Development of an optimal imaging strategy for selection of patients for affibody-based PNA-mediated radionuclide therapy

Affibody molecules are engineered scaffold proteins, which demonstrated excellent binding to selected tumor-associated molecular abnormalities in vivo and highly sensitive and specific radionuclide imaging of Her2-expressing tumors in clinics. Recently, we have shown that peptide nucleic acid (PNA)-mediated affibody-based pretargeted radionuclide therapy using beta-emitting radionuclide 177Lu extended significantly survival of mice bearing human Her2-expressing tumor xenografts. In this study, we evaluated two approaches to use positron emission tomography (PET) for stratification of patients for affibody-based pretargeting therapy. The primary targeting probe ZHER2:342-SR-HP1 and the secondary probe HP2 (both conjugated with DOTA chelator) were labeled with the positron-emitting radionuclide 68Ga. Biodistribution of both probes was measured in BALB/C nu/nu mice bearing either SKOV-3 xenografts with high Her2 expression or DU-145 xenografts with low Her2 expression. 68Ga-HP2 was evaluated in the pretargeting setting. Tumor uptake of both probes was compared with the uptake of pretargeted 177Lu-HP2. The uptake of both 68Ga-ZHER2:342-SR-HP1 and 68Ga-HP2 depended on Her2-expression level providing clear discrimination of between tumors with high and low Her2 expression. Tumor uptake of 68Ga-HP2 correlated better with the uptake of 177Lu-HP2 than the uptake of 68Ga-ZHER2:342-SR-HP1. The use of 68Ga-HP2 as a theranostics counterpart would be preferable approach for clinical translation.

The secondary targeting agent HP2 bearing a DOTA chelator was labeled with 68 Ga. Labeling was performed in 1.25 M sodium acetate buffer (pH 3.6) at 95 °C for 15 min with the radiochemical yield of 94 ± 1%. After incubation with 1200-fold molar excess of EDTA a radiochemical yield of 90 ± 1% was obtained. Purification using NAP-5 size-exclusion column provided the compound with 98.6 ± 0.3% radiochemical purity and 53 ± 5% isolated yield. Maximum specific activity of 2.9 MBq/μg (15.0 GBq/µmol, at the end of purification) was obtained.

Binding and processing by Her2-expressing cells in vitro.
Her2-binding specificity of the primary agent 68 Ga-Z HER2:342 -SR-HP1 was tested using a saturation assay in Her2-expressing SKOV3, BT474 and DU145 cells. The binding was significantly (p < 0.000001 for all cell lines) decreased when the cells were pre-incubated with the parental anti-Her2 affibody molecule (Fig. 2), demonstrating that the binding was specific and Her2-mediated. The level of cell-associated radioactivity in non-blocked SKOV3 and BT474 cells was higher than in DU145 cells, which is consistent with their Her2 expression level.
The specificity of 68 Ga-HP2 in vitro binding to Z HER2:342 -SR-HP1-pretreated Her2-expressing cells was confirmed in SKOV3, BT474 and DU145 cells (Fig. 2). The binding of 68 Ga-HP2 was significantly (p < 0.00001 for all cell lines) decreased when Her2 receptors were saturated with parental anti-Her2 affibody molecule or when Z HER2:342 -SR-HP1-treated cells were pre-incubated with a large excess of non-labeled HP2. Additionally, the binding of 68 Ga-HP2 to cells without pre-incubation with the primary agent Z HER2:342 -SR-HP1 was significantly (p < 0.00001 for all cell lines) decreased. This assay demonstrated that the pretargeting of the secondary agent was Her2-specific, PNA-mediated and depended on the pre-treatment with the primary agent.
