Preclinical Evaluation of the Copper-64 Labeled GRPR-Antagonist RM26 in Comparison with the Cobalt-55 Labeled Counterpart for PET-Imaging of Prostate Cancer

Gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of prostate cancers. This study aimed to investigate the potential of 64Cu (radionuclide for late time-point PET-imaging) for imaging of GRPR expression using NOTA-PEG2-RM26 and NODAGA-PEG2-RM26. Methods: NOTA/NODAGA-PEG2-RM26 were labeled with 64Cu and evaluated in GRPR-expressing PC-3 cells. Biodistribution of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was studied in PC-3 xenografted mice and compared to the biodistribution of [57Co]Co-NOTA/NODAGA-PEG2-RM26 at 3 and 24 h p.i. Preclinical PET/CT imaging was performed in tumor-bearing mice. NOTA/NODAGA-PEG2-RM26 were stably labeled with 64Cu with quantitative yields. In vitro, binding of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was rapid and GRPR-specific with slow internalization. In vivo, [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 bound specifically to GRPR-expressing tumors with fast clearance from blood and normal organs and displayed generally comparable biodistribution profiles to [57Co]Co-NOTA/NODAGA-PEG2-RM26; tumor uptake exceeded normal tissue uptake 3 h p.i.. Tumor-to-organ ratios did not increase significantly with time. [64Cu]Cu-NOTA-PEG2-RM26 had a significantly higher liver and pancreas uptake compared to other agents. 57Co-labeled radioconjugates showed overall higher tumor-to-non-tumor ratios, compared to the 64Cu-labeled counterparts. [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was able to visualize GRPR-expression in a murine PC model using PET. However, [55/57Co]Co-NOTA/NODAGA-PEG2-RM26 provided better in vivo stability and overall higher tumor-to-non-tumor ratios compared with the 64Cu-labeled conjugates.


Labeling, Stability, and In Vitro Characterization of [ 64 Cu]Cu-NOTA/NODAGA-PEG2-RM26
NOTA-PEG2-RM26 and NODAGA-PEG2-RM26 were successfully labeled with 64 Cu with yields and purities exceeding 98% for a molar activity of 4 MBq/nmol and 96% for 40 MBq/nmol. The compounds were stable in serum and excess of ethylenediaminetetraacetic acid (EDTA) with minimal release of free radiocopper (Table 1). Table 1. Labeling and stability of [ 64 Cu]Cu-X-RM26 (X = NOTA-PEG2, NODAGA-PEG2). Stability was checked in serum samples after 1 h incubation at 37 °C and in the presence of 1000× excess of ethylenediaminetetraacetic acid (EDTA) after 1 h incubation at RT. Data are presented as average ± standard deviation.

Labeling, Stability, and In Vitro Characterization of [ 64 Cu]Cu-NOTA/NODAGA-PEG 2 -RM26
NOTA-PEG 2 -RM26 and NODAGA-PEG 2 -RM26 were successfully labeled with 64 Cu with yields and purities exceeding 98% for a molar activity of 4 MBq/nmol and 96% for 40 MBq/nmol. The compounds were stable in serum and excess of ethylenediaminetetraacetic acid (EDTA) with minimal release of free radiocopper (Table 1). Table 1. Labeling and stability of [ 64 Cu]Cu-X-RM26 (X = NOTA-PEG 2 , NODAGA-PEG 2 ). Stability was checked in serum samples after 1 h incubation at 37 • C and in the presence of 1000× excess of ethylenediaminetetraacetic acid (EDTA) after 1 h incubation at RT. Data are presented as average ± standard deviation. The in vitro binding specificity assay demonstrated specific binding of [ 64 Cu]Cu-X-RM26 (X = NOTA-PEG 2 , NODAGA-PEG 2 ) to GRPR-expressing PC-3 cells (Figure 2). The pre-saturation of receptors by addition of a large molar excess of non-labeled peptide caused a significant reduction of [ 64 Cu]Cu-X-RM26 uptake.
Results concerning cellular processing and internalization of [ 64 Cu]Cu-X-RM26 are presented in Figure 3A. Cellular processing was similar for both conjugates. Cell-associated activity increased continuously over time, while the internalized fraction was very low at all time points, below 7% of total cell-associated activity. The cellular retention of [ 64 Cu]Cu-X-RM26 ( Figure 3B) revealed a rapid initial dissociation phase (up to 1 h) followed by a plateau. Results concerning cellular processing and internalization of [ 64 Cu]Cu-X-RM26 are presented in Figure 3A. Cellular processing was similar for both conjugates. Cell-associated activity increased continuously over time, while the internalized fraction was very low at all time points, below 7% of total cell-associated activity. The cellular retention of [ 64 Cu]Cu-X-RM26 ( Figure 3B) revealed a rapid initial dissociation phase (up to 1 h) followed by a plateau. [ 64 Cu]Cu-NOTA-PEG2-RM26 had significantly higher retention in cells compared to [ 64 Cu]Cu-NODAGA-PEG2-RM26 (43 ± 3% of cellassociated activity for NOTA-PEG2-RM26 vs 25 ± 2% for NODAGA-PEG2-RM26 after 24 h incubation). Cu-X-RM26. Cell-bound activity is normalized to the maximum uptake. Data are presented as mean value ± standard deviation. Error bars that are smaller than symbols may not be visible.
The IC50 values were in the low nanomolar range for both conjugates although pronounced chelator-dependent differences could be observed ( Figure 4). The IC50 for nat Cu-NODAGA-PEG2-RM26 showed a two-fold worse binding affinity (12.0 ± 1.0 nM) compared to nat Cu-NOTA-PEG2-RM26 (6.1 ± 0.8 nM). Cu-X-RM26. Cell-bound activity is normalized to the maximum uptake. Data are presented as mean value ± standard deviation. Error bars that are smaller than symbols may not be visible.

In Vivo Characterization of [ 64 Cu]Cu-NOTA/NODAGA-PEG2-RM26 and Comparison with 55/57 Co-Labeled Counterparts
Biodistribution of [ 64 Cu]Cu-X-RM26 (X = NOTA-PEG2, NODAGA-PEG2) was evaluated in mice bearing PC-3 xenografts at 3 and 24 h p.i. For comparison, mice were co-injected with the 57 Co-labeled counterparts. 57 Co was used as a convenient surrogate isotope for 55 Co in the biodistribution part, due to their chemical identity. It was previously demonstrated that 57 Co could be used for preclinical evaluation of radioagents that are designed to be used with 55 Co for PET [28]. [ 55 Co]Co-X-RM26 has previously shown a remarkable potential for PET imaging of GRPR expression [28].
[ 64 Cu]Cu-and [ 57 Co]Co-labeled X-RM26 displayed comparable biodistribution profiles with a fast clearance from blood and normal organs, including excretory organs ( Table 2). Tumor uptake at 3 h p.i. exceeded the uptake in normal organs for all conjugates. No significant difference was observed in tumor uptake between either the 64 Cu-and 57 Co-labeled conjugates or between the different chelators at both time points. Tracer uptake was also observed in GRPR expressing organs, excretory organs, and the gastrointestinal (GI) tract (Table 2).
Several notable differences could be observed between 64 Cu-labeled and 57 Co-labeled conjugates. The clearance of activity from blood was significantly slower for [ 64 Cu]Cu-NOTA-PEG2-RM26 in comparison to both [ 64 Cu]Cu-NODAGA-PEG2-RM26 and [ 57 Co]Co-X-RM26 at both time-points (all p-values < 0.0001). The uptake in liver tissue was also significantly higher for 64 Cu-labeled conjugates at both time-points in comparison to the 57 Co-labeled counterparts (ten-fold for [ 64 Cu]Cu-NOTA-PEG2-RM26 and four-fold for [ 64 Cu]Cu-NODAGA-PEG2-RM26, at 3 h p.i.) ( Cu]Cu-NODAGA-PEG2-RM26 (p < 0.0001). The same was observed for the 57 Co-labeled conjugates, where NOTA-PEG2-RM26 also showed significantly lower uptake (p = 0.01). At 24 h p.i. the kidney uptake was evened out and no difference was observed between the four different conjugates. For muscle and bone, no significant difference was observed between the four conjugates at both time-points (see Table 2 and Figure S1 in Supplementary Materials).  Biodistribution of [ 64 Cu]Cu-X-RM26 (X = NOTA-PEG 2 , NODAGA-PEG 2 ) was evaluated in mice bearing PC-3 xenografts at 3 and 24 h p.i. For comparison, mice were co-injected with the 57 Co-labeled counterparts. 57 Co was used as a convenient surrogate isotope for 55 Co in the biodistribution part, due to their chemical identity. It was previously demonstrated that 57 Co could be used for preclinical evaluation of radioagents that are designed to be used with 55 Co for PET [28]. [ 55 Co]Co-X-RM26 has previously shown a remarkable potential for PET imaging of GRPR expression [28].
[ 64 Cu]Cu-and [ 57 Co]Co-labeled X-RM26 displayed comparable biodistribution profiles with a fast clearance from blood and normal organs, including excretory organs ( Table 2). Tumor uptake at 3 h p.i. exceeded the uptake in normal organs for all conjugates. No significant difference was observed in tumor uptake between either the 64 Cu-and 57 Co-labeled conjugates or between the different chelators at both time points. Tracer uptake was also observed in GRPR expressing organs, excretory organs, and the gastrointestinal (GI) tract (Table 2). At 24 h p.i. the kidney uptake was evened out and no difference was observed between the four different conjugates. For muscle and bone, no significant difference was observed between the four conjugates at both time-points (see Table 2 and Figure S1 in Supplementary Materials).
The overall higher activity uptake in normal organs resulted in lower tumor-to-blood, tumor-to-liver, and tumor-to-lung ratios for [ 64 Cu]Cu-X-RM26 compared with the 57 Co-labeled counterparts at both time-points ( Figure 5 and Table S1). Tumor-to-blood ratios for [ 64 Cu]Cu-X-RM26 were three-fold lower compared with [ 57 Co]Co-X-RM26 at 3 h p.i., but without reaching statistical significance. At 24 h p.i., the tumor-to-organ ratios were generally higher for [ 57 Co]Co-X-RM26 than for [ 64 Cu]Cu-X-RM26, except for tumor-to-muscle ratios which were non-significantly higher for [ 64 Cu]Cu-X-RM26 compared with [ 57 Co]Co-X-RM26 at 3 h ( Figure 5A) and 24 h ( Figure 5B). The overall higher activity uptake in normal organs resulted in lower tumor-to-blood, tumor-toliver, and tumor-to-lung ratios for [ 64 Cu]Cu-X-RM26 compared with the 57 Co-labeled counterparts at both time-points ( Figure 5 and Table S1). Tumor-to-blood ratios for [ 64 Cu]Cu-X-RM26 were threefold lower compared with [ 57 Co]Co-X-RM26 at 3 h p.i., but without reaching statistical significance. At 24 h p.i., the tumor-to-organ ratios were generally higher for [ 57 Co]Co-X-RM26 than for [ 64 Cu]Cu-X-RM26, except for tumor-to-muscle ratios which were non-significantly higher for [ 64 Cu]Cu-X-RM26 compared with [ 57 Co]Co-X-RM26 at 3 h ( Figure 5A) and 24 h ( Figure 5B).

Imaging
PET/CT scans of PC-3 tumor-bearing mice injected with [ 64 Cu]Cu-X-RM26 or [ 55 Co]Co-X-RM26 (X = NOTA-PEG2, NODAGA-PEG2) are shown as coronal maximum intensity projection (MIP) images in Figure 6. The GRPR expression was successfully visualized in the scans obtained at 3 and 24 h p.i. and confirmed the respective findings from the biodistribution. A chelator-dependent difference was seen for both the 55 Co-and 64 Cu-labeled conjugates at 3 h p.i., where NODAGA containing conjugates showed increased uptake in the GI tract in comparison with the NOTA-labeled counterparts. Furthermore, NODAGA conjugates showed clear visualization of the gall bladder at 3 h p.i., presumably due to hepatobiliary excretion of the conjugates. At the later time-point of 24 h p.i.,

Discussion
Development of radiopharmaceuticals targeting GRPR in PC has gained increasing interest in the effort to improve the diagnostic accuracy by detection of metastases and to develop new treatment strategies [37][38][39]. Patients suffering from PC represent a very large group and even though the tumor is slowly growing in most cases, early and accurate diagnosis is essential for prognosis and the overall survival [3,4]. Radiolabeling of various GRPR antagonists has been investigated with several radionuclides and different chelators to optimize the pharmacokinetics for molecular imaging and targeted radionuclide therapy in PC [40]. The use of long-lived PET-isotopes has shown promising results due to the possibility of late time-point imaging with improved tumor-tobackground ratios and thus, image contrast [28].
It is well known, that the charge and geometry of the radiometal-chelator complex can have a major influence on the pharmacokinetic properties of labeled peptides [41][42][43]. Especially for 64 Culabeled peptides/proteins, the in vivo stability can be challenging as transchelation can occur resulting in unwanted high accumulation of radiocopper in the liver [44]. Several studies have

Discussion
Development of radiopharmaceuticals targeting GRPR in PC has gained increasing interest in the effort to improve the diagnostic accuracy by detection of metastases and to develop new treatment strategies [37][38][39]. Patients suffering from PC represent a very large group and even though the tumor is slowly growing in most cases, early and accurate diagnosis is essential for prognosis and the overall survival [3,4]. Radiolabeling of various GRPR antagonists has been investigated with several radionuclides and different chelators to optimize the pharmacokinetics for molecular imaging and targeted radionuclide therapy in PC [40]. The use of long-lived PET-isotopes has shown promising results due to the possibility of late time-point imaging with improved tumor-to-background ratios and thus, image contrast [28].
It is well known, that the charge and geometry of the radiometal-chelator complex can have a major influence on the pharmacokinetic properties of labeled peptides [41][42][43]. Especially for 64 Cu-labeled peptides/proteins, the in vivo stability can be challenging as transchelation can occur resulting in unwanted high accumulation of radiocopper in the liver [44]. Several studies have demonstrated an improved in vivo stability and performance of the NOTA chelator compared with DOTA for 64 Cu-labeling of peptides [33,45,46]. In this study, we evaluated the GRPR antagonist PEG 2 -RM26 conjugated to the macrocyclic chelators NOTA and NODAGA, labeled with [ 64 Cu]Cu and compared them with radiocobalt ( 55/57 Co) labeled counterparts (the latter was previously evaluated by our group with promising results [28]). We consider that the direct comparison of tracers in the same batch of tumor-bearing mice is a methodological advantage. The use of 57 Co instead of 55 Co allowed us to perform ex vivo biodistribution studies in dual isotope mode. This setup decreases the batch-to-batch variability of both murine physiology and target expression in cells used for tumor inoculation and should be a preferable way for preclinical comparative evaluation. Furthermore, the use of this dual-isotope approach further improves statistical power and is ethically advantageous because the number of living test subjects can be reduced, which is one of the "3R" principles of animal welfare. The risk of receptor saturation of GRPR targets should not be an issue since we used a low molar mass far from binding saturation [23,47].
