111In-Labeled anti-epidermal growth factor receptor Affibody PEP09239


Chopra A.

Publication Details



In vitro Rodents



The human epidermal growth factor receptor-2 (HER2, ErbB2) modulates its activity through a tyrosine kinase (TK) signaling pathway and is involved in the development of a variety of cancers (1, 2). Overexpression or amplification of the HER2 gene is known to occur in many cancer types (e.g., ~20% of breast cancer) and predicts a poor prognosis for the patient. Invasive methods, such as biopsies, in conjunction with immunohistochemistry and an in situ fluorescence hybridization kit (PathVysion) approved by the United States Food and Drug Administration are used 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 are not 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 imaging agent does not distinguish between benign and malignant lesions, cannot differentiate tumors that overexpress HER2 from those that have a low or no expression of 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 via chemical synthesis or produced in bacteria with the use of recombinant DNA technology (3). The Affibody scaffold consists of 3-helix peptide chains: helix chains 1 and 2 are composed of 13 randomized amino acids and contain the receptor-binding moieties of the Affibody (4), and the third helix chain functions as a stabilizer of the Affibody molecule (5). Affibody molecules have high affinity and specificity of binding to the targeted molecule and are considered to be extremely suitable for the noninvasive imaging of solid tumors (6). The radionuclide-labeled Affibody ZHER2:342 (and its derivatives), directed against the HER2 (ZHER2:342Kd = 22 pM for HER2), has been used successfully with molecular imaging techniques to screen for breast cancer patients who are likely to benefit most from treatment with trastuzumab (a monoclonal antibody that targets HER2) or lapatinib (a small-molecule drug that inhibits the TK activity of the HER2 signaling pathway) (6, 7).

To further improve the imaging properties of radiolabeled Affibodies, it was hypothesized that reducing the size of the molecule is likely to facilitate rapid clearance of the tracer from circulation, high penetration into solid tumors, ameliorate the uptake of label in nontargeted organs (such as the liver), and generate increased tumor/background ratios compared with the 3-helix molecule (8). To test the hypothesis, the stabilizing helix chain was removed from the 3-helix ZHER2:342 molecule, and the 2-helix Affibody (MUT-DS; ~4.6 kDa) was determined to have a binding affinity of 5 nM for HER2 (8). In another study, MUT-DS was conjugated with DOTA, a metal chelator, labeled with 68Ga ([68Ga]-DOTA-MUT-DS), and evaluated for the detection of human ovarian carcinoma SKOV3 cell xenograft tumors (these cells overexpress HER2) in nude mice (9). Although [68Ga]-DOTA-MUT-DS was rapidly cleared from circulation and had a high binding specificity for the tumors, it was observed that the uptake of radioactivity in the lesions was considerably lower (4.12 ± 0.83% injected dose per gram tissue (ID/g) at 2 h postinjection (p.i.)) (9) than the accumulation of label observed in the SKOV3 cell tumors with a similarly labeled HER2-binding 3-helix Affibody ([68Ga]-ABY-002; 12.4 ± 3.8% at 2 h p.i.) (10). This indicated that there was no particular advantage in using a smaller Affibody compared with the larger parent 3-helix molecule. In addition, the comparison between the two Affibody types may be inaccurate for two reasons. First, the data that were compared were obtained from two separate studies in which the two SKOV3 cell subclones used to generate the tumors in the mice may not have produced lesions of the same size. Second, the same amounts of HER2 may not have been expressed in the neoplasms (4). Therefore, to ascertain the similarities or dissimilarities between MUT-DS and ABY-002 Affibodies, it was necessary to investigate the biological characteristics of the two entities simultaneously with the use of identical handling techniques and to perform similar in vitro and in vivo studies simultaneously with the two Affibodies (4).

Rosik et al. compared the receptor targeting and biodistribution characteristics of 111In-labeled MUT-DS ( [111In]PEP09239) and 111In-labeled ABY-002 ([111In]ABY-002) in a side-by-side study of mice bearing xenograft tumors generated with SKOV3 cells (these cells express ~1.2 × 106 HER2 receptor/cell) and human colorectal LS174T cells (these cells express low levels of HER2; ~3.9 × 104 receptors/cell), respectively (4).



Synthetic DOTA-ABY-002, synthesized with the Fmoc procedure, was obtained from a commercial source (4). PEP09239 was also synthesized with the Fmoc technique on an automated peptide synthesizer, and the identity of this Affibody was confirmed with reverse-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (4). PEP09239 was subsequently conjugated with DOTA and purified with HPLC. The purity of the DOTA-PEP09239 conjugate was reported to be >97% as determined with HPLC (4).

The labeling of DOTA-PEP09239 with 111In is described elsewhere (4). The radiochemical yield (RCY) of [111In]PEP09239 was 99.4 ± 0.5%, with a specific activity of 5.1 GBq/μmol (137.83 mCi/μmol). The 111In-labeled Affibody was used in the various studies without further purification.

DOTA-ABY-002 was labeled with 111In as described by Orlova et al. (11). The RCY, radiochemical purity, and SA of [111In]ABY-002 were not reported (4).

In Vitro Studies: Testing in Cells and Tissues


The stability of [111In]PEP09239 was evaluated by exposing the 111In-labeled Affibody to 1,000-fold molar excess ethylenediamine tetracetic acid (EDTA) for 4 h at room temperature (4). Control samples were incubated in phosphate-buffered saline under the same conditions. At the end of the incubation, 96.1 ± 0.5% of [111In]PEP09239 exposed to EDTA was intact (compared with 98.5 ± 0.7% of the control) as determined with radio-thin-layer chromatography.

The binding kinetics of PEP09239 and ABY-002 were determined with surface plasmon resonance techniques, with the extracellular domain of HER2 as the target on the sensor chip (4). The dissociation constants of PEP09239 and ABY-002 were 2.1 nM and 78 pM, respectively. The Ka and Kd values for PEP09239 were 6.0 × 105 M−1s−1 and 1.3 × 10−3 s−1, respectively, and for ABY-002 these values were 4.0 × 106 M−1s−1 and 2.6 × 10−4 s−1, respectively.

The receptor binding specificity of [111In]PEP09239 was determined by exposing SKOV3 cells and LS174T cells to the labeled Affibody (10 nM) for 1 h at 37°C as described by Rosik et al. (4). Approximately 30% and 1.75% of added radioactivity was bound to the SKOV3 cells and the LS174T cells, respectively. Prior exposure of the SKOV3 cells to either non-radiolabeled PEP09239 or non-radiolabeled ABY-002 reduced the binding to ~1% of total added radioactivity. Similarly, blocking the HER2 receptors on the LS174T cells with non-radiolabeled PEP09239 or non-radiolabeled ABY-002 reduced the binding of [111In]PEP09239 to ~0.75% of total added radioactivity. These studies indicated that [111In]PEP09239 had a high binding specificity for the HER2 receptor.

In another study, SKOV3 cells were loaded with [111In]PEP09239 at 4°C for 1 h and then transferred to growth medium containing no labeled Affibody at 37°C. Subsequently, the amount of radioactivity bound to and internalized by the cells at different time points was determined (4). At 8 h after the transfer, 63 ± 1% of the label (compared with 100% at 0 h) was associated with the cells and remained relatively constant thereafter for up to 24 h. Internalization of the label from [111In]PEP09239 was slow and reached up to 21.1 ± 0.5% of the initial value at 24 h after transfer to medium containing no radioactivity.

Animal Studies



The biodistribution of [111In]PEP09239 and [111In]ABY-002 was compared in normal Naval Medical Research Institute mice (4). The animals (n = 4 mice/time point) were injected with 30 kBq (0.81 μCi) [111In]PEP09239 or [111In]ABY-002 through the tail vein and euthanized at predetermined time points ranging from 1 h p.i. to 24 h p.i. All organs of interest were removed from the animals, and the amount of radioactivity accumulated in the various tissues was determined. Data obtained from this study are shown in Table 1. A rapid clearance of radioactivity was observed from the blood and other tissues with both labeled Affibodies; the label from [111In]PEP09239 was cleared from all the tissues more rapidly than the label from [111In]ABY-002. A high reabsorption of tracer was observed in the kidneys with both tracers, a characteristic common to all HER2-binding Affibodies.

