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

Created: ; Last Update: March 28, 2012.

Chemical name:Cy5.5-Trastuzumab
Abbreviated name:Cy5.5-Trast
Agent category:Antibody
Target:Epidermal growth factor receptor-2 (EGFR-2, HER2, erbB-2/neu)
Target category:Receptor
Method of detection:Optical, near-infrared (NIR) fluorescence
Source of signal:Cy5.5
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about HER2.



Epidermal growth factor (EGF) is a 53-amino acid cytokine (6.2 kDa) secreted by ectodermic cells, monocytes, kidneys and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF with at least seven other growth factors and their transmembrane receptor kinases play important roles in cell proliferation, survival, adhesion, migration and differentiation. The EGF receptor (EGFR) family consists of four transmembrane receptors, including EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3) and HER4 (erbB-4) (2). HER1, HER3 and HER4 comprise three major functional domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain. No ligand has been clearly identified for HER2. However, HER2 can be activated as a result of ligand binding to other HER receptors with the formation of receptor homodimers and/or heterodimers (3). HER1 as well as HER2 are overexpressed on many solid tumor cells such as breast, non-small-cell lung, head and neck, and colon cancer (4-6). The high levels of HER1 and HER2 expression on cancer cells are associated with a poor prognosis (7-10).

Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets (11-13). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350-700 nm) are used. Near-infrared (NIR) fluorescence (700-1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have a wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low fluorescence background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive complement to radionuclide imaging in small animals.

Trastuzumab is a humanized IgG1 monoclonal antibody (MAb) against the extracellular domain of recombinant HER2 with an affinity constant (Kd) of 0.1 nM (14). Cardiotoxicity is the most serious complication of using trastuzumab in humans with breast cancer (15). One potential application of a radiolabeled anti-HER2 MAb is the pretreatment imaging of breast cancer patients to predict the therapeutic efficacy of trastuzumab. 111In-Trastuzumab and 68Ga-F(ab')2-trastuzumab have been developed for imaging of human breast cancer (16-19). Trastuzumab has also been successfully coupled with Cy5.5 NIR dye for optical imaging of HER2 density in tumors in mice (20).



Commercially available Cy5.5 monofunctional N-hydroxysuccinimide ester was used to conjugate trastuzumab to form Cy5.5-trastuzumab, which was purified by gel-filtration chromatography (20). The molar ratio of Cy5.5 to trastuzumab was estimated to be 2.0-2.5 by spectrometric analyses.

In Vitro Studies: Testing in Cells and Tissues


Hilger et al. (20) performed cell-binding assays with Cy5.5-trastuzumab using a human SK-BR-3 breast cancer cells (overexpressing HER2) and normal squamous cell carcinoma cell line PE/CA-PJ34 (normal HER2 expression). Incubation of 0.66 nmol Cy5.5-trastuzumab for 3 h at 4°C showed that a higher fluorescence intensity (a.u.) was observed in the SK-BR-3 cells (1x107 cells, 2133 ± 143 a.u.) than the PE/CA-PJ34 (1x107 cells, 975 ± 95 a.u.). On the other hand, Cy5.5-IgG bindings in both cell types were low (< 500 a.u.) as well as the background fluorescence with buffer alone (~200 a.u.). These results were also confirmed using fluorescence flow cytometry and laser scanning microscopy. No blocking experiment was performed.

Animal Studies



Hilger et al. (20) studied the accumulation of Cy5.5-trastuzumab in mice bearing SK-BR-3 (overexpressing HER2) or PE/CA-PJ34 (normal HER2 expression) tumors using whole-body fluorescence detection system. Cy5.5-Trastuzumab (0.66 nmol/mouse) injected intravenously into SCID mice (n = 6) bearing SK-BR-3 tumors (tumor/background = 2.2 ± 0.1) showed distinctly higher fluorescence signals in the SK-BR-3 tumors as compared with PE/CA-PJ34 tumors (tumor/background = 1.3 ± 0.2) in a different group of mice (n = 6) at 16 h after injection. There was a marked intensity of signal in the liver of mice bearing the tumors. Injection of Cy5.5 labeled control IgG in mice (n = 3) bearing the SK-BR-3 tumors revealed tumor/background ratio of 1.4. No blocking experiment was performed.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No relevant publication is currently available.


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