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Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences. (Biomedicinsk strålningsvetenskap)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. (Biomedicinsk strålningsvetenskap)
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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 (125)I 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)I after 48 h of incubation was approximately 20% when delivered as [(125)I](Z(EGFR:955))(2) and approximately 25% when delivered as [(125)I]cetuximab. [(125)I]EGF-mediated delivery gave a faster (125)I release, where almost all cell-associated radioactivity had disappeared within 24 h. 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 [(125)I](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
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97199DOI: 10.1016/j.nucmedbio.2007.05.010ISI: 000249157300003PubMedID: 17707800OAI: oai:DiVA.org:uu-97199DiVA: diva2:172026
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2011-11-03Bibliographically approved
In thesis
1. EGFR and HER2 Targeting for Radionuclide-Based Imaging and Therapy: Preclinical Studies
Open this publication in new window or tab >>EGFR and HER2 Targeting for Radionuclide-Based Imaging and Therapy: Preclinical Studies
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications.

This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of 125I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR).

In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (ZEGFR:955)2 was selected and analysed both in vitro and in vivo. In papers II, III and V, the results obtained when using (ZEGFR:955)2 were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, in vivo studies were made, demonstrating that [111In](ZEGFR:955)2 gave a tumour-specific 111In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point.

The results of these studies indicated that the affibody molecule (ZEGFR:955)2 is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (ZEGFR:955)2 might be of interest for therapy applications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 50 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 350
Keyword
Biomedicine, EGFR, HER2, affibody molecule, tumour targeting, 125I, 111In, Biomedicin
Identifiers
urn:nbn:se:uu:diva-8721 (URN)978-91-554-7197-2 (ISBN)
Public defence
2008-05-24, Fåhraeussalen, Rudbecklaboratoriet, Daghammarskjöldsväg 20, Uppsala Science Park, Uppsala, 09:15
Opponent
Supervisors
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2011-07-08Bibliographically approved
2. Cellular Studies of HER-family Specific Affibody Molecules
Open this publication in new window or tab >>Cellular Studies of HER-family Specific Affibody Molecules
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy.

Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated.

In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules.

Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation.

The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 67 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 688
Keyword
EGFR, HER2, HER3, Affibody, internalisation, tumour targeting
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-156730 (URN)978-91-554-8119-3 (ISBN)
Public defence
2011-09-24, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2011-09-02 Created: 2011-08-08 Last updated: 2016-11-22Bibliographically approved

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Nordberg, ErikaGöstring, LovisaGlimelius, BengtCarlsson, Jörgen

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