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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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Cy5.5-Ac-Cys-ZEGFR:1907

Cy5.5-ZEGFR:1907
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: September 7, 2012.

Chemical name:Cy5.5-Ac-Cys-ZEGFR:1907
Abbreviated name:Cy5.5-ZEGFR:1907
Synonym:
Agent category:Antibody, Affibody
Target:Epidermal growth factor receptor (EGFR, HER1)
Target category:Receptor
Method of detection:Optical, near-infrared (NIR) fluorescence imaging
Source of signal:Cy5.5
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about EGFR.

Background

[PubMed]

Epidermal growth factor (EGF) is a growth factor composed of 53 amino acids (6.2 kDa) that is secreted by ectodermic cells, monocytes, kidneys, and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF and 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: 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 and HER2 are overexpressed on many solid tumor cells such as breast, non-small cell lung, head and neck, and colon cancers (4-6). The high levels of HER1 and HER2 expression on cancer cells are associated with poor patient prognosis because high levels are related to increased proliferation (7-10).

Trastuzumab is a humanized IgG1 monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant (Kd) of 0.1 nM (11). Trastuzumab is approved for clinical use for anti-cancer therapies in both Europe and North America. 111In-Trastuzumab, Cy5.5-trastuzumab, and 68Ga-trastuzumab-F(ab')2 have been developed for imaging human breast cancer (12-16). However, the pharmacokinetics of the intact radiolabeled mAb, with high liver uptake and slow blood elimination, are generally not ideal for imaging. Smaller antibody fragments, such as Fab or F(ab')2, have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. A novel class of recombinant affinity ligands (Affibody molecules) for HER2, based on the Z-domain residues (58 amino acids) from one of the IgG-binding domains of staphylococcal protein A, was constructed (17). Affibody molecules exhibit high binding affinity to HER2 with Kd values <100 pM. Various radiolabeled Affibody molecules have been studied in terms of their ability to image HER2 in tumors [PubMed]. The EGFR-specific Affibody ZEGFR:1907 (6.7 kDa) was identified to have good affinity (Kd = 5.4 nM) for EGFR (HER1) and was labeled with 111In via isothiocyanate-benzoyl-diethylenetriamine pentaacetic acid (DTPA) to form 111In-Bz-DTPA-ZEGFR:1907 for single-photon emission computed tomography (18) imaging in nude mice bearing human tumors (19). To facilitate site-specific conjugation, Ac-Cys-ZEGFR:1907 (Cys at the N terminal) was synthesized and conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA) to form Ac-Cys(DOTA)-ZEGFR:1907 (DOTA-ZEGFR:1907), and the product was labeled with 64Cu (t1/2, 12.7 h) to form 64Cu-DOTA-ZEGFR:1907 for positron emission tomography imaging studies in tumor-bearing mice (20). In this chapter, Cy5.5-Ac-Cys-ZEGFR:1907 (Cy5.5-ZEGFR:1907) was prepared and evaluated in EGFR-positive tumor in mice with specific tumor accumulation of the probe using near-infrared (NIR) fluorescence imaging (21).

Synthesis

[PubMed]

Ac-Cys-ZEGFR:1907 (6.7 kDa) was prepared with an automated solid-phase peptide synthesizer, with 10% yield and >95% purity after purification with high-performance liquid chromatography (HPLC) (21). Cy5.5-Mono-maleimide and Ac-Cys-ZEGFR:1907 (1.5:1 molar ratio) were incubated in phosphate buffer (pH 7.4) for 2 h at 25°C. Cy5.5-ZEGFR:1907 (7.7 kDa) was isolated with 47% recovery yield and >95% purity after HPLC purification. Mass spectroscopy analysis confirmed 1:1 Cy5.5conjugation. Spectrum analysis showed that Cy5.5-ZEGFR:1907 exhibited similar maximal fluorescence absorption (680 nm) and emission (696 nm) characteristics similar to those of free Cy5.5 dye.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Fluorescence microscopy analysis was performed in A431 human epidermoid carcinoma cells (high EGFR expression) and MCF7 human breast cancer cells (low EGFR expression) after incubation with 100 nM Cy5.5-ZEGFR:1907 for 60 min at 37°C (21). A431 exhibited intensive fluorescence signal from the cell surface and cytoplasm. Co-incubation with 100-fold excess Ac-Cys-ZEGFR:1907 completely abrogated the signal. On the other hand, fluorescence signal from MCF7 cells was barely visible. Cy5.5-ZEGFR:1907 exhibited a Kd value of 43.6 ± 8.4 nM for the EGFR of A431 cells, as measured with flow cytometry analysis using different doses of the probe (0.1–100 nM).

Animal Studies

Rodents

[PubMed]

Miao et al. (21) performed NIR fluorescence imaging of nude mice (n = 3/group) bearing A431 or MCF7 tumors at 10, 30, 60, 120, and 240 min after intravenous injection of 0.5 nmol Cy5.5-ZEGFR:1907. The accumulation of Cy5.5-ZEGFR:1907 in A431 tumors could be clearly visualized from 10–240 min, with a maximum contrast at 120 min. The tumor/background ratio was 2.9 ± 0.1 at 240 min. Co-injection of Ac-Cys-ZEGFR:1907 (45 nmol) inhibited the tumor/background ratio to 1.8 ± 0.4 at 240 min (P < 0.05). MCF7 tumors were barely visualized, with a tumor/background ratio of 1.5 ± 0.2. Ex vivo NIR fluorescence imaging of tissues at 24 h after injection showed that the tissue with the highest signal (photons/s/cm2/steradian) was the kidney (1.2 × 108), followed by the stomach (0.7 × 108), A431 tumor (0.2 × 108), and liver (0.1 × 108). The spleen, heart, lung, blood, and muscle exhibited <0.03 × 108 photons/s/cm2/steradian. The tumor/blood, tumor/muscle, tumor/lung, and tumor/liver ratios were 8, 7, 6, and 2, respectively.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

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