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Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).ORCID iD: 0000-0003-1763-9073
Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
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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.

Place, publisher, year, edition, pages
2011. Vol. 24, no 4, 385-396 p.
Keyword [en]
Affibody, cell surface display, combinatorial protein engineering, HER3, Staphylococcus carnosus
National Category
Condensed Matter Physics
Identifiers
URN: urn:nbn:se:kth:diva-14208DOI: 10.1093/protein/gzq118ISI: 000288269600005OAI: oai:DiVA.org:kth-14208DiVA: diva2:331767
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
In thesis
1. Staphylococcal surface display in directed evolution
Open this publication in new window or tab >>Staphylococcal surface display in directed evolution
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Engineered affinity proteins have together with naturally derived antibodies becomeindispensable tools in many areas of life-science and with the increasing number ofapplications, the need for high-throughput methods for generation of such different affinityproteins is evident. Today, combinatorial protein engineering is the most successful strategy toisolate novel non-immunoglobulin affinity proteins. In this approach, generally termed directedevolution, high-complexity combinatorial libraries are created from which affinity proteins areisolated using an appropriate selection method, thus circumventing the need for detailedknowledge of the protein structure or the binding mechanism, often necessary in more rationalapproaches. Since the introduction of the phage display technology that pioneered the field ofcombinatorial engineering, several alternative selection systems have been developed for thispurpose.This thesis describes the development of a novel selection system based onstaphylococcal surface display and its implementation in directed evolution approaches. In thefirst study, the transformation efficiency to the gram-positive bacteria Staphylococcus carnosus wassuccessfully improved around 10,000-fold to a level that would allow cell surface display ofcomplex combinatorial protein libraries. In two separate studies, the staphylococcal displaysystem was investigated for the applicability in both de novo selection and affinity maturation ofaffibody molecules. First, using a pre-selection strategy with one round of phage display, ahigh-complexity affibody library was displayed on staphylococcal cells. Using fluorescenceactivatedcell sorting, binders with sub-nanomolar affinity to tumor necrosis factor-alpha(TNF-α) were isolated. Second, a combined approach using phage display for de novo selectionof first-generation affibody binders and staphylococcal display in a subsequent affinitymaturation selection was applied to generate binders with low nanomolar affinity to the humanepidermal growth factor receptor-3 (ErbB3). Moreover, in an additional study, thestaphylococcal surface display system was improved by the introduction of a protease 3Ccleavage sequence in the displayed fusion products in order to facilitate straightforwardproduction of soluble proteins for further downstream characterization.Altogether, the presented studies demonstrate that the staphylococcal selection systemindeed is a powerful tool for selection and characterization of novel affinity proteins and couldbecome an attractive alternative to existing selection techniques.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. x, 78 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:16
Keyword
affibody, combinatorial library, directed evoluation, Gram-positive bacteria. protein engineering
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11555 (URN)978-91-7415-418-4 (ISBN)
Public defence
2009-11-27, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:15 (English)
Opponent
Supervisors
Note

QC 20100726

Available from: 2009-11-20 Created: 2009-11-20 Last updated: 2012-12-14Bibliographically approved
2. 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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:1
Keyword
Affibody molecules, epidermal growth factor receptors, HER3, ErbB3, combinatorial protein engineering, combinatorial library, staphylococcal surface display
National Category
Natural Sciences
Identifiers
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)
Opponent
Supervisors
Note

QC 20131217

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

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