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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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68Ga-Labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-VENK[homoC]NKEMRNRYWEAALDPNLNNQQKRAKIRSIYDDP[homoC]-NH2 with a disulfide bridge between the two homoC

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

Created: ; Last Update: January 4, 2012.

Chemical name:68Ga-Labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-VENK[homoC]NKEMRNRYWEAALDPNLNNQQKRAKIRSIYDDP[homoC]-NH2 with a disulfide bridge between the two homoC
Abbreviated name:68Ga-DOTA-MUT-DS
Agent Category:Affibody, antibody
Target Category:Receptor
Method of detection:PET
Source of signal / contrast:68Ga
  • Checkbox In vitro
  • Checkbox Rodents
Structure is not available.



The 68Ga-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-VENK[homoC]NKEMRNRYWEAALDPNLNNQQKRAKIRSIYDDP[homoC]-NH2 (MUT-DS) conjugate with a disulfide bridge between the two homocysteines, abbreviated as 68Ga-DOTA-MUT-DS, is a 2-helix affibody derivative that was synthesized by Ren et al. for positron emission tomography (PET) of HER2-expressing tumors (1).

Affibody molecules are a group of nonimmunogenic scaffold proteins that are derived from the B-domain of staphylococcal surface protein A (2, 3). These molecules have only 58 amino acid residues (~7 kDa), which form a 3-α-helical bundle structure (3, 4). Helices 1 and 2 bundles are responsible for the high binding affinity and specificity of affibodies to their targets, while helix 3 contributes to the affibody stabilization and is not involved in receptor recognition (4). Studies have further confirmed that the binding domain in the helices 1 and 2 bundles includes 13 amino acid residues that are surface-exposed (2). Therefore, large affibody libraries have been constructed by randomization of the 13 amino acid residues, and a large set of affibody molecules against a wide variety of targets have been selected from those libraries. Of them, the affibodies specific to HER2, including ZHER2:342 and ZHER2:477, have been intensively investigated in recent years (3, 5). These molecules have been radiolabeled and tested for molecular imaging of HER2-expressing tumors.

The investigators at Stanford University first tested the feasibility of the monomeric (~7 kDa) and dimeric (~14 kDa) forms of affibody ZHER2:477 for molecular imaging (6, 7). Both forms have been labeled with various radionuclides through chelating agents. Studies have shown that smaller affibody constructs perform better in vivo in terms of tumor uptake and clearance, which prompts them to generate smaller proteins with only α-helices 1 and 2 bundles (~4 kDa) (4, 6, 7). However, simple deletion of the helix 3 leads to significantly decreased binding affinity of the proteins because of decreased helix conformation (4). The investigators then applied various strategies to improve the helix conformation of the affibody molecules, including sequence mutation, placement of disulfide bridges, and inclusion of helix-promoting amino acids (4). Although the helix conformation (~15%, the amount of α-helix represented in the secondary structure of the affibodies) of the 2-helix molecules is still much lower than that of the parent 3-helix affibodies, the investigators successfully obtained a class of 2-helix small proteins with HER2-binding affinity up to 5 nM with these strategies (1, 8, 9). One of these 2-helix proteins is MUT-DS, which has α-helices 1 and 2 bundles with a disulfide bridge being formed between the two inserted homocysteines (4). The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for imaging HER2-expressing tumors. 68Ga-DOTA-MUT-DS, 64Cu-DOTA-MUT-DS, 111In-DOTA-MUT-DS, and 18F-FBO-MUT-DS are examples of the MUT-DS derivatives (1, 8, 9).

This chapter summarizes the data obtained with 68Ga-DOTA-MUT-DS (1).



Ren et al. generated the cyclic 2-helix small protein MUT-DS with standard solid-phase peptide synthesis and I2 oxidation of the two inserted homocysteines (1). DOTA was conjugated to the C-terminus cysteine residule. The DOTA-MUT-DS conjugate was purified with high-performance liquid chromatography (HPLC). The chemical yield was 10%, with >95% purity. The retention time was 19.1 min. The measured molecular weight was 5,136.77 (calculated 5,136.53).

DOTA-MUT-DS was then radiolabeled with 68Ga through the reaction with 68GaCI3 in 0.1-N NH4OAc for 20 min at 75°C (1). After purification, the labeled 68Ga-DOTA-MUT-DS had a >95% radiochemical purity and a 35%−56% radiochemical yield. The retention time was 19.1 min on HPLC. Because the unlabeled DOTA-MUT-DS could not be separated from 68Ga-DOTA-MUT-DS, Ren et al. obtained a modest specific activity of 2.65–3.69 MBq/nmol (71.62–99.73 μCi/nmol) at the end of synthesis. The intact radiolabeled complex maintained >90% after incubation for 40 min with mouse serum at 37°C, indicating good in vitro stability.

In Vitro Studies: Testing in Cells and Tissues


The binding affinity of DOTA-MUT-DS with the extracellular domain of HER2 antigen was measured in vitro with surface plasmon resonance detection and was determined to be 4.76 nM, which is the same as unmodified MUT-DS (5 nM) (1, 4). This result suggests that DOTA conjugation did not alter the binding affinity of the 2-helix protein. DOTA-MUT-DS displayed a fast on-rate (association constant, 1.62 × 105 s-1) and a slow off-rate (dissociation constant, 7.7 × 10-4 s-1).

The HER2-targeting ability for cultured cells was evaluated with SKOV3 human ovarian cancer cells after incubation for 0.5–2 h with 6.29 kBq (0.17 μCi) 68Ga-DOTA-MUT-DS (1). The probe quickly accumulated in the SKOV3 cells and reached 1.76 ± 0.22% of applied activity at 0.5 h, which increased to 3.19 ± 0.31% at 1 h and then slightly dropped to 2.57 ± 0.48% at 2 h. In the presence of a large excess of nonradioactive ZHER2:477 (1 µg), the cell uptake of the probe was significantly inhibited at all incubation time points (P < 0.05), being only 0.17 ± 0.07% at 1 h and <0.1% at 2 h. These results suggest that 68Ga-DOTA-MUT-DS is specific to HER2.

Animal Studies



The biodistribution of 68Ga-DOTA-MUT-DS was examined in mice bearing SKOV3 human ovarian tumors after tail vein injection of 0.296 MBq (8 μCi) probe (1). Mice were euthanized at 0.5, 1, and 2 h after injection (n = 3 mice/time point). Rapid accumulation of the probe in tumors was observed at early time points (2.55 ± 0.72% injected dose per gram tissue (ID/g) at 0.5 h after injection), which continually increased to 4.04 ± 0.24% ID/g at 2 h after injection. 68Ga-DOTA-MUT-DS also displayed rapid blood clearance of 1.78 ± 0.68% at 0.5 h to 0.77 ± 0.2% and 0.60 ± 0.11% ID/g at 1 h and 2 h, respectively. The tumor/blood ratio was 1.59 ± 0.7 at 0.5 h, increased to 5.32 ± 0.18 at 1 h, and maximized at 7.72 ± 2.1 at 2 h. The tumor/muscle ratio maximized at 1 h (9.94 ± 1.29). Of the major organs, the liver uptake was 1.14 ± 0.19% ID/g at 0.5 h and decreased to 0.85 ± 0.09% ID/g at 2 h. The lung uptake was 1.76 ± 0.61% ID/g at 0.5 h and 0.67 ± 0.14% ID/g at 2 h. Kidney uptake was >200% ID/g at all time points. All other major organs showed low uptake (<1.10% ID/g at 1 h) and fast clearance.

The in vivo tumor-targeting specificity was evaluated with pretreatment of tumor-bearing mice with 17-N,N-dimethyl ethylene diamine-geldanamycin (17-DMAG; 50 mg/kg every 8 h before imaging, three times) (1). The compound 17-DMAG is known for its inhibition of HER2 signaling by binding to an Hsp90 chaperone protein. Inhibition of HER2 expression by 17-DMAG was confirmed by Ren et al. with Western blot studies. Pretreatment significantly reduced the tumor uptake of 68Ga-DOTA-MUT-DS at 2 h after injection to ~32% of the corresponding uptake in untreated tumors (4.12 ± 0.83 versus 0.74 ± 0.23% ID/g; P < 0.01). Consequently, the tumor/blood ratio was significantly decreased with 17-DMAG pretreatment (7.72 ± 2.1 versus 1.69 ± 0.16; P < 0.01). The liver and kidney uptake also decreased with 17-DMAG pretreatment (P < 0.05).

PET images were obtained from a mouse bearing a SKOV3 tumor at 0.5, 1, and 2 h after tail vein injection of 68Ga-DOTA-MUT-DS (1). The SKOV3 tumor was clearly visible with good tumor/background contrast. High accumulation of radioactivity was also seen in the kidneys. Pretreatment with 17-DMAG at 24 h before imaging made tumors hardly visible at any time points. The tumor uptake of 68Ga-DOTA-MUT-DS was significantly lower in the treated mice than in the untreated mice (n = 3 mice/group per time point; P < 0.01).

Other Non-Primate Mammals


No references are available.

Non-Human Primates


No references are available.

Human Studies


No references are available.


Ren G., Zhang R., Liu Z., Webster J.M., Miao Z., Gambhir S.S., Syud F.A., Cheng Z. A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression. J Nucl Med. 2009;50(9):1492–9. [PMC free article: PMC4216181] [PubMed: 19690041]
Friedman M., Nordberg E., Hoiden-Guthenberg I., Brismar H., Adams G.P., Nilsson F.Y., Carlsson J., Stahl S. Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor. Protein Eng Des Sel. 2007;20(4):189–99. [PubMed: 17452435]
Orlova A., Feldwisch J., Abrahmsen L., Tolmachev V. Update: affibody molecules for molecular imaging and therapy for cancer. Cancer Biother Radiopharm. 2007;22(5):573–84. [PubMed: 17979560]
Webster J.M., Zhang R., Gambhir S.S., Cheng Z., Syud F.A. Engineered two-helix small proteins for molecular recognition. Chembiochem. 2009;10(8):1293–6. [PubMed: 19422008]
Gao J., Chen K., Miao Z., Ren G., Chen X., Gambhir S.S., Cheng Z. Affibody-based nanoprobes for HER2-expressing cell and tumor imaging. Biomaterials. 2011;32(8):2141–8. [PMC free article: PMC3032351] [PubMed: 21147502]
Cheng Z., De Jesus O.P., Kramer D.J., De A., Webster J.M., Gheysens O., Levi J., Namavari M., Wang S., Park J.M., Zhang R., Liu H., Lee B., Syud F.A., Gambhir S.S. 64Cu-labeled affibody molecules for imaging of HER2 expressing tumors. Mol Imaging Biol. 2010;12(3):316–24. [PMC free article: PMC4155984] [PubMed: 19779897]
Cheng Z., De Jesus O.P., Namavari M., De A., Levi J., Webster J.M., Zhang R., Lee B., Syud F.A., Gambhir S.S. Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules. J Nucl Med. 2008;49(5):804–13. [PMC free article: PMC4154808] [PubMed: 18413392]
Miao Z., Ren G., Jiang L., Liu H., Webster J.M., Zhang R., Namavari M., Gambhir S.S., Syud F., Cheng Z. A novel (18)F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor. Eur J Nucl Med Mol Imaging. 2011;38(11):1977–84. [PMC free article: PMC4154802] [PubMed: 21761266]
Ren, G., J.M. Webster, Z. Liu, R. Zhang, Z. Miao, H. Liu, S.S. Gambhir, F.A. Syud, and Z. Cheng, In vivo targeting of HER2-positive tumor using 2-helix affibody molecules. Amino Acids, 2011. [PMC free article: PMC4172459] [PubMed: 21984380]


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