title: Functionalization of tumor necrosis factor-alpha using phage display technique and PEGylation improves its antitumor therapeutic window.
abstract: OBJECTIVE
  In this study, the optimization of antitumor therapy with tumor necrosis factor-alpha (TNF-alpha) was attempted.
  
  
  METHODS
  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.
authors.affiliation: Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Shibata
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshioka
authors.forename: Shinji
authors.initials: S
authors.lastname: Ikemizu
authors.forename: Kyoko
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Yohei
authors.initials: Y
authors.lastname: Mukai
authors.forename: Takayuki
authors.initials: T
authors.lastname: Okamoto
authors.forename: Madoka
authors.initials: M
authors.lastname: Taniai
authors.forename: Maki
authors.initials: M
authors.lastname: Kawamura
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Abe
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Takao
authors.initials: T
authors.lastname: Hayakawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagata
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Yamagata
authors.forename: Tadanori
authors.initials: T
authors.lastname: Mayumi
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Kamada
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D005260:Female; D005354:Fibrosarcoma / Q000188:drug therapy / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D019151:Peptide Library; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000737:chemistry / Q000378:metabolism; D012513:Sarcoma, Experimental / Q000188:drug therapy* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D019151:Peptide Library; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; D008239:Lysine