The radiolabeling of NOTA/NODAGA-PEG 2 -RM26 with 64 Cu gave products with high yield and radiochemical purity without the need for further purification. The final 64 Cu-chelates were found to be relatively stable against transchelation with EDTA challenge. However, activity uptake in the liver was somewhat higher for [ 64 Cu]Cu-NOTA-PEG 2 -RM26 conjugate than for other tested conjugates, which probably indicates a high degree of transchelation in accordance with previous findings [44].
The  Figure 3). Similarly, increased affinity for copper-labeled NOTA conjugate compared to NODAGA was also observed in a study from Gourni et al., who evaluated the GRPR-targeting antagonist MJ9 [48]. The same pattern was observed for the cobalt-labeled counterparts where the previously published data also showed the best affinity in vivo for the NOTA conjugate [28]. However, the affinity of the 64 Cu-labeled NODAGA-conjugate was in the subnanomolar range and hence, still adequate for molecular imaging. In agreement with the in vitro data, [ 64 Cu]Cu-NOTA-PEG 2 -RM26 showed a tendency to higher uptake in the tumor (p = 0.7) and GRPR-expressing tissues (pancreas, small intestine, and stomach) at 3 h p.i. in comparison with [ 64 Cu]Cu-NODAGA-PEG 2 -RM26. However, the difference was not significant.
Comparison of biodistribution data for the 64 Cu-labeled conjugates showed fast clearance from the blood for both conjugates, though, significantly slower for [ 64 Cu]Cu-NOTA-PEG 2 -RM26. This phenomenon was associated with higher hepatic and lower renal activity uptake of the [ 64 Cu]Cu-NOTA-PEG 2 -conjugate. A similar ratio between hepatic and renal excretion was observed for anti-HER2 affibody molecules labeled with radiocopper via NOTA and NODAGA chelators [49].
Already at 3 h p.i. both 55 Co-and 64 Cu-labeled conjugates allowed clear visualization of GRPR-expressing tumors because tumor activity uptake exceeded normal tissue uptake ( Figure 6A). Tumor-to-organ ratios did not increase with time because of the rapid washout of tumor-associated activity ( Figure 5). However, intensive clearance of activity in the GI tract content improved overall imaging contrast ( Figure 6B). The in vivo data for 55/57 Co-labeled RM26-based conjugates obtained in this study contradicts the data published by our group earlier for this conjugate [27], where tumor-to-organ ratios increased with time. We speculate that this could be attributed to batch-to-batch xenograft variability. This observation underlines our approach to compare 64 Cu-and 57 Co-labeled conjugates in the same batch of animals.
[ 64 Cu]Cu-NOTA-PEG 2 -RM26 had overall the highest uptake in the liver, which was two-fold higher compared with 64 Cu-labeled NODAGA-PEG 2 -RM26 and ten-fold higher compared with the [ 57 Co]Co-NOTA-PEG 2 -RM26 at 3 h p.i. At the 24 h time-point, the activity accumulation in the liver was still significantly higher for [ 64 Cu]Cu-NOTA-PEG 2 -RM26 in comparison with other tested agents. Despite the observed elevated liver accumulation for [ 64 Cu]Cu-NOTA-PEG 2 -RM26, the tumor-to-liver ratio was equal in comparison to [ 64 Cu]Cu-NODAGA-PEG 2 -RM26 at 3 h p.i. due to the higher tumor uptake of the NOTA analog. The observed accumulation of activity in liver tissue for [ 64 Cu]Cu-NOTA-PEG 2 -RM26 is probably due to transchelation of the 64 Cu 2+ to serum components which circulate in the blood or to superoxide dismutase that can accumulate in liver tissue. Another process that could slow down blood clearance is off-target interactions of the probe with blood plasma proteins. This phenomenon was observed both for small molecular drugs and for proteins, and it was suggested that both lipophilic/hydrophilic and charged patches could influence these interactions [50,51]. However, the relatively high liver accumulation for [ 64 Cu]Cu-NOTA-PEG 2 -RM26 at 3 h p.i. was equal or lower to other reported studies of the 64 Cu-labeled GRPR antagonists with different chelators such as DOTHA 2 , NOTA, NODAGA, and MeCoSar evaluated in a similar animal model [46,48,52,53]. We could conclude that both [ 64 Cu]Cu-NOTA/NODAGA-PEG 2 -RM26 complexes were sufficiently stable in vivo, however, less stable than their 55/57 Co-labeled counterparts as displayed in Figures 5 and 6.
As expected, our results showed that both the 55/57 Co-and 64 Cu-labeled peptides were predominantly eliminated through kidney excretion. However, a chelator dependent difference was also observed. The presence of an additional negative charge resulted in significantly higher kidney accumulation for [ 64 Cu]Cu-NODAGA-PEG 2 -RM26 and [ 57 Co]Co-NODAGA-PEG 2 -RM26 at 3 h p.i. than the NOTA-containing counterparts. At the late time-point (24 h p.i.), the observed differences in kidney uptake were evened out. The elevated kidney uptake at 24 h p.i. for [ 64 Cu]Cu-NOTA-PEG 2 -RM26 could potentially be due to the slower elimination for the reasons discussed above. This slow elimination could also be a reason for the observed elevated uptake in blood at 24 h p.i. for [ 64 Cu]Cu-NOTA-PEG 2 -RM26 in comparison with [ 64 Cu]Cu-NODAGA-RM26 and both the 57 Co-labeled peptides. Overall slow elimination of activity should result in higher dose to the patient. The choice of radionuclide for next day PET imaging is challenging. Among available radiometals with adequate half-lives, 66 Ga (9.5 h), 64 Cu (12.7 h), 86 Y (14.7 h), and 55 Co (17.5 h), 64 Cu has the lowest emission of photons and the lowest energy of emitted positrons (653 keV) that should improve imaging quality. However, this isotope has the lowest positron-abundance (17.4%) among the mentioned radiometals; that would require an injection of a 2 to 4-fold higher amount of activity to get a similar signal in the PET acquisition. 55 Co has the highest abundance of positrons (76%). It also has lower energy of emitted positrons (1498 keV) and a better ratio between emitted positrons and co-emitted gammas than for 86 Y and 66 Ga (all data are from [54]). More human data for radiometals with long half-lives for both distribution and imaging sensitivity are required for accurate comparison of the mentioned PET nuclides. However, based on preclinical data, we recently estimated the effective total patient dose for a [ 55 Co]Co-DOTATATE scan to be 4.7 mSv, which was comparable to the effective dose for [ 64 Cu]Cu-DOTATATE of 6.5 mSv [44]. This includes correction for the more than 4-fold difference in positron yield between 55 Co and 64 Cu to obtain the same equivalent number of annihilation events in identical PET scanners.
The significantly lower uptake in blood and liver for the 57 Co-labeled conjugates could be the result of better in vivo stability of the cobalt-chelator complexes and/or lower degree of their off-target interactions in comparison to the 64 Cu-labeled counterparts. This and the high tumor uptake for the 57 Co-labeled radioconjugates, especially for NODAGA-PEG 2 -RM26, resulted in overall higher tumor-to-non-tumor ratios compared to the 64 Cu-labeled counterparts, leading to increased image contrast. The tumor-to-background ratios, particularly for blood, intestine walls, muscle, and bone, are very important in diagnostic imaging of PC since the high contrast between malignant and normal tissue increases the detection rate. Advanced PC often metastasizes to lymph nodes, and bone, where detection of small distant metastases is crucial to obtain an accurate staging of the disease.

