The Use of a Non-Conventional Long-Lived Gallium Radioisotope 66Ga Improves Imaging Contrast of EGFR Expression in Malignant Tumours Using DFO-ZEGFR:2377 Affibody Molecule

Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted therapy extends survival of patients with disseminated cancers. Radionuclide molecular imaging of EGFR expression would make EGFR-directed treatment more personalized and therefore more efficient. A previous study demonstrated that affibody molecule [68Ga]Ga-DFO-ZEGFR:2377 permits specific positron-emission tomography (PET) imaging of EGFR expression in xenografts at 3 h after injection. We anticipated that imaging at 24 h after injection would provide higher contrast, but this is prevented by the short half-life of 68Ga (67.6 min). Here, we therefore tested the hypothesis that the use of the non-conventional long-lived positron emitter 66Ga (T1/2 = 9.49 h, β+ = 56.5%) would permit imaging with higher contrast. 66Ga was produced by the 66Zn(p,n)66Ga nuclear reaction and DFO-ZEGFR:2377 was efficiently labelled with 66Ga with preserved binding specificity in vitro and in vivo. At 24 h after injection, [66Ga]Ga-DFO-ZEGFR:2377 provided 3.9-fold higher tumor-to-blood ratio and 2.3-fold higher tumor-to-liver ratio than [68Ga]Ga-DFO-ZEGFR:2377 at 3 h after injection. At the same time point, [66Ga]Ga-DFO-ZEGFR:2377 provided 1.8-fold higher tumor-to-blood ratio, 3-fold higher tumor-to-liver ratio, 1.9-fold higher tumor-to-muscle ratio and 2.3-fold higher tumor-to-bone ratio than [89Zr]Zr-DFO-ZEGFR:2377. Biodistribution data were confirmed by whole body PET combined with magnetic resonance imaging (PET/MRI). The use of the positron emitter 66Ga for labelling of DFO-ZEGFR:2377 permits PET imaging of EGFR expression at 24 h after injection and improves imaging contrast.


Introduction
Several families of cell-surface receptors are overexpressed in malignant tumors. Signaling of the overexpressed receptors is frequently a driving force for malignancy. The epidermal growth factor receptor (EGFR) is one of four members of the human EGF receptor family of receptor tyrosine kinases [1]. EGFR activation normally causes signaling leading to cell proliferation, inhibition of apoptosis, and increased motility [2]. EGFR is mains on the cell surface of both tumor cells and hepatocytes in the liver and the rapid renal clearance of the probe shifts the binding equilibrium to dissociation. As a result, the liver, which is acting as a depot for ZEGFR affibody molecules, releases the labelled probe into the blood stream, in turn causing slower blood clearance compared with e.g., affibody molecules binding to HER2. As the tumor-to-blood ratio is significantly higher at 24 compared with 4 h after injection [23], research efforts aimed at developing an optimal label for ZEGFR:2377 affibody molecules have shifted to more long-lived radionuclides, such as 55/57 Co (T 1/2 = 17.5 h) [27], 64 Cu (T 1/2 = 12.7 h) [29,30], and 89 Zr (T 1/2 = 78.4 h) [31,32] as these could provide higher contrast for next-day PET imaging. These studies have provided important results including the finding that the combination of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator with either 68 Ga or 57 Co has been found to have a dramatic influence on hepatic uptake of ZEGFR:2377. This indicated that there is an EGFR-independent mechanism of the hepatic uptake that can be influenced by the charge of the chelator-metal complex [27].
Comparison of siderophore-based chelators fusarinine C (FSC) and desferrioxamine B (DFO) for labelling of ZEGFR:2377 affibody molecules with 89 Zr also revealed an obvious influence of radionuclide-chelator combinations on biodistribution properties [32]; particularly, the use of DFO for high-temperature labelling provided better contrast. For site-specific labelling of ZEGFR:2377, a maleimido derivative of desferrioxamine B (N- [5-[[4-[5-[acetyl(hydroxy)amino]-pentylamino]-4-oxobutanoyl]-hydroxyamino]pentyl]-N0-(5-aminopentyl)-N0-hydroxybutanediamide, DFO) was conjugated to a unique Cterminal cysteine of this affibody molecule ( Figure 1). ZEGFR:2377 permits saturation of EGFR expression in the liver without blocking in tumor xenografts [23]. An important feature of the ZEGFR:2377 affibody molecule is its slow internalization after binding to the EGFR [23,27,31]. Thus, the majority of the specifically bound tracer remains on the cell surface of both tumor cells and hepatocytes in the liver and the rapid renal clearance of the probe shifts the binding equilibrium to dissociation. As a result, the liver, which is acting as a depot for ZEGFR affibody molecules, releases the labelled probe into the blood stream, in turn causing slower blood clearance compared with e.g., affibody molecules binding to HER2. As the tumor-to-blood ratio is significantly higher at 24 compared with 4 h after injection [23], research efforts aimed at developing an optimal label for ZEGFR:2377 affibody molecules have shifted to more long-lived radionuclides, such as 55/57 Co (T1/2 = 17.5 h) [27], 64 Cu (T1/2 = 12.7 h) [29,30], and 89 Zr (T1/2 = 78.4 h) [31,32] as these could provide higher contrast for next-day PET imaging. These studies have provided important results including the finding that the combination of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator with either 68 Ga or 57 Co has been found to have a dramatic influence on hepatic uptake of ZEGFR:2377. This indicated that there is an EGFR-independent mechanism of the hepatic uptake that can be influenced by the charge of the chelator-metal complex [27].
