19 The Generation and Characterisation of LO1: A Unique IgG Monoclonal Natural Antibody Against Oxidised LDL (OxLDL)

Abstract

Background Although current evidence implicates IgG natural antibodies against oxLDL in atherogenesis, it has previously been difficult to isolate these as monoclonals. We set out to make IgG monoclonal natural antibodies from a LDLR-/- mouse for mechanistic studies and translational applications. Methodology and Results One antibody we obtained is a unique IgG3k antibody designated mAb LO1. This reacts with copper-oxidised but not native LDL. Further characterisation revealed that LO1 reacts in vitro with malondialdehyde-conjugated LDL (MDA-LDL), a known LDL oxidation product implicated in plaque vulnerability. Sequencing of LO1 heavy and light chain variable regions showed it to be almost entirely germ-line, consistent with it being a natural antibody. Studies using a subsequently generated neutralising anti-idiotype single chain antibody (H3) against LO1 indicated that it is likely to react with a conformational epitope involving discrete ApoB peptide sequences. Immunocytochemical staining demonstrated that LO1 binds epitopes in mouse and human atherosclerotic lesions. In culprit human carotid endarterectomy tissue, LO1 but not IgG3k control stained intracellular deposits in macrophages and occasional extracellular deposits adjacent to the edge of the lipid necrotic core. This pattern is consistent with the distribution of heavily oxidised LDL in vulnerable plaque. LO1 was successfully labelled, and function retained, with a NIRF imaging agent. We are also molecularly expressing various LO1 constructs to be used in imaging studies. Conclusions LO1 provides a promising tool for the analysis in detail of the function of an individual IgG autoantibody in relation to atherosclerosis and the targeting of oxidised LDL in vivo