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Journal of Biochemistry Advance Access originally published online on October 23, 2007
Journal of Biochemistry 2008 143(1):9-19; 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; 2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST); 3Institute of Glycotechnology, Tokai University, Kanagawa 259-1292; and 4Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan

*To whom correspondence should be addressed. Tel: +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

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. (2000) Cancer Immunol. Immunother. 49, 243; Sachdev et al. (2003) Cancer Res. 63, 627]. 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 pre-incubated with mAbs suggested that (i) 1H7 and 3B7 bind to IGF-IR but do not bind to its structurally related IR, (ii) either the VL–VH or VH–VL sequence order does not apparently affect specificity for IGF-IR and (iii) 1H7 and 3B7 bind the independent epitopes, located in or near the N-terminal (440–514) and C-terminal (62–184) 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

Abbreviations: Ab, antibody; ABTS, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid); BSA, bovine serum albumin; CBB, Coomassie brilliant blue; CDR, complementarity determining region; cfu, colony formation unit; CH1, constant domain 1 of antibody heavy chain; Con A, concanavalin A; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; FR, framework region; Fv, variable fragment; GR, glutathione reductase; HRP, horseradish peroxidase; IGF-IR, insulin-like growth factor I receptor; IPTG, isopropyl-β-D-thiogalactopyranoside; IR, insulin receptor; mAb, monoclonal Ab; OVA, ovalbumin; PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride; rhIGFIR, recombinant extracellular domain of human IGF-IR; rhIR, recombinant extracellular domain of human IR; RNase A, ribonuclease A; RT-PCR, reverse transcription PCR; scFv, single-chain variable fragment; SPR, surface plasmon resonance; TBS, Tris-buffered saline; VH, variable region of antibody heavy chain; VL, variable region of antibody light chain


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