Comparison of siderophore-based chelators fusarinine C (FSC) and desferrioxamine B (DFO) for labelling of ZEGFR:2377 affibody molecules with 89 Zr also revealed an obvious influence of radionuclide-chelator combinations on biodistribution properties [32]; particularly, the use of DFO for high-temperature labelling provided better contrast. For site-specific labelling of ZEGFR:2377, a maleimido derivative of desferrioxamine B (N- [5-[[4-[5-[acetyl(hydroxy)amino]-pentylamino]-4-oxobutanoyl]-hydroxyamino]pentyl]-N0-(5-aminopentyl)-N0-hydroxybutanediamide, DFO) was conjugated to a unique Cterminal cysteine of this affibody molecule ( Figure 1). Several studies have shown that DFO is also a suitable chelator for radiolabeling of targeting agents with 67 Ga or 68 Ga [33][34][35]. DFO contains three hydroxamate groups, which provide six oxygen donors for the metal binding [36]. This is a good match for Ga 3+ , which is typically hexacoordinated. We have recently shown that ZEGFR:2377 affibody molecules labelled with 68 Ga via DFO as a chelator have a tendency to demonstrate lower liver uptake and significantly increased tumor uptake when compared with ZEGFR:2377-DFO labelled with 89 Zr at 3 h after injection [28]. This resulted in a higher tumor-to-liver ratio for the 68 Ga-labelled conjugate. The use of Ga 3+ instead of Zr 4+ decreases the positive charge on C-terminus of ZEGFR:2377, which is favorable for biodistribution.
The use of long-lived radionuclides for radiolabeling of biomolecules undergoing slow distribution in vivo enables high-resolution imaging at later time points after injection. Gallium-66 ( 66 Ga, T1/2 =9.49 h, β + = 56.5%, EC = 43.5%) is a long-lived positronemitting isotope of gallium [37]. The half-life of 66 Ga makes it possible to perform nextday PET imaging, further improving imaging contrast. 66 Ga has been tested for studies of some slow dynamic processes (such as lymphatic transport) using PET [38] and for Several studies have shown that DFO is also a suitable chelator for radiolabeling of targeting agents with 67 Ga or 68 Ga [33][34][35]. DFO contains three hydroxamate groups, which provide six oxygen donors for the metal binding [36]. This is a good match for Ga 3+ , which is typically hexacoordinated. We have recently shown that ZEGFR:2377 affibody molecules labelled with 68 Ga via DFO as a chelator have a tendency to demonstrate lower liver uptake and significantly increased tumor uptake when compared with ZEGFR:2377-DFO labelled with 89 Zr at 3 h after injection [28]. This resulted in a higher tumor-to-liver ratio for the 68 Ga-labelled conjugate. The use of Ga 3+ instead of Zr 4+ decreases the positive charge on C-terminus of ZEGFR:2377, which is favorable for biodistribution.
The use of long-lived radionuclides for radiolabeling of biomolecules undergoing slow distribution in vivo enables high-resolution imaging at later time points after injection. Gallium-66 ( 66 Ga, T 1/2 =9.49 h, β + = 56.5%, EC = 43.5%) is a long-lived positron-emitting isotope of gallium [37]. The half-life of 66 Ga makes it possible to perform next-day PET imaging, further improving imaging contrast. 66 Ga has been tested for studies of some slow dynamic processes (such as lymphatic transport) using PET [38] and for imaging of tumor angiogenesis using a monoclonal antibody [39]. The use of a 66 Ga-labelled somatostatin analogue as an imaging agent for somatostatin receptor positive tumors has also been Pharmaceutics 2021, 13, 292 4 of 18 reported [40]. Recently, first preclinical imaging of GRPR-expression in prostate cancer using [ 66 Ga]Ga-NOTA-PEG2-RM26 has been reported [41].
The goal of this study was to test the hypothesis that the use of 66 Ga would permit specific imaging of EGFR-expressing xenografts 24 h after injection and enhance the tumorto-blood ratio when compared with imaging using the short-lived 68 Ga radioisotope. For this purpose, labelling of DFO-ZEGFR:2377 affibody molecules with the positron emitting 66 Ga radionuclide was optimized. The stability and In Vitro targeting properties of [ 66 Ga]Ga-DFO-ZEGFR:2377 were investigated and compared to targeting properties of previously studied 68 Ga and 89 Zr counterparts. In vivo targeting properties of a novel [ 66 Ga]Ga-DFO-ZEGFR:2377 were compared directly with the properties of [ 68 Ga]Ga-DFO-ZEGFR:2377 and [ 89 Zr]Zr-DFO-ZEGFR:2377 at 3 and 24 h after injection, respectively. Such direct comparison is preferable to a comparison with historical controls as the targeting properties of all conjugates are therefore subject to the same animal batch-dependent biological parameters.

Materials and Methods
High quality Milli-Q water was used to prepare all buffer solutions, and the buffers were purified from metal contamination using Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA, USA).
Gallium-68 ( 68 Ga) was obtained by fractionated elution of the 68 Ge/ 68 Ga generator (Eckert and Ziegler AG, Berlin, Germany) with 0.1 M HCl. The eluate with the highest radioactivity concentration was used for labelling. Zirconium-89 ( 89 Zr, solution in 1 M oxalic acid) was purchased from PerkinElmer (Waltham, MA, USA).
The radioactivity was measured using a gamma spectrometer with a NaI(Tl) detector (2480 WIZARD, Wallac Oy, Turku, Finland). The labelling yield and radiochemical purity of labelled conjugates were measured using the instant thin-layer chromatography (ITLC) (ITLC-SG, Agilent Technologies, Santa Clara, CA, USA). The strips were developed with 0.2 M citric acid, pH 2.0 and 5.0 for 68 Ga and 89 Zr conjugates, respectively. Distribution of the radioactivity among the strips was measured with a Cyclone Phosphor Storage Screen using OptiQuant software (Packard Instrument Company, Meriden, CT, USA) for data processing as well as Fujifilm Bioimaging Analyzers (BAS) 1800II using MultiGauge V3.0 analysis software (Tokyo, Japan). Radiolabelled bioconjugates were purified for further studies using NAP-5 size exclusion columns (Cytiva, Uppsala, Sweden) pre-equilibrated with phosphate-buffered saline (PBS) with 1% bovine serum albumin. In Vitro cell studies were performed using the EGFR-expressing A431 and PC3 cells, both obtained from the American Type Culture Collection (ATCC).
The anti-EGFR affibody molecule containing C-terminal cysteine was produced as described earlier [23]. Maleimido-DFO (Figure 1), was site-specifically conjugated to Cterminal cysteine of ZEGFR:2377 to provide DFO-ZEGFR:2377 according to the method described by Garousi and co-workers [31]. The identity of conjugates was confirmed by mass spectrometry as described earlier [31]. The purity of the conjugate was greater than 95%, as determined using reversed phase HPLC according to a method described earlier [31].
Statistical treatment and linear regression analysis were performed using GraphPad Prism software version 5.00 for Windows (GraphPad Software, San Diego, CA, USA). A two-tailed unpaired t-test was used for comparison of the two sets of data. The difference was considered significant when the p value was less than 0.05.

Production of 66 Ga
Gallium-66 ( 66 Ga) was produced using the 66 Zn(p,n) 66 Ga reaction by irradiating isotopically enriched (99.07%) 66 Zn target with 14.3 MeV protons, as described earlier [41] with 1 M [ 66 Zn]Zn(NO 3 ) 2 in 0.3 M HNO 3 as a target material. Irradiations (70-75 min,~25 µA) were performed with a GE PETtrace cyclotron (GE Healthcare, Uppsala, Sweden) using a liquid target developed for a cyclotron production of 68 Ga. Separation of [ 66 Ga]GaCl 3 was performed using the GE FASTlab Developer platform using methodology developed for 68 Ga purification [41]. The product was collected here in several 300 µL fractions and the eluate with the highest radioactivity concentration was used for labelling.
In Vitro stability of [ 66 Ga]Ga-DFO-ZEGFR:2377 was evaluated in PBS and also in the presence of 1000-fold molar excess of ethylenediaminetetraacetic acid (EDTA). Namely, after purification, samples of freshly labelled conjugate (1.4 µg, 50 µL) were mixed with EDTA (60 µg, 2 mg/mL in PBS) to obtain a 1000-fold molar excess of EDTA and incubated at room temperature for 1 h. Control samples were mixed with an equal volume of PBS. The experiment was performed in triplicate.
For a binding specificity test, a set of nine dishes containing approximately 10 6 cells/dish was used for each data point. To saturate the EGFR, a 100-fold molar excess of anti-EGFR antibody cetuximab or non-labelled DFO-ZEGFR:2377 were used as blocking agents in three control dishes. Cells were incubated with blocking agents at room temperature for 15 min. Then, [ 66 Ga]Ga-DFO-ZEGFR:2377 (5 nM) was added to all dishes followed by incubation for 1 h at 37 • C. After incubation, the medium was aspired. Next, the cells were washed with cold serum-free medium, treated with 2 mL trypsin-EDTA solution per dish at 37 • C, and collected. Radioactivity of cells was measured. The experiments were performed in triplicate.
To evaluate the binding affinity of [ 66 Ga]Ga-DFO-ZEGFR:2377 to EGFR receptors, kinetics of labelled conjugate binding to-and their dissociation from A431 cells were measured using a LigandTracer Yellow instrument (Ridgeview Instruments AB, Vänge, Sweden). A431 cells were seeded on a local area of a cell culture dish (NunclonTM, Size 100620, NUNC A/S, Roskilde, Denmark). The measurements were performed at room temperature to prevent internalization. Uptake curves were recorded at 0.33, 1, and 3 nM of [ 66 Ga]Ga-DFO-ZEGFR:2377 conjugate. Thereafter, the radioactive medium was withdrawn, fresh non-radioactive medium was added, and the dissociation curve was recorded. The data were analyzed using the InteractionMap software (Ridgeview Diagnostics AB, Uppsala, Sweden) in order to calculate the association rate, dissociation rate, and dissociation constant at equilibrium (K D ). Analysis was performed in duplicates.
