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Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för onkologi, radiologi och klinisk immunologi, Enheten för biomedicinsk strålningsvetenskap.ORCID-id: 0000-0001-6120-2683
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2008 (Engelska)Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, nr 5, s. 1388-1402Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K(d)=5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K(d), was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K(d)=2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.

Ort, förlag, år, upplaga, sidor
2008. Vol. 376, nr 5, s. 1388-1402
Nyckelord [en]
Affibody, EGFR, tumor targeting, phage display selection, affinity maturation
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-17042DOI: 10.1016/j.jmb.2007.12.060ISI: 000254585900014PubMedID: 18207161OAI: oai:DiVA.org:uu-17042DiVA, id: diva2:44813
Tillgänglig från: 2008-06-16 Skapad: 2008-06-16 Senast uppdaterad: 2017-12-08Bibliografiskt granskad

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Orlova, AnnaTolmachev, Vladimir

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