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Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule
Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia.
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2007 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 34, no 6, 609-618 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison.

Methods: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125) I, labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers.

Results: [(125) I](Z(EGFR:955))(2) and [(125) I]cetuximab gave a maximum cellular uptake of I-125 within 4 to 8 h of incubation, while [(125) I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of 125 1 after 48 It of incubation was similar to 20% when delivered as [(125) I](Z(FGFR:955))(2) and similar to 25% when delivered as [I-125] cetuximab. [(125) I]EGF-mediated delivery gave a faster (125) I release, where almost all cell-associated radioactivity had disappeared within 24 It. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding Of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site.

Conclusion: The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [I-125](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine.

Place, publisher, year, edition, pages
2007. Vol. 34, no 6, 609-618 p.
Keyword [en]
A431; affibody molecule; EGFR; internalization; radionuclide; retention
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8277DOI: 10.1016/j.nucmedbio.2007.05.010ISI: 000249157300003Scopus ID: 2-s2.0-34547830391OAI: oai:DiVA.org:kth-8277DiVA: diva2:13556
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2010-07-23Bibliographically 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. xii, 102 p.
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
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Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2010-07-23Bibliographically approved

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Brismar, HjalmarStåhl, Stefan

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