111In-Labeled DOTA-conjugated 6-aminohexanoic linker-containing variant of anti-epidermal growth factor receptor 2 Affibody ZHER2:342 (ABY-003)


Chopra A.

Publication Details



In vitro Rodents



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 through chemical synthesis or produced in bacteria with the use of recombinant technology (1). Because of their small size and high chemical and thermal stability, these molecules are used as radiolabeled probes for the targeted detection and treatment of malignant tumors as discussed elsewhere (1-3).

The epidermal growth factor receptor 2 (HER2) is considered to be an important Affibody target because it is believed to promote the development of the malignant phenotype, it plays a role in the development of resistance to anticancer drugs and radiation therapy, it is overexpressed in several different cancer tumor cells, and it often indicates a poor prognosis for the patient (4). Several radionuclide-labeled Affibodies and their derivatives, such as ZHER2:342, have been evaluated to detect tumors expressing HER2 in preclinical studies as discussed by Tolmachev (5). Earlier, a derivative of the ZHER2:342 Affibody, designated ABY-002, which has the radiometal chelator 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA) linked directly to the N-terminal valine of the molecule was constructed, radiolabeled with 111In or 68Ga, and evaluated for the imaging of malignant tumors that express HER2 (6). Although radiolabeled ABY-002 (both with 111In or 68Ga) could detect these tumors under in vivo conditions, the ZHER2:342 Affibody derivative exhibited a reduced affinity for HER2 (KD = 65 pM) compared to the parent Affibody (KD = 22 pM).

From this study, it was concluded that the reduced affinity of ABY-002 for the receptor is probably due to a steric hindrance from the DOTA moiety, which is located very close to the HER2 binding site of the molecule. Tolmachev et al. decided to introduce a linker between the DOTA moiety and the HER2 binding sequence and evaluate whether this modification reduced or abolished the steric obstruction that may reduce the affinity of the Affibody for the receptor (7). The investigators placed an aliphatic linker (6-aminohexanoic acid) between DOTA and the valine on the N-terminal of ABY-002, labeled the modified Affibody (ABY-003) with 111In ([111In]-ABY-003), and evaluated the biodistribution and tumor detection properties of [111In]-ABY-003 in mice bearing HER2-expressing LS174T cell xenograft tumors (a human colon adenocarcinoma cell line showing a moderate expression level of HER2) or SKOV-3 cell xenograft tumors (a human ovarian carcinoma cell line showing a high expression level of HER2) (7).



The synthesis and 111In labeling of the ABY-003 Affibody have been described by Tolmachev et al. (7). The radiochemical yield of [111In]-ABY-003 was 98.9 ± 0.9% (n = 22 synthesis procedures), and the final product was used in all experiments without further purification. The specific activity of the labeled Affibody was reported to be up to 14 GBq/μmol (0.476 mCi/μmol).

In some studies, 111In-labeled ABY-002 ([111In]-ABY-002) was used for comparison purposes (7). The labeling of ABY-002 was performed in the same manner as for ABY-003. The radiochemical purity, radiochemical yield, and specific activity of [111In]-ABY-002 was not reported.

In Vitro Studies: Testing in Cells and Tissues


The in vitro stability of [111In]-ABY-003 was tested by incubating the labeled Affibody with or without 500-fold molar excess ethylenediamine acetic acid (EDTA) in phosphate-buffered saline for 6 h at room temperature (7). At the end of incubation, instant thin-layer chromatography analysis showed that 97.6 ± 0.2% of 111In remained associated with the Affibody in the test samples (with EDTA) versus 99.6 ± 0.3% in the controls (without EDTA), indicating that the [111In]-ABY-003 had high stability at room temperature.

The binding of ABY-003 to the extracellular domain of HER2 (HER2-ECD) and to the chimeric HER2/Fc fusion protein (HER2/Fc; has the HER2-ECD fused to the Fc region of human IgG (8)) was determined with the Biacore technique and compared with the binding of ABY-002 and ABY-004, the unmodified parent molecule of ABY-002 (7). The KD values of ABY-004, ABY-002, and ABY-003 for HER2-ECD were reported to be 45 ± 11 pM, 118 ± 5 pM, and 135 ± 3 pM, respectively. With HER2/Fc, these values were 58 ± 2 pM, 139 ± 8 pM, and 145 ± 5 pM for ABY-004, ABY-002, and ABY-003, respectively.

The in vitro HER2 binding specificity of [111In]-ABY-003 was tested by exposing SKOV-3 cells (~1.2 × 106 HER2 receptors/cell) to the labeled Affibody (1 nM) for 1 h at 37°C (7). Under these conditions, 43 ± 2% of the radioactivity remained associated with the cells at the end of the incubation period. When the cells were preincubated with excess unlabeled His6-ZHER2:342 (concentration not reported), which is another HER2-binding derivative of the ZHER2:342 Affibody, the amount of radioactivity that remained associated with the cells was reduced to 0.12 ± 0.05% (P < 0.0001). This indicated that [111In]-ABY-003 bound specifically to the HER2 on the cells.

