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Bacterial display systems for engineering of affinity proteins
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins.

Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro.

In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application.

The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules.

In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2014. , s. 89
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:18
Emneord [en]
Combinatorial protein engineering, staphylococcal display, Affibody, biparatopic, VEGFR2, nanobody, E. coli display, autotransporter
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-156420ISBN: 978-91-7595-374-8 (tryckt)OAI: oai:DiVA.org:kth-156420DiVA, id: diva2:767023
Disputas
2014-12-19, FD5, AlbaNova Universitetscentrum, KTH, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Swedish Research CouncilVinnovaSwedish Foundation for Strategic Research
Merknad

QC 20141203

Tilgjengelig fra: 2014-12-03 Laget: 2014-11-28 Sist oppdatert: 2014-12-03bibliografisk kontrollert
Delarbeid
1. Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Åpne denne publikasjonen i ny fane eller vindu >>Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Vise andre…
2014 (engelsk)Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 4, s. 7518-Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases.

HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-156529 (URN)10.1038/srep07518 (DOI)000346404200002 ()2-s2.0-84922784130 (Scopus ID)
Forskningsfinansiär
Swedish Foundation for Strategic Research , RBa08-0067Swedish Research Council
Merknad

Updated from "Manuscript" to "Article". QC 20141218

Tilgjengelig fra: 2014-11-28 Laget: 2014-11-28 Sist oppdatert: 2017-12-05bibliografisk kontrollert
2. Efficient blocking of VEGFR2-mediated signaling using biparatopic Affibody constructs
Åpne denne publikasjonen i ny fane eller vindu >>Efficient blocking of VEGFR2-mediated signaling using biparatopic Affibody constructs
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-156532 (URN)
Merknad

QS 2014

Tilgjengelig fra: 2014-11-28 Laget: 2014-11-28 Sist oppdatert: 2014-12-03bibliografisk kontrollert
3. Surface display of a single-domain antibody library on Gram-positive bacteria
Åpne denne publikasjonen i ny fane eller vindu >>Surface display of a single-domain antibody library on Gram-positive bacteria
Vise andre…
2013 (engelsk)Inngår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 70, nr 6, s. 1081-1093Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

Emneord
Bacterial display, Combinatorial protein engineering, Nanobodies, Phage display, Recombinant antibodies
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-111859 (URN)10.1007/s00018-012-1179-y (DOI)000315343600009 ()23064703 (PubMedID)
Forskningsfinansiär
Swedish Research Council, 2009-5758Vinnova
Merknad

QC 20130411

Tilgjengelig fra: 2013-01-14 Laget: 2013-01-14 Sist oppdatert: 2017-12-06bibliografisk kontrollert
4. An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains
Åpne denne publikasjonen i ny fane eller vindu >>An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains
2014 (engelsk)Inngår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 13, s. 179-Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Background: Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. Results: The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection using FACS from a 1: 100,000 background yielded around 20,000-fold enrichment in a single round and high viability of the isolated bacteria after sorting. Conclusions: The new expression vectors are promising for combinatorial engineering of Affibody molecules and the strategy for small-scale production of soluble recombinant proteins has the potential to increase throughput of the entire discovery process.

Emneord
Affibody molecule, Bacterial display, Directed evolution, Combinatorial protein engineering, AIDA-I, Autotransporter, FACS, Secreted protein production, E. coli, Phage display
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-156531 (URN)10.1186/s12934-014-0179-z (DOI)000349043900001 ()2-s2.0-84924087937 (Scopus ID)
Forskningsfinansiär
Swedish Research Council, 621-2012-5336
Merknad

QC 20150303

Tilgjengelig fra: 2014-11-28 Laget: 2014-11-28 Sist oppdatert: 2017-12-05bibliografisk kontrollert

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