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111In-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid- Affibody ZHER2:342-pep2

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

Created: ; Last Update: June 27, 2007.

Chemical name:111In-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-Affibody ZHER2:342-pep2
Abbreviated name:111In-DOTA-ZHER2:342-pep2
Agent category:Antibody fragment, Affibody
Target:EGF HER2 receptor
Target category:Receptor
Method of detection:SPECT, gamma planar
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 cytokine that comprises 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 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 based on the 58-amino-acid Z-domain residues from one of the IgG-binding domains of staphylococcal protein A (17). Affibody molecules exhibit high binding affinity to HER2 with Kd values of <100 pM. Various radiolabeled Affibody molecules have been studied in terms of their ability to image HER2 in tumors [PubMed]. A synthetic Affibody molecule was successfully produced as DOTA-ZHER2:342-pep2 for labeling with 111In. 111In-DOTA-ZHER2:342-pep2 has been evaluated in nude mice bearing human SKOV-3 ovarian cancer cells (18).



DOTA-ZHER2:342-pep2 was prepared by standard peptide synthesis (18). DOTA-ZHER2:342-pep2 in 0.2 M ammonium acetate buffer (pH 5.25) was mixed with a predetermined amount of 111InCl3. The mixture was incubated for 30 or 60 min at different temperatures (room temperature, 37°C, 50°C, and 90°C). Under all conditions tested, the labeling efficiency of 111In incorporation was 96% and 98% after incubation for 30 or 60 min at room temperature, respectively; 98% for both times at 37°C; 99% for both times at 50°C; and 99% for 30 min at 90°C. Specific activities of the preparations were not mentioned.

In Vitro Studies: Testing in Cells and Tissues


Orlova et al. (18) performed binding experiments with DOTA-ZHER2:342-pep2 with use of a Biacore sensor chip immobilized with extracellular domain of HER2 (HER2ECD) or chimeric HER2/Fc fusion protein. The Kd values of DOTA-ZHER2:342-pep2 were calculated to be 65 pM for HER2ECD and 78 pM for chimeric HER2/Fc fusion protein. The Kd values of His6-ZHER2:342 were 20 pM for HER2ECD and 29 pM for chimeric HER2/Fc fusion protein. Hence, the binding affinity of the synthetic Affibody molecule DOTA-ZHER2:342-pep2 was approximately two-fold lower than the recombinant Affibody molecule His6-ZHER2:342.

In vitro binding specificity tests showed that binding of 111In-DOTA-ZHER2:342-pep2 to SKOV-3 cells expressing HER2 was receptor-mediated because saturation of receptors by preincubation with non-labeled His6-ZHER2:342 significantly decreased binding of 111In-DOTA-ZHER2:342-pep2 (P < 0.0001). The antigen binding capacity of 111In-DOTA-ZHER2:342-pep2 was 82–86%. After an initial reduction, the cell-bound radioactivity remained constant at ~90% of the initially bound activity for up to 24 h when the cells were incubated with 111In-DOTA-ZHER2:342-pep2. In contrast, cells incubated with 125I-His6-ZHER2:342 lost ~40% of the original cell-associated radioactivity during the first 4 h and an additional 20% at 29 h.

Animal Studies



Orlova et al. (18) monitored biodistribution using single-photon emission computed tomography (SPECT) analysis after injection of therapeutic doses of 0.1 MBq (2.7 μCi) 111In-DOTA-ZHER2:342-pep2 in nude mice bearing tumor xenografts. Tumor radioactivity was measured 4 h after tracer injection. The tumor xenograft with the highest uptake was the human breast cancer cell line BT474 (39.9% injected dose per gram (ID/g)), followed by the human ovarian cancer cell line SKOV-3 (13.3% ID/g), the human breast cancer cell line MCF7 (12.4% ID/g), the human breast cancer cell line MDA-MB-231 (4.4% ID/g), and the human Ramos B lymphoma cell line with no HER2 expression (0.1% ID/g). The uptake values in these tumors were proportional to their HER2 expression levels. Pretreatment with ZHER2:342 significantly inhibited the 111In-DOTA-ZHER2:342-pep2 uptake values in all HER2-expressing tumors. The kidney was the only organ that had a higher accumulation than the tumors (256% ID/g at 4 h). The tumor/blood ratio was 8 ± 1 at 1 h and 121 ± 33 at 72 h. SPECT gamma imaging revealed that the SKOV-3 tumors were clearly visualized in mice injected with 111In-DOTA-ZHER2:342-pep2. In mice pretreated with ZHER2:342, the tumors could not be visualized with 111In-DOTA-ZHER2:342-pep2. Furthermore, the tumors could not be visualized by injection of a control-labeled nonspecific peptide (111In-DOTA-Ztaq4:5).

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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