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Development of molecular recognition by rational and combinatorial engineering
KTH, School of Biotechnology (BIO), Molecular Biotechnology. (Biotherapy and Bacterial Display)
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 [en]
Affibody molecule, albumin binding domain, protein engineering, phage display, amyloid beta peptide, TNF, HSA
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-10038ISBN: 978-91-7415-238-8 (print)OAI: oai:DiVA.org:kth-10038DiVA: diva2:201734
Public defence
2009-03-27, sal E3, Osquarsbacke 14, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note
QC 20100722Available from: 2009-03-10 Created: 2009-03-05 Last updated: 2011-11-24Bibliographically approved
List of papers
1. 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: 2010-07-28Bibliographically approved
2. 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: 2010-07-28Bibliographically approved
3. Engineering of a femtomolar affinity binding protein to human serum albumin
Open this publication in new window or tab >>Engineering of a femtomolar affinity binding protein to human serum albumin
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2008 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 8, 515-527 p.Article in journal (Refereed) Published
Abstract [en]

We describe the development of a novel serum albumin binding protein showing an extremely high affinity (K(D)) for HSA in the femtomolar range. Using a naturally occurring 46-residue three-helix bundle albumin binding domain (ABD) of nanomolar affinity for HSA as template, 15 residues were targeted for a combinatorial protein engineering strategy to identify variants showing improved HSA affinities. Sequencing of 55 unique phage display-selected clones showed a strong bias for wild-type residues at nine positions, whereas various changes were observed at other positions, including charge shifts. Additionally, a few non-designed substitutions appeared. On the basis of the sequences of 12 variants showing high overall binding affinities and slow dissociation rate kinetics, a set of seven 'second generation' variants were constructed. One variant denoted ABD035 displaying wild-type-like secondary structure content and excellent thermal denaturation/renaturation properties showed an apparent affinity for HSA in the range of 50-500 fM, corresponding to several orders of magnitude improvement compared with the wild-type domain. The ABD035 variant also showed an improved affinity toward serum albumin from a number of other species, and a capture experiment involving human serum indicated that the selectivity for serum albumin had not been compromised from the affinity engineering.

Keyword
affinity/combinatorial protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-10016 (URN)10.1093/protein/gzn028 (DOI)000257789400005 ()18499681 (PubMedID)2-s2.0-47649128420 (Scopus ID)
Note
QC 20100722Available from: 2009-03-04 Created: 2009-03-04 Last updated: 2011-11-08Bibliographically approved
4. Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro
Open this publication in new window or tab >>Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro
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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.

Keyword
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:nbn:se:kth:diva-18865 (URN)10.1042/ba20090085 (DOI)000270769000003 ()2-s2.0-70350521639 (Scopus ID)
Note
QC 20100525 QC 20111124Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-11-24Bibliographically approved
5. A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
Open this publication in new window or tab >>A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
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2008 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 4, 247-255 p.Article in journal (Refereed) Published
Abstract [en]

Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.

Keyword
affibody/cell surface display/combinatorial protein engineering/Gram-positive bacteria/Staphylococcus carnosus
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7846 (URN)10.1093/protein/gzm090 (DOI)000254295200004 ()
Note
QC 20100722Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-12-06Bibliographically approved

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