Cellular processing of 68 Ga-Z HER2:342 -SR-HP1 by SKOV3 and DU145 cells after interrupted incubation is shown in Fig. 3. The retention of the radiolabeled primary agent on the cell membrane was higher in high-expressing SKOV3 cells than DU145 cells with low Her2 expression (77 ± 1% for SKOV3 vs. 31 ± 2% for DU145 at 3 h time point). The internalization of 68 Ga-Z HER2:342 -SR-HP1 was low in both cell lines. The internalized fraction in SKOV3 cells at 4 h was 11 ± 1% and in DU145 at 3 h was 4 ± 1%, which is in agreement with the previously reported values for the 111 In-labeled analogue 14 (in SKOV3 cells at 4 h it was 8.0 ± 0.3%).
Cellular processing of the Z HER2:342 -SR-HP1: 68 Ga-HP2 complex had a similar pattern as the processing of the primary agent alone (Fig. 3). Initial release from the membrane after 1 h was followed by a plateau resulting in high retention of radioactivity by SKOV3 cells (ca. 80% of initially bound activity at 4 h). However, in DU145 cells with low Her2 expression the complex was rapidly released from the membrane with only 10% of total cell-associated activity at 1 h.
The binding strength of nat Ga-Z HER2:342 -SR-HP1 to Her2-expressing SKOV3 cells was compared to the binding strength of nat In-Z HER2:342 -SR-HP1 in a competitive binding assay using 111 In-DOTA-Z HER2:2395 21 as the displacement radioligand. The IC 50 values for nat Ga-Z HER2:342 -SR-HP1 and nat In-Z HER2:342 -SR-HP1 were determined to be 24 ± 2 nM and 22 ± 1 nM, respectively (SI Fig. 1). No difference between the IC 50 values was observed, which suggested that the affinity of gallium-labeled Z HER2:342 -SR-HP1 is similar to the affinity of the indium-labeled conjugate.
Animal studies. Initial assessment of 68 Ga-HP2 biodistribution was performed in healthy NMRI mice. The biodistribution of 68 Ga-HP2 and 177 Lu-HP2 in NMRI mice at 1 and 2 h p.i. (SI Table 1) showed rapid and predominantly renal clearance of probes. The data for the 177 Lu-HP2 biodistribution was in a good agreement with the previously reported values 12 . As it was earlier observed for 111 In-HP2 and 177 Lu-HP2, the radionuclide had a In vitro binding specificity of the secondary agent 68 Ga-HP2 pretargeting to Her2-expressing SKOV3, BT474 and DU145 cells. In the control groups, Her2 was blocked by adding 500-fold molar excess of non-labeled anti-Her2 Z HER2:342 affibody molecule, HP1 was blocked by adding 150-fold excess of non-labeled HP2. In the third control, no Z HER2:342 -SR-HP1 was added. The data are presented as an average value from 3 samples ± SD.
To compare two approaches, direct targeting and pretargeting, the biodistribution of 68 Ga-labeled primary and secondary agents was evaluated in BALB/C nu/nu mice bearing SKOV3 (high Her2 expression) and DU145 (low Her2 expression) xenografts (SI Fig. 3, Table 1). In both approaches, a high dose of the primary agent Z HER2:342 -SR-HP1 (100 µg) was administered to saturate Her2 receptors and provide maximum discrimination between high and low Her2-expressing tumors 22 . The pretargeting protocol used in this study has been previously optimized for the affibody-based PNA-mediated therapy using 177 Lu-HP2 19 . It was found that 16 h between the injections of primary and secondary agents and 3.5 µg of the secondary agent provided the highest tumor accumulation, low kidney uptake and optimal dose delivered to the tumor.
In the direct targeting approach the biodistribution of 68 Ga-labeled primary probe, 68 Ga-Z HER2:342 -SR-HP1, at 1 h p.i. was typical of affibody molecules, i.e. rapid clearance from blood and normal tissues and a high level of renal reabsorption. Tumor-associated radioactivity in the SKOV3 group was about three times higher compared to the DU145 group (4.3 ± 0.9 vs. 1.3 ± 0.3% ID/g, p = 0.0006).
In the pretargeting approach, injection of the unlabeled primary agent 16 h before the administration of 68 Ga-HP2 resulted in approximately twelve-fold higher tumor uptake in the SKOV3 group compared to the DU145 group (6.3 ± 1.5 vs. 0.5 ± 0.2% ID/g) at 1 h p.i. Pretargeting provided significantly (p < 0.0001, one-way ANOVA test) higher tumor-to-organ ratios, i.e. six-fold higher tumor-to-blood, four-fold higher tumor-to-liver and fifty four-fold higher tumor-to-kidney ratios, compared to direct targeting in mice bearing SKOV3 xenografts.
To further evaluate if pretargeted imaging with 68 Ga-HP2 could be used for the prediction of tumor uptake of therapeutic 177 Lu-HP2, the biodistribution of 177 Lu-HP2 was studied alongside with 68 Ga-HP2 in the same mice (Table 1, SI Fig. 2). In SKOV3-bearing mice pretargeted with Z HER2:342 -SR-HP1, tumor uptake of 177 Lu-HP2 was about twice higher than 68 Ga-HP2 (12 ± 3 vs. 6.3 ± 1.5% ID/g, p = 0.00006, one-way ANOVA test), while in DU145 tumors with low Her2 expression no difference in uptake was observed. No statistically significant differences (p > 0.05, one-way ANOVA test) between uptake of 177 Lu-HP2 and 68 Ga-HP2 was observed in healthy organs and tissues in SKOV3 group.
Imaging. PET/CT imaging confirmed the results of the biodistribution studies. Both direct targeting using 68 Ga-Z HER2:342 -SR-HP1 and pretargeting using Z HER2:342 -SR-HP1 and 68 Ga-HP2 were able to visualize Her2-expressing xenografts in mice (Fig. 4). In agreement with the ex vivo data, the uptake of radioactivity in SKOV3 xenografts was higher than in DU145 xenografts. In the direct targeting approach high accumulation of Tumor-to-organ ratio  68 Ga-HP2 and 177 Lu-HP2 (3.5 µg total) injections. The uptake is expressed as % ID/g and presented as an average value from 4 mice ± SD (5 mice ± SD for the pretargeting groups). Data for GI tract with content and carcass are presented as % of injected dose per whole sample. One-way ANOVA with Bonferroni's multiple comparisons test was performed to find significant differences. a Significant difference between 68 Ga-Z HER2:342 -SR-HP1 and 68 Ga-HP2 uptake in SKOV3 group. b Significant difference in 68 Ga-HP2 uptake between SKOV3 and DU145 groups. c Significant difference between 68 Ga-HP2 and 177 Lu-HP2 uptake in SKOV3 group. d Significant difference in 177 Lu-HP2 uptake between SKOV3 and DU145 groups. radioactivity was observed in kidneys, while the pretargeting provided decreased kidney uptake and increased tumor uptake of radioactivity. No noticeable uptake in other organs was detected.

Discussion
Clinical PET/CT imaging using 68 Ga-labeled ABY-025 demonstrated that this affibody molecule provides non-invasive whole-body quantification of Her2 expression in patients with metastatic breast cancer 17 . We have recently demonstrated that affibody-mediated pretargeted radionuclide therapy using 177 Lu successfully delayed tumor growth and doubled median survival of mice bearing SKOV3 xenografts. Importantly, no acute toxicity or kidney damage was observed 19 . Promising results of pretargeted radionuclide therapy and clinical applicability of affibody molecules for imaging prompted us to develop a theranostic methodology for the affibody-based pretargeting treatment, where PET/CT imaging would guide the selection of patients for therapy. The most straightforward way would be to base the decision concerning therapy on the results of quantitative imaging of Her2-expression using 68 Ga-ABY-025. The assumption would be that the patients with high expression would have high tumor accumulation of the therapeutic probe. However, this approach does not take into account the differences in structures of ABY-025 and Z HER2:342 -SR-HP1-DOTA leading to different capacity in penetrating the vasculature, potential differences in their accumulation in tumors and the differences in probe dosing between imaging and therapy. Another strategy would be the pretargeted theranostics using the positron-emitting radionuclide gallium-68. Such a strategy could be realized in two ways depending on the placement of the label. When 68 Ga is placed on Z HER2:342 -SR-HP1, PET/CT imaging would provide assessment of tumor accumulation of the primary agent and identification of patients with sufficiently high accumulation. Alternatively, the 68 Ga label could be placed on the secondary agent. In that case, unlabeled primary agent is injected the day before the diagnostic secondary agent 68 Ga-HP2. Patients with high tumor uptake of radioactivity would then proceed to pretargeted treatment. The first approach is simpler logistically, however, the second might better reflect the whole pretargeted delivery of a radionuclide to tumors.
