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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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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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18F-Labeled N-(4-fluorobenzylidene)oxime-monomeric ZHER2:477

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

Created: ; Last Update: January 4, 2012.

Chemical name:18F-N-(4-fluorobenzylidene)oxime-monomeric ZHER2:477Image F18ZHER2477.jpg
Abbreviated name:18F-FBO-ZHER2:477
Agent Category:Affibody, antibody
Target Category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:18F
  • Checkbox In vitro
  • Checkbox Rodents
Structure of the agents by Cheng et al. (1).



The 18F-labeled N-(4-fluorobenzylidene)oxime (FBO)-monomeric ZHER2:477 conjugate, abbreviated as 18F-FBO-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 by the 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 of 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 of 50 nM (ZHER2:4, the first generation) to 22 pM (8).

The investigators at Stanford University first tested the feasibility of the monomeric and dimeric forms of anti-HER2 affibody ZHER2:477 for molecular imaging. 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) (1). The investigators have also coupled 64Cu to the affibody through DOTA, leading to the development of imaging agents 64Cu-DOTA- ZHER2:477 and 64Cu-DOTA-(ZHER2:477)2 (9). Interestingly, these studies showed that smaller affibody constructs performed better in vivo in terms of tumor uptake and clearance in spite of the lower affinity in vitro. 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). 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 (refer to MUT-DS derived agents in MICAD).

This series of chapters summarizes the data obtained with the ZHER2:477 derivatives, and this chapter presents the data obtained with 18F-FBO-ZHER2:477 (1).



The monomeric affibody ZHER2:477 with a distal C-terminal cysteine residue (purity, >95%) is commercially available. The agent 18F-FBO-ZHER2:477 was synthesized in three steps (1). The bifunctional linker 2-(aminooxy)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)acetamide hydrochloride was first prepared by reacting N-(2-aminoethyl)malemide with 2-(tert-butoxycarbonylaminooxy)acetic acid under carbodiimide-mediated coupling conditions. The linker consisted of two orthogonal groups, a thiol-reactive maleimide group for conjugation to the cysteine residue and an 18/19F-aldehyde–reactive aminooxy group. The yield was 80%. The linker was then selectively conjugated to the affibody molecule to generate aminooxy-functionalized ZHER2:477 (ZHER2:477-ONH2; yield = 82%; molecular weight (MW) = 6,962.58 Da). As the last step, ZHER2:477-ONH2 was conjugated with 4-fluorobenzaldehyde (4-FBA), which resulted in the nonradioactive FBO-ZHER2:477. The recovery yield was 70%–90%, and the measured MW was 7,068.94 Da for the nonradioactive FBO-ZHER2:477. Similarly, conjugation of the ZHER2:477-ONH2 with 4-18F-FBA led to the generation of radioactive 18F-FBO-ZHER2:477. For radiolabeling, the coupling yield was 30%–40%, and the overall radiochemical yield ranged from 13% to 18% (end of synthesis, corrected for decay). The specific activity and purity of 18F-FBO-ZHER2:477 were 13.0–20.7 MBq/nmol (50–80 μCi/μg) and >95%, respectively, at end of synthesis. The total synthesis time was ~100 min.

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). The measurements showed that the binding affinities of ZHER2:477-ONH2 and FBO-ZHER2:477 were 400 and 430 pM, respectively (180 and 200 pM for the dimeric forms (ZHER2:477)2-ONH2 and FBO-(ZHER2:477)2, respectively). The association constants were in the range of 105–106 (association time, >2 min), and the dissociation constants were in the range of 10-4–10-5 (disassociation time, >15 min), indicating that the on rate was very fast and the off rate was very slow.

The HER2-targeting ability for cultured cells was evaluated with SKOV3 human ovarian cancer cells (0.2 × 106 cells/well) after incubation for 2 h with 1.11−6.29 kBq (0.03–0.17 μCi) 18F-FBO-ZHER2:477. 18F-FBO-ZHER2:477 quickly accumulated in the SKOV3 cells and reached the value of 0.00476 cell counts per minute [cpm]/medium cpm/μg of protein/μL at 0.5 h, which was maintained up to 2 h. In the presence of a large excess of nonradioactive ZHER2:477 (final concentration, 0.28 μM), the cell uptake of 18F-FBO-ZHER2:477 was inhibited significantly at all incubation time points (P < 0.05), showing a value of only 0.00329 from the unblocked amount of 0.00476 at 0.5 h, which suggests that the agent is specific to HER2 (1).

