title: Construction of TNF-binding proteins by grafting hypervariable regions of F10 antibody on human fibronectin domain scaffold.
abstract: Tumor necrosis factor (TNF) plays a key role in the pathogenesis of various diseases. To study the possibility of constructing TNF-binding proteins by grafting hypervariable regions of immunoglobulins (CDR), we have replaced amino acid sequences of loops from the tenth type III domain of human fibronectin ((10)Fn3) by amino acid sequences of CDR from the light and heavy chains of the anti-TNF antibody F10. The assessment of TNF-binding properties of the resulting proteins by ELISA has revealed the highest activity of Hd3 containing sequences CDR-H1 and CDR-H2 of the antibody F10 and of Hd2 containing sequences CDR-H1 and CDR-H3. The proteins constructed by us on the fibronectin domain scaffold specifically bound TNF during Western blotting and also weakened its cytotoxic effect on L929 line cells. The highest neutralizing activity was demonstrated by the proteins Hd2 and Hd3, which induced, respectively, 10- and 50-fold increase in the EC(50) of TNF.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. lpetr65@yahoo.com
authors.forename: L E
authors.initials: LE
authors.lastname: Petrovskaya
authors.forename: L N
authors.initials: LN
authors.lastname: Shingarova
authors.forename: E A
authors.initials: EA
authors.lastname: Kryukova
authors.forename: E F
authors.initials: EF
authors.lastname: Boldyreva
authors.forename: S A
authors.initials: SA
authors.lastname: Yakimov
authors.forename: S V
authors.initials: SV
authors.lastname: Guryanova
authors.forename: V N
authors.initials: VN
authors.lastname: Novoseletsky
authors.forename: D A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D022801:Complementarity Determining Regions; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000906:Antibodies; D022801:Complementarity Determining Regions; D005353:Fibronectins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha