Nanobody-Facilitated Multiparametric PET/MRI Phenotyping of Atherosclerosis

Abstract

OBJECTIVES This study sought to develop an integrative positron emission tomography (PET) with magnetic resonance imaging (MRI) procedure for accurate atherosclerotic plaque phenotyping, facilitated by clinically approved and nanobody radiotracers. BACKGROUND Noninvasive characterization of atherosclerosis remains a challenge in clinical practice. The limitations of current diagnostic methods demonstrate that, in addition to atherosclerotic plaque morphology and composition, disease activity needs to be evaluated. METHODS We screened 3 nanobody radiotracers targeted to different biomarkers of atherosclerosis progression, namely vascular cell adhesion molecule (VCAM)-1, lectin-like oxidized low-density lipoprotein receptor (LOX)-1, and macrophage mannose receptor (MMR). The nanobodies, initially radiolabeled with copper-64 ( 64 Cu), were extensively evaluated in Apoe –/– mice and atherosclerotic rabbits using a combination of in vivo PET/MRI readouts and ex vivo radioactivity counting, autoradiography, and histological analyses. RESULTS The 3 nanobody radiotracers accumulated in atherosclerotic plaques and displayed short circulation times due to fast renal clearance. The MMR nanobody was selected for labeling with gallium-68 ( 68 Ga), a short-lived radioisotope with high clinical relevance, and used in an ensuing atherosclerosis progression PET/MRI study. Macrophage burden was longitudinally studied by 68 Ga-MMR–PET, plaque burden by T2-weighted MRI, and neovascularization by dynamic contrast-enhanced (DCE) MRI. Additionally, inflammation and microcalcifications were evaluated by fluorine-18 ( 18 F)-labeled fluorodeoxyglucose ( 18 F-FDG) and 18 F-sodium fluoride ( 18 F-NaF) PET, respectively. We observed an increase in all the aforementioned measures as disease progressed, and the imaging signatures correlated with histopathological features. CONCLUSIONS We have evaluated nanobody-based radiotracers in rabbits and developed an integrative PET/MRI protocol that allows noninvasive assessment of different processes relevant to atherosclerosis progression. This approach allows the multiparametric study of atherosclerosis and can aid in early stage anti-atherosclerosis drug trials.

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

“…[ 18 F]FB-cAbVCAM-1-5 has a low myocardial uptake with a high lesion-to-myocardium ratio, which makes it promising for the detection of coronary atherosclerosis in patients compared to [ 18 F]FDG, but further studies are needed for clinical use. Senders et al developed a 64 Cu-labelled cAbVCAM1-5 in a rabbit model of atherosclerosis whose uptake was negatively associated with the vessel wall area [116]. This result was consistent with the study of Iiyama et al showing that the expression of VCAM-1 is expressed predominantly by the endothelium in early lesions [120].…”

“…In vivo: ApoE −/− on HD and atherosclerotic rabbits [116] Raman spectroscopy Biofunctional gold nanoprobe (BFNP) + Raman reporters Antibody…”

Recent Advances in the Molecular Imaging of Atherosclerosis

“…More recently, Senders et al developed a multiparametric PET/MRI phenotyping of atherosclerosis using three camelid single-domain antibody fragments (nanobodies), one directed against vascular cell adhesion mol-ecule-1 (VCAM-1) and two other targeting the SRs, macrophage MR (MMR or CD206), and LOX-1. 99 Nanobodies are the smallest Fabs (12-15 kDa) derived from heavy-chain-only antibodies naturally occurring in camelids. The three nanobodies radiolabeled with 64 Cu and injected in ApoE À/À mice showed accumulation in the aortic root and arch, where advanced lesions develop by in vivo micro-PET/CT imaging, which was further confirmed ex vivo.…”

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

“…In order to visualise the recruitment of inflammatory cells in atherosclerotic plaques with PET imaging, we used a Nanobody (Nb)-based tracer (cAbVCAM1-5) targeting the vascular cell adhesion molecule-1 (VCAM-1) [14,15]. Nbs are small antigen-binding fragments derived from heavy-chain-only antibodies and proved to have ideal characteristics for PET imaging [16,17].…”