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Affibody molecules for proteomic and therapeutic applications
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
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 [en]
Affibody, protein engineering, phage display, amyloid beta peptide, transferrin, CD25, IL-2 receptor, proteomics
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4674ISBN: 978-91-7178-901-3 (print)OAI: oai:DiVA.org:kth-4674DiVA: diva2:13368
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
List of papers
1. Affibody-mediated transferrin depletion for proteomics applications
Open this publication in new window or tab >>Affibody-mediated transferrin depletion for proteomics applications
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2007 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 2, no 11, 1389-1398 p.Article in journal (Refereed) Published
Abstract [en]

An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.

Keyword
Affibody ligand; Affinity resin; Protein engineering; Proteomics; Transferrin
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8125 (URN)10.1002/biot.200700053 (DOI)
Note
QC 20100728Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2010-07-28Bibliographically approved
2. Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid
Open this publication in new window or tab >>Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid
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2009 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 52, no 2, 159-166 p.Article in journal (Refereed) Published
Abstract [en]

Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSIF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSIF were desired. Affibody(R) molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20 % of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSIF proteins were chosen for the design of a CSF-specific depletion setup. Affibody(R) molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83 % for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSIF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted: CSIF and the achieved data were compared. Enhanced identification of lower abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.

Keyword
Affibody (R) molecule; affinity resin; biomarker; cerebrospinal fluid (CSF); MS; proteomics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8126 (URN)10.1042/BA20080028 (DOI)000263202500008 ()2-s2.0-60549084220 (Scopus ID)
Note
QC 20100728. Uppdaterad från manuskript till artikel (20100728). Tidigare titel: Development of affinity columns for the removal of high-abundant proteins in cerebrospinal fluidAvailable from: 2008-03-19 Created: 2008-03-19 Last updated: 2017-12-14Bibliographically approved
3. Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides
Open this publication in new window or tab >>Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides
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2007 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 128, no 1, 162-183 p.Article in journal (Refereed) Published
Abstract [en]

Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.

Keyword
Affibody ligand; Amyloid beta peptide; Serum depletion; Phage display; Protein engineering
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8127 (URN)10.1016/j.jbiotec.2006.09.013 (DOI)000243733700016 ()
Note
QC 20100722Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2017-12-14Bibliographically approved
4. Stabilization of a beta-hairpin in monomeric Alzheimer´s amyloid beta-peptide inhibits amyloid formation
Open this publication in new window or tab >>Stabilization of a beta-hairpin in monomeric Alzheimer´s amyloid beta-peptide inhibits amyloid formation
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2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 13, 5099-5104 p.Article in journal (Refereed) Published
Abstract [en]

According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Bound Aβ(1–40) features a β-hairpin comprising residues 17–36, providing the first high-resolution structure of Aβ in β conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Aβ. ZAβ3 stabilizes the β-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Aβ hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZAβ3 acts as a stoichiometric inhibitor of Aβ fibrillation. The selected Aβ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates.

Keyword
Aβ-peptide, engineered binding protein, molecular recognition, protein structure, nuclear magnetic resonance
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8128 (URN)10.1073/pnas.0711731105 (DOI)000254723700027 ()2-s2.0-42449111198 (Scopus ID)
Note
QC 20100722Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2017-12-14Bibliographically approved
5. Generation of Affibody (R) ligands binding interieukin-2 receptor alpha/CD25
Open this publication in new window or tab >>Generation of Affibody (R) ligands binding interieukin-2 receptor alpha/CD25
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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.

Keyword
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:nbn:se:kth:diva-8129 (URN)10.1042/BA20070261 (DOI)000256563200004 ()2-s2.0-44849128916 (Scopus ID)
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: 2017-12-14Bibliographically approved

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Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
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  • en-GB
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  • nn-NO
  • nn-NB
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  • Other locale
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Output format
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