Nanobodies Targeting Mouse/Human VCAM1 for the Nuclear Imaging of Atherosclerotic Lesions

Abstract

Rationale: A noninvasive tool allowing the detection of vulnerable atherosclerotic plaques is highly needed. By combining nanomolar affinities and fast blood clearance, nanobodies represent potential radiotracers for cardiovascular molecular imaging. Vascular cell adhesion molecule-1 (VCAM1) constitutes a relevant target for molecular imaging of atherosclerotic lesions. Objective: We aimed to generate, radiolabel, and evaluate anti-VCAM1 nanobodies for noninvasive detection of atherosclerotic lesions. Methods and Results: Ten anti-VCAM1 nanobodies were generated, radiolabeled with technetium-99m, and screened in vitro on mouse and human recombinant VCAM1 proteins and endothelial cells and in vivo in apolipoprotein E–deficient (ApoE −/− ) mice. A nontargeting control nanobody was used in all experiments to demonstrate specificity. All nanobodies displayed nanomolar affinities for murine VCAM1. Flow cytometry analyses using human human umbilical vein endothelial cells indicated murine and human VCAM1 cross-reactivity for 6 of 10 nanobodies. The lead compound cAbVCAM1-5 was cross-reactive for human VCAM1 and exhibited high lesion-to-control (4.95±0.85), lesion-to-heart (8.30±1.11), and lesion-to-blood ratios (4.32±0.48) ( P <0.05 versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE −/− mice were successfully identified by single-photon emission computed tomography imaging. 99m Tc-cAbVCAM1-5 binding specificity was demonstrated by in vivo competition experiments. Autoradiography and immunohistochemistry further confirmed cAbVCAM1-5 uptake in VCAM1-positive lesions. Conclusions: The 99m Tc-labeled, anti-VCAM1 nanobody cAbVCAM1-5 allowed noninvasive detection of VCAM1 expression and displayed mouse and human cross-reactivity. Therefore, this study demonstrates the potential of nanobodies as a new class of radiotracers for cardiovascular applications. The nanobody technology might evolve into an important research tool for targeted imaging of atherosclerotic lesions and has the potential for fast clinical translation.

New Targets of Molecular Imaging in Atherosclerosis: Prehension of Current Status

“…1). 18,19,26 Similarly, VCAM-1 ligand conjugatedGd was also reported for detecting early atherogenesis with MRI ( Table 1, Fig. 1).…”

“…muscle cells during the initiation of atherogenesis. 11,12 This is stage 1 where VCAM-1, ICAM-1, and P-selectin [13][14][15][16][17][18][19] (Table 1) are the main targets for imaging early stage atherosclerosis. At stage 2, fatty streaks formation by macrophages displays scavenger function during immune responses.…”

“…1). 13 In addition, 18 F-FDG detects highly increased metabolic activity associated with atherosclerosis since the metabolic activities of cells are increased in the immune response area. 27,28,32 On the other hand, cathepsin protease activity and matrix metalloproteinases activity have been the main targets applied in fluorescent imaging, where diverse imaging probes were developed and used in apoE-/-mice.…”

“…2,23 These adhesion molecule and extracellular matrix targets, which are parenchymal components of atherosclerosis, can increase the success of imaging and many studies have in fact shown their application in pre-clinical and clinical trials. However, these cathepsin protease activity and matrix metalloproteinases activity targeted molecules, as well as 18 F-FDG are stage 3 or stage 4 imaging molecules, making them more suited for diagnosis rather than prognosis. As these methods also have increased background signals in the case of atherosclerosis, it is necessary to increase the contrast so that accurate imaging information can be extracted.…”

“…46,47 Recently, human carotid atherosclerosis imaging was evaluated by PET/CT multimodality using the 18 F-galacto-RGD probe. 48 During atherogenesis, arterial vessels undergo fibrosis and deposit minerals in the plaque area.…”