The Near Infra-Red (NIRF) molecular imaging of oxidised LDL in atherosclerosis with the native autoantibody LO1, and its molecularly expressed cysteine-tagged Fab construct (LO1-Fab-cys)

Purpose: Oxidation of low density lipoprotein in the arterial wall is a key molecular event on the pathogenesis of atherosclerosis. We aimed to develop a NIRF-based antibody-targeted molecular imaging approach for identifying oxidised LDL (oxLDL) in vivo. Methods and results: LO1 is a naturally occurring IgG3κ autoantibody isolated from an LDL receptor deficient hypercholesterolaemic mouse. It reacts with malondialdehyde-conjugated LDL (MDA-LDL), an epitope expressed in oxidised LDL and which is recognised as reflecting vulnerability in human atherosclerotic lesions. Immunohistochemical (IHC) staining of biotinylated LO1 showed colocalisation to advanced mouse atherosclerotic plaque and to necrotic core within culprit human carotid atherosclerotic plaque. We used the immunoglobulin SH groups to successfully label LO1 with a Near Infra Red Fluorescence (NIRF) agent for in vivo imaging studies. Using two optical imaging techniques (IVIS Spectrum combined with hybrid high resolution CT as well as Fluorescence Molecular Tomography (FMT)) we demonstrated that LO1 sensitively and specifically identifies atherosclerotic lesions in vivo in the aorta of the LDLr-/- atherosclerotic mouse model but not in control mice. LO1 also demonstrated superior specificity to lesional regions of interest (ROIs) than a caged MMP probe (MMP-sense). En face immunohistochemistry on aortae from mice injected with labelled LO1 and an anti CD-31 antibody showed localisation of the targeting antibody to the sub-endothelium of the aortic arch and root. LO1 was successfully sequenced and sequence verified as germline. It was then expressed in HEK cells as a chimeric Fab (∼50kD), with murine VH and VL and human CH1 and CL. The terminus of CH1 extends to the THTC of the hinge region to provide the free cysteine for NIRF labelling. In vitro characterisation of the construct revealed maintained function following affinity purification on an anti-human CH1 column and labelling with a NIRF agent. Initial in vivo work confirms localisation to atherosclerotic lesions within the LDLr-/- model. Conclusion: LO1 and its molecularly expressed human chimeric construct (LO1-Fab-cys) provide useful tools for imaging and therapeutic targeting of oxidised LDL in atherosclerosis. Further imaging studies will be enabled by the development new technologies such as the combined Optical Fluorescence Domain Imaging and Near Infrared Fluorescence (OFDI/NIRF) intravascular catheter.