Molecular imaging has great potential for characterization of therapeutically important targets in malignancies [19
], including HER2/neu [6
]. Because HER2/neu expression levels in metastases may be quite different from the primary tumor [21
], assessment of the specific expression of these receptors on cancer cells in various locations may be useful in selecting appropriate therapy [22
]. However, the development of HER2/neu-specific imaging probes for routine clinical use has not yet been successful.
In our previous work [15
], we labeled Affibody molecules with 18
F - widely available and a nearly pure (97%) positron emitter with a relatively short, but clinically useful, half-life of 110 minutes. However, 18
F production requires a cyclotron, and the radiolabeling procedures involving this radionuclide require several low-yielding steps that are often time consuming. Therefore, in this study, we evaluated the potential of 68
] as an imaging agent in xenografts with different HER2/neu expression levels.
Gallium-68 presents an attractive alternative as a radionuclide for PET imaging. It can be obtained from commercially available 68Ge-68Ga generators, which have a half-life of 271 days and, thus, can provide an almost constant supply of 68Ga for over one year. The labeling procedures are well established and relatively straightforward. Also, when prorated, the unit cost of 68Ga from generators is reasonable when amortized over a year, which makes it even more attractive for busy nuclear medicine departments, particularly those with limited or no access to cyclotrons.
Most of the efforts in developing tracers for HER2/neu focused on using anti-HER2/neu antibodies. However, antibodies are rather bulky proteins typically characterized by limited tumor penetration and slow clearance from the circulation. A successful molecular imaging probe requires high affinity and specificity binding to tumor-associated structures with minimal non-specific accumulation in normal tissues and rapid clearance from the background. Optimally, it should easily penetrate the tumor parenchyma and rapidly clear from the blood to increase the tumor to background ratio. Recently, significant progress has been made in protein display technology that has led to the discovery of many small proteins and peptides [24
]. Among them, Affibody molecules have been shown to meet most of the aforementioned requirements providing a suitable platform for development of new imaging probes [14
Our current results demonstrated the high affinity of 68
-Affibody for HER2/neu receptors on MDAMB-361 cells in vitro.
was similar to those found with other Affibody radioconjugates and was comparable to antibodies used clinically for imaging and therapy (e.g., the reported affinity of trastuzumab to HER2/neu is 5 nM) [27
The in vivo
pharmacokinetics data were also promising. The elimination half-life estimated by measuring radioactivity in blood samples at several time points after injection was approximately 21 min. This relatively fast clearance resulted in high tumor-to-blood ratios as well as high-contrast PET images of 68
-Affibody molecules as early as 1 hour post tracer injection. It is noteworthy that the obtained tumor-to-blood ratios were higher than for other imaging probes including 68
fragment of Herceptin, as well as full length antibodies and antibody fragments labeled with 124
Zr and, 86
Because of the fast blood clearance, the images showed high tumor accumulation as compared with other tissues (except the kidneys) in the high- and intermediate-expressing tumors. In highly HER2/neu-expressing BT474 tumors the uptake reached 31±7.0 %ID/g as soon as 1 hour post tracer injection, and is was higher than the accumulation levels recently described by Ren et al. [30
] and Tolmachev et a [16
] for SKOV3 tumors. The absolute tumor accumulation reported by these authors was only 4.12±2.2 %ID/g (2 hours post injection of an Affibody-based, 2-helix 68
Ga-DOTA-MUT-DS molecule) and 8.9±1.0 %ID/g (45 min post injection of 68
Ga-ABY-002). It should be noted that SKOV3 cells express even more HER2/neu receptors than BT474 (ELISA results from our laboratory: 335 ng/mg for SKOV3 versus
260 ng/mg for BT474). These results are in line with our previous data obtained with radio-fluorinated probe [17
]. It should be stressed that correction for the uptake for nonspecific binding of His6-ZTaq-Cys-DOTA-68Ga was not necessary since the accumulation of this non-HER2-specific tracer was approximately at the same level as surrounding tissues, and tumors were not detectable. We have previously shown a strong association between the uptake of 18
F-FBEM– ZHER2:342-Affibody tracer in the panel of breast cancer tumor xenografts with different HER2/neu expression [17
]. Also, McLarty et al., using mice bearing tumors with a wide range of receptor levels, showed a correlation between 111In-DTPA-trastuzumab accumulation and HER2/neu density in individual tumors [32
In this work, we investigated the correlation between HER2/neu expression determined by PET imaging in vivo
and the biodistribution data for each particular tumor model following injection of 68
-Affibody and by comparison to HER2/neu protein concentration measured in the same tumors ex vivo
by ELISA. Based on our experience with 18
F-labeled Affibody molecules and considering the short half-life of gallium, the biodistribution and imaging studies were done at 1 and 3 hours post tracer injection. There was no indication of label instability since the tracer was rapidly cleared from the blood and liver. By analyzing the biodistribution and imaging data, we were also able to correlate the tracer uptake with number of HER2/neu receptors. Unlike results reported for antibodies by McLarty et al. [32
], who had to apply appropriate corrections for non-specific tumor uptake and blood flow, we found a strong linear correlation between PET values [%ID/g] and biodistribution data that also corresponded to HER2/neu level in each tumor. It is important to note that using gallium-labeled agents the estimation of receptor expression may be limited for the very low receptor expressing tumors. This is due to the greater positron range of 68
Ga (1.9 MeV) compared for example to 18
F that could lead to poorer spatial resolution and erroneous assignment of the ROI. The intrinsic resolution of small-animal PET scanners has improved to a level where the positron range of the radionuclide eventually could influence the effective spatial resolution. Jorn van Dalen et al.
showed that the image resolution of a PET scanner with a 1.5 mm intrinsic system resolution is decreased by using PET emitters with large positron ranges such as 68
Ga and 124
]. This could impact: (i) the detection of small lesions, (ii) assessment of low receptor expression, (iii) the ability to distinguish lesions that are close together, as well as (iv) quantification of tumor uptake. Therefore, the positron energy and range must be considered when planning small-animal imaging. On the other hand from clinical point of view, Yang et al.
demonstrated that the conventional FWHM of 18
F and that of 68
Ga are indistinguishable in soft tissues (3.01 mm vs. 3.09 mm) [34
]. This implies that with the spatial resolution of 5-7 mm for current clinical scanners, image quality using 68
Ga-labeled tracers should be comparable to 18
We observed very high radioactivity accumulation in the kidney. These values are comparable to those previously reported for gallium-labeled Affibody molecules [16
]. Use of 68
Ga as well as other metal positron emitters including 64
Cu and 89
Zr, leads to residualization that results in trapping of radionuclides in lysosomes after internalization and degradation of the tracer [35
]. Because of this phenomenon, the kidney is the major dose-limiting organ for this tracer. However, this is not a major diagnostic limitation since renal lesions are uncommon in breast cancer.
Positively charged amino acids, such as lysine and arginine are now commonly used for renal protection in clinical trials of peptide receptor-targeted radionuclide therapy and may be useful in this setting as well [37
]. Studies of the possible effect of positively charged amino acids on accumulation of radioactivity in the kidney following 68
Affibody administration are, however, outside the scope of this paper. In conclusion, our results indicate that differences in HER2/neu expression levels can be detected in vivo
by PET imaging with 68
-Affibody. This generator-produced agent may, therefore, be useful in the characterization HER2/neu-positive tumors as an alternative to 18
F-labeled compounds especially where access to a cyclotron is limited.