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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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111In-1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide-Affibody ZHER2:2395-Cys

111In-[MMA-DOTA-Cys61]-Z2395-C
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: November 19, 2009.

Chemical name:111In-1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide-Affibody ZHER2:2395-Cys
Abbreviated name:111In-[MMA-DOTA-Cys61]-Z2395-C, 111In-DOTA-Z2395-C
Synonym:
Agent category:Antibody fragment, Affibody
Target:Epidermal growth factor (EGF) HER2 receptor
Target category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT), gamma planar imaging
Source of signal:111In
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about HER2.

Background

[PubMed]

Epidermal growth factor (EGF) is a cytokine of 53 amino acids (6.2 kDa) and 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 a poor prognosis (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). 111In-Trastuzumab, Cy5.5-trastuzumab, and 68Ga-trastuzumab-F(ab')2 have been developed for imaging of 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 was constructed on the basis of the Z-domain residues (58 amino acids) from one of the IgG-binding domains of staphylococcal protein A (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]. A cysteine molecule was introduced at the C-terminus of Affibody molecules for site-specific coupling with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111In labeling (111In-[MMA-DOTA-Cys61]-Z2395-C). 111In-[MMA-DOTA-Cys61]-Z2395-C has been evaluated in nude mice bearing human colon adenocarcinoma tumors (18).

Synthesis

[PubMed]

Affibody Z2395-C was reduced with dithiothreitol in 0.02 M ascorbic acid for 18 h at 37°C (18). The reduced Z2395-C was incubated with MMA-DOTA in 1:1 molar ratio in ammonium acetate buffer (pH 5.5) for 18 h at 37°C. The coupling efficiency was 93% as determined by LC-MS analysis. There was ~1 chelator per antibody. MMA-DOTA-Z2395-C was isolated with column chromatography. MMA-DOTA-Z2395-C (25 µg) was mixed with 7.5 MBq (0.2 mCi) 111InCl3 and incubated for 60 min. The labeling efficiency of 111In incorporation was >93%. Specific activity of the preparation was 7 GBq/µmol (189 mCi/µmol). 111In-[MMA-DOTA-Cys61]-Z2395-C was found to be stable after incubation for 3 h in the presence of 1,000-fold molar excess of ethylenediaminetetraacetic acid.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Ahlgren et al. (18) performed binding experiments with Z2395-C with the use of a Biacore sensor chip immobilized with extracellular domain of HER2 protein. The KD value of Z2395-C was calculated to be 27 pM. The KD value of ZHER2:342 was 22 pM. Hence, the binding affinity of Z2395-C was about the same as the parent Affibody molecule ZHER2:342. In vitro binding specificity tests showed that binding of Z2395-C to SKOV-3 cells expressing HER2 was receptor-mediated because saturation of receptors by preincubation with non-labeled ZHER2:342 significantly decreased binding of 111In-[MMA-DOTA-Cys61]-Z2395-C. The cell-bound radioactivity remained at 79% of the initially bound activity for up to 24 h when the cells were incubated with 99mTc-Z2395-C.

Animal Studies

Rodents

[PubMed]

Ahlgren et al. (18) performed ex vivo biodistribution studies of 0.1 MBq (2.7 μCi) 111In-[MMA-DOTA-Cys61]-Z2395-C in nude mice bearing LS174T xenograft tumors. 99mTc-Z2395-C (0.14 nmol) was injected intravenously into each mouse. The initial tracer accumulation in the LS174T tumors was 12% injected dose per gram (ID/g) at 1 h and 10% ID/g at 4 h after injection. The radioactivity level in tumors was higher than in other organs and tissues (the lung, liver, spleen, and intestines) except in the kidneys (117–200% ID/g) at these time points. Blood levels were ~0.73% ID/g at 1 h and 0.1% ID/g at 4 h. The biodistribution was characterized by rapid clearance of radioactivity from blood. Tumor/blood ratios were 18 and 116 at 1 and 4 h after injection, respectively. Preadministration of ZHER2:342 (600 µg (83 nmol)) decreased tumor accumulation by >95% (P = 0.005) at 4 h after injection.

Single-photon emission computed tomography analysis was performed in nude mice bearing the LS174T tumors after injection of 3 MBq (81 μCi) 111In-[MMA-DOTA-Cys61]-Z2395-C. The tumor was clearly visualized at 1 h along with the kidneys. In mice pretreated with ZHER2:342, the tumors were marginally visualized with 111In-[MMA-DOTA-Cys61]-Z2395-C. Tumor/muscle ratios were 11.8 and 2.9 with blocking.