Cellular processing of bound [ 66 Ga]Ga-DFO-ZEGFR:2377 was evaluated using A431 and PC3 cell lines. The cells (10 6 cells/dish) were incubated with 5 nM of [ 66 Ga]Ga-DFO-ZEGFR:2377 at 37 • C. After 1, 2, 4, 8, and 24 h incubation, the internalized fraction was determined by the acid wash method [44]. The membrane-bound conjugate was removed from cells by treatment with 4 M urea solution in a 0.1 M glycine buffer, pH 2.5, for 5 min on ice. The cell debris containing the internalized conjugates was detached by treatment with 1 M NaOH. Radioactivity of the samples was measured and the percentage of membrane-bound, internalized, and total radioactivity was calculated. The experiments were performed in triplicate.

In Vivo Studies
Animal studies were planned and performed in agreement with EU Directive 2010/63/EU for animal experiments and Swedish national legislation concerning protection of laboratory animals. Experiments were approved by the Ethics Committee for Animal Research in Uppsala (C4/16). Biodistribution studies were performed in female BALB/C nu/nu mice purchased from Scanbur A/S (Karlslunde, Denmark).
EGFR-expressing xenografts were established by subcutaneous injection of 10 7 A431 cells in the hind legs of mice. The tumors were grown for 10 days. At the time of the experiment, the average animal weight was 18 ± 1 g and the average tumor weight was 0.5 ± 0.2 g. The animals were randomized into groups of four mice for each data point.
The injected activity was selected based on results of previous studies in order to provide a counting statistics error of less than 3% for samples with the lowest uptake, and a dead time of less than 20% for the standards of injected activity. The injected protein dose was determined in earlier studies to be optimal for a partial saturation of EGFR in the liver without saturation of receptors in the tumor [23]. To obtain the desirable protein dose, non-labelled DFO-ZEGFR:2377 was added to conjugates during formulation.
For biodistribution measurements, the mice were euthanized by an intraperitoneal injection of an anesthetic solution (20 µL of solution per gram of body weight: ketamine, 10 mg/mL; Xylazine, 1 mg/mL) followed by heart puncture. Blood, salivary glands, lung, liver, spleen, colon, kidneys, tumor, muscle, and bone were collected and weighed. Then, the organ radioactivity was measured and uptake values of organs were calculated as a percentage of injected dose per gram tissue (%ID/g).
Small animal PET imaging was performed to obtain qualitative visual confirmation of the results of ex vivo measurements. Three mice with A431 xenografts were injected with 1 MBq of [ 66 Ga]Ga-DFO-ZEGFR:2377 (38 µg) intravenously. Whole body PET and MRI measurements were performed using a 3 Tesla nanoScan PET/MRI system (Mediso Medical Imaging Systems Ltd., Budapest, Hungary) at 3, 6, and 24 h p.i. The net measurement time was one hour for the PET scans started 3 and 6 h after injection or 1.5 h for the Pharmaceutics 2021, 13, 292 7 of 18 scans performed 24 h p.i. Measured data were reconstructed using the Tera-Tomo™ 3D reconstruction engine which takes into account the matching MR images for correction of positron range. Mice were kept under general anesthesia (0.06% sevoflurane; 50%/50% medical oxygen: air) during PET/MRI acquisitions made 3 and 6 h p.i. Mice were euthanized prior to the scans 24 hours p.i. MR images were measured using a T1-weighted gradient echo sequence prior each PET scan. Acquisition time was 4 min 2 sec for 21 slices. Parameters were as follows: coronal slices (1 mm thickness), FOV 80 × 60 mm, acquisition matrix 256 × 192, resolution in plane 0.313 × 0.313 mm, 4 accumulations, repetition time 300 ms, echo time 4.5 ms, flip angle 45 o , receiver bandwidth 40 000 Hz. Additionally, one mouse was pre-injected with 10 mg cetuximab 24 h before injection of 1 MBq of [ 66 Ga]Ga-DFO-ZEGFR:2377, and imaging was performed 3 h p.i.

Nuclide Production and Radiolabelling
66 Ga was produced with end-of-bombardment (EOB) yields up to 0.50 GBq, which corresponds to saturation yields of 0.25 GBq/µA. The isolated [ 66 Ga]GaCl 3 activity considering all fractions was~320-340 MBq, of which the fractions used for radiolabeling had activity concentrations ranging within 160-280 MBq/mL The 66 Ga labelling of ZEGFR:2377 containing DFO as a chelator was performed at 85 • C. The radiochemical yield was 64.5 ± 3.5% and the isolated yield was 61.5 ± 0.7%. The radiochemical purity of the radiolabeled affibody molecules after purification using NAP-5 columns was greater than 99%. The specific activity of [ 66 Ga]Ga-DFO-ZEGFR:2377 was 0.3 MBq/µg (molar activity 2.43 GBq/µmol).
After the purification, In Vitro stability test of DFO-ZEGFR:2377 labelled with 66 Ga was performed in PBS and in the presence of 1000-fold molar excess of EDTA. The release of the 66 Ga when challenged by EDTA was less than 4% compared to control samples incubated in PBS.

