Construction of TNF-binding proteins by grafting hypervariable regions of F10 antibody on human fibronectin domain scaffold

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 fibro...

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Bibliographic Details
Published in:Biochemistry (Moscow) 2012, Vol.77 (1), p.62-70
Main Authors: Petrovskaya, L. E., Shingarova, L. N., Kryukova, E. A., Boldyreva, E. F., Yakimov, S. A., Guryanova, S. V., Novoseletsky, V. N., Dolgikh, D. A., Kirpichnikov, M. P.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino acids
Animals
Antibodies - chemistry
Antibodies - genetics
Antibodies - metabolism
Binding proteins
Binding sites
Biochemistry
Biomedical and Life Sciences
Biomedicine
Bioorganic Chemistry
Cell Line
Chemical properties
Complementarity Determining Regions
Enzyme-Linked Immunosorbent Assay
Fibronectins
Fibronectins - chemistry
Fibronectins - genetics
Fibronectins - metabolism
Humans
Immunoglobulins
Life Sciences
Mice
Microbiology
Molecular Sequence Data
Monoclonal antibodies
Plasmids - chemistry
Protein Binding
Proteins
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Tumor necrosis factor
Tumor Necrosis Factor-alpha - chemistry
Tumor Necrosis Factor-alpha - metabolism
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Summary: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.
ISSN:0006-2979
1608-3040
DOI:10.1134/S0006297912010075