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Engineered High-Affinity Affibody Molecules Targeting Platelet-Derived Growth Factor Receptor beta In Vivo
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2011 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 407, no 2, 298-315 p.Article in journal (Refereed) Published
Abstract [en]

Platelet-derived growth factor receptor (PDGFR) B is a marker of stromal pericytes and fibroblasts and represents an interesting target for both diagnosis and therapy of solid tumors. A receptor-specific imaging agent would be a useful tool for further understanding the prognostic role of this receptor in vivo. Affibody molecules constitute a class of very small binding proteins that are highly suited for in vivo imaging applications and that can be selected to specifically recognize a desired target protein. Here we describe the isolation of PDGFRB-specific Affibody molecules with subnanomolar affinity. First-generation Affibody molecules were generated from a large naive library using phage display selection. Subsequently, sequences from binders having a desired selectivity profile and competing with the natural ligand for binding were used in the design of an affinity maturation library, which was created using a single partially randomized oligonucleotide. From this second-generation library, Affibody molecules with a 10-fold improvement in affinity (K-d =0.4-0.5 nM) for human PDGFR beta and a 4-fold improvement in affinity (K-d = 6-7 nM) for murine PDGFRO were isolated and characterized. Complete reversible folding after heating to 90 degrees C, as demonstrated by circular dichroism analysis, supports tolerance to labeling conditions for molecular imaging. The binders were highly specific, as verified by dot blot showing staining reactivity only with human and murine PDGFR beta, but not with human PDGFR alpha, or a panel of control proteins including 16 abundant human serum proteins. The final binder recognized the native conformation of PDGFR beta expressed in murine NIH-3T3 fibroblasts and human AU565 cells, and inhibited ligand-induced receptor phosphorylation in PDGFR beta-transfected porcine aortic endothelial cells. The PDGFR beta-specific Affibody molecule also accumulated around tumoral blood vessels in a model of spontaneous insulinoma, confirming a potential for in vivo targeting.

Place, publisher, year, edition, pages
2011. Vol. 407, no 2, 298-315 p.
Keyword [en]
Affibody molecule, PDGFR, tumor targeting, affinity maturation, stroma targeting
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-151971DOI: 10.1016/j.jmb.2011.01.033ISI: 000288925100008PubMedID: 21277312OAI: oai:DiVA.org:uu-151971DiVA: diva2:412609
Available from: 2011-04-26 Created: 2011-04-20 Last updated: 2017-12-11Bibliographically approved

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