Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-111

Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N′,N′′-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DOTAGA (1,4,7,10-tetraazacyclododececane,1-(glutaric acid)−4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z08698 and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged 111In-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

SCIEnTIfIC RepoRts | (2019) 9:655 | DOI: 10.1038/s41598-018-36827-w N-terminus of Z 08698 to investigate the pure influence of the C-terminal charge. In vitro and in vivo properties of all radio-conjugates were investigated in order to select the conjugate providing the best imaging properties for SPECT-imaging of HER3 expression in vivo.

Results
Production, conjugation, purification and characterization. The HER3-binding affibody Z 08698 was recombinantly produced in E. coli, recovered with cation exchange chromatography, coupled to maleimide derivatives of NOTA, NODAGA, DOTA and DOTAGA and subjected to reverse-phase high performance liquid chromatography (RP-HPLC) as a final step for remnant chelator removal and separation from unconjugated protein.
The purity, as determined by RP-HPLC, exceeded 98% for all four conjugates ( Supplementary Fig. S1). Molecular mass determination with electrospray ionization mass spectrometry (ESI-MS) confirmed the identity of the conjugated proteins and the observed masses were in good agreement with the theoretical (Table 1 and Supplementary Figure S2). Notably, the proteins conjugated to NOTA and NODAGA exhibited additional peaks which is likely the result of chelated metal contaminants.
The alpha-helical content, thermal stability and refolding of the conjugates were investigated with circular dichroism spectroscopy. The thermal denaturation curves for the constructs are shown in Supplementary  Fig. S3A and the associated melting temperatures are presented in Table 1. Following thermal denaturation, complete refolding of all four conjugates was evident from comparison of spectra obtained at 20 °C before and after denaturation (Supplementary Figure S3B). Kinetic data acquired from surface plasmon resonance (SPR) analysis are presented in Table 1. K D values refer to the monovalent affinity for human HER3 according to a Langmuir 1:1 model. Representative sensorgrams with fitted curves for each conjugate are shown in Supplementary Fig. S4.
Labeling and Stability. Results for radiolabeling of Z 08698 -X with indium-111 are shown in Table 2. For radiolabeling, the conjugates were dissolved in ammonium acetate (0.2 M, pH 5.5) and incubated with 20 MBq indium-chloride for 40 minutes at 85 °C. 111 In-Z 08698 -NODAGA and 111 In-Z 08698 -DOTAGA were successfully labeled with almost quantitative yields. Radiochemical yields for 111 In-Z 08698 -NOTA and 111 In-Z 08698 -DOTA were lower. Purification with NAP5 size-exclusion columns resulted in high radiochemical purity. Despite differences in labeling efficiency, all conjugates demonstrated high metal/chelator complex stability when challenged in human serum at physiological temperature (Table 2).
In vitro studies. In vitro studies were performed using BxPC-3 (pancreatic cancer) and DU145 (prostate cancer) cells. To demonstrate binding specificity, HER3 receptors were pre-saturated with 500-fold molar excess of a non-labeled HER3-tageting affibody molecule before incubation with the radiolabeled conjugates. Pre-saturation resulted in significantly decreased uptake of the radiolabeled conjugates. Hence, all tested conjugates retained receptor-specific binding after labeling as shown in Fig. 2. Overall, the uptake of all conjugates was lower for DU145 cells compared to BxPC-3 cells.
Data regarding cellular processing are presented in Fig. 3. Cells were continuously incubated with 0.1 nM of the radiolabeled conjugates and, cell-membrane bound and internalized activity was analyzed at pre-determined timepoints. Overall, the association of all radiolabeled conjugates to the cells was rapid but the internalization rate was low. The internalized fraction in BxPC-3 cells was higher for 111 In-Z 08698 -NOTA and 111 In-Z 08698 -DOTA compared to 111 In-Z 08698 -NODAGA and 111 In-Z 08698 -DOTAGA. After 24 h continuous incubation, the internalized portion of the activity was over 30% of cell-associated activity for the NOTA-and DOTA-conjugates and around 10% for the NODAGA-and DOTAGA-conjugates for BxPC-3 cells. In DU145 cells, internalization was around 20% of cell-associated activity at 24 h without any significant difference between the conjugates.  Real-time kinetics of binding to HER3 expressing BxPC-3 cells. Association rates (k a ), dissociation rates (k d ) and equilibrium dissociation constants (K D ) were analyzed on living cells in real-time using LigandTracer yellow (Ridgeview Instruments AB). Results are shown in Table 3. For all conjugates, K D -values were in the picomolar range. 111 In-Z 08698 -DOTAGA showed the highest affinity and 111 In-Z 08698 -NOTA showed the lowest affinity in this assay.   Tables 4 and 5. The general biodistribution pattern was characteristic for affibody molecules (fast blood clearance and renal excretion) and for anti-HER3-affibody molecules in particular (elevated uptake in lung, liver, stomach, salivary glands and intestines). Tumor uptake 4 h pi was in the range of 3-4%ID/g and no significant difference in tumor uptake was observed between the tested conjugates. Retention of activity in tumors over time was high and no significant changes of activity uptake were observed between 4 and 24 h pi for any conjugate. Activity concentration in blood was highest for 111 In-Z 08698 -NOTA and it was significantly higher than the concentration of the other conjugates at both time points.
All conjugates demonstrated elevated initial uptake in organs with endogenous expression of mErbB3 (salivary gland, lung, stomach, intestines and especially liver). Hepatic uptake of 111 In-Z 08698 -DOTA and 111 In-Z 08698 -DOTAGA was significantly lower than for 111 In-Z 08698 -NOTA. The liver uptake of 111 In-Z 08698 -DOTAGA was the lowest at both studied time points. The difference was most pronounced at 4 h pi where uptake of the DOTAGA-conjugate was nearly two-fold lower than for the NOTA-conjugate, which demonstrated the highest hepatic uptake. 111 In-Z 08698 -NOTA had also the highest uptake in lungs, stomach, spleen, muscle and bone at 4 h pi. Significant reduction of activity accumulation was observed in liver, lung and blood for all conjugates over time. Still,111 In-Z 08698 -NOTA demonstrated significantly higher uptake in liver, blood and lung than all other conjugates at 24 h.
The decrease of activity uptake in normal tissue together with good retention in tumors manifested in an increase of tumor-to-organ ratios between 4 and 24 h pi for the majority of the studied tissues, except spleen. Tumor-to-organ ratios did not differ significantly between 111 In-Z 08698 -NODAGA, 111 In-Z 08698 -DOTA and 111 In-Z 08698 -DOTAGA. However, at 24 h pi 111 In-Z 08698 -NOTA presented almost two-fold lower tumor-to-blood-ratio than the other conjugates. 111 In-Z 08698 -DOTAGA was the only conjugate providing significantly better tumor-to-organ ratios than 111 In-Z 08698 -NOTA for liver, spleen and muscle at both time points.
SPECT-Imaging. SPECT-CT imaging of 111 In-Z 08698 -X confirmed the results of the biodistribution and images are displayed in Fig. 4. HER3-expressing BxPC-3 xenografts could be clearly visualized. Uptake related to natural expression of mErbB3 could be observed in salivary glands, GI-tract and liver. 111 In-Z 08698 -NOTA had the highest activity accumulation in liver at both timepoints.

Discussion
The development of therapeutic agents against HER3 urges simultaneous development of reliable methods for patient stratification. Radionuclide-based molecular imaging provides a non-invasive and repeatable method to identify patients who might benefit from targeted therapy 17,18,30 . Radiolabeled monoclonal antibodies against HER3 have recently demonstrated the capacity to image overexpression and evaluate HER3 receptor occupancy in patients with HER3-positive tumor lesions 31,32 . However, the rather large size (150 kDa) and the slow in vivo kinetics of monoclonal antibodies result in a moderate imaging contrast 33 . In addition, unspecific accumulation in tumors due to the enhanced permeability and retention effect reduces sensitivity and specificity of monoclonal antibodies as imaging probes 34 . The use of affibody molecules for imaging of HER3 expression could provide a promising alternative.
Affibody molecules are small affinity proteins (58 amino acids) and even small modifications of these targeting molecules can have significant impact on their biodistribution and tumor-to-non-tumor ratios which could be a useful instrument to optimize their imaging properties. The only difference between the affibody molecules used in this study was the chelator conjugated to the C-terminus. Earlier data have shown that even such small differences might cause noticeable modification in biodistribution of radiolabeled affibody molecules 26,27 .
Previous studies demonstrated that there are two characteristics of the radiometal-chelator complex having major impact on the behavior of anti-HER2 affibody molecules: complex charge and geometry [26][27][28] . The charge of the radiometal/chelator complex can vary depending on the valency of the metal and the number of available charged groups of the chelator 35 . The geometry of the formed complex is influenced by the size of the radiometal, which may require co-ligands for stable chelation [35][36][37] . Indium is a trivalent metal and its complexes with NOTA and DOTA are well studied 35,37 . Indium is hexa-coordinated with NOTA in a distorted prismatic structure and octa-coordinated with DOTA resulting in a square antiprism-like structure 37,38 . Their respective derivatives NODAGA and DOTAGA are assumed to form similar complexes 37 . Hexa-coordination of indium with NOTA results in an overall positively charged complex (Fig. 1). 111 In-NODAGA and 111 In-DOTA complexes are neutral in charge, but differ in size and structure. Lastly, the DOTAGA chelator carries an additional carboxylic arm resulting in a negatively charged complex. While the charge and geometry of radiometal complexes are important per se, their interplay with amino acids in the binding site is also essential. The binding site of affibody scaffold 111 In-Z 08698 -NOTA 111 In-Z 08698 -NODAGA 111 In-Z 08698 -DOTA 111 In-Z 08698 -DOTAGA k a (1/M*s) 10 × 10 4 ± 2 × 10 4 1.7 × 10 5 ± 0.8 × 10 5 1.5 × 10 5 ± 0.4 × 10 5 1.3 × 10 5 ± 0.8 × 10 5 contains 13 amino acids of 58 (22%), and physicochemical properties of side chains of binding amino acids may strongly influence off-target interactions. Modifications of binding sites of anti-HER2 and anti-HER3 affibody molecules had significant influence on blood clearance rate and hepatic uptake, even when the same labelling approach was used 22,39 . Therefore it was necessary to evaluate if observations made for anti-HER2 affibody molecules are still valid for anti-HER3 counterparts. Despite of differences in size and denticity of radiometal-chelator complexes all conjugates were stably radiolabeled with indium-111. However, labeling yields were much higher for the NODAGA and DOTAGA-conjugated variants. Difference in labeling yields did not correlate with size of the chelator (triaza vs. tetraaza), chelator's denticity or presence of impurities. Impurities were observed in the ESI-MS spectra of NOTA and NODAGA, possibly due to chelation of iron and copper. It could be speculated that the accessibility of the chelator was influenced by the length of the linker between the affibody molecule and the chelator. Nevertheless, such steric hindrance did not affect the targeting properties of the conjugates. All radiolabeled conjugates bound to the target with high affinity. When comparing the binding affinity of 111 In-Z 08698 -NOTA with its previously investigated (HE) 3  Analysis of cellular processing demonstrated that the overall internalization rate was slow, even though 111 In-Z 08698 -NOTA and 111 In-Z 08698 -DOTA showed somewhat higher internalization rate in BxPC-3 cells compared to the other conjugates. However, no differences in internalization pattern were seen between conjugates   in DU145 cells. Since members of the human epidermal growth factor receptor family act through dimerization, cellular processing and internalization rate can be influenced by varying expression levels of the target receptor and potential presence of dimerization partners. This is particularly important in the case of HER3 which is dependent on hetero-dimerization with other HER-family members for signaling, i.e. HER2 and EGFR 1 . Hence, different levels of HER co-expression can explain small observed differences between cell lines. In addition, it has been previously suggested that the radiometal-chelator complex can influence the cellular processing of affibody molecules 26,27 . The overall in vivo distribution patterns of the tested conjugates were comparable with the previously studied anti-HER3 affibody molecule (HE) 3 -Z 08698 -NOTA labeled with indium-111, gallium-68 and radiocobalt 20,21,29 . Tumor uptake at 4 h pi was in the range of 3-4%ID/g and the retention of activity in tumors was good, therefore it can be concluded that the choice of chelator had no significant influence on tumor uptake. Despite 20-fold difference in affinity the initial tumor uptake was similar. This could reflect that other factors such as tumor size, shape or vascularization have an effect on tumor uptake. Nevertheless, 111 In-Z 08698 -DOTAGA conjugates had the best tumor retention, which correlated with the best affinity found for this conjugate. Blood clearance of 111 In-Z 08698 -NOTA was significantly slower than for the other conjugates. In addition, its concentration in blood was notably higher than for the previously studied (HE) 3 -containing variants (0.1-0.27%ID/g) 20,21,29 . The relatively slow blood clearance of 111 In-Z 08698 -NOTA might be a result of weak interaction with blood proteins, which was previously balanced by the presence of the hydrophilic (HE) 3 -tag 20 . Overall, it can be concluded that the choice of the radiometal-chelator complex influenced blood clearance, affinity and tumor retention.
