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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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99mTc(CO)3-Anti-vascular cell adhesion molecule-1 nanobody cAbVCAM1-5

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: July 26, 2012.

Chemical name:99mTc(CO)3-Anti-vascular cell adhesion molecule-1 nanobody cAbVCAM1-5
Abbreviated name:99mTc-cAbVCAM1-5
Agent category:Antibody fragment, nanobody
Target:Vascular cell adhesion molecule-1 (VCAM-1)
Target category:Adhesion molecule
Method of detection:Single-photon emission computed tomography (SPECT), gamma planar imaging
Source of signal:99mTc
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  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about VCAM-1.



Endothelial cells are important cells in inflammatory responses (1, 2). Bacterial lipopolysaccharide (LPS), virus, inflammation, and tissue injury increase tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and other cytokine and chemokine secretion. Leukocyte emigration from blood is dependent on their ability to adhere to endothelial cell surfaces. Inflammatory mediators and cytokines induce chemokine secretion from endothelial cells and other vascular cells and increase their expression of cell surface adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), integrins, and selectins. Chemokines are chemotactic toward leukocytes and toward sites of inflammation and tissue injury. The movement of leukocytes through endothelial junctions into the extravascular space is highly orchestrated through various interactions with different adhesion molecules on endothelial cells (3).

VCAM-1 is found in very low amounts or is undetectable on the cell surface of resting endothelial cells and other vascular cells, such as smooth muscle cells and fibroblasts (4-8). VCAM-1 binds to very late antigen-4 (VLA-4) integrin on the cell surface of leukocytes. IL-1 and TNFα increase expression of VCAM-1 and other cell adhesion molecules on vascular endothelial cells, which leads to leukocyte adhesion to the activated endothelium. Furthermore, VCAM-1 expression is also induced by oxidized low-density lipoproteins under atherogenic conditions (9). Overexpression of VCAM-1 by atherosclerotic lesions plays an important role in their progression toward vulnerable plaques, which may erode and rupture. Microbubbles targeted with monoclonal antibody against VCAM-1 are being developed as a noninvasive agent for VCAM-1 expression in vascular endothelial cells during different stages of inflammation in atherosclerosis (10). Nanobodies are the smallest intact antigen-binding fragments (15 kDa) isolated from heavy-chain camelid antibodies with efficient and specific tumor targeting (11-13). Broisat et al. (14) evaluated ten anti-VCAM-1 nanobodies, of which cAbVCAM1-5 was the lead nanobody. cAbVCAM1-5 is cross-reactive for human VCAM-1 (hVCAM1) and mouse VCAM-1 (mVCAM1) with nanomolar affinities. cAbVCAM1-5 was radiolabeled with 99mTc using tricarbonyl chemistry (99mTc-cAbVCAM1-5) for noninvasive in vivo single-photon emission computed tomography (SPECT) imaging of atherosclerotic lesions in apolipoprotein E (ApoE)-deficient (ApoE–/–) mice.



cAbVCAM1-5 were produced as hexahistidine-tagged proteins in Escherichia coli (14). A solution of [99mTc(H2O)3(CO)3]+ and nanobody cAbVCAM1-5 (pH 7–8) was incubated for 90 min at 60°C, with a radiochemical purity of >95%. The specific activity and yield of 99mTc-cAbVCAM1-5 were not reported. 99mTc-cAbVCAM1-5 was >98% intact for up to 6 h at 20°C in phosphate-buffered saline (PBS). 99mTc-cAbBcII10 was prepared similarly as a control.

In Vitro Studies: Testing in Cells and Tissues


Binding experiments with cAbVCAM1-5 with the use of a Biacore sensor chip immobilized with mVCAM1 or hVCAM1 protein were performed (14). The Kd values of cAbVCAM1-5 were calculated to be 2.0 nM and 6.5 nM for mVCAM1 and hVCAM1, respectively. cAbBcII10 showed no binding to mVCAM1 or hVCAM1.

Broisat et al. (14) performed cell uptake assays with 99mTc-cAbVCAM1-5 (5 nmol) using mouse bEND5 endothelial cells. The assays were performed after incubation for 90 min at 37°C. High levels of radioactivity accumulated in bEND5 cells that were activated with TNFα compared to non-stimulated bEND5 cells (P < 0.05). Co-incubation with 500-fold excess unlabeled cAbVCAM1-5 almost completely inhibited the radioactivity in TNFα-activated bEND5 cells. Flow cytometry analysis also showed binding of cAbVCAM1-5 to TNFα-activated mouse and human endothelial cells, whereas cAbBcII10 showed no binding.

Animal Studies



Broisat et al. (14) performed ex vivo biodistribution studies in ApoE–/– mice (n = 6/group) and control C57Bl/6J mice (n = 4/group) at 3 h after injection of 99mTc-cAbVCAM1-5 or 99mTc-cAbBcII10. The accumulation of both tracers was highest in the kidneys (>222% ID/g) in both types of mice. The VCAM-1–positive organs from the ApoE–/– mice and the control mice, such as the spleen (9.2% versus 7.4% ID/g), thymus (1.7% versus 1.5% ID/g), and bone marrow (10.7% versus 7.9% ID/g), showed higher accumulation of 99mTc-cAbVCAM1-5 than the control 99mTc-cAbBcII10 (0.1–1.0% ID/g) (P < 0.05). 99mTc-cAbVCAM1-5 accumulation in the spleen, bone marrow, lymph node, thymus, liver, heart, and aortic arch lesions was significantly blocked (P < 0.05) by co-injection of 100-fold excess unlabeled cAbVCAM1-5 in ApoE–/– mice. The accumulation of 99mTc-cAbVCAM1-5 correlated with the volume of the atherosclerotic lesions as measured with immunochemistry analysis (P < 0.001), whereas no correlation with 99mTc-cAbBcII10 was found.

SPECT imaging at 3 h after injection showed that the aortic arch/blood ratio for 99mTc-cAbVCAM1-5 was higher (P < 0.05) in ApoE–/– mice (n = 6; 2.5 ± 0.2) than C57Bl/6J mice (n = 4; 1.2 ± 0.2), whereas little difference was observed with 99mTc-cAbBcII10 (1.0 ± 0.2 versus 0.8 ± 0.1) (14). The aortic arch lesions, kidneys, and urinary bladder were clearly visualized in the ApoE–/– mice. Co-injection of 100-fold excess unlabeled cAbVCAM1-5 abrogated the visualization of the lesions in the aortic arch but not the kidneys.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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