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.

References

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Carpenter G., Cohen S. Epidermal growth factor. J Biol Chem. 1990;265(14):7709–12. [PubMed: 2186024]
2.
Yarden Y. The EGFR family and its ligands in human cancer: signalling mechanisms and therapeutic opportunities. Eur J Cancer. 2001;37 Suppl 4:S3–8. [PubMed: 11597398]
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Rubin I., Yarden Y. The basic biology of HER2. Ann Oncol. 2001;12 Suppl 1:S3–8. [PubMed: 11521719]
4.
Grunwald V., Hidalgo M. Developing inhibitors of the epidermal growth factor receptor for cancer treatment. J Natl Cancer Inst. 2003;95(12):851–67. [PubMed: 12813169]
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Mendelsohn J. Anti-epidermal growth factor receptor monoclonal antibodies as potential anti-cancer agents. J Steroid Biochem Mol Biol. 1990;37(6):889–92. [PubMed: 2285602]
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Yasui W., Sumiyoshi H., Hata J., Kameda T., Ochiai A., Ito H., Tahara E. Expression of epidermal growth factor receptor in human gastric and colonic carcinomas. Cancer Res. 1988;48(1):137–41. [PubMed: 2446740]
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Ang K.K., Berkey B.A., Tu X., Zhang H.Z., Katz R., Hammond E.H., Fu K.K., Milas L. Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res. 2002;62(24):7350–6. [PubMed: 12499279]
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Costa S., Stamm H., Almendral A., Ludwig H., Wyss R., Fabbro D., Ernst A., Takahashi A., Eppenberger U. Predictive value of EGF receptor in breast cancer. Lancet. 1988;2(8622):1258. [PubMed: 2903994]
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Ethier S.P. Growth factor synthesis and human breast cancer progression. J Natl Cancer Inst. 1995;87(13):964–73. [PubMed: 7629883]
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Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology. 2001;61 Suppl 2:1–13. [PubMed: 11694782]
11.
Carter P., Presta L., Gorman C.M., Ridgway J.B., Henner D., Wong W.L., Rowland A.M., Kotts C., Carver M.E., Shepard H.M. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A. 1992;89(10):4285–9. [PMC free article: PMC49066] [PubMed: 1350088]
12.
Perik P.J., Lub-De Hooge M.N., Gietema J.A., van der Graaf W.T., de Korte M.A., Jonkman S., Kosterink J.G., van Veldhuisen D.J., Sleijfer D.T., Jager P.L., de Vries E.G. Indium-111-labeled trastuzumab scintigraphy in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. J Clin Oncol. 2006;24(15):2276–82. [PubMed: 16710024]
13.
Lub-de Hooge M.N., Kosterink J.G., Perik P.J., Nijnuis H., Tran L., Bart J., Suurmeijer A.J., de Jong S., Jager P.L., de Vries E.G. Preclinical characterisation of 111In-DTPA-trastuzumab. Br J Pharmacol. 2004;143(1):99–106. [PMC free article: PMC1575276] [PubMed: 15289297]
14.
Garmestani K., Milenic D.E., Plascjak P.S., Brechbiel M.W. A new and convenient method for purification of 86Y using a Sr(II) selective resin and comparison of biodistribution of 86Y and 111In labeled Herceptin. Nucl Med Biol. 2002;29(5):599–606. [PubMed: 12088731]
15.
Smith-Jones P.M., Solit D., Afroze F., Rosen N., Larson S.M. Early tumor response to Hsp90 therapy using HER2 PET: comparison with 18F-FDG PET. J Nucl Med. 2006;47(5):793–6. [PMC free article: PMC3193602] [PubMed: 16644749]
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Smith-Jones P.M., Solit D.B., Akhurst T., Afroze F., Rosen N., Larson S.M. Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors. Nat Biotechnol. 2004;22(6):701–6. [PubMed: 15133471]
17.
Wikman M., Steffen A.C., Gunneriusson E., Tolmachev V., Adams G.P., Carlsson J., Stahl S. Selection and characterization of HER2/neu-binding affibody ligands. Protein Eng Des Sel. 2004;17(5):455–62. [PubMed: 15208403]
18.
Ahlgren S., Orlova A., Rosik D., Sandstrom M., Sjoberg A., Baastrup B., Widmark O., Fant G., Feldwisch J., Tolmachev V. Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules. Bioconjug Chem. 2008;19(1):235–43. [PubMed: 18163536]
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