Even though the choice of chelator did not affect the tumor uptake, the results of the current study suggest that the radiometal-chelator complex greatly influences the uptake in organs with endogenous expression of HER3, particularly in liver, both in initial uptake (4 h pi) and retention (24 h pi). It was hypothesized earlier that hepatic uptake of affibody molecules is mediated by two mechanisms: a primary receptor specific mechanism and a secondary off-target mechanism possibly related to lipophilicity and charged surface groups of the affibody scaffold 29,40 . Previous reports have also shown that the increase of negative charge on the N-or C-terminus significantly reduces the hepatic uptake of affibody molecules 27,28 . This was in agreement with the observation that the uncharged cobalt-NOTA complex provided lower hepatic uptake than positively charged indium-and gallium-NOTA complexes when conjugated to (HE) 3 -Z 08698 20,21,29 . The results of the current study support this hypothesis. 111 In-Z 08698 -DOTAGA carrying a negative charge at the C-terminus had the lowest uptake in liver and significantly lower compared with the 111 In-NOTA conjugate. Figure 5 illustrates the differences in hepatic uptake and tumor-to-liver ratio at 4 and 24 h pi. 111 In-Z 08698 -NODAGA and 111 In-Z 08698 -DOTA have neutral charge at the C-terminus and both showed lower uptake in liver than 111 In-Z 08698 -NOTA, but their hepatic uptake did not differ significantly from each other. 111 In-Z 08698 -DOTAGA also showed the lowest uptake in organs with endogenous expression of mErbB3 at 4 h pi, which may be related to a reduced bioavailability of the conjugate due to rapid blood clearance. As a result, 111 In-Z 08698 -DOTAGA is considered the most favorable variant among the studied conjugates. The differences in uptake between conjugates also reflected on the tumor-to-organ ratios. Tumor-to-organ-ratios for 111 In-Z 08698 -DOTAGA were significantly higher than 111 In-Z 08698 -NOTA for liver, spleen, stomach, muscle and bone at 4 and 24 h pi. Hence, introduction of a negative charge at the C-terminus of the anti-HER3 affibody molecules improved the contrast in these organs. This was furthermore supported by the SPECT-CT images (Fig. 4). At both time points, the xenografts could be clearly visualized, but the best imaging contrast was provided by 111 In-Z 08698 -DOTAGA 24 h pi.

Conclusions
The biodistribution of anti-HER3 affibody molecules is influenced by the combination of chelator and radiometal. It can be concluded that an increased negative charge at the C-terminus of anti-HER3 affibody molecules decreases the activity uptake in liver by reducing unspecific uptake. SPECT-CT imaging of 111 In-Z 08698 -X (X = NOTA, NODAGA, DOTA, DOTAGA) confirmed the results of the biodistribution. Thus, the 111 In-Z 08698 -DOTAGA conjugate is considered superior to the other variants for imaging of HER3 overexpression in vivo.

Material and Methods
All data generated or analysed during this study are included in this published article (and its Supplementary Information files) and are available from the corresponding author on reasonable request. HER3 expressing cell lines, BxPC-3 (pancreatic cancer) and DU145 (prostate cancer), were purchased from American Type Tissue Culture Collection (ATTC via LGC Promochem, Borås, Sweden) and cultured in RMPI-1640 media supplemented with 1% penicillin and 1% L-glutamine (all from Biochrom, Berlin, Germany) and 10% fetal bovine serum (Sigma-Aldrich, Germany) at 37 °C with 5% CO 2 . Trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer) was used to detach cells.
