Florey Department of Neuroscience and Mental Health - Theses
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ItemNovel single-chain antibody-targeted conjugates for detection of chronic inflammation in positron emission tomography( 2017)Chronic inflammatory diseases are a global health concern, with 36 million deaths worldwide in 2008 being attributed to chronic inflammatory diseases (Lozano et al. 2012). These chronic inflammatory diseases can be subcategorised into autoimmune diseases, metabolic disorders, neurodegenerative diseases, allergic and cardiovascular diseases. Many of these diseases have an early asymptomatic phase making them difficult to diagnose and treat early. In order to allow early intervention and develop more effective therapeutics, better diagnostic tools are required. Many different types of innate and adaptive immune cells are involved in the complex process of chronic inflammation. Activated platelets play a major role in lesion and thrombus development in the chronic inflammatory disease, atherosclerosis. Activated platelets have also shown to be a risk factor in other chronic inflammatory and neuroinflammatory diseases such as Alzheimer’s disease and multiple sclerosis and have only relatively recently been demonstrated to have an active function in progressing these diseases. Molecular imaging of activated platelets is an appealing way to detect acute thrombotic events, and inflammatory processes associated with chronic diseases. Using a previously generated a single-chain antibody (scFv) binding specifically to the platelet surface receptor GPIIb/IIIa in its activated, ligand-bound form (LIBS) we have designed and tested PET radiotracers for the in vivo detection of activated platelets. Both anti-LIBS-scFv and control-scFv were conjugated to N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB), a radioactive NHS ester compound. Afterwards binding to activated-platelets in vitro and in vivo was examined. In vitro formed platelet clots were incubated with different concentrations of the labeled scFv. After incubation with radiolabelled LIBS-scFv, clots retained 2.5 x greater radioactivity in comparison to clots incubated with control-scFv. In vivo experiments were performed in a mouse model of acute thrombosis where a platelet rich clot is formed in the carotid artery using FeCl3. High uptake was observed in injured vessel compared to non- injured vessel. Presence of activated-platelets was determined by histology subsequently. An alternative site-specific radiolabelling strategy was also investigated, as [18F]SFB radiolabelling resulted in a loss of scFv binding activity. N-[2-(4[18F]fluorobenzamido) ethyl]maleimide ([18F]FBEM) radiolabelling of both anti-LIBS-scFv and control-scFv resulted in minimal loss of binding activity and improved specific activity. PET is a powerful imaging modality that can be customised to detect activated platelets in vivo. Both [18F]SFB and [18F]FBEM radiolabelled anti-LIBS-scFv had high specific uptake in the target thrombus of the mouse model, indicating the suitability of anti-LIBS-scFv immunoPET as a diagnostic tool for detecting activated platelets associated with chronic inflammation. Radiolabeled LIBS-scFv could potentially identify patients with serious inflammatory disease earlier than currently possible, thereby allowing early initiation of treatment to reverse chronic inflammatory effects and improve the quality of life for those suffering from these chronic conditions.https://minerva-access.unimelb.edu.au/handle/11343/212/most-popular/item