Improved Detection of Molecular Markers of Atherosclerotic Plaques Using Sub-Millimeter PET Imaging

Abstract

Since atherosclerotic plaques are small and sparse, their non-invasive detection via PET imaging requires both highly specific radiotracers as well as imaging systems with high sensitivity and resolution. This study aimed to assess the targeting and biodistribution of a novel fluorine-18 anti-VCAM-1 Nanobody (Nb), and to investigate whether sub-millimetre resolution PET imaging could improve detectability of plaques in mice. The anti-VCAM-1 Nb functionalised with the novel restrained complexing agent (RESCA) chelator was labelled with [18F]AlF with a high radiochemical yield (>75%) and radiochemical purity (>99%). Subsequently, [18F]AlF(RESCA)-cAbVCAM1-5 was injected in ApoE−/− mice, or co-injected with excess of unlabelled Nb (control group). Mice were imaged sequentially using a cross-over design on two different commercially available PET/CT systems and finally sacrificed for ex vivo analysis. Both the PET/CT images and ex vivo data showed specific uptake of [18F]AlF(RESCA)-cAbVCAM1-5 in atherosclerotic lesions. Non-specific bone uptake was also noticeable, most probably due to in vivo defluorination. Image analysis yielded higher target-to-heart and target-to-brain ratios with the β-CUBE (MOLECUBES) PET scanner, demonstrating that preclinical detection of atherosclerotic lesions could be improved using the latest PET technology.

VCAM-1 Target in Non-Invasive Imaging for the Detection of Atherosclerotic Plaques

“…[ 18 F]AlF(RESCA)-cAbVCAM1-5 accumulated in atherosclerotic lesions in the aortic arch of ApoE −/− mice, but also unfortunately in the bone structure, which is probably due to the uptake of degradation products of the tracer by unclear biological processes ( Figure 6). This bone uptake is difficult to avoid because the imaging must be performed at a precise time to ensure the lowest blood background signal and the highest probe signal, but this time correlates with the time of the formation of radio-metabolites [115].…”

“…Figure 6. Images from the study of Bridoux et al [115]. Detection of atherosclerotic lesions in the mouse aortic arch (Ao), near the heart (H), by in vivo β-CUBE imaging system after administration of [ 18 In summary, VCAM-1 imaging enables us to detect atherosclerotic plaques, stratify the risk and evaluate a new therapy in atherosclerosis in animal studies.…”

Nanobody: a promising toolkit for molecular imaging and disease therapy

“…Intriguingly, among the various radio-labeled nanobody 99m Tc-labeled cAbVCAM1-5 showed the highest lesion uptake, followed by the 68 Ga-and 18 F-labeled tracers, demonstrating that radioisotope did have a significant impact on the biodistribution of nanobodies [46]. Meanwhile, plaques detectability was improved by using restrained complexing agents (RESCA) as the radioisotope chelators, which allowed faster 18 F-labelling and yielded significantly higher plaque-to-brain and plaque-to-heart ratios [60]. VCAM-1 is a good target for the detection of existing atherosclerosis due to its highest abundance among atherosclerosis-related adhesion molecules.…”

Radiolabeling Strategies of Nanobodies for Imaging Applications

“…For this purpose, the radiofluoride is attached to a suitable metal, in particular aluminum, which itself is bound to an appropriate chelator conjugated to a targeting vehicle, altogether resulting in a stable complex [ 48 ]. Such an Al 18 F-labeling strategy has also been applied to the three nanobodies 2Rs15d, cAbVCAM-1−5 and NbV4m119, with the latter addressing the complement receptor of the immunoglobulin superfamily (CRIg) expressed on Kupffer cells [ 15 , 49 , 50 , 51 ]. Cleeren et al established a new restrained complexing agent (RESCA) in order to facilitate the chelation reaction with aluminum mono[ 18 F]fluoride ([ 18 F]{AlF} 2+ ) at room temperature, which is particularly suited for heat-sensitive biomolecules, e.g., nanobodies ( Figure 10 ) [ 49 , 50 ].…”