Targeting of Vascular Cell Adhesion Molecule 1 with an Ultrasound Contrast Agent Bearing Designed Ankyrin Repeat Proteins as Targeting Ligands

Kosareva, Alexandra. Targeting of Vascular Cell Adhesion Molecule 1 with an Ultrasound Contrast Agent Bearing Designed Ankyrin Repeat Proteins as Targeting Ligands. 2021, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/84355/

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Atherosclerosis is an inflammatory disease of the large arteries that progresses silently over decades until it causes stroke and myocardial infarction. In order to prevent such late complications, an early and reliable diagnosis of atherosclerosis is clinically needed. Vascular cell adhesion molecule 1 (VCAM-
1) is expressed on the surface of endothelial cells throughout the pathogenesis of atherosclerosis and can therefore be used as a biomarker. Hence, non-invasive imaging of the expression of VCAM-1 could be used for early detection and risk stratification. Contrast enhanced ultrasound molecular imaging (CEUMI) of VCAM-1 was shown to be possible in mouse models of atherosclerosis. However, the ultrasound contrast agents that were used so far have relied on biotin-streptavidin conjugation chemistry and full-size antibodies as targeting agents, which are expensive, potentially immunogenic and may have low targeting efficiency. For clinical translation, there is thus an urgent need for the development of ultrasound contrast agents that use smaller, easy to produce and highly specific ligands. Designed Ankyrin Repeat Proteins (DARPins) are multipurpose affinity reagents that have not just been proven to recognize targets with exceptional specificity and selectivity, but that also may overcome the aforementioned limitations. Therefore, the aim of this work is to develop and characterize an ultrasound contrast agent carrying a DARPin ligand targeted to VCAM-1.
DARPin binders were selected from a large library though specific screening assays. After several screening rounds, 5 high affinity binders were identified and characterized. In vitro flow chamber experiments were performed with the purpose of predicting in vivo performance of the five selected binders. Subsequently, in a mouse model of hind limb inflammation, it could be shown that successful imaging of VCAM-1 expression in a low shear stress environment is possible using microbubbles bearing DARPins as ligands targeted to VCAM-1. However, the individual in vivo performance of the DARPin ligands was not predicted by the flow chamber studies. In a second step, three of the binders were selected to be tested in a pathologically relevant mouse model of atherosclerosis in the aortic arch. In this high-shear environment there were no differences in signal from microbubbles bearing DARPin ligands targeted to VCAM-1 versus a microbubble bearing a control DARPin ligand. Subsequent flow chamber studies performed using pulsatile shear stress and flow chamber experiments testing the off-rate of VCAM-1 targeted microbubbles bearing DARPin ligands indicate that a high off-rate could be primarily responsible for the lack of selective attachment.
Advisors:Kaufmann, Beat and Handschin, Christoph and von zur Mühlen, Constantin
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Cardiovascular Molecular Imaging (Kaufmann)
UniBasel Contributors:Kaufmann, Beat and Handschin, Christoph
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14401
Thesis status:Complete
Number of Pages:92
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss144011
edoc DOI:
Last Modified:22 Oct 2021 04:30
Deposited On:21 Oct 2021 09:42

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