Preclinical Evaluation of [155/161Tb]Tb-Crown-TATE—A Novel SPECT Imaging Theranostic Agent Targeting Neuroendocrine Tumours

Terbium radioisotopes (149Tb, 152Tb, 155Tb, 161Tb) offer a unique class of radionuclides which encompass all four medicinally relevant nuclear decay modalities (α, β+, γ, β−/e−), and show high potential for the development of element-matched theranostic radiopharmaceuticals. The goal of this study was to design, synthesise, and evaluate the suitability of crown-TATE as a new peptide-conjugate for radiolabelling of [155Tb]Tb3+ and [161Tb]Tb3+, and to assess the imaging and pharmacokinetic properties of each radiotracer in tumour-bearing mice. [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE were prepared efficiently under mild conditions, and exhibited excellent stability in human serum (>99.5% RCP over 7 days). Longitudinal SPECT/CT images were acquired for 155Tb- and 161Tb- labelled crown-TATE in male NRG mice bearing AR42J tumours. The radiotracers, [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE, showed high tumour targeting (32.6 and 30.0 %ID/g, respectively) and minimal retention in non-target organs at 2.5 h post-administration. Biodistribution studies confirmed the SPECT/CT results, showing high tumour uptake (38.7 ± 8.0 %ID/g and 38.5 ± 3.5 %ID/g, respectively) and favourable tumour-to-background ratios. Blocking studies further confirmed SSTR2-specific tumour accumulation. Overall, these findings suggest that crown-TATE has great potential for element-matched molecular imaging and radionuclide therapy using 155Tb and 161Tb.