In Vitro Studies
The specificity of [ 66 Ga]Ga-DFO-ZEGFR:2377 binding to EGFR expressing cells was tested by pre-saturation of receptors. Saturation of the receptors with a large excess of anti-EGFR antibody cetuximab and non-labelled DFO-ZEGFR:2377 affibody molecule significantly (p < 5 × 10 −7 ) decreased the binding of the radiolabelled affibody molecules to EGFR-expressing cell lines A431 as well as PC3 cells ( Figure 2). Therefore, the specificity test demonstrated that the binding of the tracer to both EGFR-expressing cell lines was EGFRmediated. In addition, cell-associated activity was proportional to the EGFR expression level ( Figure 2).
The data concerning the kinetic evaluation of binding and affinity of [ 66 Ga]Ga-DFO-ZEGFR:2377 to A431 cells are presented in Table 1 and Figure S1. According to LigandTracer measurements, the best fit of the binding to A431 cell line was achieved using a 1:2 model. Interaction Map calculations showed a major strong interaction with K D1 = 342 ± 14 pM and a weaker minor interaction with K D in the nanomolar range (K D2 = 29.2 ± 0.2 nM).
The processing of bound [ 66 Ga]Ga-DFO-ZEGFR:2377 by A431 and PC3 cell lines was studied by the acid wash method. The data are presented in Figure 3, whereby the labelled conjugate showed a rapid binding in the first 2 h with relatively slow internalization as it is typical for affibody molecules. The internalized fraction was less than 22% for both cell lines.  Table 1 and Figure S1. According to LigandTracer measurements, the best fit of the binding to A431 cell line was achieved using a 1:2 model. Interaction Map calculations showed a major strong interaction with KD1 = 342 ± 14 pM and a weaker minor interaction with KD in the nanomolar range (KD2 = 29.2 ± 0.2 nM). The processing of bound [ 66 Ga]Ga-DFO-ZEGFR:2377 by A431 and PC3 cell lines was studied by the acid wash method. The data are presented in Figure 3, whereby the labelled conjugate showed a rapid binding in the first 2 h with relatively slow internalization as it is typical for affibody molecules. The internalized fraction was less than 22% for both cell lines.    The data concerning the kinetic evaluation of binding and affinity of [ 66 Ga]Ga-DFO-ZEGFR:2377 to A431 cells are presented in Table 1 and Figure S1. According to LigandTracer measurements, the best fit of the binding to A431 cell line was achieved using a 1:2 model. Interaction Map calculations showed a major strong interaction with KD1 = 342 ± 14 pM and a weaker minor interaction with KD in the nanomolar range (KD2 = 29.2 ± 0.2 nM). The processing of bound [ 66 Ga]Ga-DFO-ZEGFR:2377 by A431 and PC3 cell lines was studied by the acid wash method. The data are presented in Figure 3, whereby the labelled conjugate showed a rapid binding in the first 2 h with relatively slow internalization as it is typical for affibody molecules. The internalized fraction was less than 22% for both cell lines.

In Vivo Studies
The in vivo specificity of [ 66 Ga]Ga-DFO-ZEGFR:2377 binding to EGFR in tumor xenografts was evaluated by pre-saturation of receptors using the monoclonal anti-EGFR antibody cetuximab, which binds to the same epitope as ZEGFR:2377 ( Figure 4). Tumor uptake in the blocked group was significantly (p < 0.005) reduced compared with the unblocked group, indicating highly EGFR specific accumulation of [ 66 Ga]Ga-DFO-ZEGFR:2377 in EGFR-expressing tumors. In addition, the liver uptake showed significant reduction (p < 0.005) in the blocked group compared with the unblocked group. xenografts was evaluated by pre-saturation of receptors using the monoclonal anti-EGFR antibody cetuximab, which binds to the same epitope as ZEGFR:2377 (Figure 4). Tumor uptake in the blocked group was significantly (p < 0.005) reduced compared with the unblocked group, indicating highly EGFR specific accumulation of [ 66 Ga]Ga-DFO-ZEGFR:2377 in EGFR-expressing tumors. In addition, the liver uptake showed significant reduction (p < 0.005) in the blocked group compared with the unblocked group.  Table 2. The clearance of radioactivity from blood and non-target tissues over time was quite fast. For example, the blood concentration was notably less than 2% at 3 h after injection, and there was a significant (p < 0.05) reduction of radioactivity accumulation in blood, liver, spleen, and kidneys between 3 and 24 h after injection ( Table 2). A remarkable feature was the relatively rapid kidney clearance. There was no significant difference in tumor uptake at 3 h (6.5 ± 1.3% ID/g) compared to 6 h (5.1 ± 0.5% ID/g) after injection, but there was a significant (p < 0.05) reduction of tumor uptake at 24 h (3.7 ± 0.5% ID/g) compared with 3 and 6 h p.i. The trend of slower reduction of tumor uptake accompanied by faster clearance from blood, liver, and kidneys over time resulted in significantly (p < 0.05) higher tumor-to-blood, tumor-to-liver, and tumor-to-kidney ratios at 24 h compared with 3 and 6 h ( Table 2).  