Materials and Methods
[ 57 Co]Co-chloride was purchased from PerkinElmer (Upplands Vasby, Sweden). 55 Co and 64 Cu were produced in-house at Odense University Hospital as previously described [44,55]. In one instance, the 64 Cu was purchased at DTU NuTech, Technical University of Denmark. The GRPR antagonists NOTA-PEG 2 -RM26 and NODAGA-PEG 2 -RM26 were synthesized as described earlier [26]. Buffers for radiolabeling were produced in-house from chemicals supplied by Merck (Darmstadt, Germany) and were pretreated with Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) to remove metal contaminants. Other chemicals were purchased from Sigma-Aldrich Sweden (Upplands Vasby, Sweden). Radioactive samples were measured in an automated gamma-counter (2470 Wizard Automatic Gamma Counter, Perkin-Elmer).
PC-3 human PC cell line expressing GRPR was purchased from ATCC, LGC Promochem. Cells were cultured in RPMI-1640 media supplemented with 10% fetal calf serum (Sigma), PEST (penicillin 100 IU/mL, streptomycin 100 g/mL), and 2 mM l-glutamine (all from Biochrom AG, Berlin, Germany). This medium is referred to in the text as complete medium. Trypsin-EDTA (0.05% trypsin, 0.02% EDTA in buffer) was purchased from Biochrom AG.

In Vitro Studies
GRPR expressing PC-3 PC cells were used for in vitro studies. To evaluate in vitro binding specificity of [ 64 Cu]Cu-X-RM26 to GRPR, a group of cell dishes was incubated with an excess amount (300-fold) of non-labeled peptide for 10 min at RT to block GRPR receptors prior to the addition of the radioactive solution containing [ 64 Cu]Cu-X-RM26 (1 nM). After 1 h incubation at 37 • C, the cells were washed with serum-free media and detached using trypsin-EDTA solution. Measurements of cell-associated activity were done against standards in an automated gamma-counter. Cellular processing was evaluated by incubating PC-3 cells with 1 nM solution of [ 64 Cu]Cu-X-RM26 at 37 • C. At predetermined time points (1,2,4,8, and 24 h after the start of incubation), the membrane-bound and internalized activity were collected using the acid wash method [23]. To assess the cellular retention of activity, PC-3 cells were incubated for 1 h at 4 • C with 1 nM solution of [ 64 Cu]Cu-X-RM26. The radioactive media was replaced by fresh complete media, and the cells were incubated at 37 • C. At predetermined time-points (1, 2, 4, 8, and 24 h), membrane-bound and internalized activity fractions were collected using the previously described acid wash method. The samples were measured in an automated gamma-counter. The half-maximal inhibitory concentration (IC 50 ) was determined for nat Cu-X-RM26 using the universal BN radioligand 125 I-Tyr 4 -BBN (Perkin Elmer). PC-3 cells were incubated with 125 I-Tyr 4 -BBN (0.1 pmol/well) at 4 • C for 5 h in the presence of increasing concentrations of nat Cu-X-RM26 (0.5, 2, 5, 50, 200, and 600 nM). Following incubation, cells were collected and the cell-associated activity was measured in an automated gamma-counter.

In Vivo Studies
All animal experiments were planned and performed in accordance with the national legislation on the protection of laboratory animals, and the study plans were approved by the Animal Experiments Inspectorate in Denmark (approval number 2016-15-0201-01027).
For biodistribution studies, 16 female BALB/c nu/nu mice (age 15-16 weeks) bearing PC-3 xenografts (inoculated subcutaneously with 8 × 10 6 PC-3 cells three weeks before the experiment) were randomized into groups of four. The mice were intravenously injected into the tail vein with a mixture of [ 64 Cu]Cu-X-RM26 and [ 57 Co]Co-X-RM26 and were euthanized at 3 and 24 h p.i. Injected activity was adjusted to 300 kBq/mouse for 64 Cu-and 40 kBq/mouse for 57 Co-labeled conjugates. The total injected peptide mass was adjusted to 45 pmol/mouse (in 100 µL). Blood, kidney, pancreas, liver, lung, bone, muscle, tumor, spleen, stomach, small intestines, and the rest of the GI tract with content were collected, weighed, and their activity content was measured in a gamma-counter. The 64 Cu activities were determined from measurements performed on the day of the experiment, while the 57 Co activities were measured after two weeks to allow the 64 Cu to decay. Tissue uptake of the radiopeptides was calculated as a percent of injected dose per gram tissue (%ID/g), with exception of the GI tract for which tissue uptake was calculated as %ID per whole sample.

