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18F-Labeled Cys-ZHER2:342, an anti-epidermal growth factor receptor-2 Affibody

, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: August 9, 2012.

Chemical name:18F-Labeled Cys-ZHER2:342, an anti-epidermal growth factor receptor-2 Affibody
Abbreviated name:[18F]-Cys-ZHER2:342
Agent Category:Antibody
Target:Epidermal growth factor receptor 2 (HER2)
Target Category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:18F
  • Checkbox In vitro
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Structure not available in PubChem.



The epidermal growth factor receptor-2 (HER2, ErbB2) modulates its activity through a tyrosine kinase signaling pathway and is involved in the development of various cancers, including those of the lung and the breast (1, 2). Overexpression or amplification of the HER2 gene is known to occur in a high percentage of cancer cases (e.g., ~20% of breast cancer (BC)) and predicts a poor prognosis for the patient. Invasive methods such as biopsies in conjunction with immunohistochemistry and fluorescence in situ hybridization are often employed to assess the HER2 status of the primary and metastasized neoplastic tumors; however, because of sampling bias and tumor heterogeneity, results obtained with these procedures may not be reliable (2). In the clinic, 18F-labeled fluorodeoxyglucose ([18F]-FDG) is commonly used with positron emission tomography (PET) to detect and determine the tumor burden of a patient, but this technique does not distinguish between benign and malignant lesions, cannot differentiate tumors that overexpress HER2 from those that have a low expression or do not express the receptor, and often identifies inflammation as a false-positive neoplasm (1).

An Affibody molecule is a chain of 58 amino acids (~6.5 kDa) that contains a modified B domain of the staphylococcal protein A and can be obtained by chemical synthesis or produced in bacteria with the use of recombinant DNA technology (3). Because of their small size and high chemical and thermal stability, there is much interest to radiolabel these molecules and use them for the targeted detection and treatment of malignant tumors as discussed in detail elsewhere (3-5). Orlova et al. generated an anti-EGFR2 Affibody molecule (ZHER2:342) with a picomolar affinity for the receptor and showed that 125I-labeled ZHER2:342 can be used to visualize xenograft SKOV-3 cell tumors (these cells express the HER2) with gamma planar imaging in mice (6). Kramer-Marek et al. envisioned that, because 18F is routinely available in the clinic, 18F-labeled ZHER2:342 can be used to detect and monitor (after therapy) cancerous lesions that express HER2 (7). For the site-specific labeling of ZHER2:342 with 18F, a cysteine residue was attached to the C-terminus of the Affibody (Cys-ZHER2:342), and the derivative was conjugated with N-(2-(4-[18F]fluorobenzamido)ethyl)maleimide ([18F]FBEM) to obtain [18F]-Cys-ZHER2:342 (7). Subsequently, the radiofluorinated conjugate was characterized in vitro and evaluated in vivo to assess the expression of HER2 in xenograft tumors in mice (7).



The expression of ZHER2:342 in Escherichia coli as a His-tagged protein and its purification from the microbes are described elsewhere (6). The Affibody was labeled with [18F]FBEM as detailed by Kiesewetter et al. (8). The radiochemical yield of the labeling reaction was 10.5 ± 1.9% (n = 39 reactions; average time was 117 min/reaction), and the specific activity of the labeled Affibody was 2.51 ± 0.92 MBq/153.8 pmol (93.0 ± 34.2 μCi/153.8 pmol). The radiochemical purity and stability of the labeled product was not reported.

In Vitro Studies: Testing in Cells and Tissues


The affinity constant (Kd), the association rate constant (ka), and the dissociation rate constant (kd) of ZHER2:342 for HER2 were reported to be ~22 pmol/L, ~4.8 × 106 M–1s–1, and ~1.1 × 10–4s–1, respectively, as determined with surface plasmon resonance techniques (6).

The in vitro binding characteristics of [18F]-Cys-ZHER2:342 were studied with displacement and saturation cell-binding assays using SKOV-3 cells (7). For the displacement assay, the cells were incubated with 216 nM [18F]-Cys-ZHER2:342 in the presence of increasing concentrations of the non-radiolabeled Affibody (1.2 nM–120 μM). This study demonstrated that increasing concentrations of the unlabeled Affibody could displace [18F]-Cys-ZHER2:342 from the receptor expressed on the SKOV-3 cells (7). The Kd of [18F]-Cys-ZHER2:342 for the HER2 expressed on the SKOV-3 cells was reported to be 15 nM as determined with the saturation binding assay (7).

Animal Studies



The biodistribution of [18F]-Cys-ZHER2:342 was investigated in mice bearing BT474 cell tumors (these cells originate from human mammary gland ductal carcinoma) (8). The animals (n = 4–5 anesthetized mice) were injected with 5.5–6.3 MBq (203.5–233.1 μCi; 461.4–633.2 pmol) of the labeled Affibody through the tail vein and euthanized at 2 h postinjection (p.i.). All organs of interest, including the tumors, were harvested from the animals to determine the amount of radioactivity accumulated in the various tissues. Results obtained from this study were calculated as the percent of injected dose per gram tissue (% ID/g). Maximum uptake of label was observed in the tumors (23% ID/g), followed by the kidneys (10% ID/g). All other organs showed an accumulation of <5% ID/g. No blocking studies were reported.

For imaging studies, the anesthetized mice (n = 5 animals) were injected with [18F]-Cys-ZHER2:342 as described above, and whole-body PET scans of the animals were acquired at 1 h p.i (8). Regions-of-interest (ROI) were marked on the images, and the accumulation values for the various tissues and the tumors were calculated as detailed elsewhere (8). From the images it was apparent that maximum accumulation of label was in the tumors, followed by the kidneys. An analysis of the ROI data showed that the tumor/muscle ratio was 22, and the background activity in the other tissues was very low. From this study, the investigators concluded that the tracer can be used for the detection of HER2-positive tumors. No blocking studies were reported.

Kramer-Marek et al. studied the biodistribution of [18F]-Cys-ZHER2:342 in athymic nude mice bearing xenograft SKOV-3 cell tumors to assess the utility of this labeled Affibody to monitor the expression of HER2 in cancerous tumors (7). For the biodistribution studies, the mice (n = 3–6 animals/time point) were injected with 1.8–2.2 MBq (66.6–81.4 μCi; 245.3–306.6 pmol) [18F]-Cys-ZHER2:342, and the animals were euthanized at time points ranging from 1 h p.i. to 6 h p.i. The blood, tumors, and various organs were subsequently collected from the animals to determine the uptake of radioactivity in the different tissues. The amount of radioactivity present in select tissues at select time points is presented in Table 1.

Table 1: Biodistribution of radioactivity from [18F]-Cys-ZHER2:342 in select tissues at select time points in mice bearing xenograft SKOV-3 cell tumors (7).

TissueAccumulated radioactivity (% ID/g)
Time points (p.i.)
1 h3 h6 h
Blood1.31 ± 0.750.25 ± 0.090.16 ± 0.08
Kidney14.08 ± 0.052.36 ± 0.111.43 ± 0.29
Tumor9.73 ± 1.916.95 ± 0.936.80 ± 0.01
Liver5.04 ± 0.690.68 ± 0.180.41 ± 0.05
Bone2.15 ± 0.402.07 ± 2.270.60 ± 0.04

From the biodistribution study, it was apparent that at 6 h p.i. maximum radioactivity was present in the tumors, and all other organs, except the kidneys, accumulated <1.0% ID/g of radioactivity (Table 1). At 4 h p.i., the tumor/blood, tumor/kidney, tumor/bone, and tumor/muscle ratios were 69 ± 27, 4 ± 0.8, 5 ± 2.6, and 311 ± 167, respectively. To establish the in vivo receptor binding specificity of [18F]-Cys-ZHER2:342, the animals (n = 3–6 mice/group) bearing SKOV-3 cell tumors were injected with 6.1 nmol unlabeled Cys-ZHER2:342 45 min before the administration of [18F]-Cys-ZHER2:342 (7). For use as controls, another group of mice bearing U251 cell tumors (these cells do not express HER2) were injected with [18F]-Cys-ZHER2:342 alone. At 2 h p.i., the animals were euthanized, and all the organs of interest, as well as the tumors, were removed to determine the amount of label accumulated in the various tissues. A significantly reduced accumulation of label was observed in the tumors of animals given a blocking dose of unlabeled Cys-ZHER2:342 (~6.5% ID/g in mice without blocking versus ~2.0% ID/g in blocked animals; P value not reported). All other organs showed almost no change in uptake of the label. This showed that the 18F-labeled Affibody had a binding specificity for the HER2 expressed primarily in the SKOV-3 cell tumors. Pre-incubation with trastuzumab did not inhibit the binding of [18F]-Cys-ZHER2:342 to the SKOV-3 cells, indicating that the two agents targeted distinctly separate epitopes on the extracellular domain of the HER2 (7).

It has been observed that [18F]-Cys-ZHER2:342 is superior to [18F]-FDG as a tracer for the detection of breast cancer tumors and for the evaluation of the HER2 status of metastasized lesions (1). In another study, it was shown that [18F]-Cys-ZHER2:342 can be used with PET to determine the changes in the expression of HER2 in BT474 xenografts tumors in mice, and the investigators suggested that determination of the HER2 levels of a tumor may be a good strategy to predict the response of a cancerous lesion to treatment with trastuzumab (2).

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

Supplemental Information


No information is currently available.

NIH Support

Studies reported in this chapter were funded in part by contracts from N01-CO-12400, N01-CO-12401, and HHSN261200800001E from the National Cancer Institute (NCI), National Institutes of Health (NIH). The research was also supported by the Center for Cancer Research, an Intramural Research Program of the NCI, Imaging Probe Development Center, National Heart, Lung and Blood Institute, National Institute of Biomedical Imaging and Bioengineering, NIH.


Kramer-Marek G., Bernardo M., Kiesewetter D.O., Bagci U., Kuban M., Omer A., Zielinski R., Seidel J., Choyke P., Capala J. PET of HER2-Positive Pulmonary Metastases with 18F-ZHER2:342 Affibody in a Murine Model of Breast Cancer: Comparison with 18F-FDG. J Nucl Med. 2012;53(6):939–46. [PubMed: 22582046]
Kramer-Marek G., Gijsen M., Kiesewetter D.O., Bennett R., Roxanis I., Zielinski R., Kong A., Capala J. Potential of PET to predict the response to trastuzumab treatment in an ErbB2-positive human xenograft tumor model. J Nucl Med. 2012;53(4):629–37. [PubMed: 22410461]
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