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Cellular Studies of HER-family Specific Affibody Molecules
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
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 [en]
EGFR, HER2, HER3, Affibody, internalisation, tumour targeting
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-156730ISBN: 978-91-554-8119-3 (print)OAI: oai:DiVA.org:uu-156730DiVA: diva2:433093
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
List of papers
1. Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule
Open this publication in new window or tab >>Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule
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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.

Keyword
A431, Affibody molecule, EGFR, Internalization, Radionuclide, Retention
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-97199 (URN)10.1016/j.nucmedbio.2007.05.010 (DOI)000249157300003 ()17707800 (PubMedID)
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
2. Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects
Open this publication in new window or tab >>Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects
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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.

Keyword
111In, A431, Affibody molecule, Alexa488, Cetuximab, CypHer5E, EGFR, Internalization, Radionuclide, Retention
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-130667 (URN)10.3892/ijo_00000551 (DOI)000275794300002 ()20198317 (PubMedID)
Available from: 2010-09-10 Created: 2010-09-10 Last updated: 2017-12-12Bibliographically approved
3. Differences in radiosensitivity between three HER2 overexpressing cell lines
Open this publication in new window or tab >>Differences in radiosensitivity between three HER2 overexpressing cell lines
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2008 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 35, no 6, 1179-91 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin(R) treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. METHODS: The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from (211)At decays using the HER2-binding affibody molecule (211)At-(Z(HER2:4))(2) as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. RESULTS: SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of (211)At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from (211)At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. CONCLUSION: There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy.

Keyword
Cancer, Tumor, Radiation, Therapy
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-104387 (URN)10.1007/s00259-007-0713-x (DOI)000255856800018 ()18193218 (PubMedID)
Available from: 2009-05-28 Created: 2009-05-28 Last updated: 2017-12-13Bibliographically approved
4. 17AAG-induced internalisation of HER2- specific Affibody molecules
Open this publication in new window or tab >>17AAG-induced internalisation of HER2- specific Affibody molecules
2016 (English)In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 12, no 4, 2574-2580 p.Article in journal (Refereed) Published
Abstract [en]

The geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) is known to induce internalisation and degradation of the otherwise internalisation-resistant human epidermal growth factor receptor 2 (HER2) receptor. In the present study, 17-AAG was used to increase internalisation of the HER2-specific Affibody molecule ABY-025. The cellular redistribution of halogen-labelled At-211-ABY-025 and radiometal-labelled In-111-ABY-025 following treatment with 17-AAG was studied. 17-AAG treatment of SKOV-3 human ovarian carcinoma and SKBR-3 human breast carcinoma cells to some extent shifted the localisation of In-111-ABY-025 from the cell surface to intracellular compartments in the two cell lines. ABY-025 labelled with the high-linear energy transfer emitter At-211 was also internalised to a higher degree; however, due to its physiological properties, this nuclide was excreted faster. The results indicate that 17-AAG may be used to facilitate cell-specific intracellular localisation of a suitable cytotoxic or radioactive agent coupled to ABY-025 in HER2-overexpressing cells.

Keyword
HER2; 17-AAG; Affibody molecule; radiometal; radiohalogen
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-279516 (URN)10.3892/ol.2016.4990 (DOI)000385579200050 ()27698830 (PubMedID)
Available from: 2016-03-02 Created: 2016-03-02 Last updated: 2017-11-30Bibliographically approved
5. Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
Open this publication in new window or tab >>Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
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2011 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 24, no 4, 385-396 p.Article in journal (Refereed) Published
Abstract [en]

Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

Keyword
Affibody, cell surface display, combinatorial protein engineering, HER3, Staphylococcus carnosus
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-147024 (URN)10.1093/protein/gzq118 (DOI)000288269600005 ()21177282 (PubMedID)
Available from: 2011-02-23 Created: 2011-02-23 Last updated: 2017-12-11Bibliographically approved
6. Cellular effects of HER3-specific affibody molecules
Open this publication in new window or tab >>Cellular effects of HER3-specific affibody molecules
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 6, e40023- p.Article in journal (Refereed) Published
Abstract [en]

Recent discoveries have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. Although practically devoid of kinase activity, signaling via this receptor is often seen in tumours resistant to EGFR or HER2 therapy. Here, we show that two HER3-specific affibody molecules, Z5416 and Z5417, reduce heregulin-induced cell growth of the breast cancer cells MCF-7 and, to a lesser extent, SKBR‑3 cells. These affibody molecules have earlier been shown to block binding of the natural ligand heregulin (HRG) to HER3, which was confirmed here in cellular studies. Further, both Z5416 and Z5417 blocked HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, but only HER3 phosphorylation in SKBR-3 cells which have constantly active HER2.. These findings demonstrate that Z5416 and Z5417 exert an anti-proliferative effect on two breast cancer cells with either high or low HER2 expression, by inhibiting HRG-induced phosphorylation of HER3. The promising results presented in this study indicate that the HER3-binding affibody molecules may be suitable candidates for future therapy of cancers in which the interaction between HER3 and HRG plays an important role.

Keyword
HER3, heregulin, Affibody, proliferation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-156734 (URN)10.1371/journal.pone.0040023 (DOI)000305892100176 ()
Available from: 2011-08-08 Created: 2011-08-08 Last updated: 2017-12-08Bibliographically approved

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