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Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
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2007 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 4, 189-199 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed.

Place, publisher, year, edition, pages
2007. Vol. 20, no 4, 189-199 p.
Keyword [en]
Affibod, EGFR, phage display, selection, targeting
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-97198DOI: 10.1093/protein/gzm011ISI: 000246966800005PubMedID: 17452435OAI: oai:DiVA.org:uu-97198DiVA: diva2:172025
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2011-01-28Bibliographically approved
In thesis
1. EGFR and HER2 Targeting for Radionuclide-Based Imaging and Therapy: Preclinical Studies
Open this publication in new window or tab >>EGFR and HER2 Targeting for Radionuclide-Based Imaging and Therapy: Preclinical Studies
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications.

This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of 125I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR).

In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (ZEGFR:955)2 was selected and analysed both in vitro and in vivo. In papers II, III and V, the results obtained when using (ZEGFR:955)2 were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, in vivo studies were made, demonstrating that [111In](ZEGFR:955)2 gave a tumour-specific 111In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point.

The results of these studies indicated that the affibody molecule (ZEGFR:955)2 is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (ZEGFR:955)2 might be of interest for therapy applications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 50 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 350
Biomedicine, EGFR, HER2, affibody molecule, tumour targeting, 125I, 111In, Biomedicin
urn:nbn:se:uu:diva-8721 (URN)978-91-554-7197-2 (ISBN)
Public defence
2008-05-24, Fåhraeussalen, Rudbecklaboratoriet, Daghammarskjöldsväg 20, Uppsala Science Park, Uppsala, 09:15
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2011-07-08Bibliographically approved

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