Targeting the lectin-like low density lipoprotein 1 scavenger receptor using synthetic protein reagents called Affimers

Atherosclerosis is a chronic inflammatory condition characterised by the development of fat and lipid-enriched plaques or lesions within arterial blood vessel walls. Such atherosclerotic plaques can rupture, causing arterial blockage leading to heart attacks, strokes and peripheral arterial disease. Current treatments for atherosclerosis (e.g. statins) reduce the cellular production of cholesterol and indirectly reduce cholesterol accumulation but do not directly target plaque formation. Lectin-like oxidised low-density lipoprotein (LOX-1) is a vascular membrane protein receptor which binds oxLDL particles and promotes atherosclerosis. Our hypothesis is that targeting the interaction between LOX-1 and oxLDL could be beneficial in directly blocking lipid and fat accumulation within plaques. To test this idea, we screened synthetic proteins called Affimers for binding to the LOX-1 extracellular domain and tested their ability to bind LOX-1 and block oxLDL recognition, uptake and accumulation. Five different Affimers with LOX-1-specificity were identified. These recombinant polyhistidine and cysteine-tagged Affimers were expressed in E. coli, purified and modified using maleimide chemistry for a range of applications. Using immobilised ELISA, we found that LOX-1-specific Affimers recognised the carboxy-proximal LOX-1 C-type lectin-like domain, which is also the oxLDL-binding domain. Two of these Affimers also exhibited limited cross-reactivity with mouse LOX-1. To evaluate Affimer effects on LOX-1-regulated cellular function, we used a tetracycline-inducible human HEK293 T-REx™ cell line which expresses FLAG-tagged LOX-1. Immunofluorescence analysis shows that Affimers display a dose�dependent inhibition of labelled oxLDL uptake by LOX-1-expressing cells. Affimers were also seen
to modulate LOX-1-regulated and oxLDL-dependent signal transduction. LOX-1-dependent and oxLDL-stimulated activation of the canonical MAPK pathway, specifically ERK1/2 phosphorylation, was blocked by pre-incubation with Affimer. Such studies show that Affimers can specifically target LOX-1 and functional properties implicated in pro-atherogenic responses. The studies in this work support the view that Affimers have potential for applications in the study and treatment of atherosclerosis via biotechnological, diagnostic, and therapeutic approaches.