In this study, we performed a side-by-side comparison of direct targeting using radiolabeled primary agent 68 Ga-Z HER2:342 -SR-HP1 and pretargeting using radiolabeled secondary agent 68 Ga-HP2 for imaging of Her2 expression. To address a clinically relevant problem of discrimination between tumors with high and low Her2 expression we compared these two approaches in mice bearing SKOV3 (high Her2) and DU145 (low Her2) tumor xenografts.
The primary Z HER2:342 -SR-HP1 and secondary HP2 probes carrying a DOTA chelator were labeled with 68 Ga with good yields. Minor release of 68 Ga under pre-purification EDTA challenge from both primary (ca. 10%) and secondary (ca. 5%) probes could be due to the presence of weak chelating sites that compete with DOTA for 68 Ga. Despite this initial release of 68 Ga, neither of the conjugates 68 Ga-Z HER2:342 -SR-HP1 and 68 Ga-HP2 showed any signs of label loss in vivo (e. g. elevated bone uptake).
Gallium-labeled primary probe 68 Ga-Z HER2:342 -SR-HP1 retained binding specificity to Her2-expressing cells (Fig. 2a). Pretargeting specificity of the secondary probe 68 Ga-HP2 was also confirmed (Fig. 2b). Ga-Z HER2:342 -SR-HP1 had the same half maximal inhibitory concentration (IC 50 ) (Supplementary Information (SI) Fig. 1) as In-Z HER2:342 -SR-HP1 used in earlier studies 14 . An important factor for the success of pretargeting is the availability of the primary probe on the cell surface for the reaction with the secondary probe. The retention of the primary probe 68 Ga-Z HER2:342 -SR-HP1 on the surface of SKOV3 cells was high, which is in accordance with the previously reported data for 111 In-Z HER2:342 -SR-HP1 14 and is typical for affibody molecules.
Labeling chemistry and the choice of radionuclide might have a profound effect on the biodistribution of peptides and small targeting molecules 12,[23][24][25][26] . In our previous studies the primary agent Z HER2:342 -SR-HP1-DOTA was labeled with 111 In 14 .
Although both indium and gallium are trivalent metals, they have a different ionic radius and their complexes with DOTA have different coordination geometry. The complex of indium with the amide derivative of DOTA has a square-antiprismatic geometry with the amide oxygen involved in chelation, where indium is octacoordinated. The gallium-DOTA complex has a pseudo-octahedral geometry and gallium is hexacoordinated 27 . This leaves a free carboxyl group noninvolved in coordination and a different charge distribution compared to the complex with indium. Despite these differences, the uptake of the gallium-labeled primary agent in SKOV-3 xenografts (4.3 ± 0.9% ID/g) was similar to the indium-labeled analogue 14 (5.9 ± 2.4% ID/g).
The secondary pretargeting agent HP2-DOTA was previously radiolabeled with 111 In and 177 Lu 12 . It was found that the radionuclide had a substantial effect on the biodistribution of this molecule. 177 Lu-HP2 had a faster clearance from blood and normal organs, except kidneys, compared to 111 In-HP2 already at 1 h p.i.
