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Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
Vise andre og tillknytning
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.

sted, utgiver, år, opplag, sider
2014. Vol. 4, s. 7518-
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-156529DOI: 10.1038/srep07518ISI: 000346404200002Scopus ID: 2-s2.0-84922784130OAI: oai:DiVA.org:kth-156529DiVA, id: diva2:767025
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
Inngår i avhandling
1. Bacterial display systems for engineering of affinity proteins
Åpne denne publikasjonen i ny fane eller vindu >>Bacterial display systems for engineering of affinity proteins
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
Combinatorial protein engineering, staphylococcal display, Affibody, biparatopic, VEGFR2, nanobody, E. coli display, autotransporter
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-156420 (URN)978-91-7595-374-8 (ISBN)
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

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