Indium-111 was purchased in form of 111 In-chloride from Mallinckrodt Pharmaceuticals (Staines-upon-Thames, United Kingdom). Radioactivity content of samples in in vitro and in vivo experiments was measured with a 3-inch NaI(Tl) detector (1480 Wizard; Wallac Oy, Turku, Finland). All animal studies were performed in accordance with the national legislation on protection of laboratory animals and approved by the Ethics Committee for Animal Research in Uppsala, Sweden.
Results are presented as average with standard deviation. Statistical significance (p < 0.05) was evaluated by unpaired, two-tailed t-test using GraphPad Prism (version 7.03 for Windows, GraphPad Software, San Diego, CA, USA).
Production, conjugation and purification. The HER3-binding affibody Z HER3:08698 was produced and purified according to similar procedures as described previously 20 .
The Z 08698 was produced in E. coli BL21*(DE3) (Thermo Fisher Scientific) in an overnight culture at 25 °C after induced expression with 100 μM IPTG (Isopropyl β-D-1-thiogalactopyranoside) at an OD 600 of 0.8. Following cell lysis with French press, the supernatant was heated to 90 °C for 10 min with subsequent incubation on ice for 20 min, followed by centrifugation to remove precipitated proteins. The affibody molecule was purified on an ÄKTAexplorer (GE Healthcare, Uppsala, Sweden) using a 1 ml Resource S cation exchange column (GE Healthcare), running in 20 mM mM MES (2-(N-morpholino)ethanesulfonic acid, pH 6) and eluted by 20 mM MES with 1 M NaCl (pH 6). The buffer of the eluate was changed to 20 mM NH 4 Ac (pH 5.5) and the proteins were freeze-dried.
The proteins were dissolved in 20 mM NH 4 Ac (pH 5.5) and reduced with a molar concentration of tris(2-carboxyethyl)phosphine (TCEP) equal to the protein concentration for 30 min at 37 °C. The proteins were incubated at 37 °C for 90 min with ten-fold molar excess of maleimide derivatives of DOTA, DOTAGA, NOTA and NODAGA (CheMatech) for site-specific conjugation to the C-terminal cysteine on the affibody. Metal ion contaminations were removed from all buffers with Chelex 100 resin (Bio-Rad Laboratories).
After the site-specific conjugation, reverse-phase high performance liquid chromatography (RP-HPLC) on a 1200 series HPLC system using a Zorbax 300SB-C18 semi-preparative column (Agilent Technologies, Santa Clara, CA) was used for purification. Water with 0.1% trifluoroacetic acid was used as running buffer and an acetonitrile gradient was used for elution.
Characterization. The purity of the four conjugates was determined using RP-HPLC and an analytical Zorbax 300SB-C18 column (Agilent Technologies) with a 20-40% acetonitrile elution gradient over 20 min with a flow rate of 1 ml/min. Circular dichroism spectroscopy was performed using a Chirascan spectropolarimeter (Applied Photophysics, United Kingdom) with an optical path length of 1 mm, to analyze the alpha-helical content, thermal stability and refolding capacity of the four conjugates at a concentration of 0.25 mg/ml. The thermal stability was evaluated by Figure 5. Activity uptake in liver (left) and tumor-to-liver ratios for all 111 In-Z 08698 -X conjugates over time studied in female Balb/c nu/nu mice bearing BxPC-3 xenografts. Significant differences between conjugates are marked with asterisks.
SCIEnTIfIC RepoRts | (2019) 9:655 | DOI:10.1038/s41598-018-36827-w measuring the change in ellipticity at 221 nm during heating (5 °C/min) from 20 to 90 °C. The melting temperatures (T m ) were approximated from the data acquired from variable temperature measurements (VTM) by curve fitting using a Boltzmann Sigmoidal model (GraphPad Prism, version 7). The refolding capacity was assessed by comparing spectra obtained from measurements at wavelengths in the range 195-260 nm at 20 °C, before and after thermal denaturation. ESI-MS with a 6520 Accurate-Mass Q-TOF LC/MS (Agilent Technologies) was used for confirmation of molecular masses of the purified conjugates.