A cell internalization study with [111In]-ABY-003 revealed that 37.9 ± 0.2% of radioactivity was taken up by the cells after 24 h at 37°C (7). This rate of internalization was similar to that observed with [111In]-ABY-002 (9). This indicated that placement of the aliphatic linker between the DOTA and the ZHER2:342 Affibody did not affect the cellular internalization of the Affibody.

After an interrupted incubation, the retention of [111In]-ABY-003 by SKOV-3 cells was 84.8 ± 0.0% and 69.2 ± 0.3% at 24 h and 72 h, respectively, versus retention levels of 90.0 ± 0.6% and 79.1 ± 0.4% for [111In]-ABY-002 at the same time points (7). This indicated that the cellular retention of [111In]-ABY-002 was slightly superior to that of its derivative.

Animal Studies



The biodistribution of [111In]-ABY-003 was compared with that of [111In]-ABY-002 in normal Naval Medical Research Institute mice (7). The animals (n = 4 mice/group) were subcutaneously injected with a total of 1 μg protein adjusted with an appropriate amount of unlabeled Affibody (specific activity, 213 MBg/μmol (5.75 mCi/μmol)), and the mice were euthanized at predetermined time points ranging from 1 h to 24 h postinjection (p.i.) to determine radioactivity content of the various tissues. Both labeled Affibodies were cleared rapidly from the blood circulation and whole body of the animals. Although there was low accumulation of radioactivity in the liver with both radiolabeled Affibodies, accumulation of label from [111In]-ABY-003 was observed in this organ at all the time points to be significantly higher (P < 0.05) than that of [111In]-ABY-002. Except for the kidneys, the accumulation of radioactivity was low in the different organs with both labeled Affibodies. This study indicated that [111In]-ABY-003 had a biodistribution pattern similar to that of the parent Affibody (ABY-002) in rodents.

In another study, the biodistribution of [111In]-ABY-003 was studied at 4 h p.i. as described above in mice (n = 4 animals/group) bearing LS174T cell xenograft tumors (7). The biodistribution pattern of radioactivity in the various organs of these mice was similar to that observed with the NMRI mice as detailed above. The tumor showed an accumulation of 9.1 ± 0.5% injected dose per gram tissue (% ID/g). With [111In]-ABY-003, the tumor/blood, tumor/liver, and tumor/muscle ratios were 18 ± 4, 6 ± 1, and 70 ± 36, respectively. The uptake of radioactivity in tumors of mice pretreated with excess unlabeled ZHER2:342 Affibody (~6.6 nM) was 0.5 ± 0.2% ID/g (P <0.000%), indicating that [111In]-ABY-003 bound specifically to the HER2-expressing tumors.

The biodistribution of [111In]-ABY-003 was also investigated in mice bearing SKOV-3 cell xenograft tumors (n = 4 animals/group), and data obtained from this study were compared to data obtained from a similar study performed with [111In]-ABY-002 (7). Both labeled Affibodies showed a similar biodistribution pattern in the animals. The only difference was that the SKOV-3 cell tumors showed a higher accumulation of radioactivity from [111In]-ABY-003 (14 ± 2% ID/g) than did the LS174T lesions (9.1 ± 0.5% ID/g; see above). At 4 h p.i., the tumor/heart, tumor/lung, tumor/liver, tumor/spleen, and tumor/muscle ratios were 48 ± 16, 26 ± 3, 9 ± 1, 31 ± 6, and 110 ± 40, respectively, which were higher than the ratios obtained with [111In]-ABY-002. For details, see Table 4 in Tolmachev et al. (7).

Mice bearing SKOV-3 cell tumors (n = 3 animals) were injected with [111In]-ABY-003 to acquire whole-body gamma planar images at 4 h p.i (7). The xenograft lesions and the kidneys were clearly visible in the images, but the other organs were not visualized. Therefore, high-contrast images of tumors expressing HER2 were generated with the labeled ABY-003 Affibody.

From these studies, the investigators concluded that the introduction of a linker between the metal chelator and the N-terminus of the ZHER2:342 Affibody had little influence on the biological properties of the molecule (7). The rapid clearance of [111In]-ABY-003 from the blood and whole body of the mice resulted in a higher tumor/organ ratio of radioactivity, although in general, compared to [111In]-ABY-002, a lower accumulation of label was observed in the various with [111In]-ABY-003.

Other Non-Primate Mammals


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Non-Human Primates


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Human Studies


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Supplemental Information


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