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Affibody Molecules Targeting VEGFR2 - Two Turns Off and Four Turns On

Time: Fri 2020-05-15 10.00

Location:, Stockholm (English)

Subject area: Biotechnology

Doctoral student: Rezan Güler , Proteinvetenskap, John Löfblom

Opponent: Professor Dario Neri, ETH Zurich

Supervisor: Associate Professor John Löfblom, Proteinvetenskap; Professor Stefan Ståhl, Proteinvetenskap

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The notion of employing proteins as drugs traces back several decades. As recombinant DNA technology emerged, it became a powerful tool for the tailoring of protein traits via genetic approaches - so-called protein engineering. The application of such tools to develop proteins that bind specific molecular targets has seen remarkable clinical success, and today seven out of the ten top-selling drugs in the world belong to this class of proteins. A well-investigated protein scaffold for generating novel target-binding moieties is the small staphylococcal protein A-derived affibody molecule. This thesis revolves around the engineering of affibody-based binding proteins that aim to influence the signaling network in the biological process of blood vessel formation, so-called angiogenesis. The first study in this thesis describes the engineering of a heterodimeric affibody molecule that targets the principal regulating receptor of angiogenesis, vascular endothelial growth factor receptor-2 (VEGFR2). Two separate affibody molecules that bind adjacent VEGFR2 epitopes were previously fused into one biparatopic construct, leading to a remarkable increase in apparent affinity. Further, the biparatopic protein here demonstrated inhibition of vascular endothelial growth factor A (VEGF-A) binding to VEGFR2, and consequently inhibition of VEGFR2 phosphorylation, proliferation and in vitro sprouting of endothelial cells. In the second study, the aim was to evaluate the biparatopic protein as a molecular imaging probe for in vivo visualization of VEGFR2 expression in a glioblastoma multiforme (GBM) brain tumor-model. The results displayed significantly higher probe uptake in tumor compared to normal brain tissue, with a two-hour post injection tumor-to-brain ratio of 78. In the third study, the goal was to mimic the ability of the natural ligand to agonize VEGFR2 via receptor dimerization, and also simulate presentation as extracellular matrix (ECM) bound factors. To this end, the dimeric antagonist was reformatted into a tetrameric construct, hypothesized to bridge two receptor units, and fused to recombinant spider silk. Interestingly, whereas the tetramer displayed agonistic effects both in soluble and immobilized form, the activity of the dimer shifted from antagonistic to agonistic when immobilized. In the fourth study a combined in silico and directed evolution approach was used to increase the thermal stability and hydrophilicity of the biparatopic protein. The final construct demonstrated an increase in melting temperature of about 15°C, complete refolding after heat-induced denaturation and decreased uptake in normal tissues when evaluated in mouse biodistribution studies. 

In conclusion, this thesis covers the development, characterization and engineering of VEGFR2-binding affibody molecules, aimed for use in research, therapy and diagnosis.