Endre søk
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
Affibody AB, Bromma.
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.ORCID-id: 0000-0003-0578-4003
Vise andre og tillknytning
2007 (engelsk)Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, nr 4, s. 189-199Artikkel i tidsskrift (Fagfellevurdert) 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 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.

sted, utgiver, år, opplag, sider
2007. Vol. 20, nr 4, s. 189-199
Emneord [en]
affibody; EGFR; phage display; selection; targeting
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-8276DOI: 10.1093/protein/gzm011ISI: 000246966800005Scopus ID: 2-s2.0-34347380371OAI: oai:DiVA.org:kth-8276DiVA, id: diva2:13555
Merknad
QC 20100723Tilgjengelig fra: 2008-04-25 Laget: 2008-04-25 Sist oppdatert: 2017-12-14bibliografisk kontrollert
Inngår i avhandling
1. Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications
Åpne denne publikasjonen i ny fane eller vindu >>Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications
2008 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2008. s. xii, 102
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2008:2
Emneord
Affibody, affinity maturation, phage display selection, EGFR, molecular imaging, protein engineering, tumor targeting
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-4710 (URN)978-91-7178-890-0 (ISBN)
Disputas
2008-05-16, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Veileder
Merknad
QC 20100723Tilgjengelig fra: 2008-04-25 Laget: 2008-04-25 Sist oppdatert: 2010-07-23bibliografisk kontrollert

Open Access i DiVA

Fulltekst mangler i DiVA

Andre lenker

Forlagets fulltekstScopus

Personposter BETA

Brismar, HjalmarStåhl, Stefan

Søk i DiVA

Av forfatter/redaktør
Friedman, MikaelaBrismar, HjalmarStåhl, Stefan
Av organisasjonen
I samme tidsskrift
Protein Engineering Design & Selection

Søk utenfor DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric

doi
urn-nbn
Totalt: 393 treff
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf