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64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7-β max tris(acetic acid)-10-acetate mono(N-ethylmaleimide amide)-monomeric ZHER2:477

, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: November 30, 2011.

Chemical name:64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7-β max tris(acetic acid)-10-acetate mono(N-ethylmaleimide amide)-monomeric ZHER2:477Image Cu64ZHER2477.jpg
Abbreviated name:64Cu-DOTA-ZHER2:477
Agent Category:Affibody, antibody
Target Category:Receptor
Method of detection:PET
Source of signal / contrast:64Cu
  • Checkbox In vitro
  • Checkbox Rodents
Structure of the agents by Cheng et al. (1).



The 64Cu-1,4,7,10-tetraazacyclododecane-1,4,7-β max tris(acetic acid)-10-acetate mono(N-ethylmaleimide amide) (Mal-DOTA)-monomeric ZHER2:477 conjugate, abbreviated as 64Cu-DOTA-ZHER2:477, is an affibody derivative synthesized by Cheng et al. for positron emission tomography (PET) of HER2-expressing tumors (1).

Affibody molecules are a group of nonimmunogenic scaffold proteins that derive from the B-domain of staphylococcal surface protein A (2, 3). In the past several years, affibodies have drawn significant attention for developing imaging and therapeutic agents because of their unique features (3, 4). First, affibodies are small, with only 58 amino acid residues (~7 kDa) (3, 5). The small size allows affibodies to be generated with solid-phase peptide synthesis and to be cleared quickly from kidneys. Second, affibodies have a high binding affinity and specificity to their targets. Their binding affinity can be further improved by generating multimeric constructs through the solvent-exposed termini of affibody Z-domain. The anti-HER2 monomeric affibody ZHER2:4 is an example that has a binding affinity of ~50 nM, but its dimeric form, (ZHER2:4)2, exhibits an improved binding affinity up to ~3 nM (6). Third, affibodies lack cysteine residues and disulfide bridges in structure, and they fold rapidly. These features make it possible to chemically synthesize fully functional molecules and to introduce unique cysteine residues or chemical groups into affibodies for site-specific labeling. Several anti-HER2 affibody derivatives have been synthesized in this way. The imaging agent HPEM-His6-(ZHER2:4)2-Cys was generated by radiobrominating the dimeric (ZHER2:4)2 through the cysteine residues that were introduced to the C-terminus of (ZHER2:4)2 (7). Several affibody derivatives (e.g., 68Ga-DOTA-ZHER2:342-pep2, 111In-DOTA-ZHER2:342-pep2, 111In-benzyl-DOTA-ZHER2:342, and 111In-benzyl-DTPA-ZHER2:342) were synthesized by coupling a chelating agent with a specifically protected site group of the ZHER2:342 peptide chain (3). Furthermore, these small affibody proteins can be selected and optimized with a strategy of sequence mutation and affinity maturation, and an example selected with this strategy is the anti-HER2 affibody ZHER2:342, which has an increased affinity from 50 nM to 22 pM (8).

The investigators at the Stanford University first tested the feasibility of the monomeric and dimeric forms of affibody ZHER2:477 for molecular imaging. ZHER2:477 is a commercially available anti-HER2 affibody. Both forms of the ZHER2:477 molecule were radiofluorinated with an 18F-labeled prosthetic group of 4-18F-fluorobenzaldehyde (18F-FBO-ZHER2:477 and 18F-FBO-(ZHER2:477)2, respectively) (9). The investigators have also coupled 64Cu to the affibody through DOTA, leading to the development of imaging agents of 64Cu-DOTA- ZHER2:477 and 64Cu-DOTA-(ZHER2:477)2 (1). Interestingly, these studies showed that smaller affibody constructs performed better in vivo in terms of tumor uptake and clearance. The investigators then generated a class of small proteins consisting of two α-helix bundles of the 3-helix affibody by deleting the helix 3 because the binding domain localizes in the α-helices 1 and 2 bundles (5). 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 (10-12). The helix conformation of MUT-DS has been shown to be improved with the placement of a disulfide bridge. MUT-DS showed a binding affinity to HER2 in the low-nM range. The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for both imaging and therapy of HER2-expressing tumors.

This series of chapters summarizes the data obtained with the ZHER2:477 derivatives, and this chapter presents the data obtained with 64Cu-DOTA-ZHER2:477 (1).



The monomeric affibody ZHER2:477 with a purity of >95% is commercially available. The maleimide-functionalized chelator, Mal-DOTA, was synthesized with DOTA-mono-NHS-tris(tBu)ester, triethylamine, and N-(2-aminoethyl)maleimide-trifluoroacetic acid salt. The affibody ZHER2:477 was then modified at the cysteine residue of the C terminus with Mal-DOTA, which generated the conjugate DOTA-ZHER2:477 (1). The molecular weight of DOTA-ZHER2:477 was 7,277.50 Da with 25% recovery yield after purification. The conjugate was radiolabeled with 64Cu by incubation with 64CuCl2 for 1 h at 40°C. The product, 64Cu-DOTA-ZHER2:477, was purified on a PD-10 column and analyzed with radioanalytical high-performance liquid chromatography (HPLC).

The final product, 64Cu-DOTA-ZHER2:477, had a radiochemical purity of >95% and a specific activity of 29.1 MBq/nmol (786.5 µCi/nmol). HPLC analysis of the 64Cu-DOTA-ZHER2:477 showed a single peak with a retention time of 24.0 min. Octanol–water partition coefficient measurements showed a logP value of 0.018 ± 0.006, indicating intermediate hydrophilicity of the 64Cu-DOTA-ZHER2:477 (1).

In Vitro Studies: Testing in Cells and Tissues


Binding affinity with the extracellular domain of HER2 antigen was measured in vitro with surface plasmon resonance detection (1). Unlabeled DOTA-ZHER2:477 showed a binding affinity of 1.5 nM (100 pM for DOTA-(ZHER2:477)2) with association constants in the range of 105 and dissociation constants in the range of 10−4–10−5, indicating that the on rate was very fast and the off rate was very slow. Binding affinity for 64Cu-DOTA-ZHER2:477 was not measured.

HER2 expression in SKOV3 human ovarian and MDA-MB-435 human breast cancer cells and tumor samples was verified with Western blot analysis (1). SKOV3 cells had high HER2 expression, whereas MDA-MB-435 cells expressed minimum HER2. The HER2 expression was 10–15-fold higher in SKOV3 tumors than in MDA-MB-435 tumors.

Animal Studies



Biodistribution studies were performed in nude mice bearing SKOV3 tumors (n = 3 mice/time point) (1). Mice were injected via the tail vein with 0.481–0.925 MBq (13–25 µCi) 64Cu-DOTA-ZHER2:477 and euthanized at 1, 4, and 20 h after injection, respectively. The radioactivity in ex vivo tissues was expressed as a percentage of the injected radioactive dose per gram of tissue (% ID/g). 64Cu-DOTA-ZHER2:477 exhibited rapid accumulation with moderate activity in the SKOV3 tumors at the early time point (3.86 ± 0.58% ID/g at 1 h), which increased to 6.12 ± 1.44% ID/g at 4 h and then dropped to 3.65 ± 0.69% ID/g over 20 h after injection. The tumor/blood ratios were 5.28 ± 0.91, 12.44 ± 3.39, and 5.12 ± 0.87, and the tumor/muscle ratios were 5.29 ± 1.61, 9.47 ± 2.46, and 7.23 ± 0.39 at 1, 4, and 20 h, respectively.

Comparison between 64Cu-DOTA-ZHER2:477 and its dimeric form 64Cu-DOTA-(ZHER2:477)2 showed that 64Cu-DOTA-ZHER2:477 had much higher tumor uptake than 64Cu-DOTA-(ZHER2:477)2 at all time points (P < 0.05). Both forms displayed rapid blood clearance, with 0.75 ± 0.22% ID/g and 2.27 ± 0.27% ID/g blood uptake at 1 h for 64Cu-DOTA-ZHER2:477 and 64Cu-DOTA-(ZHER2:477)2, respectively. Extremely high renal uptake and retention were found for both forms, but the renal uptake for monomeric 64Cu-DOTA-ZHER2:477 was 1–1.5-fold higher than that for 64Cu-DOTA-(ZHER2:477)2. The renal uptake values at 1, 4, and 20 h were 206.26 ± 22.36%, 237.51 ± 34.49%, and 129.69 ± 14.43% ID/g for 64Cu-DOTA-ZHER2:477 and 114.33 ± 11.22%, 75.21 ± 5.74%, and 37.95 ± 5.55% ID/g for 64Cu-DOTA-(ZHER2:477)2, respectively. Although relatively high liver uptake was observed for both agents, it was much lower than the renal uptake. Both forms exhibited relatively low uptake in all other normal organs.

PET imaging was performed in a mouse bearing SKOV3 and MDA-MB-435 tumors at 1, 4, and 20 h after tail vein injection of 64Cu-DOTA-ZHER2:477 (1). SKOV3 tumor was clearly visible with high tumor/background contrast at 1–20 h after injection. Only low uptake was observed in the MDA-MB-435 tumor, which makes it difficult to be visualized. Consistent with the biodistribution findings, high activity accumulation was observed in the kidneys.

To understand the relationship between 64Cu-DOTA-ZHER2:477 and Herceptin (trastuzamab), mice bearing SKOV3 tumors (n = 6 mice) were pretreated with 300 µg Herceptin or phosphate-buffered saline via tail vein injection. At 48 h after pretreatment, the mice were injected with 64Cu-DOTA-ZHER2:477 (2.59–3.14 MBq (70–85 μCi)) and imaged at 1, 3.5, and 24 h. Pretreatment with Herceptin reduced the uptake of 64Cu-DOTA-ZHER2:477 in SKOV3 tumors, indicating that Herceptin blocks the binding of 64Cu-DOTA-ZHER2:477. Blocking studies were not performed for the dimeric form.

In conclusion, the 64Cu-labeled dimer and monomer exhibited rapid tumor accumulation and blood clearance, which are favorable for imaging (1). Although the dimeric form exhibited higher HER2-binding affinity than did the monomeric form, the dimeric form showed much lower SKOV3 tumor accumulation and tumor/normal organ ratios than that of the monomeric form. The blocking results with Herceptin and affibody molecules are controversial in the literature, and the use of different affibody clones may be partially responsible for the controversy (4, 6). Very high kidney uptake and retention for both agents are likely to be a concern for their clinical use, and further optimization is necessary (1).

Other Non-Primate Mammals


No references are available.

Non-Human Primates


No references are available.

Human Studies


No references are available.


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