In this study, 68 Ga-HP2 was excreted from blood slower than 177 Lu-HP2 (Table 1 and SI Table 1) but faster than 111 In-HP2 at 1 h p.i. 12 . In general, the uptake of 68 Ga-HP2 in normal organs and tissues was higher than of 177 Lu-HP2 at both 1 and 2 h p.i. (SI Table 1). High kidney uptake of 68 Ga-HP2 (9 ± 2 vs. 4.6 ± 0.5% ID/g for 177 Lu-HP2 at 1 h, p = 0.008) also suggested higher reabsorption rate of this molecule in kidneys compared to 177 Lu-HP2. As discussed above, the difference in biodistribution could be attributed to the differences in ionic radii of the radionuclides and geometries of metal-DOTA complex. Lutetium has a larger ionic radius than gallium and the complex of Lu 3+ and DOTA derivatives predominantly exists in a square antiprism geometry with different bond length compared to the indium-DOTA complex 21 . This phenomenon was also observed in preclinical studies of other theranostic pairs, e.g. 68 Ga/ 177 Lu-PSMA 28 and 67 Ga/ 111 In/ 177 Lu-NeoBOMB1 29 in direct targeting or 68 Ga/ 111 In-IMP288 in different pretargeting systems 30,31 . Due to the differences in biodistribution in this mouse model, PET imaging with 68 Ga-HP2 would not precisely predict the uptake of the therapeutic 177 Lu-HP2, however, it should be further evaluated if these differences would translate to humans.
In this study, both direct use of 68 Ga-Z HER2:342 -SR-HP1 and pretargeted imaging using 68 Ga-HP2 allowed discrimination between tumors with high and low Her2 expression (Table 1, Fig. 4). Direct targeting provided 3.3-fold difference between uptake in SKOV-3 and DU-145 xenografts, while pretargeting provided 11.8-fold difference. In clinical studies using 68 Ga-labeled anti-Her2 affibody molecule, Standard Uptake Values (SUV) in lesions with confirmed Her2-status were approximately five times higher in Her2-positive metastases than in Her2-negative 17 . Notably, the clinical study indicated that among the metastases defined as Her2-positive by biopsies and immunohistochemistry the SUVs ranged from 6 to more than 40 at 2 h after tracer injection. In clinical practice, a single whole-body scan performed early after tracer injection would then be able to characterize each individual metastasis on a continuous scale regarding Her2 protein expression. Access to this directly obtainable information on clonal heterogeneity and tumor burden might lead to more optimal and earlier treatment decisions. The dramatically improved tumor-to kidney uptake ratio obtained with pretargeting in this study is promising. In the clinical study using direct imaging tumor-to-kidney ratios ranging from 1/10 to 1/1 were recorded, which appears to be much lower than in mice, suggesting that pretargeting could reduce kidney uptake even further in humans and indicates that kidney-sparing treatment using endogenous radiation with a theranostic approach might be one of the available therapeutic options.
Overall, imaging using 68 Ga-HP2 provided better discrimination between tumors with high and low Her2 expression and better reflected the tumor uptake of the therapeutic counterpart, 177 Lu-HP2. This method should be considered as the most promising for clinical translation.

Methods
Buffers used for labeling were prepared from high-quality Milli-Q water and purified from metal contamination using Chelex 100 resin (Bio-Rad Laboratories, USA). 111 InCl 3 was purchased from Mallinckrodt Sweden AB (Stockholm, Sweden). Carrier-free 177 LuCl 3 was purchased from PerkinElmer (Waltham, MA, USA). Radioactivity was measured using an automated gamma-spectrometer with a NaI(TI) detector (1480 Wizard, Wallac, Finland). Her2-expressing SKOV3, BT474 and DU145 cells were purchased from the American Type Culture Collection (ATCC) and were cultured in complete RPMI-medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin in a humidified incubator with 5% CO 2 at 37 °C, unless stated otherwise.
