Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding Affibody molecule
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
Affibody AB, Bromma.
Affibody AB, Bromma.
Show others and affiliations
2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 5, p. 1388-1402Article in journal (Refereed) 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, Kd, 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.

Place, publisher, year, edition, pages
2008. Vol. 376, no 5, p. 1388-1402
Keyword [en]
Affibody; EGFR; tumor targeting; phage display selection; affinity maturation
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8279DOI: 10.1016/j.jmb.2007.12.060ISI: 000254585900014Scopus ID: 2-s2.0-39149087035OAI: oai:DiVA.org:kth-8279DiVA, id: diva2:13558
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications
Open this publication in new window or tab >>Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A.

In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging.

Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. p. xii, 102
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:2
Keyword
Affibody, affinity maturation, phage display selection, EGFR, molecular imaging, protein engineering, tumor targeting
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4710 (URN)978-91-7178-890-0 (ISBN)
Public defence
2008-05-16, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2010-07-23Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textScopus

Authority records BETA

Ståhl, Stefan

Search in DiVA

By author/editor
Friedman, MikaelaStåhl, Stefan
By organisation
Molecular Biotechnology
In the same journal
Journal of Molecular Biology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 146 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf