Selection of affibody molecules to the ligand-binding site of the insulin-like growth factor-1 receptor.
Affibody molecules binding to the site of hormone interaction in IGF-1R (insulin-like growth factor-1 receptor) were successfully selected by phage-display technology employing a competitive-elution strategy during biopanning, whereby release of receptor-bound phagemids was accomplished by competition with IGF-1 (insulin-like growth factor-1). In non-competitive selections, the elution of receptor-bound phagemids was performed by imidazole or low-pH incubation, which also resulted in the isolation of affibody molecules that could bind to the receptor. An ELISA-based assay showed that the affibody molecules generated by IGF-1 competition during elution, in addition to affibody molecules generated in the non-competitive selections, could compete with IGF-1 for binding to the receptor. The affinities of the isolated variants to IGF-1R-overexpressing MCF-7 cells were determined and ranged from high nanomolar to 2.3 nM. The most promising variant, Z4:40, was shown to recognize IGF-1R efficiently in several different contexts: in analyses based on flow cytometry, fluorescence microscopy and receptor pull-down from cell extracts. In addition, when Z4:40 was added to the medium of MCF-7 cells that were dependent on IGF-1 for efficient growth, it was found to have a dose-dependent growth-inhibitory effect on the cells. Applications of affibody-based reagents for quantitative and qualitative analyses of IGF-1R status, as well as applications of affibody-based reagents for therapy, are discussed.
Li
Jingjing
J
Department of Molecular Biotechnology, Royal Institute of Technology, 106 91 Stockholm, Sweden.
Lundberg
Emma
E
Vernet
Erik
E
Larsson
Barbro
B
Höidén-Guthenberg
Ingmarie
I
Gräslund
Torbjörn
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D017526:Receptor, IGF Type 1 / Q000737:chemistry* / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D057127:Single-Chain Antibodies; D017526:Receptor, IGF Type 1