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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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99mTc-Mercaptoacetyl-Ser-Ser-Ser-Affibody ZHER2:342

, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: May 5, 2008.

Chemical name:99mTc-Mercaptoacetyl-Ser-Ser-Ser-Affibody ZHER2:342
Abbreviated name:99mTc-MaSSS-ZHER2:342
Agent category:Antibody fragment, Affibody
Target:EGF HER2 receptor
Target category:Receptor
Method of detection:SPECT, gamma planar
Source of signal:99mTc
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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). Trastuzumab is approved for clinical use for 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 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]. Mercaptoacetyl-Gly-Gly-Gly (MAG3) was used a chelating linker for coupling 99mTc to ZHER2:342 Affibody (18). 99mTc-MAG3-ZHER2:342 has been evaluated in nude mice bearing human colon adenocarcinoma tumors, resulting in high tumor/blood and tumor/muscle ratios. However, 99mTc-MAG3-ZHER2:342 exhibited a high hepatobiliary clearance, which resulted in a high radioactivity (30% of the injected dose) in the intestines at 4 h after injection. Mercaptoacetyl-Ser-Ser-Ser (MaSSS) was used a chelating linker for coupling 99mTc to ZHER2:342 Affibody (19) to reduce hepatobiliary clearance. 99mTc-MaSSS-ZHER2:342 has been evaluated in nude mice bearing human SKOV-3 ovarian carcinoma tumors.



MaSSS-ZHER2:342 Affibody was prepared by standard solid phase peptide synthesis. 99mTc as pertechnetate was added to a solution of MaSSS-ZHER2:342 containing SnCl2 (19). The mixture was incubated for 60 min at room temperature. The labeling efficiency of 99mTc incorporation was 97.0 ± 1.2% with >99% purity. 99mTc-MaSSS-ZHER2:342 was purified by size-exclusion chromatography. Specific activities of the preparations were not reported. 99mTc-MaSSS-ZHER2:342 was found to be stable after incubation at 37°C for 1 h in murine blood serum (>99%) or solution containing 300-fold excess cysteine (97.1%) at 37°C for 2 h.

In Vitro Studies: Testing in Cells and Tissues


Engfeldt et al. (19) performed binding experiments with MaSSS-ZHER2:342 with use of a Biacore sensor chip immobilized with extracellular domain of HER2 protein. The Kd value of MaSSS-ZHER2:342 was calculated to be 400 pM. The Kd value of ZHER2:342 was 80 pM. Hence, the binding affinity of the synthetic Affibody molecule MaSSS-ZHER2:342 was 4-fold lower than the parent Affibody molecule ZHER2:342. In vitro binding specificity tests showed that binding of 99mTc-MaSSS-ZHER2:342 to SKOV-3 cells expressing HER2 was receptor-mediated because saturation of receptors by preincubation with non-labeled ZHER2:342 significantly decreased binding of 99mTc-MaSSS-ZHER2:342. The antigen binding capacity of 99mTc-MaSSS-ZHER2:342 was 69.3 ± 2.5%. The cell-bound radioactivity remained 77% of the initially bound activity at 8 h and ~65% at 24 h when the cells were incubated with 99mTc-MaSSS-ZHER2:342.

Animal Studies



Engfeldt et al. (19) performed biodistribution studies of 0.1 MBq (2.7 μCi) 99mTc-MaSSS-ZHER2:342 in nude mice (n = 4) bearing SKOV-3 xenografts. 99mTc-MaSSS-ZHER2:342 (0.14 nmol) was injected s.c. to each mouse. The initial tracer accumulation in the SKOV-3 tumors was 11.5 ± 0.5% injected dose per gram (ID/g) at 4 h after injection. The radioactivity level in tumors was higher than in other organs and tissues except the kidneys (33.6 ± 1.7% ID/g) at 4 h. Blood level was 0.15% ID/g. Tumor/blood and tumor/muscle ratios were ~80 and ~600, respectively. 99mTc-MaSSS-ZHER2:342 was 83% intact in the blood at 15 min after injection. Biodistribution studies were also performed in normal NMRI mice (n = 4) without bearing any tumors at 4 h after injection. The results showed low accumulation in all organs and tissues except with high 99mTc-MaSSS-ZHER2:342 accumulations in the kidneys (~20% ID/g) and intestines (~10% ID/g). The kidney accumulation was higher than 99mTc-MAG3-ZHER2:342 (~7% ID/g) and the accumulation in the intestines was lower than 99mTc-MAG3-ZHER2:342 (~30% ID/g). Single-photon emission computed tomography (SPECT) analysis was performed in nude mice (n = 3) bearing the SKOV-3 tumors after i.v. injection of 3 MBq (81 μCi) 99mTc-MaSSS-ZHER2:342. Tumor was clearly visualized at 6 h along with the kidneys with tumor/muscle ratio of 38. No blocking experiment was performed.

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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