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Construction of TNF-Binding Proteins by Grafting Hypervariable Regions of F10 Antibody on Human Fibronectin Domain Scaffold

L. E. Petrovskaya1*, L. N. Shingarova1, E. A. Kryukova1, E. F. Boldyreva1, S. A. Yakimov1, S. V. Guryanova1, V. N. Novoseletsky2, D. A. Dolgikh1, and M. P. Kirpichnikov1,2

1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 330-6983; E-mail: lpetr65@yahoo.com

2Biological Faculty, Lomonosov Moscow State University, 119993 Moscow, Russia

* To whom correspondence should be addressed.

Received May 19, 2011; Revision received August 17, 2011
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 (10Fn3) 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 EC50 of TNF.
KEY WORDS: tumor necrosis factor, TNF-binding proteins, fibronectin domain, CDR grafting, ELISA, cytotoxicity test

DOI: 10.1134/S0006297912010075