Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Selection and characterization of HER2/neu-binding affibody ligands
KTH, Superseded Departments, Biotechnology.
Show others and affiliations
2004 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 5, 455-462 p.Article in journal (Refereed) Published
Abstract [en]

Affibody® (affibody) ligands that are specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) have been selected by phage display technology from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. The predominant variants from the phage selection were produced in Escherichia coli, purified by affinity chromatography, and characterized by biosensor analyses. Two affibody variants were shown to selectively bind to the extracellular domain of HER2/neu (HER2-ECD), but not to control proteins. One of the variants, denoted His6-ZHER2/neu:4, was demonstrated to bind with nanomolar affinity (∼50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His 6-ZHER2/neu:4 affibody showed specific binding to native HER2/neu, overexpressed on the SKBR-3 tumor cell line. Such affibody ligands might be considered in tumor targeting applications for radionuclide diagnostics and therapy of adenocarcinomas such as breast and ovarian cancers.

Place, publisher, year, edition, pages
2004. Vol. 17, no 5, 455-462 p.
Keyword [en]
Affibody, HER2, Ligand, Phage display, Selection
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:kth:diva-5177DOI: 10.1093/protein/gzh053ISI: 000223949100006Scopus ID: 2-s2.0-4644247278OAI: oai:DiVA.org:kth-5177DiVA: diva2:7985
Note
QC 20100929Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Rational and combinatorial protein engineering for vaccine delivery and drug targeting
Open this publication in new window or tab >>Rational and combinatorial protein engineering for vaccine delivery and drug targeting
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting.

In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines.

In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.

Keyword
Biomedicine, affibody, affinity chromatography, biotin, combinatorial, delivery, phage display, Biomedicin
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:kth:diva-231 (URN)91-7178-003-3 (ISBN)
Public defence
2005-06-03, Sal FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2012-03-22

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Authority records BETA

Ståhl, Stefan

Search in DiVA

By author/editor
Wikman, MariaStåhl, Stefan
By organisation
Biotechnology
In the same journal
Protein Engineering Design & Selection
Microbiology in the medical area

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 84 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf