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111In-Labeled affibody ABY-025 targeting epidermal growth factor receptor 2

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

Created: ; Last Update: August 27, 2010.

Chemical name:111In-Labeled affibody ABY-025 targeting epidermal growth factor receptor 2
Abbreviated name:[111In]-ABY-025
Agent Category:Antibody
Target:Epidermal growth factor receptor 2 (HER2)
Target Category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT); gamma planar imaging
Source of signal / contrast:111In
  • Checkbox In vitro
  • Checkbox Rodents
  • Checkbox Non-human primates
Structure not available in PubChem.



An overexpressed or a mutated transmembrane epidermal growth factor receptor (EGFR; for details regarding this receptor family, see Hollmen and Elenius (1)) that modulates its physiological functions (cell growth, proliferation, migration, etc.) through an intracellular tyrosine kinase (TK) signaling pathway is known to be involved in the development of various cancers (2). As a consequence, one of the strategies used for the in vivo detection (by imaging) or treatment of cancers that are known to involve the EGFR includes the use of anti-EGFR antibodies (Abs) and monoclonal antibodies (mAbs) or their modified derivatives (3, 4). For use as imaging agents, Abs, mAbs, or their derivatives are designed to bind a radionuclide that serves as a source of the detection signal (4). However, their large size (~150 kDa) does not allow Abs and mAbs to penetrate deep into large tumors, and long blood circulating half-life times result in low sensitivity and the generation of a high background during imaging, thereby limiting their use for the detection or radiotherapy of malignant tumors (5, 6). To circumvent the limitations observed with the Abs and the mAbs, many small Ab or mAb derivatives such as the affibody molecules (ABs) have been generated (6, 7). The ABs can be up to ~7 kDa in size and have the same antigen specificity and binding characteristics as the native Ab from which they are derived. Various Abs have been labeled with radionuclides and evaluated for the detection of cancers (7).

The ZHER2:342 affibody, which is based on the Z domain of the staphylococcus protein A, is directed against the human EGFR2 (HER2) and has been investigated extensively (5). The non-binding surface domain of ZHER2:342 was then altered to make it suitable for chemical synthesis and to enhance the stability and hydrophilicity of the molecule (5). The resulting AB, ZHER2:2891, which had 11 surface assessable amino acids substituted in the parent AB, was radioiodinated and shown to have an accumulation of ~10% of the injected activity per gram tissue (% IA/g) in tumors on mice at 4 h after an intravenous injection, indicating that it was a suitable tracer for the detection of malignant tumors in a murine model (8). In an effort to further improve the stability and hydrophilic properties of ZHER2:2891, a 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) moiety was conjugated to the unique C-terminal cysteine of the parent AB to generate a second-generation AB designated ABY-025 (5). The new AB was then labeled with 111In ([111In]-ABY-025) and evaluated for its in vitro cell binding characteristics and for biodistribution and tumor imaging properties in mice bearing xenograft tumors (derived from SKOV-3, a human ovarian adenocarcinoma cell line that expresses the HER2 receptor) (5). These studies were performed to confirm that the various changes made to the native AB to produce ABY-025 had not changed the in vivo accuracy of the second generation AB for the detection of HER2 and also to confirm there was no change in the tumor and normal uptake properties of ABY-025.

Other sources of information

Human EGFR Gene (Gene ID: 1956)

Protein and mRNA sequence of human EGFR variant 1

EGFR in OMIM (Online Mendelian Inheritance in Man)

EGFR signaling pathways (NCI-Nature Pathways Interaction Database)

Anti-EGFR antibodies in PubMed

EGFR tyrosine kinase inhibitors in PubMed

Related chapters in MICAD



The synthesis of [111In]-ABY-025 has been described by Ahlgren et al. (5). The yield of the labeling reaction was >95%. The labeled AB had a specific activity of 15.1 GBq/μmol (407.7 mCi/μmol) with a radiochemical purity of >97% as determined with size-exclusion chromatography. The tracer was reported to be stable for at least 24 h after labeling.

The biodistribution characteristics of [111In]-ABY-025 were compared with those of [111In]-DOTA-ZHER2:2395-C (9) and [111In]-DOTA-ZHER2:342-pep2 in mice bearing SKOV-3 cell xenograft tumors (5). However, the radiochemical yield and purity, stability, and specific activity of [111In]-DOTA-ZHER2:2395-C and [111In]-DOTA-ZHER2:342-pep2 used for the biodistribution study were not reported.

In Vitro Studies: Testing in Cells and Tissues


The HER2 binding specificity of [111In]-ABY-025 was confirmed in an in vitro blocking experiment with SKOV-3 cells, SKBR-3 cells (of human breast adenocarcinoma origin and overexpressing HER2), and NCI-N87 cells (of human gastric carcinoma origin and expressing normal levels of HER2) using unlabeled ABY-025 (5). Pretreatment of the different cell types with unlabeled ABY-025 was shown to reduce the cell binding of [111In]-ABY-025, indicating that the cellular binding of the tracer was specific to HER2.

In another experiment, the different cell lines were exposed to [111In]-ABY-025 for 4 h at 4°C, washed with buffer, and incubated at 37°C for >24 h (5). After 24 h, 81.0 ± 2.0%, 76.0 ± 1.0%, and 58.0 ± 1.0% of radioactivity was shown to remain bound to the SKOV-3, NCI-N87, and SKBR-3 cells, respectively. Cell internalization of the radiolabel was determined to be 12.0%, 13.0 ± 1.0%, and 2.5% for the SKOV-3, NCI-N87, and SKBR-3 cells, respectively. The amount of radioactivity bound to all the cell lines remained almost constant from 2 h to 24 h after exposure to the radiochemical.

Animal Studies



Ahlgren et al. studied the comparative biodistribution of [111In]-ABY-025, [111In]-DOTA-ZHER2:2395-C, and [111In]-DOTA-ZHER2:342-pep2 in BALB/c nu/nu mice (n = 4 animals/group) bearing SKOV-3 xenograft tumors (5). The animals were injected intravenously with the respective radiochemicals and euthanized at predetermined time points post-injection (p.i.). At 4 h p.i., all three radiochemicals were reported to have a high accumulation in the tumors (11.0 ± 3.0% IA/g, 11.0 ± 2.0% IA/g, and 14.0 ± 1.0% IA/g for [111In]-ABY-025, [111In]-DOTA-ZHER2:2395-C, and [111In]-DOTA-ZHER2:342-pep2, respectively) compared with other soft tissues ranging from 0.07 ± 0.01% IA/g (muscle) to 0.93 ± 0.08% IA/g (gastrointestinal tract) for the three tracers. As expected, the kidneys showed very high uptake (ranging from 181.0 ± 17.0% IA/g to 184 ± 48% IA/g for the three radiochemicals) because this is the route of elimination for these labeled compounds. The tumor/muscle ratio was reported to increase from 27.0 ± 7.0 at 0.5 h to 255 ± 51 at 3 days p.i. (compared with ratios ranging from 6.0 ± 1.0 (blood) to 25.0 ± 7.0 (pancreas) at 0.5 h p.i. to 11.0 ± 2.0 (liver) to 474.0 ± 528 (small intestine) at 3 days p.i.).

To investigate the in vivo receptor binding specificity of [111In]-ABY-025, the mice were injected subcutaneously with unlabeled His6-ZHER2:342, the parent AB of ABY-025 (8), 45 min before injecting the radiolabel (5). Tumors from these animals showed a six-fold lower uptake of radioactivity compared with tumors from animals without pretreatment of the unlabeled AB. This indicated that [111In]-ABY-025 had a high specificity for binding to the HER2 receptor. The major organs of the animals showed an accumulation of <3%IA/g radioactivity and animals pre-treated with the non-radioactive AB showed an approximate 50% lower uptake of [111In]-ABY-025.

Gamma-camera images from mice bearing the SKOV-3 cell tumors were acquired 0.5 h and 4 h after an intravenous injection of [111In]-ABY-025 (5). The xenograft tumors were clearly visible in the images acquired at 0.5 h p.i., and the tumor/contralateral thigh ratio was reported to be 7.0 ± 1.0. This ratio was reported to increase to 25.0 ± 12.0 at 4 h p.i., indicating a quick clearance of radioactivity from blood and the other tissues of the animals. Control animals with A431 cell xenograft tumors (derived from a human epidermoid carcinoma; does not express HER2) did not show any uptake of radioactivity.

Groups of rats (n = 8 animals per group) given five consecutive injections of three different doses of ABY-025 (2, 6, and 30 μg, respectively) every 3 weeks did not develop any immunogenicity against the AB (5). Only 1 of 24 animals showed a very low-level immune response after the second injection, and the antibody level remained almost constant even after the three additional treatments of ABY-025. This indicated that the AB was not immunogenic in rats at the doses used for the study.

In another study, the pharmacokinetics of [111In]-ABY-025 was investigated in rats (n = 3 animals/gender) (5). Each animal was intravenously administered the tracer at a dose of 0.424 mg/kg body weight (2.0 kBq/μg (54 pCi/μg)), and blood samples were taken from the animals at fixed time points ranging from 2 min to 240 min. A biphasic decrease in concentration of the label was observed in both genders of the rats, and the mean initial and terminal half-lives in the animals were determined to be 9–14 min and 52–58 min, respectively.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


The pharmacokinetics of [111In]-ABY-025 were also investigated in cynomolgus macaques (Macaca fascicularis; n = 3 animals/gender) (5). The tracer was administered at a dose of 0.424 mg/kg body weight (1.6 kBq/μg (43.5 pCi/μg)), and blood samples were taken as described for the rat study above. Similar to the rats, a biphasic decrease in the concentration of the tracer was observed in the macaques, and the mean initial and terminal half-lives were determined to be 12–19 min and 104–123 min, respectively, in these animals.

From the various studies performed with [111In]-ABY-025, the investigators concluded that the radiochemical can be used for the detection of HER2-expressing xenograft tumors in a murine model. In addition, the AB was suitable for further development because it did not evoke an immune response in rats and macaques (5).

Human Studies


No references are currently available.

Supplemental Information


No information is currently available.


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