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Journal of Biochemistry Advance Access published online on October 23, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm192
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© 2007 The Japanese Biochemical Society

Construction and Characterization of Single-chain Antibodies against Human Insulin-like Growth Factor-I Receptor from Hybridomas Producing 1H7 or 3B7 Monoclonal Antibody

Yu Kusada1,2, Toru Morizono1, Ayano Matsumoto-Takasaki2,3, Keiko Sakai2,3, Shuma Sato1,2, Hideki Asanuma1,2, Atsushi Takayanagi2,4 and Yoko Fujita-Yamaguchi1,2,3,*

1Department of Applied Biochemistry, Tokai University School of Engineering, Kanagawa 259-1292, Japan
2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Japan
3Institute of Glycotechnology, Tokai University, Kanagawa 259-1292, Japan
4Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan

*To whom correspondence should be addressed: Yoko Fujita-Yamaguchi, Address: 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan. Phone: +81-463-58-1211 (ex. 4188), Fax: +81-463-50-2012, E-mail: yamaguch{at}keyaki.cc.u-tokai.ac.jp

Received September 18, 2007; Accepted September 19, 2007


  Abstract

Summary

Recombinant antibody consisting of the single chain variable fragment (scFv) of 1H7 monoclonal antibody against insulin-like growth factor-I receptor (IGF-IR) and human IgG1 Fc domain, scFv-Fc, has been found to exhibit inhibitory effects on breast cancer growth in vitro and in vivo (Li et al. Cancer Immunol. Immunother. 49, 243, 2000; Sachdev et al. Cancer Res. 63, 627, 2003). Various types of scFvs from hybridomas producing 1H7 or 3B7 mAb were constructed using conventional phage display technology to further characterize the specificity and affinity of anti-IGF-IR mAbs. Binding studies performed using either phage antibodies or soluble scFv proteins to IGF-IR or insulin receptor (IR) and IGF-IR preincubated with mAbs suggested that 1) 1H7 and 3B7 bind to IGF-IR but do not bind to its structurally-related IR, 2) either the VL-VH or VH-VL sequence order does not apparently affect specificity for IGF-IR, and 3) 1H7 and 3B7 bind the independent epitopes, located in or near the N-terminal (62-184) and C-terminal (440-514) domains of the {alpha} subunit, respectively. This study not only revealed new information on binding regions for two anti-IGF-IR mAbs but also provided the scFv genes as tools for further manipulation of the affinity or development of new IGF-IR-targeted cancer therapeutics.

Key Words: epitope specificity, insulin-like growth factor I receptor, insulin receptor, phage display, single-chain antibody


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