Production and purification of PNA-based probes have been described previously in Westerlund et al. 13 and Altai et al. 12 . Briefly, the PNA-based probes HP1 and HP2 were synthesized manually using solid phase synthesis with commercially available building blocks. The primary agent, Z HER2:342 -SR-HP1, was produced by site-specifically attaching HP1 to the anti-HER2 affibody using a sortase A mediated ligation strategy. Z HER2:342 -SR-HP1 and the secondary agent HP2 were both purified using reversed phase HPLC to a final purity of ≥95% and kept lyophilized at −20 °C until use.
Labeling. 68 Ge/ 68 Ga generator (Cyclotron Co., Obninsk, Russia) was eluted with 0.1 M HCl (prepared from 30% ultrapure HCl from Merck). The generator was eluted with 400 μL fractions of 0.1 M HCl. Fraction 3 containing the maximum radioactivity (ca. 60% of total) was used for labeling.
The labeling yield and purity were measured by radio-ITLC eluted with 0.2 M citric acid.
In vitro studies. Her2-expressing cell lines used for in vitro studies were SKOV3 (1.6 × 10 6 receptors/cell) 33 , BT474 (1.2 × 10 6 receptors/cell) 34 and DU145 (5 × 10 4 receptors/cell) 35 . Cells were seeded in 3 cm petri dishes (ca. 10 6 cells/dish), a set of three dishes was used for each data set. In Her2 binding specificity assay, two sets of dishes were used. A 500-fold excess of non-labeled anti-Her2 Z HER2:342 Affibody molecule (1000 nM) was added to the control group of cell dishes to saturate Her2 receptors 5 min before adding the labeled compound. Then 68 Ga-Z HER2:342 -SR-HP1 (2 nM) was added to both groups of dishes. The cells were incubated for 1 h in a humidified incubator at 37 °C. Then the medium was collected, the cells were washed and detached by trypsin, the radioactivity in medium and cells was measured to calculate the percent of cell-bound radioactivity.
Pretargeting specificity assay was performed using four sets of cell dishes. To demonstrate the pretargeting, one set of cells was incubated with Z HER2:342 -SR-HP1 (1 nM) for 1 h at 37 °C and washed. Radiolabeled 68 Ga-HP2 SCIENTIfIC RepORtS | (2018) 8:9643 | DOI:10.1038/s41598-018-27886-0 (10 nM) was added and cells were incubated for 1 h at 37 °C. To show that the pretargeting was Her2-mediated, the second set of cell dishes was incubated with a 500-fold excess of Z HER2:342 (1000 nM) for 5 min before the addition of Z HER2:342 -SR-HP1. Radiolabeled 68 Ga-HP2 (10 nM) was added and cells were incubated for 1 h at 37 °C. To demonstrate that pretargeting was PNA-mediated, the third set of cell dishes was incubated with Z HER2:342 -SR-HP1 followed by incubation with a 150-fold excess of non-labeled HP2 (150 nM) for 30 min and then the radiolabeled 68 Ga-HP2 (10 nM) was added followed by 1 h incubation. In the fourth set the cells were incubated only with 68 Ga-HP2 (10 nM) to assess non-specific binding. After incubation with 68 Ga-HP2 the medium was collected, the cells were washed and detached by trypsin to calculate the percent of cell-bound radioactivity.
Cellular retention and processing of 68 Ga-Z HER2:342 -SR-HP1 by SKOV3 and DU145 cells was studied during interrupted incubation by an acid-wash method 36 . Cells were incubated with 68 Ga-Z HER2:342 -SR-HP1 (1 nM for SKOV3, 0.25 nM for DU145 cells) for 1 h at 4 °C. Then the medium was removed, the cells were washed, new medium was added and the cells were placed in a humidified incubator at 37 °C. At 0.5, 1, 2, 3 and 4 h the medium was collected, cells were washed and treated with 0.2 M glycine buffer containing 4 M urea, pH 2.0, for 5 min on ice. The acidic solution was collected and cells were additionally washed with glycine buffer. The cells were then incubated with 0.5 mL of 1 M NaOH at 37 °C for 10 min and collected with 1 mL of 1 M NaOH. The radioactivity in acidic fractions was considered as membrane-bound, and in the alkaline fractions as internalized.
