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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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64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-epidermal growth factor receptor-binding fibronectin domain E13.4.3'

64Cu-DOTA-FnE13.4.3'
, PhD
National for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: August 9, 2012.

Chemical name:64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-EGFR-binding fibronectin domain E13.4.3'
Abbreviated name:64Cu-DOTA-FnE13.4.3', 64Cu-FnE13.4.3'
Synonym:
Agent category:Protein
Target:Epidermal growth factor receptor (EGFR, HER1)
Target category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal:64Cu
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about fibronectin.

Background

[PubMed]

Epidermal growth factor (EGF) is a 53-amino-acid growth factor (6.2 kDa) that is secreted by ectodermic cells, monocytes, kidneys, and duodenal glands (1). EGF stimulates the 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, 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 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, a humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 (11), was labeled as 111In-trastuzumab (12-14). C225, an anti-EGFR (HER1), mouse-human chimeric, IgG1 mAb, also known as erbitux and cetuximab, was labeled as 99mTc-EC-C225 (15, 16) and 64Cu-DOTA-cetuximab for imaging EGFR expression on solid tumors with the use of single-photon emission computed tomography (SPECT) and positron emission tomography (PET), respectively. However, the pharmacokinetics of the intact radiolabeled mAb, with high liver uptake and slow blood elimination, are generally not ideal for imaging (17, 18). Smaller antibody fragments, such as scFv, Fab, or F(ab')2, have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. Nanobodies (15 kDa) and affibodies (50 kDa) exhibit efficient and specific tumor targeting but with high kidney accumulation (19-21). An engineered fibronectin scaffold with EGFR-binding domain (FnE13.4.3', 10 kDa) was radiolabeled with 64Cu via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugation to form 64Cu-DOTA-FnE13.4.3' as a PET probe for in vivo imaging of EGFR in tumor-bearing nude mice (22). FnE13.4.3' does not contain the integrin binding sequence of Arg-Gly-Asp (RGD) (23).

Synthesis

[PubMed]

EGFR-binding FnE13.4.3' (92 amino acids) with amino acid residue 63 mutated to arginine to create a single primary amine at the N-terminus was purified from the transfected bacteria (22). A C-terminal His6 tag was added for purification and identification. DOTA-FnE13.4.3' was prepared by incubation of FnE13.4.3' and DOTA-N-hydroxysuccinimide ester in 1:20 molar ratio for 60 min at room temperature. DOTA-FnE13.4.3' was purified with high-performance liquid chromatography and mass spectroscopy analysis supported a 1:1 DOTA to protein conjugation. Radiolabeling was performed by mixing 64CuCl2 with DOTA- FnE13.4.3' in sodium acetate buffer at pH 5.5. The mixture was heated at 37°C for 60 min. 64Cu-DOTA-FnE13.4.3' was purified with column chromatography. The radiochemical purity was >96%, with a specific activity of 15 MBq/nmol (0.41 mCi/nmol) at the end of purification. The labeling yield was ~90%. 64Cu-DOTA-FnE13.4.3' was >98% intact in 50% human or mouse serum at 37°C for 24 h. EGFR non-binding 64Cu-DOTA-FnWT' was also prepared similarly.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

64Cu-DOTA-FnE13.4.3' (40 nM) exhibited significantly higher accumulation of radioactivity in EGFR(+) A431 human epidermoid carcinoma cells than 64Cu-DOTA-FnWT' and buffer (P < 0.005) (22). Unlabeled FnE13.4.3' (800 nM) blocked the accumulation by >65% (P < 0.03). Using five cell lines with different expression levels of EGFR for 64Cu-DOTA-FnE13.4.3' binding, there was significant correlation between 64Cu-DOTA-FnE13.4.3’' accumulation and EGFR expression levels (P < 0.017). Saturation binding assays showed the binding affinity (Kd) values were 5 ± 2 nM and 17 ± 10 nM for FnE13.4.3' and DOTA-FnE13.4.3', respectively.

Animal Studies

Rodents

[PubMed]

Hackel et al. (22) performed ex vivo biodistribution studies of 1 MBq (0.03 mCi) 64Cu-DOTA-FnE13.4.3' (0.25 nmol) or 1 MBq (0.03 mCi) 64Cu-DOTA-FnWT' (0.1 nmol) in A431 tumor-bearing nude mice (n = 3) at 1 h after injection. Accumulation of 64Cu-DOTA-FnE13.4.3' into A431 tumor was 2.4 ± 1.0% injected dose/gram (ID/g). The kidney accumulation was 200% ID/g. The accumulation levels in other normal tissues were low (<1% ID/g) except those for the liver (3% ID/g) and lung (7% ID/g). The tumor/blood and tumor/muscle ratios were 8.9 and 10.3, respectively. 64Cu-DOTA-FnWT' showed low accumulation in the tumor (0.6 ± 0.1% ID/g) but a similar biodistribution profile in the non-tumor tissues. No blocking studies were performed with unlabeled FnE13.4.3'.

Whole-body PET imaging studies were performed in A431 or MDA-MB-435 tumor-bearing nude mice at 1, 2, 4, and 24 h after injection of 1 MBq (0.03 mCi) 64Cu-DOTA-FnE13.4.3' (n = 5) or 1 MBq (0.03 mCi) 64Cu-DOTA-FnWT' (n = 3) (22). The A431 tumor was clearly visualized with 64Cu-DOTA-FnE13.4.3' but not with 64Cu-DOTA-FnWT' at 1 h after injection. Tumor accumulation level (estimated from the PET images) of 64Cu-DOTA-FnE13.4.3' was 3.4 ± 1.0% ID/g at 1 h with a tumor/muscle ratio of 8.6. At 24 h, the tumor accumulation level of 64Cu-DOTA-FnE13.4.3' was 2.7 ± 0.6% ID/g with a tumor/muscle ratio of 6.9. Tumor accumulation level of 64Cu-DOTA-FnWT' was 0.8 ± 0.2% ID/g at 1 h with a tumor/muscle ratio of 1.8. Intense accumulation in the kidneys and urinary bladder was observed. 64Cu-DOTA-FnE13.4.3' exhibited <1% ID/g in MDA-MB-435 tumors (low EGFR expression). No blocking studies were performed.

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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