The affinity of the conjugates to human HER3 was investigated using single-cycle kinetics on a BIAcore T200 system (GE Healthcare) using a CM5 sensor chip with immobilized His-hHER3 and mFc-hHER3 (Sino Biological) with immobilization levels of 1500 and 2500 resonance units (RU) respectively. Measurements were performed in duplicates. Five concentrations of each conjugate were sequentially injected in a single cycle with a contact time of 100 seconds for DOTA-, DOTAGA-and NODAGA-conjugated affibody and 50 seconds for NOTA-conjugated affibody, for each concentration. The acquired sensorgrams were analyzed using a Langmuir 1:1 kinetic model. Stability of the radiolabeled conjugates was evaluated by incubating 2.5 µg of 111 In-Z 08698 -X (here and further "radiolabeled affibody molecules") at 37 °C with 100 µl human serum for 24 h. The samples were thereafter analyzed with ITLC.

Real-time ligand-binding kinetics. The kinetics of radiolabeled affibody molecules binding to living
BxPC-3 cells was measured in real time using LigandTracer yellow (Ridgeview Instruments AB, Vänge, Sweden) at room temperature, as previously described 41 . Concentrations of the radiolabeled affibody molecules were between 0.3 nM and 10 nM.
In vitro studies. Binding specificity and cellular processing of radiolabeled conjugates were investigated using BxPC-3 and DU145 cells as described previously 20 . All experiments were performed in triplicates. Cells were seeded in 35 mm cell dishes one day prior to the experiment in a density of 10 6 cells/dish (BxPC-3) or 0.7 × 10 6 cells/dish (DU145).
For investigation of binding specificity, HER3 receptors were pre-saturated by addition of 50 nM unlabeled Z 08698 to half of the dishes. After incubation for 15 min at room temperature, 0.1 nM of 111 In-Z 08698 -X was added to all dishes and cells were incubated for 1 hour at 37 °C. Afterwards, detached cells were collected and radioactivity content was measured.
For cellular processing, cells were incubated continuously with 0.1 nM of 111 In-Z 08698 -X at 37 °C. At pre-determined time points, a set of 3 dishes was analyzed. To collect the membrane bound radioactivity, cells were incubated with glycine buffer (0.2 M, pH 2.5, 4 M urea) on ice. In order to collect the internalized radioactivity, cells were further incubated at 37 °C with NaOH (1 M) for 30 min at 37 °C. Samples were measured in the automated gamma counter.
In vivo biodistribution. Biodistribution of 111 In-Z 08698 -X was investigated in female Balb/c nu/nu mice bearing BxPC-3 xenografts. To establish tumors, 5 × 10 6 cells per mouse were implanted 17 days prior to the study. At the day of experiment, the average mouse weight was 18 ± 1 g and average tumor weight was 0.09 ± 0.05 g.
Mice were intravenously injected with 2 µg of 111 In-Z 08698 -X (30 kBq) in 100 µl of 1% BSA/PBS. Non-labeled protein was used to adjust the amount of injected protein. Groups of 4 mice per data point were sacrificed 4 h and 24 h pi (pre-injected intra-peritoneal with Ketalar-Rompun solution, 10 mg/mL Ketalar and 1 mg/mL Rompun; 20 μL solution/gram of body weight). Blood samples were collected by heart puncture. Samples of lung, salivary glands, liver, stomach, small intestine, spleen, kidneys, muscle, bone and tumor were collected, weighed and measured for radioactivity content in the automated gamma counter. Radioactivity uptake was calculated as percent injected dose per gram (%ID/g). Results for carcass and gastrointestinal tract are expressed as %ID.
Imaging. Whole body SPECT/CT scans of the mice injected with 111 In-Z 08698 -X (2 µg, ~1.5 MBq) were performed at 4 and 24 h pi using nanoScan SPECT/CT (Mediso Medical Imaging Systems Ltd., Hungary). For the 4 h pi scan, the mice were placed under general anesthesia by administration of a mixture of Sevoflurane (3.5%), oxygen and medical air. CT was acquired at the following parameters: 50 keV energy peak, 670 μA, 480 projections, 5.26 min acquisition time. SPECT was carried out using 111 In energy peaks of 245.4 keV and 171.3 keV, window width of 20%, matrix of 256 × 256, acquired for 1 h. CT raw files were reconstructed using Nucline 2.03 Software (Mediso Medical Imaging Systems, Hungary). SPECT raw data were reconstructed using Tera-Tomo ™ 3D SPECT reconstruction technology. Images are presented as maximum intensity projection (MIP) in RGB (red, green and blue) color scale.