Table 2. The clearance of radioactivity from blood and non-target tissues over time was quite fast. For example, the blood concentration was notably less than 2% at 3 h after injection, and there was a significant (p < 0.05) reduction of radioactivity accumulation in blood, liver, spleen, and kidneys between 3 and 24 h after injection ( Table 2). A remarkable feature was the relatively rapid kidney clearance. There was no significant difference in tumor uptake at 3 h (6.5 ± 1.3% ID/g) compared to 6 h (5.1 ± 0.5% ID/g) after injection, but there was a significant (p < 0.05) reduction of tumor uptake at 24 h (3.7 ± 0.5% ID/g) compared with 3 and 6 h p.i. The trend of slower reduction of tumor uptake accompanied by faster clearance from blood, liver, and kidneys over time resulted in significantly (p < 0.05) higher tumor-to-blood, tumor-to-liver, and tumor-to-kidney ratios at 24 h compared with 3 and 6 h ( Table 2). Tables 3 and 4 showed the data regarding the biodistribution and tumor-to-organ ratio of [ 68 Ga]Ga-DFO-ZEGFR:2377 and [ 89 Zr]Zr-DFO-ZEGFR:2377 in A431 xenograft-bearing mice at 3 and 24 h after injection, respectively.  Table 2. Biodistribution and tumor-to-organ ratio for [ 66 Ga]Ga-DFO-ZEGFR:2377 in BALB/C nu/nu mice bearing A431 xenografts at 3, 6, and 24 h after injection. Data are expressed as the percentage of administered activity (injected probe) per gram of tissue (% ID/g) after intravenous injection of the probe. The data are presented as the average (n = 4) and SD.       Figure 7. [ 66 Ga]Ga-DFO-ZEGFR:2377 had significantly (p < 0.05) higher uptake in tumors, but significantly lower uptake in the liver, spleen, and kidneys ( Figure 7A). In particular, the 66 Ga-labelled counterpart demonstrated significantly (p < 0.05) higher tumorto-blood (10.7 ± 2.7 vs. 5.9 ± 1.3), tumor-to-liver (2.7 ± 0.7 vs. 0.9 ± 0.2), tumor-to-kidney (0.088 ± 0.014 vs. 0.009 ± 0.002), tumor-to-muscle (19 ± 3 vs. 10 ± 1) and tumor-to-bone (4.6 ± 1.7 vs. 2.0 ± 0.3) ratios than [ 89 Zr]Zr-DFO-ZEGFR:2377 ( Figure 7B).  Figure 7. [ 66 Ga]Ga-DFO-ZEGFR:2377 had significantly (p < 0.05) higher uptake in tumors, but significantly lower uptake in the liver, spleen, and kidneys ( Figure 7A). In particular, the 66 Ga-labelled counterpart demonstrated significantly (p < 0.05) higher tumor-to-blood (10.7 ± 2.7 vs. 5.9 ± 1.3), tumor-to-liver (2.7 ± 0.7 vs. 0.9 ± 0.2), tumor-to-kidney (0.088 ± 0.014 vs. 0.009 ± 0.002), tumor-to-muscle (19 ± 3 vs. 10 ± 1) and tumor-to-bone (4.6 ± 1.7 vs. 2.0 ± 0.3) ratios than [ 89 Zr]Zr-DFO-ZEGFR:2377 ( Figure 7B).   Figure 7. [ 66 Ga]Ga-DFO-ZEGFR:2377 had significantly (p < 0.05) higher uptake in tumors, but significantly lower uptake in the liver, spleen, and kidneys ( Figure 7A). In particular, the 66 Ga-labelled counterpart demonstrated significantly (p < 0.05) higher tumor-to-blood (10.7 ± 2.7 vs. 5.9 ± 1.3), tumor-to-liver (2.7 ± 0.7 vs. 0.9 ± 0.2), tumor-to-kidney (0.088 ± 0.014 vs. 0.009 ± 0.002), tumor-to-muscle (19 ± 3 vs. 10 ± 1) and tumor-to-bone (4.6 ± 1.7 vs. 2.0 ± 0.3) ratios than [ 89 Zr]Zr-DFO-ZEGFR:2377 ( Figure 7B). The ability to use [ 66 Ga]Ga-DFO-ZEGFR:2377 for specifically visualizing EGFR-expressing xenografts was confirmed using microPET/MRI ( Figure 8A-C). Mice bearing A431 xenografts were imaged 3, 6, and 24 h after injection of [ 66 Ga]Ga-DFO-ZEGFR:2377. EGFR-expressing xenografts on the right hind leg were clearly visualized after injection at all three time points. The imaging results were in good agreement with the biodistribution data. There was a tendency towards reduced liver uptake with time, thus resulting in a higher tumorto-liver ratio at the latest time point. The only normal organ with a high radioactivity accumulation was the kidneys. Otherwise, the uptake of radioactivity in the tumor appreciably exceeded uptake when compared with other normal organs, thus providing a clear, high-contrast image of an EGFR-expressing xenograft. In the control mouse, EGF receptors were saturated by pre-injection of a large molar excess of the anti-EGFR antibody cetuximab. The activity uptake in tumor was noticeably reduced, which confirmed EGFR-specificity of [ 66 Ga]Ga-DFO-ZEGFR:2377 using in vivo imaging (Figure 8, panel D).

Uptake, %ID/g Tumor-to-Organ
with a high radioactivity accumulation was the kidneys. Otherwise, the uptake of radioactivity in the tumor appreciably exceeded uptake when compared with other normal organs, thus providing a clear, high-contrast image of an EGFR-expressing xenograft. In the control mouse, EGF receptors were saturated by pre-injection of a large molar excess of the anti-EGFR antibody cetuximab. The activity uptake in tumor was noticeably reduced, which confirmed EGFR-specificity of [ 66 Ga]Ga-DFO-ZEGFR:2377 using in vivo imaging (Figure 8, panel D).

Discussion
The use of small scaffold proteins, such as affibody molecules, offers an advantage in radionuclide molecular imaging compared with the use of monoclonal antibodies due to the potential for affibody molecules to provide higher contrast [45]. Consequently, the sensitivity of such imaging is also higher. Typically, high-contrast imaging is achieved on the same day as the injection [21,45], and short-lived positron emitters (e.g., 18 F and 68 Ga) are generally suitable for affibody molecule labelling. Being positron emitters, the use of such radionuclides additionally enable higher sensitivity and resolution imaging by PET vs. SPECT. However, imaging of EGFR is somewhat different. Expression of EGFR in normal tissues (most importantly in the liver) and reversible binding of affibody molecules to EGFR causes slower clearance and necessitates next-day imaging in order to obtain adequate contrast. Thus, the half-life of a nuclide for labelling should be sufficiently long to prevent decay prior to a tracer's optimal imaging time.
The use of a positron-emitting radionuclide remains to be desirable due to advantages of PET as a radionuclide imaging modality. 66 Ga is one of a few positron emitting nuclides that meet the requirement of a sufficiently long half-life, reasonably

Discussion
The use of small scaffold proteins, such as affibody molecules, offers an advantage in radionuclide molecular imaging compared with the use of monoclonal antibodies due to the potential for affibody molecules to provide higher contrast [45]. Consequently, the sensitivity of such imaging is also higher. Typically, high-contrast imaging is achieved on the same day as the injection [21,45], and short-lived positron emitters (e.g., 18 F and 68 Ga) are generally suitable for affibody molecule labelling. Being positron emitters, the use of such radionuclides additionally enable higher sensitivity and resolution imaging by PET vs. SPECT. However, imaging of EGFR is somewhat different. Expression of EGFR in normal tissues (most importantly in the liver) and reversible binding of affibody molecules to EGFR causes slower clearance and necessitates next-day imaging in order to obtain adequate contrast. Thus, the half-life of a nuclide for labelling should be sufficiently long to prevent decay prior to a tracer's optimal imaging time.
The use of a positron-emitting radionuclide remains to be desirable due to advantages of PET as a radionuclide imaging modality. 66 Ga is one of a few positron emitting nuclides that meet the requirement of a sufficiently long half-life, reasonably abundant positron decay branching ratio and feasibility of production by low-energy cyclotrons available to the PET community [46] (Table S1). In addition to the selection of a radionuclide with a suitable half-life, the selection of suitable labelling chemistry is an essential factor for development of an imaging probe. Multiple studies have demonstrated that a combination of radionuclide and chelator can profoundly influence the biodistribution of affibody molecules and therefore the imaging contrast [22,47]. For example, the use of the versatile and commonly used chelator DOTA for labelling of ZEGFR:2377 with 68 Ga resulted in higher uptake in the liver vs. tumor, thus making imaging of frequently encountered hepatic metastases impossible [27]. On the other hand, the use of DFO as a chelator provided a targeting conjugate with higher uptake in tumor than in liver [28]. Therefore, DFO was selected for labelling with 66 Ga in this study.
This study demonstrated that DFO-ZEGFR:2377 can be labelled with 66 Ga providing radiochemical purity sufficient for clinical application. A production of sufficient amount of radionuclide is essential for future clinical translation. In the case of 68 Ga-labelled PSMAbinders and somatostatin analogues, an injected activity of 100-120 MBq is adequate for clinical applications. Our current experimental protocol for production of 66 Ga enables production of about 300 MBq with a radioactivity concentration suitable for labelling. With an isolated yield of 61.5 ± 0.7%, [ 66 Ga]Ga-DFO-ZEGFR:2377 with activity of 160-180 MBq could be produced after single irradiation. In terms of positron emission such activity would be equivalent to 100-115 MBq of 68 Ga. Furthermore, the production rate of 66 Ga can be appreciable increased by rising the proton beam current form 25 to 40 µA on the existing liquid technology, or by use of solid targets. Taking into account that a clinically relevant injected mass of DFO-ZEGFR:2377 should be on the order of several milligrams to prevent sequestering of the imaging probe by the liver, we consider the current specific activity as sufficient for clinical translation. [ 66 Ga]Ga-DFO-ZEGFR:2377 retained specific binding to EGFR-expressing cell lines since this binding was saturable with both non-labelled ZEGFR:2377 or anti-EGFR antibody cetuximab ( Figure 2). According to InteractionMap analysis, the binding of [ 68 Ga]Ga-DFO-ZEGFR:2377 to A431 cells was characterized by high affinity (K D1 = 342 ± 15 pM) interaction, which was predominant (Table 1). There was also a weaker (K D1 = 29.2 ± 0.2 nM) but much less abundant interaction. It has been previously shown that the appearance of a weaker interaction is associated with the presence of dimerized EGFR on cancer cells' surface [48]. The internalization of [ 66 Ga]Ga-DFO-ZEGFR:2377 conjugate by cancer cells after binding was slow (Figure 3), similarly to the pattern observed for [ 68 Ga]Ga-DFO-ZEGFR:2377 [28].
