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Generation and characterization of Affibody molecules targeting HER3
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-1763-9073
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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:1
Keyword [en]
Affibody molecules, epidermal growth factor receptors, HER3, ErbB3, combinatorial protein engineering, combinatorial library, staphylococcal surface display
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:kth:diva-137972ISBN: 978-91-7501-955-0 (print)OAI: oai:DiVA.org:kth-137972DiVA: diva2:680055
Public defence
2014-01-24, FR 4, AlbaNova, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20131217

Available from: 2013-12-17 Created: 2013-12-17 Last updated: 2014-01-22Bibliographically approved
List of papers
1. 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
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-14208 (URN)10.1093/protein/gzq118 (DOI)000288269600005 ()
Funder
Swedish Research Council, 2009-5758
Note

QC 20100726 Uppdaterad från manuskript till artikel i tidskrift (20110408)

Available from: 2010-07-26 Created: 2010-07-26 Last updated: 2017-12-12Bibliographically approved
2. 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 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.

Keyword
Epidermal-Growth-Factor, Breast-Cancer Cells, Oncogene Product, Antitumor Action, Egf Receptor, Kinase, Ligand, Erbb2, Therapy, Family
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-99416 (URN)10.1371/journal.pone.0040023 (DOI)000305892100176 ()2-s2.0-84863110374 (Scopus ID)
Funder
Swedish Research Council, 2009-5758
Note

QC 20120731

Available from: 2012-07-31 Created: 2012-07-30 Last updated: 2017-12-07Bibliographically approved
3. Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification
Open this publication in new window or tab >>Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, e62791- p.Article in journal (Refereed) Published
Abstract [en]

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

Place, publisher, year, edition, pages
Public Library Science, USA, 2013
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-124294 (URN)10.1371/journal.pone.0062791 (DOI)000318852400011 ()2-s2.0-84877609907 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer Society
Note

QC 20130701

Available from: 2013-07-01 Created: 2013-06-28 Last updated: 2017-12-06Bibliographically approved

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