Cellular retention and processing of Z HER2:342 -SR-HP1: 68 Ga-HP2 adduct by SKOV3 and DU145 cells was studied analogously. The cells were incubated with Z HER2:342 -SR-HP1 (1 nM) for 1 h at 4 °C, then the medium was removed, the cells were washed, 68 Ga-HP2 (10 nM) was added, and the cells were incubated for 30 min at 4 °C. Then the medium was removed, the cells were washed, new medium was added and the cells were placed in a humidified incubator at 37 °C. At 0.5, 1, 2, 3 and 4 h a group of three dishes was removed from the incubator and treated as described above.
To evaluate the relative binding strength of nat Ga-Z HER2:342 -SR-HP1 and nat In-Z HER2:342 -SR-HP1, the half maximal inhibitory concentration (IC 50 ) was measured using 111 In-DOTA-Z HER2:2395 in SKOV3 cells. The cells were incubated with nat Ga-or nat In-Z HER2:342 -SR-HP1 (0-200 nM) in the presence of 1 nM 111 In-DOTA-Z HER2:2395 for 4 h at 4 °C. After incubation the medium was collected, the cells were washed and collected using trypsin to calculate cell-associated activity. The IC 50 values were determined using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).
Animal studies. Animal studies were planned in agreement with Swedish national legislation concerning protection of laboratory animals and were approved by the Ethics Committee for Animal Research in Uppsala (Permit C 4/2016). All experiments were performed in accordance with the guidelines of the European Community Council Directives 86/609/EEC.
For comparative biodistribution of 68 Ga-HP2 and 177 Lu-HP2 a dual-isotope approach was used. Eight female NMRI mice (24 ± 1 g) were intravenously (i.v.) injected with a mixture of 68 Ga-HP2 and 177 Lu-HP2 (1 μg in 100 μL of 2% BSA in PBS/mouse, 70 kBq for 68 Ga-HP2, 45 kBq for 177 Lu-HP2). At 1 and 2 h mice were anesthesized by an intraperitoneal injection of Ketalar and Rompun solution, followed by euthanasia through cervical dislocation. Blood and organs were collected, weighed and the total radioactivity corresponding to the sum of 68 Ga and 177 Lu signals was measured using an open protocol. One day after the first radioactivity measurement (when 68 Ga has decayed), the 177 Lu radioactivity was measured in all samples using the same protocol. Subtraction of the 177 Lu decay-corrected radioactivity from the total signal measured on the first day was considered as 68 Ga radioactivity. The percent of injected dose per gram of sample (%ID/g) was calculated. Statistical analysis (two-tailed paired t test) was performed using Microsoft Excel 2016 (Microsoft, Redmond, WA, USA).
For tumor implantation, 10 7 SKOV3 cells or 5 × 10 6 DU145 cells were subcutaneously injected on the left hind leg of female BALB/c nu/nu mice. The biodistribution experiments were performed two weeks after cell implantation. The average animal weight was 18 ± 1 g in SKOV3 group, 19 ± 1 g in DU145 group. The average tumor weight was 0.08 ± 0.03 g for SKOV3 xenografts, 0.06 ± 0.03 g for DU145 xenografts.
PET imaging was performed using Triumph ™ Trimodality system (Gamma Medica). The CT scan was performed at the following parameters: field of view (FOV), 8 cm; magnification, 1.48; one frame and 512 projections for 2.13 min. PET data were acquired in list mode during 30 min and reconstructed using OSEM-3D. CT raw files were reconstructed by filter back projection using Nucline 2.03 Software (Mediso Medical Imaging Systems, Hungary). PET and CT dicom files were analyzed using PMOD v 3.12 software (PMOD Technologies, Switzerland).