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111In-2-(4,7,10-Tris(carboxymethyl)-1,4,7,10-tetracyclododecan-1-yl)pentanedioic acid-Trastuzumab

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

Created: ; Last Update: September 13, 2012.

Chemical name:111In-2-(4,7,10-Tris(carboxymethyl)-1,4,7,10-tetracyclododecan-1-yl)pentanedioic acid-Trastuzumab
Abbreviated name:111In-DOTAGA-Trastuzumab
Agent category:Antibody
Target:Epidermal growth factor receptor (EGFR, HER2)
Target category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT), gamma planar imaging
Source of signal:111In
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Click on protein, nucleotide (RefSeq), and gene for more information about HER2.



Epidermal growth factor (EGF) is a growth factor composed of 53 amino acids (6.2 kDa), and it 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 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). 111In-Trastuzumab, Cy5.5-trastuzumab, 89Zr-trastuzumab, and 68Ga-trastuzumab-F(ab')2 have been developed for imaging of human breast cancer (12-17). Moreau et al. (18) conjugated 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetracyclododecan-1-yl)pentanedioic acid (DOTAGA), which is a derivative of 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), to trastuzumab and labeled it with 111In as 111In-DOTAGA-trastuzumab for single-photon emission computed tomography (SPECT) imaging of HER2 expression in tumors.



Trastuzumab (20 nmol) was incubated with DOTAGA-anhydride (400 nmol) in phosphate-buffered saline (PBS) (pH, 7.4) for 30 min at 25°C (18). DOTAGA-Trastuzumab was isolated with ultrafiltration. There were 2.6 DOTAGA moieties per conjugate (148.9 kDa), as confirmed with mass spectroscopy. DOTAGA-Trastuzumab (0.83 nmol) was mixed with 125 MBq (3.4 mCi) 111InCl3 in ammonium acetate buffer (pH, 5.7) and incubated for 3 h at 37°C, with 66% labeling yield after removing free 111In3+ ions with ETDA treatment and isolation with ultrafiltration. The radiochemical purity was >97%. The specific activity was 150 MBq/nmol (4.1 mCi/nmol) at the end of synthesis. 111In-DOTAGA-Trastuzumab was >97% intact after incubation with 2,000-fold excess diethylenetriamine pentaacetic acid in PBS for up to 5 d.

In Vitro Studies: Testing in Cells and Tissues


Moreau et al. (18) performed saturation binding experiments with 111In-DOTAGA-trastuzumab using the HER2-positive HCC1954 breast tumor cell line. The binding affinity (Kd) value was 5.5 ± 0.6 nM. 111In-DOTAGA-Trastuzumab exhibited an immunoreactivity of 65%.

Animal Studies



Moreau et al. (18) performed SPECT imaging scans in female nude mice (n = 4) bearing BT-474 human breast tumors at 24, 48, and 72 h after injection of 15 MBq (0.41 mCi) 111In-DOTAGA-trastuzumab (0.17 nmol). The tumors were clearly visualized at these time points. Region of interest analysis showed that the tumor accumulation of radioactivity was 11.0 ± 5.6% injected dose (ID), 13.1 ± 7.6% ID, and 14.0 ± 8.1% ID at 24, 48, and 72 h after injection, respectively. The radioactivity was located mainly in the rim of the tumor, with a lower radioactivity in the necrotic center of the tumor. The effective half-life (radioactive decay of 111In and biological excretion) of 111In-DOTAGA-trastuzumab in the mice was found with the image analysis to be 53.5 ± 4.0 h. The biological half-life (biological excretion) was calculated to be 268 ± 92 h. Ex vivo tissue biodistribution study was performed at 72 h after the last SPECT scan. Tumor accumulation was 66.9 ± 0.9% ID/g or 13.2 ± 8.2% ID. The highest radioactivity level was in the blood (10.0% ID/g), followed by the ovary (8.0% ID/g), spleen (6.9% ID/g), kidney (6.3% ID/g), lung (5.6% ID/g), liver (4.3% ID/g), heart (3.6% ID/g), and gastrointestinal tract (1.8% ID/g). Co-injection with 100-fold excess unlabeled trastuzumab reduced tumor accumulation by 73% at 72 h after injection, with little effect on the blood radioactivity.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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