 60 times and 18 times compared with those of mTNF-alpha-K90R, wTNF-alpha and ran-PEG-wTNF-alpha, respectively. sp-PEG-mTNF-alpha-K90R may, thus, be a potential systemic anti-tumor 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.
Yoshioka
Yasuo
Y
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Japan. y-yoshioka@nihs.go.jp
D000818:Animals; D000970:Antineoplastic Agents; D001435:Bacteriophages; D016503:Drug Delivery Systems; D015195:Drug Design; D006801:Humans; D008239:Lysine; D019151:Peptide Library; D011092:Polyethylene Glycols; D040901:Proteomics / Q000379:methods; D014409:Tumor Necrosis Factor-alpha
D000970:Antineoplastic Agents; D019151:Peptide Library; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; D008239:Lysine