Imaging
Whole body PET/CT scans were performed using a Siemens Inveon preclinical scanner (Siemens Healthcare, Knoxville, USA) on PC-3 xenografted male NOD-scid mice (in-bread, age 12-13 weeks). The mice (n = 2/group) were anesthetized with a mixture of 1.5-2% isoflurane and 100% oxygen and injected via the tail vein with either 2.0-2.9 MBq (0.24-0.32 nmol) of [ 55 Co]Co-X-RM26 or 3.1-3.7 MBq (0.18-0.23 nmol) of [ 64 Cu]Cu-X-RM26, respectively. At 3 and 24 h p.i., the mice were anesthetized and PET/CT scanned with PET acquisition times of 15 and 30 min, respectively. All mice were awakened from anesthesia between longitudinal scans and allowed to roam freely in cages with unrestricted access to food and water. The CT scans were performed with 2 bed positions, 270 projections in 360 degrees' rotation, and with bin 4. CT and PET images were co-registered and the CT-based attenuation corrected PET data were reconstructed using an OSEM3D/MAP algorithm (4 OSEM3D iterations, 16 MAP subsets, and 18 MAP iterations, target resolution 1.5 mm). PET and CT data were analyzed using the Inveon Research Workplace (Siemens Healthcare) and presented as maximum intensity projections (MIPs) adjusted to display a color scale from 0 to the maximum tumor uptake value in the actual scan.

Statistics
Data were analyzed using GraphPad Prism (version 7.03, GraphPad Software Inc., San Diego, CA, USA) by explorative statistics using one-way ANOVA with Holm-Sidak correction for multiple comparisons to determine significant statistical differences (p < 0.05). All p-values given are adjusted for multiple comparisons. The IC 50 values were calculated by nonlinear regression using GraphPad Prism. In vitro data are presented as mean values including standard deviation (SD). In vivo data as mean including standard error of means (SEM).

Conclusions
In this study, we prepared the GRPR-targeting antagonists [ 64 Cu]Cu-NOTA-PEG 2 -RM26 and [ 64 Cu]Cu-NODAGA-PEG 2 -RM26 with high yields and high radiochemical purities. Both radiolabeled peptides showed high affinities for GRPR in vitro. Ex vivo biodistribution and PET/CT images showed high accumulation in GRPR-expressing PC-3 tumors for both conjugates resulting in favorable tumor-to-background ratios. [ 64 Cu]Cu-NOTA-PEG 2 -RM26 had an overall higher uptake in non-targeted tissues resulting in decreased tumor-to-background ratios. The head-to-head comparison of 64 Cu-and 55/57 Co-labeled conjugates showed comparable pharmacokinetic profiles, though [ 55/57 Co]Co-NOTA/NODAGA-PEG 2 -RM26 presented important advantages by significantly higher tumor-to-background ratios at the early time-points. Hence, the [ 55/57 Co]Co-NOTA-PEG 2 -RM26 showed the overall best in vivo characteristics. However, all tested radioconjugates were able to visualize GRPR-expression at early and later time-points with high image contrast. These results indicate that for PET imaging, [ 55 Co]Co-NOTA/NODAGA-PEG 2 -RM26 are preferred to the 64 Cu-labeled counterparts due to the increased in vivo stability and overall higher tumor-to-non-tumor ratios, thus providing a suitable candidate for clinical translation.  Table S1: Tumor-to-organ ratios for biodistribution data.