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Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.ORCID iD: 0000-0001-6120-2683
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2010 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 36, no 4, 757-763 p.Article in journal (Refereed) Published
Abstract [en]

Tumor cell internalization of targeting agents is of interest, since internalization influences the local retention time of a radionuclide and thereby imaging quality in PET and SPECT and effects of radionuclide therapy. In cases where nuclear methods are not applicable at the cellular level, quantitative fluorescent techniques are useful as described in this article. Two fluorescence-based methods to study cellular internalization were applied: the CypHer and the Alexa488-quenching methods, both utilized in fluorescence microscopy and flow cytometry. Two EGFR-binding Affibody molecules were analyzed in A431 cells: the monomer Z1907 and the dimer (Z1907)2. EGF, cetuximab and non-specific Affibody molecules were used as controls. For comparison, internalization of 111In-labeled Z1907 was studied with the acid wash internalization assay. The Cypher method is straightforward, but requires equal labeling of all compounds for accurate quantification. The Alexa488-quenching method is preferable since it is independent of the dye-to-protein ratio. According to this method, about 45% of EGF and 19-24% of the bound Affibody molecules and cetuximab were internalized within one hour. Similar results were seen with 111In-Z1907 in the acid wash method, while (Z1907)2 was not removed by acid and thus could not be studied this way. The fluorescence-based Alexa488-quenching method is well suited to quantitatively analyze internalization of targeting agents, also those that resist acid wash. The internalized fraction showed that both the monomeric and dimeric Affibody molecules are expected to give good uptake and thereby good retention of metallic radionuclides which will render good tumor to background values.

Place, publisher, year, edition, pages
2010. Vol. 36, no 4, 757-763 p.
Keyword [en]
111In, A431, Affibody molecule, Alexa488, Cetuximab, CypHer5E, EGFR, Internalization, Radionuclide, Retention
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-130667DOI: 10.3892/ijo_00000551ISI: 000275794300002PubMedID: 20198317OAI: oai:DiVA.org:uu-130667DiVA: diva2:350265
Available from: 2010-09-10 Created: 2010-09-10 Last updated: 2015-03-24Bibliographically approved
In thesis
1. 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 688
EGFR, HER2, HER3, Affibody, internalisation, tumour targeting
National Category
Medical and Health Sciences
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)
Available from: 2011-09-02 Created: 2011-08-08 Last updated: 2011-11-03Bibliographically approved

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