Anti-oxidised low density lipoprotein antibodies for the serology and imaging of atherosclerosis

Abstract

Atherosclerosis and its related major adverse cardiovascular events represent a major worldwide morbidity and mortality burden. The paradigm of identifying ‘vulnerable plaques’ leading to ‘vulnerable patients’ remains challenging despite recent advances in cardiovascular medicine. The project aimed to develop an antibody-based strategy for the translational development of imaging oxidised low density lipoprotein (LDL), a major constituent of vulnerable atherosclerotic plaque. It also aimed to simultaneously develop anti-oxidised LDL antibody-based serological assays for the stratification of individual cardiovascular risk independently of classical risk factors. In this thesis, I first discuss the development of a number of assays for the measurement of anti-oxidised LDL antibodies in serum and plasma. I then describe our findings, using a subpopulation of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) to discover that IgG anti-Malondialdehyde (MDA)-LDL antibody levels are stable, independent biomarkers of freedom from adverse cardiovascular events over a 5.5 year follow up. This is followed by describing LO1 and LO9, both murine monoclonal antibodies, isolated in our laboratory, against MDA-LDL and adherent native LDL respectively. We characterise and sequence both, and take LO1 further into development as an imaging agent. I then progress to discussing the utility of directly labelled LO1 (LO1-750) as a near infra-red fluorescence (NIRF) imaging agent. We use two animal models (Ldlr-/- and rabbit atherosclerosis models), utilising three different modalities (IVIS Spectrum/ computed tomography (CT), Fluorescence molecular tomography (FMT)/CT and intravascular 2D NIRF/intravascular ultrasound (IVUS)). I further discuss the generation of a partially humanised tagged molecular construct (LO1-Fab-Cys), as a first step towards the molecular expression and humanisation of LO1 in an optimised form for targeting of oxidised LDL in humans. Moreover I detail the characterisation of LO9 by delineating in a series of experiments its allosteric epitope on adherent LDL, and describe a potential pathological role for it. We label LO9 with a NIRF agent, and show LO9 localisation in vitro and in vivo in mouse studies.