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Cellular Effects of HER3-Specific Affibody Molecules
Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).ORCID iD: 0000-0003-1763-9073
Affibody AB, Stockholm, Sweden.
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2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 6, e40023- p.Article in journal (Refereed) Published
Abstract [en]

Recent studies 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. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.

Place, publisher, year, edition, pages
2012. Vol. 7, no 6, e40023- p.
Keyword [en]
Epidermal-Growth-Factor, Breast-Cancer Cells, Oncogene Product, Antitumor Action, Egf Receptor, Kinase, Ligand, Erbb2, Therapy, Family
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-99416DOI: 10.1371/journal.pone.0040023ISI: 000305892100176ScopusID: 2-s2.0-84863110374OAI: diva2:542315
Swedish Research Council, 2009-5758

QC 20120731

Available from: 2012-07-31 Created: 2012-07-30 Last updated: 2013-12-17Bibliographically approved
In thesis
1. Generation and characterization of Affibody molecules targeting HER3
Open this publication in new window or tab >>Generation and characterization of Affibody molecules targeting HER3
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the field of oncology, the ability to target specific tumor cells using highly selective targeting molecules is an attractive and emerging concept. In this context, the epidermal growth factor receptor HER3 has proven central to the biology behind many different human cancers and inhibition of the signaling mediated by this receptor could provide antitumoral effects. Consequently, this receptor has emerged as a suitable target for imaging, functional blocking or delivery of toxic payloads. A promising targeting-molecule for such applications is the small non-immunoglobulin derived Affibody molecule. The work upon which this thesis is based, revolves around HER3 with the aim to generate and characterize Affibody molecules targeting this receptor.


In the first study, HER3-specific Affibody molecules were generated by combinatorial protein engineering using a combined approach where first generation binders were isolated from a phage-displayed naive library, followed by affinity maturation of these binders using a focused staphylococcal surface-displayed library and flow-cytometric cell sorting. This engineering strategy enabled the successful isolation of HER3-specific Affibody molecules with subnanomolar affinities for the receptor and the ability to compete with the natural ligand heregulin (HRG) for binding to HER3. In the second study, the cellular effects of these Affibody molecules were characterization in vitro. The results demonstrated that the ability to inhibit HRG-binding to the receptor translated into inhibition of ligand-induced phosphorylation of HER3, HER2 as well as the downstream signaling molecules Akt and Erk. As a result, the HER3-specific Affibody molecules also inhibited HRG-induced cell growth of two different breast cancer cell lines in vitro. These promising results, suggested that the HER3-targeting Affibody molecules could have a therapeutic effect in tumors that are dependent on ligand-induced signaling of HER3. However, due to the relatively low expression level of HER3 on tumor cells, we explored two different engineering approaches of the HER3-specific Affibody molecules in order to potentially improve its tumor targeting ability. One approach was to construct bispecific Affibody molecules where a HER3- and a HER2-specific Affibody molecule were fused on each side of an albumin-binding domain (ABD). In the third study, one such bispecific construct was shown to have increased ability to inhibit ligand-induced phosphorylation of HER2 and retained ability to inhibit HRG-induced activation of HER3, as compared to the monomeric anti-HER3 Affibody. Another strategy was to further increase the affinity of the HER3-specific Affibody molecules towards the receptor through a semi-rational affinity maturation approach. In the fourth study, a staphylococcal displayed affinity maturation library was screened by FACS using an off-rate selection procedure. This approach resulted in the successful isolation of picomolar HER3-binders with improved potency of inhibiting HRG-induced cell growth as compared to a first generation binder. Moreover, in the fifth study, in vivo characterization of these HER3-specific Affibody molecules was performed in both normal and xenograft mice. The results suggested specific targeting of HER3 in vivo and provided the first evidence of successful tumor imaging using a HER3-specific Affibody. Taken together, the work included in this thesis describes (to our knowledge) the first non-immunoglobulin derived affinity protein targeting HER3, with promising features for both therapeutic and imaging applications.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. vii, 87 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:1
Affibody molecules, epidermal growth factor receptors, HER3, ErbB3, combinatorial protein engineering, combinatorial library, staphylococcal surface display
National Category
Natural Sciences
urn:nbn:se:kth:diva-137972 (URN)978-91-7501-955-0 (ISBN)
Public defence
2014-01-24, FR 4, AlbaNova, Stockholm, 10:00 (English)

QC 20131217

Available from: 2013-12-17 Created: 2013-12-17 Last updated: 2014-01-22Bibliographically approved

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Malm, MagdalenaStåhl, StefanLöfblom, John
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