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Generation and Evaluation of Bispecific Affibody Molecules for Simultaneous Targeting of EGFR and HER2
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Division of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH).
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
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2012 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 9, 1802-1811 p.Article in journal (Refereed) Published
Abstract [en]

Co-expression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here we have generated six such bispecific targeting proteins, which each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly4Ser)3 or (Ser4Gly)3, and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the monomeric affibody molecules was favorable to promote the binding to respective target. Interestingly, bispecific constructs containing the novel (Ser4Gly)3 linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly4Ser)3. It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimerization and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.

Place, publisher, year, edition, pages
2012. Vol. 23, no 9, 1802-1811 p.
Keyword [en]
HER2, EGFR, bispecifik, dual targeting
National Category
Medical and Health Sciences
Research subject
Biomedical Radiation Science
Identifiers
URN: urn:nbn:se:uu:diva-160172DOI: 10.1021/bc3000645ISI: 000308833600011OAI: oai:DiVA.org:uu-160172DiVA: diva2:449057
Available from: 2011-10-19 Created: 2011-10-17 Last updated: 2017-12-08Bibliographically approved
In thesis
1. 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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