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Generation of Affibody (R) ligands binding interieukin-2 receptor alpha/CD25
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital.
(Affibody AB, Bromma)
Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital.
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2008 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 50, no 2, 97-112 p.Article in journal (Refereed) Published
Abstract [en]

Affibody (R) molecules specific for human IL-2R alpha, the IL-2 (interieukin-2) receptor a subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody (R) molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody (R) molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody (R) molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody (R) molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody (R) molecules bound to CD4(+) CD25(+) PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody (R) molecules as targeting agents for medical imaging and for therapeutic applications is discussed.

Place, publisher, year, edition, pages
2008. Vol. 50, no 2, 97-112 p.
Keyword [en]
Affibody (R) molecule; interleukin-2 receptor alpha (IL-2R alpha/CD25); phage display; peripheral-blood mononuclear cell (PBMC); protein engineering
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8129DOI: 10.1042/BA20070261ISI: 000256563200004Scopus ID: 2-s2.0-44849128916OAI: oai:DiVA.org:kth-8129DiVA: diva2:13367
Note
QC 20100728. Uppdaterad från in press till published (20100728). Tidigare titel: Generation of Affibody ligands binding the IL-2 receptor alphaAvailable from: 2008-03-19 Created: 2008-03-19 Last updated: 2010-07-29Bibliographically approved
In thesis
1. Affibody molecules for proteomic and therapeutic applications
Open this publication in new window or tab >>Affibody molecules for proteomic and therapeutic applications
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology.

In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis.

Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro.

In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. ix, 73 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:3
Keyword
Affibody, protein engineering, phage display, amyloid beta peptide, transferrin, CD25, IL-2 receptor, proteomics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4674 (URN)978-91-7178-901-3 (ISBN)
Public defence
2008-04-11, F3, KTH, Lindstedsvägen 26, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100729Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2010-07-29Bibliographically approved

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