An accurate assessment of HER2 expression is crucial for the selection of patients that may benefit from HER2-targeted therapies. Currently, ex vivo techniques, such as immunohistochemistry and fluorescence in situ hybridization, are used to estimate HER2 expression before recommending HER2-tageted molecular therapies, of which the most common is treatment with trastuzumab. However, these screening approaches have significant limitations. New methods are needed to allow for non-invasive or minimally invasive evaluation of HER2 expression in vivo that can be repeated in individual patients and can provide an assessment of global HER2 expression in both primary tumors and distant metastasis. It is critical that these methods are also capable of monitoring efficacy in response to targeted therapy as measured by receptor levels on tumors. Thus, the high resolution, sensitivity, and quantitative nature of PET imaging offer an ideal alternative for assessing HER2 expression status. Radiotracers suitable for non-invasive imaging of HER2 in vivo have been previously reported. Robinson et al. developed and labeled with 124
I a divalent antibody (C6.5) fragment that effectively functions in the imaging of HER2-positive tumors with PET. Moreover, they validated a method for using a clinical PET/computed tomography scanner to quantify tumor uptake [17
]. The p185HER2
in xenografts could be distinguished by microPET imaging using a different size and format of anti-p185HER2
antibody fragments [30
]. Smith-Jones et al. [16
] used an F(ab′) fragment of trastuzumab labeled with 111
In and 68
Ga for SPECT and PET, respectively. However, the targeting agent they used cannot be applied to monitor response to trastuzumab, which is the current standard treatment for HER2-positive breast cancer, because the probe will compete for the same binding sites on HER2 receptors as non-labeled trastuzumab. Therefore, a decrease in signal will not correlate with decreased expression.
The Affibody molecules described by Orlova et al. [31
] and Steffen et al. [32
] offer a trastuzumab-compatible alternative for HER2 imaging. Using 125
–Affibody molecules, tumor-specific accumulation of radioactivity was demonstrated, and a high-contrast visualization of HER2-positive xenografts in mice was obtained 6 h post-injection.
More recently, promising results using the synthetic Affibody molecule 111
-tetraacetic acid) for SPECT imaging were also reported by the same group [22
]. In this study, biodistribution analysis revealed very high accumulation of the new tracer in tumor only 1 h post-injection (23% ID/g). Consequently, high-contrast images as soon as 1 h post-injection using a Gamma Camera could be obtained. The images obtained after treatment with 17-AAG (heat-shock protein inhibitor) showed significant changes in the observed signal. The same DOTA-modified constructs can also be labeled with a positron emitter, 68
Ga. Considering the very efficient and straightforward labeling procedure and the relatively easy access to 68
Ga from commercially available generators, this approach presents an attractive alternative for PET imaging. However, we are not aware of any publication describing use of such conjugates.
Mume et al. [33
] used maleimide chemistry for site-specific radiboromination of (ZHER2:4)2
-Cys. The resulting conjugate was shown to bind specifically to the HER2-expressing cell-line, SKOV-3, and the biodistribution studies confirmed its ability to accumulate in SKOV-3 xenografts. This preferential tumor binding combined with fast clearance from normal organs resulted in high tumor-to-normal tissue ratios at 4 h post-injection.
Cysteine-based chelators were incorporated by Tran et al. [34
] to ZHER2:342
for site specific labeling with 99m
Tc. Again, the Affibody molecule-based radioconjugates, in this case 99m
, were shown to accumulate specifically in SKOV-3 xenografts and, because of rapid clearance from the blood and other organs (except kidneys), allowed clear images of tumors by Gamma-camera at 6 h post-injection.
In this work, we have further demonstrated the feasibility of using Affibody molecules to monitor HER2 expression. By labeling ZHER2:342
–Cys Affibody molecule with 18
F, we have created a new tracer that will allow the application of a well-established PET methodology to quantify HER2 expression in vivo. The radionuclide chosen for the imaging agent offers several distinct advantages: (1) broad availability, (2) high positron yield (almost 100%), (3) a half-life (T1/2
109.7 min) that closely matches the relatively short biological lifetime of Affibody molecules.
