Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth (original) (raw)
Abstract
Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (αIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 αIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92–98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the αIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble αIGF-IR scFvs, a prototype αIGF-IR scFv and its alternative type αIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. αIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A–agarose chromatography. Levels of αIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that αIGF-IR scFv-Fc is a dimeric antibody. αIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 108 M−1, which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of αIGF-IR scFv-Fc (500 μg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the αIGF-IR scFv-Fc is a first-generation recombinant αIGF-IR for the potential development of future αIGF-IR therapeutics.
Access this article
Subscribe and save
- Starting from 10 chapters or articles per month
- Access and download chapters and articles from more than 300k books and 2,500 journals
- Cancel anytime View plans
Buy Now
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Instant access to the full article PDF.
Similar content being viewed by others
Author information
Authors and Affiliations
- Department of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, USA e-mail: yyamaguchi@coh.org Tel.: +1-626-301-8247 Fax: +1-626-301-8280, , , , , , US
Shu-Lian Li, Shu-Jian Liang, Ning Guo, Anna M. Wu & Yoko Fujita-Yamaguchi
Authors
- Shu-Lian Li
- Shu-Jian Liang
- Ning Guo
- Anna M. Wu
- Yoko Fujita-Yamaguchi
Additional information
Received: 21 January 2000 / Accepted: 7 March 2000
Rights and permissions
About this article
Cite this article
Li, SL., Liang, SJ., Guo, N. et al. Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth.Cancer Immunol Immunother 49, 243–252 (2000). https://doi.org/10.1007/s002620000115
- Issue date: June 2000
- DOI: https://doi.org/10.1007/s002620000115