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Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0002-9282-0174
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2009 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, 93-103 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For Z(TNF alpha:185), subnanomolar affinity (K-D = 0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the Z(TNF-alpha:185) affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dinners with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

Place, publisher, year, edition, pages
2009. Vol. 54, 93-103 p.
Keyword [en]
affibody molecule, albumin-binding-domain-based affibody-screening, ELISA (ABAS ELISA), biospecifc interaction analysis (BIA), immobilized-metal-ion affinity chromatography (IMAC), phage display, tumour necrosis factor-alpha (TNF-alpha), rheumatoid-arthritis, protein, affinity, disease, diagnosis, selection, therapy, domain
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-18865DOI: 10.1042/ba20090085ISI: 000270769000003Scopus ID: 2-s2.0-70350521639OAI: oai:DiVA.org:kth-18865DiVA: diva2:336912
Note
QC 20100525 QC 20111124Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Development of molecular recognition by rational and combinatorial engineering
Open this publication in new window or tab >>Development of molecular recognition by rational and combinatorial engineering
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Combinatorial protein engineering, taking advantage of large libraries of protein variants and powerful selection technology, is a useful strategy for developing affinity proteins for applications in biotechnology and medicine. In this thesis, two small affinity proteins have been subjected to combinatorial protein engineering to improve or redirect the binding. In two of the projects, a three-helix protein domain based on staphylococcal protein A has been used as scaffold to generate so called Affibody molecules capable of binding to key proteins related to two diseases common among elderly people.

In the first project, Affibody molecules were selected using phage display technology for binding to Ab-peptides, believed to play a crucial role in Alzheimer’s disease, in that they can oligomerize and contribute to the formation of neural plaques in the brain. The selected Affibody molecules were found to efficiently capture Ab from spiked human plasma when coupled to an affinity resin. The structure of the complex was determined by nuclear magnetic resonance (NMR) and demonstrated that the original helix 1 in the two Affibody molecules was unfolded upon binding, forming intermolecular b-sheets that stabilized the Ab peptide as buried in a tunnel-like cavity. Interestingly, the complex structure also revealed that the Affibody molecules were found to homo-dimerize via a disulfide bridge and bind monomeric Ab-peptide with a 2:1 stoichiometry. Furthermore, Affibody molecule-mediated inhibition of Ab fibrillation in vitro, suggested a potential of selected binders for future therapeutic applications.

In the second project, two different selection systems were used to isolate Affibody molecules binding to tumor necrosis factor alpha (TNF), which is involved in inflammatory diseases such as rheumatoid arthritis. Both selection systems, phage display and Gram-positive bacterial display, could successfully generate TNF-binding molecules, with equilibrium dissociation constants (KD) in the picomolar to nanomolar range. Initial characterization of the binding to TNF was evaluated by competitive binding studies between the Affibody molecules and clinically approved TNF antagonists (adaliumumab, infliximab and etanercept) and demonstrated overlapping binding sites with both adaliumumab and etanercept. Furthermore, linkers of different lengths were introduced between Affibody moieties, in dimeric and trimeric constructs that were evaluated for their ability to block the binding between TNF and a recombinant form of its receptor. In the dimeric constructs, a linker length of 20-40 amino acids seemed to have an advantage compared to shorter and longer linkers, and the tested trimeric construct could block the TNF binding at even lower concentration. The results provided valuable information for the design of future Affibody-based molecules that could be investigated in therapeutic or medical imaging applications.

In the third project aiming to generate a protein domain with capacity to influence the pharmacokinetics of protein therapeutics, a natural serum albumin-binding domain (ABD) was subjected to an engineering effort aiming at improving the affinity to human serum albumin (HSA), a protein with an exceptional long half-life in serum (19 days). First-generation affinity improved ABD variants were selected using phage display technology from a constructed ABD library. After additional rational engineering of such first generation variants, one variant with a 10,000-fold improved affinity to HSA (KD ≈ 120 fM) was obtained. Furthermore, characterization of this molecule also demonstrated improved affinity to several other serum albumins. When used as a gene fusion partner, this affinity-maturated variant denoted ABD035, should have the potential to extend the half-life of biopharmaceuticals in humans, and several other animal species.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 94 p.
Series
TRITA BIO-Report 2009:2, ISSN 1654-2312 ; 2009 : 2
Keyword
Affibody molecule, albumin binding domain, protein engineering, phage display, amyloid beta peptide, TNF, HSA
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-10038 (URN)978-91-7415-238-8 (ISBN)
Public defence
2009-03-27, sal E3, Osquarsbacke 14, Stockholm, 10:00 (Swedish)
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Supervisors
Note
QC 20100722Available from: 2009-03-10 Created: 2009-03-05 Last updated: 2011-11-24Bibliographically approved

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