Table 1: Biodistribution of radioactivity from [111In]PEP09239 and [111In]ABY-002 in normal NMRI mice


Table 1: Biodistribution of radioactivity from [111In]PEP09239 and [111In]ABY-002 in normal NMRI mice.

The biodistribution of [111In]PEP09239 and [111In]ABY-002 was also investigated in outbred BALB/c nu/nu mice bearing SKOV-3 cell and LS714T cell xenograft tumors on the right hind legs (4). The animals (n = 4 mice/time point) were injected with 30 kBq (0.14 nmol) [111In]PEP09239 or [111In]ABY-002 in 100 μl PBS through the tail vein. The rodents were euthanized at 1 h p.i. and 4 h p.i. to determine the amount of radioactivity accumulated in the various tissues. At 1 h p.i., the uptake of label from [111In]ABY-002 in the SKOV3 cell tumors (high expression of HER2) was 1.7-fold higher (17.8 ± 0.4% ID/g) than the uptake from [111In]PEP09239 (10.6 ± 0.4% ID/g; P < 0.005), and almost no change in uptake of the tracers was observed for up to 4 h p.i. With the LS174T tumors (low expression of HER2), the accumulation of radioactivity with [111In]ABY-002 (8.6 ± 4.0% ID/g) was ~3-fold higher than with [111In]PEP09239 (2.9 ± 0.6% ID/g) at 1 h p.i. At 4 h p.i., the uptake of label from [111In]ABY-002 in the LS174T cell tumors was ~8-fold higher than with [111In]PEP09239 (because with [111In]PEP09239 the amount of radioactivity in the LS174T cell tumors was observed to decrease by 4 h p.i.).

To ascertain the HER2 binding specificity of [111In]PEP09239, mice bearing SKOV3 cell tumors and LS174T cell tumors (n = 4 animals/tumor type) were subcutaneously pretreated with 115.4 nmol (for SKOV3 cell tumors) or 76.9 nmol (for LS174T cell tumors) non-radioactive ZHER2:342; 40 min later, the animals were intravenously injected with the labeled 2-helix Affibody (4). Pretreatment of the animals with excess non-labeled ZHER2:342 resulted in a significantly lower (P < 0.05) accumulation of radioactivity in both the tumor types; however, no reduced uptake of tracer was apparent in any other organs. These results indicated that [111In]PEP09239 had a high binding specificity for HER2.

For PET imaging studies (to confirm the biodistribution data), mice bearing SKOV3 cell tumors and LS174T cell tumors were injected with 0.6 MBq (16.2 μCi) [111In]PEP09239 or [111In]ABY-002, and the animals were euthanized at 4 h p.i (4). The urinary bladders of the animals were removed immediately before imaging, and gamma planar images of the rodents were acquired for 60 min. From the images it was clear that both tumor types were clearly visible only in rodents injected with [111In]ABY-002, but the amount or tracer accumulated in the LS174T cell lesions was lower than that in the SKOV3 cell tumors. With [111In]PEP09239, only the SKOV3 cell tumors were clearly visible, and the LS174T cell lesions were only faintly visible in the animals.

From these studies, the investigators concluded that radioactivity from the 2-helix [111In]PEP09239 Affibody was cleared rapidly from circulation and normal tissues of the rodents compared with the label from [111In]ABY-002 (4). In addition, although the uptake of label from [111In]PEP09239 in the neoplastic lesions with a high expression of HER2 was lower than with [111In]ABY-002, the tumor/blood ratio with the former labeled Affibody was higher than that with the latter labeled Affibody (4). However, [111In]ABY-002 appeared to be superior to [111In]PEP09239 for the visualization of tumors that have a low expression of HER2 (4).

Other Non-Primate Mammals


No reference is currently available.

Non-Human Primates


No reference is currently available.

Human Studies


No reference is currently available.

Supplemental Information


No information is currently available.


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