The cysteine residue on the C-terminal of ZHER2:342
–Cys allowed for the application of a well-defined labeling chemistry using [18
F]fluorine-labeled maleimide. Cai et al. [18
] described the radiosynthesis of N
F]FBEM) and applied this molecule to the radiolabeling of an arginine–glycine–aspartic acid peptide for imaging of αv
integrin expression. We developed an alternative synthesis of [18
F]FBEM that employed the radiosynthesis of [18
F]fluorobenzoic acid and then coupling to aminoethylmaleimide using diethylcyanophosphate. This procedure was based on our previous experience in the radiosynthesis of fluoropaclitaxel [28
]. The preparation of the [18
F]FBEM was completed in a two-pot, three-step reaction sequence and provided sufficient product for coupling to the reduced ZHER2:342
–Cys Affibody molecule. The terminal cysteine caused the stock ZHER2
–Cys Affibody molecules to exist as a mixture of monomeric and dimeric forms. Initially, we attempted to reduce the disulfide bonds with Tris(2-carboxyethyl)phosphine (TCEP), but we observed that the TCEP reacted with the limited amount of [18
F]FBEM and precluded successful conjugation. We then adapted the procedure of Mume et al. [33
] that used DTT to reduce the protein and then removed the excess DTT using a NAP-5 column before conjugation. These conditions have proven quite reproducible for the preparation of conjugated Affibody molecules.
The affinity observed when the radiotracer was bound to HER2 receptors on SKOV-3 and SKBR-3 cells in vitro was comparable to that of typical antibodies used clinically for imaging and therapy (e.g., the reported affinity of trastuzumab to HER2 is 5 nM) [35
]. Our studies of the radiotracer binding to tumor cells expressing different levels of HER2 showed that the total cell-associated radioactivity was (1) correlated with the level of receptor expression, (2) reduced by pre-treatment of the cells with non-labeled Affibody molecules, and (3) was not affected by pre-treatment with trastuzumab. Consequently, our radioconjugate might be used to assess the level of receptor expression and possibly to monitor the changes of its expression in tumors also in patients treated with trastuzumab.
The results of our in vivo studies were very promising. The calculated elimination half-life was about 36 min, which definitely facilitated the imaging studies, whereas clearance of antibody fragments is in the range of a few hours [36
], and trastuzumab requires about 25 days [37
]. High accumulation of the radioactivity in HER2-positive tumors as compared with the background was observed as early as 20 min after injection as shown by PET imaging reached the plateau after 40 min and remained at that level for the duration of the experiments. For imaging purposes, this ratio at such a short time post-injection is much better than the previously published data with other HER2-targeting agents that demonstrated much weaker tumor accumulation and worse signal-to-background ratio [17
]. Four hours later, the ratio increased to 69, which resulted in further improvement of the contrast between tumor and the surrounding tissues, as shown by the whole body scans (Fig. , upper panels). By that time, only the bowel and the urinary bladder indicated any accumulation of radioactivity. These results were confirmed by the biodistribution studies indicating that 1 h post-injection, the concentration of radioactivity (% ID/g) in tumor was 7.5 times higher than that in the blood. Two hours after injection, the tumor uptake was higher than the uptake in any other organs, and levels remained steady over the course of the study. Pretreatment with non-radiolabeled Affibody molecules decreased the tumor accumulation to the levels observed in HER2-negative tumors, which was comparable to that found in the normal organs, with the exception of the kidneys, where the uptake was significantly higher. However, the radioactivity in the kidneys decreased rapidly because of excretion of the tracer to the urinary bladder. This rapid clearance of radioactivity from the kidneys distinguishes our radioconjugate from other Affibody-based radiotracers that (or their radioactive degradation products) tend to accumulate in the kidneys. On the other hand, [18
produced a higher level of radioactivity in the bone as a result of possible dehaloganation. Nevertheless, according to extrapolation of pre-clinical biodistribution data using the Olinda simulation package [38
], several PET scans per year could be carried out using the typical dose of 185 MBq without exceeding the radiation doses to the patient that are allowed for routine diagnostic purposes.
Our results suggest that the [18F]FBEM-ZHER2–Affibody radioconjugate described herein can be used to assess HER2 expression in vivo by PET imaging and, therefore, might be used to monitor possible changes of receptor expression in response to therapeutic interventions. The combination of a radionuclide commonly used in the clinic and an Affibody molecule characterized by a relatively low molecular weight (compared to antibodies or single chain antibody fragments), high affinity to the target receptors, and fast clearance from the blood and normal organs makes it an optimal candidate for clinical applications.