Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
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
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 2550 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 ils discussed.
Place, publisher, year, edition, pages
2007. Vol. 20, no 4, 189-199 p.
affibody; EGFR; phage display; selection; targeting
IdentifiersURN: urn:nbn:se:kth:diva-8276DOI: 10.1093/protein/gzm011ISI: 000246966800005ScopusID: 2-s2.0-34347380371OAI: oai:DiVA.org:kth-8276DiVA: diva2:13555
QC 201007232008-04-252008-04-252010-07-23Bibliographically approved