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64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid-human serum albumin-Ac-Cys-ZEGFR:1907

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

Created: ; Last Update: September 27, 2012.

Chemical name:64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid-human serum albumin-Ac-Cys-ZEGFR:1907
Abbreviated name:64Cu-DO3A-HSA-ZEGFR:1907
Agent category:Antibody, Affibody
Target:Epidermal growth factor receptor (EGFR, HER1)
Target category:Receptor
Method of detection:Positron emission tomography
Source of signal:64Cu
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Click on protein, nucleotide (RefSeq), and gene for more information about EGFR.



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 in 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 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-acetic acid-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 (20) imaging studies in tumor-bearing mice (21). Although high specific tumor accumulation was observed with 111In- and 64Cu-labeled ZEGFR:1907, very high kidney accumulation (>100% injected dose per gram (ID/g)) was also observed. Addition of human serum albumin (HSA) to Affibody ZHER2:342 dramatically reduced kidney accumulation and improved tumor accumulation (22). Therefore, Hoppmann et al. (23) prepared 64Cu-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-human serum albumin-ZEGFR:1907 (64Cu-DO3A-HSA-ZEGFR:1907) for evaluation showing good PET tumor contrast with low kidney accumulation in tumor-bearing mice.

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Ac-Cys-ZEGFR:1907 was prepared with automated solid-phase peptide synthesizer (23). DOTA-N-hydroxysuccinimide ester and HSA (100:1 molar ratio) were incubated in borate buffer (pH 8.5) for 2 h at 4°C to form DO3A-HSA, which was isolated with PD-10 column chromatography. DO3A-HSA was then reacted with sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) in a molar ratio of 1:5 for 1 h at 4°C. The resulting bioconjugate was further purified with PD-10 column chromatography. Ac-Cys-ZEGFR:1907 was conjugated in a site-specific manner to the DOTA and Sulfo-SMCC modified HSA via the cysteine residue. The reaction was performed in a molar ratio of 1:5 of DO3A-HSA-SMCC and Ac-Cys-ZEGFR:1907 (22 nmol) for 6 h at room temperature. DO3A-HSA-ZEGFR:1907 was purified with ultracentrifugation. There were an average of three DO3A moieties and two Ac-Cys-ZEGFR:1907 moieties per HSA protein as determined with MALDI-TOF-MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Approximately 2.4 nmol DO3A-HSA-ZEGFR:1907 was radiolabeled with 64Cu by addition of 37 MBq (1 mCi) 64CuCl2 in 0.1 N sodium acetate buffer (pH 6.0) and incubated for 1 h at 39°C. 64Cu-DO3A-HSA-ZEGFR:1907 was isolated with PD-10 column chromatography, with >70% radiolabeling yield. 64Cu-DO3A-HSA-ZEGFR:1907 exhibited a specific activity of ~14 MBq/nmol (0.38 mCi/nmol) at the end of synthesis.

In Vitro Studies: Testing in Cells and Tissues


64Cu-DO3A-HSA-ZEGFR:1907 accumulated in SAS human oral squamous carcinoma cells with 26.8 ± 2.5% incubation dose at 37°C for 2 h (23). Co-incubation with excess Ac-Cys-ZEGFR:1907 reduced the accumulation to 9.6 ± 1.5% incubation dose. The Kd value of 64Cu-DO3A-HSA-ZEGFR:1907 for EGFR of SAS cells was not determined.

Animal Studies



PET static imaging analysis was performed in nude mice (n = 4) bearing SAS tumors at 1, 4, 24, 48, and 72 h after intravenous injection of 6.7–7.8 MBq (0.18–0.21 mCi) 64Cu-DO3A-HSA-ZEGFR:1907 (~0.5 nmol) (23). The tumor was clearly visualized at 24–72 h. Quantification analysis showed that the tumor accumulation levels were 3.3 ± 0.2% ID/g, 5.2 ± 0.4% ID/g, 7.0 ± 1.1% ID/g, 7.7 ± 1.1% ID/g, and 7.5 ± 1.0% ID/g at 1, 4, 24, 48, and 72 h, respectively. At 24 h, the liver and kidney accumulation levels were 11.3 ± 1.1% ID/g and 2.5 ± 1.7% ID/g, respectively. The kidney accumulation (2.5% ID/g) of 64Cu-DO3A-HSA-ZEGFR:1907 was drastically reduced compared with 64Cu-DOTA -ZEGFR:1907 (88.4% ID/g) (21). At 1–72 h, the blood levels were <5% ID/g, and the muscle levels were <2% ID/g. No blocking and ex vivo biodistribution studies were reported.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

5R01 CA119053, P50 CA114747


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