Abstract
Cardiovascular diseases are the leading cause of death globally, accounting for approximately 32% of all deaths. These diseases, such as ischemic heart disease, stroke, and venous thromboembolism, are characterized by thrombosis, which is the formation of blood clots that block blood vessels, leading to oxygen and nutrient deprivation. Atherosclerosis, the
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development of plaque that narrows blood vessels, is a major contributor to thrombosis. Current diagnostic methods like angiography are not suitable to detect vascular stenosis and microvascular thrombosis. Also, treatments of thrombosis, either via mechanical or pharmaceutical interventions, have adverse effects. Early recognition of endothelial dysfunction and thrombus markers is crucial for timely intervention and effective treatment. Targeting these markers could improve diagnosis but also enable drug delivery.
For fifty years, antibodies have been investigated for targeting purposes. Unlike conventional antibodies, antibodies from amongst others cartilaginous fish, camels, llamas, and alpacas naturally lack a light chain. The targeting, variable domains of these heavy-chain-only antibodies (VhHs) are known as nanobodies. Compared to conventional antibodies (150 kD), nanobodies are small (15 kD) which allows for improved tissue penetration, binding of conformational epitopes, and reduced immunogenicity due to the lack of the Fc region. In recent years, nanobodies have gained momentum targeting moiety. As therapeutic agent, Caplacizumab/Cablivi received market authorization in 2018 as the first nanobody-based therapy, whereas several physiologically interfering nanobodies have been investigated in phase I and II trials since then. As diagnostic agents, no nanobody-conjugates have received market authorization yet, but they have shown promise in nuclear imaging of tumors and real-time visualization during surgery. The fast blood clearance of nanobodies and rapid tissue penetration enable specific imaging at early time points after administration.
This thesis highlights the versatility and potential applications of nanobodies in the field of cardiovascular diseases. Hereto, nanobodies were developed against endothelial targets and thrombus components. Chapter 2 describes the development of a nanobody that interferes with the physiological function of thrombomodulin, a molecule important for balancing hemostasis. The nanobody was found to enhance tPA-mediated fibrinolysis in thrombomodulin-enriched human whole blood under flow. Chapter 3 describes the development of a non-physiologically active nanobody, targeting Von Willebrand factor in thrombi, conjugated to the catalytic domain of urokinase Plasminogen Activator (uPA), allowing for thrombus degradation. In preclinical mouse models of acute ischemic stroke (AIS), this so-called fusion protein showed reduced cerebral lesion volumes in both fibrin-rich and platelet-rich thrombus models, suggesting its potential as a treatment for thrombotic stroke. Chapter 4 compares two methods for conjugating nanobodies to carriers of interest. It demonstrates that only click chemistry methodology provides a fixed stoichiometry and maintains the nanobody's binding affinity to its target. Consequently, click chemistry was used to conjugate a nanobody targeting fibrin to a liposome in chapter 5, creating a nanobody-nanoparticle carrier platform called NANOCAR. NANOCAR was tested in an ex vivo model for mural thrombosis, a vascular disease characterized by thrombus formation inside blood vessels. The study demonstrated that NANOCAR specifically binds to vascular fibrin depositions, providing a promising approach for assessing mural thrombosis.
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