title: A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification.
abstract: Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein bothin vitroandin vivoSpecifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retainin vitromolecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNFin vivowas also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF.
authors.affiliation: From the Laboratory of Biopharmaceutical Research and.|Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.|Laboratory of Cellular and Molecular Physiology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe, 650-8586, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Inoue
authors.affiliation: From the Laboratory of Biopharmaceutical Research and.|Laboratories of Biomedical Innovation and.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ando
authors.affiliation: From the Laboratory of Biopharmaceutical Research and kamada@nibiohn.go.jp.|Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.|Global Center for Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Kamada
authors.affiliation: From the Laboratory of Biopharmaceutical Research and.|Laboratories of Biomedical Innovation and.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Taki
authors.affiliation: From the Laboratory of Biopharmaceutical Research and.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Niiyama
authors.affiliation: From the Laboratory of Biopharmaceutical Research and.|Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Mukai
authors.affiliation: Center for Research and Education on Drug Discovery, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan, and.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tadokoro
authors.affiliation: Center for Research and Education on Drug Discovery, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan, and.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Maenaka
authors.affiliation: From the Laboratory of Biopharmaceutical Research and.|Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Nakayama
authors.affiliation: From the Laboratory of Biopharmaceutical Research and.|Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kado
authors.affiliation: From the Laboratory of Biopharmaceutical Research and.|Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Inoue
authors.affiliation: Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.|Global Center for Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.|Toxicology and Safety Science, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
authors.affiliation: From the Laboratory of Biopharmaceutical Research and tsunoda@pharm.kobegakuin.ac.jp.|Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.|Laboratory of Cellular and Molecular Physiology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe, 650-8586, Japan.|Laboratories of Biomedical Innovation and.|Global Center for Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Tsunoda
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000627:therapeutic use; D001327:Autoimmune Diseases / Q000188:drug therapy; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism; D015195:Drug Design; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D051379:Mice; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000737:chemistry*; D009154:Mutation; D011092:Polyethylene Glycols / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering; D055503:Protein Multimerization; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000037:antagonists & inhibitors*; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics*
chemical_list: D000893:Anti-Inflammatory Agents; D016207:Cytokines; D050505:Mutant Proteins; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols
keywords: PEGylation; R1antTNF; TNF; autoimmune disease; biomaterials; drug design; molecular stability; protein chemical modification; protein design; protein engineering; single chain; thermal shift assay; tumor necrosis factor (TNF)