Saturation of EGFR in A431 xenografts resulted in significant (p < 0.05) reduction in [ 66 Ga]Ga-DFO-ZEGFR:2377 uptake in tumors (Figures 4 and 8, Panels A and D). This demonstrated that EGFR-specific targeting was preserved in vivo.
The biodistribution data ( Table 2) demonstrated that uptake of [ 66 Ga]Ga-DFO-ZEGFR:2377 at 3 h p.i. was already higher than in the majority of other organs. However, the renal uptake was higher than the tumor uptake, which is attributed to an efficient re-absorption in proximal tubuli. The high renal uptake is typical for affibody molecules [22]. However, clinical studies with HER2-targeting affibody molecules [49] demonstrated that the high renal uptake does not complicate imaging of metastases in the lumbar area, including adrenal metastases. An interesting finding was a rapid clearance of activity from kidneys (i.e., six-fold between 3 and 24 h). This is unusual for radiometal-labelled affibody molecules. In the case of [ 111 In]In-DOTA-ZEGFR:2377, [23] the renal activity reduced only 1.3-fold between 4 and 24 h p.i.; in the case of [ 57 Co]Co-DOTA-ZEGFR:2377 [27], the reduction between 3 and 24 h was 1.2-fold. The renal retention of activity was much lower for [ 66 Ga]Ga-DFO-ZEGFR:2377 in this study (Table 2 and Figure 8A-C). Such a rapid clearance suggests that the residualizing properties of the [ 66 Ga]Ga-DFO-label are not very strong. Accordingly, there was also some release of [ 66 Ga]Ga-DFO-ZEGFR:2377 activity from tumors (6.5 ± 1.3% ID/g at 3 h p.i. and 3.7 ± 0.5% ID/g at 24 h p.i.). However, the retention of activity in tumor was sufficient to provide a significant (p < 0.05) increase in tumor-to-blood and tumor-to-liver and tumor-to-kidney ratios at 24 h compared with 3 h p.i. (Table 2). This is essential as blood-borne activity contributes to background in all tissues, and the liver often harbors metastases. Thus, the use of a long-lived radionuclide permits improvement in the imaging contrast and therefore, the sensitivity of the imaging of EGFR.
Expression of EGFR in the liver of young rodents can differ depending on their age [50]. These changes are difficult to control. Accordingly, an injected protein dose that was optimal in one study might result in an excessive breakthrough of a radioligand through the "liver barrier" and partial saturation of receptors in tumor leading to decreased tumor uptake in another study. Particularly, the tumor uptake of [ 68 Ga]Ga-DFO-ZEGFR:2377 in this study was nearly two-fold lower than in the previous one [28]. This resulted in a notable reduction of tumor-to-blood and tumor-to-muscle ratios compared with previous data. However, the comparison between [ 66 Ga]Ga-DFO-ZEGFR:2377, [ 68 Ga]Ga-DFO-ZEGFR:2377 and [ 89 Zr]Zr-DFO-ZEGFR:2377 in this study was performed head-to-head, in the same batch of tumor-bearing mice. This permits a reliable comparison between tested compounds since the batch-to-batch variability of the animal physiology or expression of EGFR in normal organs influences all imaging probes in a similar fashion. For [ 68 Ga]Ga-DFO-ZEGFR:2377, a 3 h p.i. time point was selected for comparison. This is nearly three half-lives of the radionuclide. Performing imaging any later would necessitate injection of higher activity, which would increase an absorbed dose burden to patients. Data from this study showed ( Figure 7B) that imaging at 24 h using [ 66 Ga]Ga-DFO-ZEGFR:2377 would result in appreciably higher tumor-to-blood, tumor-to-liver and tumor-to-bone ratios compared with [ 89 Zr]Zr-DFO-ZEGFR:2377 at the same time point. 89 Zr has a sufficiently long half-life (78.4 h) to permit imaging at 24 h p.i. This radionuclide is commercially available, which simplifies its application for imaging. DFO is a commonly used chelator for labelling of proteins with 89 Zr [51]. We have demonstrated that the use of [ 89 Zr]Zr-DFO-ZEGFR:2377 permits higher contrast in imaging of EGFR expression in xenografts than 89 Zr-labelled antibody cetuximab [31]. However, our earlier studies [28] have demonstrated that 3 h p.i., [ 68 Ga]Ga-DFO-ZEGFR:2377 provides higher tumor-to-organ ratio. This study has demonstrated that [ 66 Ga]Ga-DFO-ZEGFR:2377 provides significantly (p < 0.05) higher tumor-to-blood, tumor-to-liver, tumor-to-kidneys, tumor-to-muscle, and tumor-to-bone ratios than [ 89 Zr]Zr-DFO-ZEGFR:2377 at 24 h p.i. ( Figure 7B). Thus, 66 Ga is a more suitable radionuclide for imaging using DFO-ZEGFR:2377 at 24 h after injection than 89 Zr.

Conclusions
The Thus, selection of a radionuclide with an optimal half-life and chemical properties can appreciably improve imaging properties of EGFR-targeting affibody molecules.