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 0.37–1.11 MBq (10–30 μCi) 18F-FBO-ZHER2:477 and then euthanized at different time points after injection. The radioactivity in ex vivo tissues was expressed as a percentage of the injected radioactive dose per gram of tissue (% ID/g). Rapid and high accumulation of 18F-FBO-ZHER2:477 in SKOV3 tumors was observed at early time points (4.77 ± 0.78% ID/g at 0.5 h after injection), and accumulation decreased to 3.61 ± 0.29% ID/g at 3 h after injection. At 3 h after injection, the tumor/blood and tumor/muscle ratios were 12.72 ± 2.94 and 28.03 ± 8.28, respectively.

Compared to the dimeric form (refer to the chapter of 18F-FBO-(ZHER2:477)2 in MICAD), 18F-FBO-ZHER2:477 showed significantly higher tumor/normal tissue (blood, muscle, liver, and lung) ratios at all time points. Both monomeric and dimeric forms displayed relatively rapid blood clearance, with blood uptake values of 3.81 ± 0.50% and 1.97 ± 0.24% ID/g at 0.5 h (P < 0.05), respectively, and 1.50 ± 0.35% and 1.56 ± 0.29% ID/g at 1 h after injection, respectively. In the kidneys, both forms displayed very high levels of uptake, especially at the early time points (>20% ID/g at 0.5 h after injection), and the monomeric form had a comparatively higher renal uptake than the dimeric form. On the contrary, the dimeric form showed significantly higher levels of uptake in the liver and muscle at 0.5 h after injection than the monomeric form, but both forms displayed similar levels of uptake at later time points.

PET imaging was performed in mice bearing SKOV3 tumors at 0.5, 1, 2, and 4 h after tail vein injection of 18F-FBO-ZHER2:477 (0.444–0.814 MBq (12–22 μCi)) or 18F-FBO-(ZHER2:477)2 (1.85–2.035 MBq (50–55 μCi)) (1). The tumor was visible with both forms, especially at later time points, but a better tumor/background contrast was observed with the monomeric form. 18F-FBO-ZHER2:477 had approximately two-fold higher tumor uptake than 18F-FBO-(ZHER2:477)2. High levels of activity accumulation were observed in the kidneys and the intestinal tract for both forms.

To test the in vivo HER2-targeting specificity of 18F-FBO-ZHER2, nude mice bearing SKOV3 tumors (n = 3 mice/each group) were pretreated with either 500 μg trastuzumab or 300 μg affibody ZHER2:477 (1). Mice were then given 18F-FBO-ZHER2:477 at 72 h after trastuzumab injection and at 1 h after ZHER2:477 injection. Mice without pretreatment were used as a control. The biodistribution at 1 h after 18F-FBO-ZHER2:477 injection was examined. Pretreatment with ZHER2:477 specifically reduced the tumor uptake of 18F-FBO-ZHER2:477 (1.73 ± 0.37% ID/g) to ~36% of the control tumor uptake (4.77 ± 0.88%; P < 0.05). No significant difference was observed for the uptake in normal tissues. Pretreatment with trastuzumab specifically inhibited the uptake of 18F-FBO-ZHER2:477 (2.25 ± 0.49% ID/g) to ~47% of the control tumor uptake (4.77 ± 0.88%; P < 0.05), and again no inhibition of the uptake was observed in normal organs.

In conclusion, studies by Cheng et al. showed that 18F-FBO-ZHER2:477 rapidly localized in SKOV3 tumors and exhibited good tumor uptake, retention, tumor/muscle ratio, and high specificity to HER2 (1). Compared to its dimeric form, 18F-FBO-ZHER2:477 showed better in vivo performance, although it had a lower affinity with HER2 and a lower cell uptake in vitro. 18F-FBO-ZHER2:477 is more promising than its dimeric form as an agent for HER2 imaging (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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