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Effects of an EGFR-binding affibody molecule on intracellular signaling pathways
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
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2010 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 36, no 4, 967-972 p.Article in journal (Refereed) Published
Abstract [en]

Effects on intracellular signaling were studied in cells treated with the affibody molecule (Z(EGFR:955))(2) that targets the epithelial growth factor receptor (EGFR). EGFR is over-expressed in many types of cancers and plays a fundamental role in cell signaling and it is of interest to find targeting agents capable of blocking the receptor. The clinically approved antibody cetuximab (Erbitux (R)) and the natural ligand EGF were included as reference molecules. Two EGFR-rich cell lines, A-431 and U-343, were exposed to the three targeting agents and lysed. The cell lysates were immunoprecipitated with the receptors, or directly separated by SDS-Pace. Autophosphorylation of the receptors and phosphorylation of the downstream signaling proteins Erk and Akt, were evaluated by Western blotting. Although the three different agents compete for the same binding site on EGFR, they influenced the signaling differently. The affibody molecule did not induce autophosphorylation of EGFR or my other receptor in the EGFR-family but, in spite of this, induced phosphorylation of Erk in both cell lines and Akt in the A-431 cells. Thus, the results suggest that the signaling pattern induced by (Z(EGFR:955))(2) is only partly similar to that induced by cetuximab. This makes the affibody molecule a potentially interesting alternative to cetuximab for EGFR-targeted therapy since it might give different therapy-related effects on tumor cells and different side effects on normal tissues.

Place, publisher, year, edition, pages
2010. Vol. 36, no 4, 967-972 p.
Keyword [en]
A-431, affibody, Akt, epithelial growth factor receptor, Erk, signaling, U-343
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97201DOI: 10.3892/ijo_00000576ISI: 000275794300027OAI: oai:DiVA.org:uu-97201DiVA: diva2:172028
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically 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. EGFR- and HER2-Binding Affibody Molecules: Cellular studies of monomeric, dimeric and bispecific ligands
Open this publication in new window or tab >>EGFR- and HER2-Binding Affibody Molecules: Cellular studies of monomeric, dimeric and bispecific ligands
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Abnormal expression and signaling of the ErbB receptors is associated with the development and progression of several forms of cancer. In this thesis, new ErbB-targeting affibody molecules are evaluated regarding their cellular effects in vitro. Since ligand binding to an ErbB receptor might have an impact on the cell it is important to be aware of these effects as they may have consequences for the continued growth of the tumor when used in vivo. The affibody molecules are intended for tumor targeting with the prospect of clinical use in imaging or therapy.

Three types of affibody molecules were studied, HER2-binding, EGFR-binding and bispecific binders that target both EGFR and HER2. The HER2-targeting (ZHER2:342)2 showed promising characteristics. It sensitized SKBR-3 cells to irradiation and decreased cell growth to the same extent as the clinically approved antibody Herceptin. The monomeric version, ZHER2:342, did not induce any large effects on intracellular signaling or biological outcome. This makes (ZHER2:342)2 interesting for therapy purposes, while ZHER2:342 may be better suited for imaging.

The bispecific affibody molecules were all able to simultaneously bind to both EGFR and HER2, but none of the six constructs resulted in any large effects on cellular outcome. Interestingly, all three monovalent binders are more functional when positioned at the N-terminal part of the construct and the (S4G)3 linker renders higher affinity of the bispecific binders compared to (G4S)3. Tumors that co-express several ErbB receptors are often more aggressive and associated with a worse prognosis, suggesting that the total ErbB expression pattern might be more informative than the expression level of one receptor regarding cancer prognosis and prediction of response to targeted therapies. Bispecific ligands could thus be used as imaging agents with prognostic value. Another aspect of dual targeting is the possibility of increased tumor specificity since tumors are more likely than healthy tissue to express high amounts of two receptors.

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 ; 715
Keyword
EGFR, HER2, Affibody molecule, cell signaling, receptor dimerization
National Category
Medical and Health Sciences
Research subject
Biomedical Radiation Science
Identifiers
urn:nbn:se:uu:diva-160173 (URN)978-91-554-8195-7 (ISBN)
Public defence
2011-12-03, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2011-11-11 Created: 2011-10-17 Last updated: 2011-11-23Bibliographically approved

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Biomedical Radiation SciencesDepartment of Oncology, Radiology and Clinical ImmunologyLudwig Institute for Cancer ResearchOncology
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