Specific Targeting of Atherosclerotic Plaques in ApoE-/- Mice Using a New Camelid sdAb Binding the Vulnerable Plaque Marker LOX-1

Molecular imaging has the potential to provide quantitative information about specific biological aspects of developing atherosclerotic lesions. This requires the generation of reliable, highly specific plaque tracers. This study reports a new camelid single-domain antibody fragment (sdAb) targeting the Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a biomarker for the detection and molecular phenotyping of vulnerable atherosclerotic plaques. A camelid sdAb was generated and selected for high affinity binding to LOX-1. Ex vivo biodistribution and in vivo single photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies were performed in wild-type mice and in fat-fed atherosclerotic apolipoprotein E-deficient mice with Tc-99m-labeled sdAbs. Gamma-counting and autoradiography analyses were performed on dissected aorta segments with different degrees of plaque burden. The specificity of the LOX-1-targeting sdAb was evaluated by blocking with unlabeled sdAb or by comparison with a nontargeting Tc-99m-labeled control sdAb. We generated a sdAb binding LOX-1 with a K-D of 280 pM +/- 62 pM affinity. After Tc-99m-labeling, the tracer had radiochemical purity higher then 99 % and retained specificity in in vitro binding studies. Tracer blood clearance was fast with concomitant high kidney retention. At 3 h after injection, uptake in tissues other than plaques was low and not different than background, suggesting a restricted expression pattern of LOX-1. Conversely, uptake in aortic segments increased with plaque content and was due to specific LOX-1 binding. In vivo SPECT/CT imaging 160 min after injection in atherosclerotic mice confirmed specific targeting of LOX-1-expressing aortic plaques. The LOX-sdAb specifically targets LOX-1-expressing atherosclerotic plaques within hours after injection. The possibility to image LOX-1 rapidly after administration combined with the favourable biodistribution of a sdAb are beneficial for molecular phenotyping of atherosclerotic plaques and the generation of a future prognostic tracer.