Introduction
Peptide receptor radionuclide therapy (PRRT) with complementary diagnostic imaging is a highly effective approach for the treatment of advanced neuroendocrine tumours (NETs) and has shown high response rates in patients previously refractory to conventional somatostatin therapy [1][2][3]. This approach involves the systemic administration of therapeutic radiopharmaceuticals which enable the selective localisation of ionising radiation to tumour sites through peptide-based molecular recognition of cell-surface receptors. The successful application of PRRT in the clinic is exemplified by [ 177 Lu]Lu-DOTA-TATE (Lutathera ® ), which has shown high patient efficacy and was approved by the U.S. Food and Drug Administration (FDA) in 2018 following the NETTER-1 Phase III clinical trial. This treatment is suitable for early stage (G1-G2) progressive, well-differentiated gastroenteropancreatic (GEP) NETs [4]. A complete course of treatment (4 × 7.4 GBq, 8-week cycle) with [ 177 Lu]Lu-DOTA-TATE achieves a median progression free survival of 28.4 months [4] and shows improvements in median overall survival (OS) by 11.7 months compared to the control group; however, this difference was not statistically significant. Additional treatment options are required for patients who experience relapse, show poor tolerance, or do not respond to treatment [5][6][7].
Targeted alpha therapy (TAT) utilising α-emitting radionuclides ( 225 Ac, 227 Th, 230 U, 213 Bi, 211 At) is being intensively pursued in the emerging radiopharmaceutical sector as a potential alternative to conventional β-therapy for treatment of systemic malignancies, with several Phase II clinical trials underway ([ 225 Ac]Ac-DOTA-TATE) [8,9]. The high potency of alpha radiation has achieved tumour regression in patients who did not respond to 177 Lu treatments [10,11]. To fully capitalise on the potential benefits offered by TAT, accurate personalised dosimetry is crucial. Terbium radionuclides ( 149 Tb, 152 Tb, 155 Tb, 161 Tb) are particularly attractive in this context, owing to their diverse nuclear decay characteristics (α, PET, SPECT, β − /e − , respectively) and myriad of half-lives (4.12 h, 17.5 h, 5.32 d, 6.95 d, respectively), which may allow advancement of element-matched theranostic radiopharmaceuticals [10][11][12]. From this quartet of radioisotopes, 155 Tb and 161 Tb are a particularly appealing theranostic pair, with 155 Tb (t 1/2 = 5.32 d) being suitable for single photon emission computed tomography (SPECT) diagnostics and 161 Tb (t 1/2 = 6.95 d) being suitable for β-therapy. 161 Tb shares similar decay characteristics to 177 Lu and is being pursued as a more potent therapeutic radionuclide owing to the additional Meitner-Auger electron (MAE) emissions and low energy γ-ray emissions which are suitable for post-therapy whole-body SPECT scintigraphy [13,14]. Furthermore, 155 Tb also generates several MAEs in its decay scheme which are of sufficient energy and intensity for therapeutic applications, making 155 Tb a potential standalone theranostic radionuclide. In addition, expanding the repertoire of imaging radionuclides with longer half-lives (i.e., 155 Tb (t 1/2 = 5.32 d)) than the conventional clinically applied isotopes (i.e., 18 F (t 1/2 = 110 min), 68 Ga (t 1/2 = 68 min), 99m Tc (t 1/2 = 6 h)) is another attractive feature of terbium radioisotopes and would enable diagnostic imaging studies at later timepoints which may be beneficial for determining the ultimate biological fate of long-lived therapeutic radiopharmaceuticals ( 225 Ac, 227 Th, 230 U).
In a previous study, we introduced crown [15] as a new chelating ligand with impressive characteristics for coordinating large radiometal ions, specifically 225 Ac. Crown was shown to form stable, kinetically inert metal complexes with [ 225 Ac]Ac 3+ under mild conditions (ambient temperature, 10 min); a clear advantage over the conventional gold-standard chelator DOTA which requires high temperatures and/or long reaction times to achieve quantitative radiolabelling [15]. Further studies of the corresponding bioconjugate, crown-αMSH, established the in vivo viability of the 225 Ac-labelled chelator, demonstrating effective targeting of subcutaneous melanoma tumours and maintaining high in vivo stability in preclinical studies [15]. Ongoing studies involving an 255 Ac-labelled bioconjugate for TAT of NETs are currently in progress [16].
To further explore the scope of this promising chelator, a new peptide-chelate bioconjugate targeting NETs, referred to as crown-TATE herein, was designed and synthesised. In this research, we investigate for the first time the suitability of crown-TATE for radiolabelling of both [ 155 Tb]Tb 3+ and [ 161 Tb]Tb 3+ , and assess the kinetic inertness of the resulting complexes using human serum stability studies. Preclinical assessments of both [ 155 Tb]Tbcrown-TATE and [ 161 Tb]Tb-crown-TATE were undertaken using longitudinal quantitative SPECT/CT in tumour-bearing mice to establish the imaging potential for each of these radiotracers. Full biodistribution studies were performed on both blocked and unblocked groups to determine the in vivo performance of this new bioconjugate for targeting NETs. Altogether, this study demonstrates the capability of crown-TATE to chelate 155 Tb and 161 Tb effectively and stably, and its feasibility to selectively target AR42J NETs in mice. Overall, this research confirms the in vivo viability of these radiotracers as imaging agents for use in conjunction with 225 Ac TAT or as standalone 155 Tb/ 161 Tb theranostics.

Synthesis of Crown-TATE
The majority of advanced, progressive NETs show overexpression of somatostatin receptors (SSTRs) on the cell surface and are therefore attractive targets for radionuclide imaging and therapy [1,2]. In humans, SSTRs are comprised of five subtypes (SSTR1-5), which are expressed in numerous organ systems including the gastrointestinal tract, pancreas, lungs, and renal organs. Several classes of SSTR2 targeting peptides have been developed including agonists (DOTA-TOC, DOTA-TATE) and antagonists (DOTA-LM3), both of which show high receptor binding affinity with low non-target organ toxicities [13,[17][18][19]. The clinically applied bioconjugate DOTA-TATE (DOTA-[Tyr 3 ]-octreotate) utilises a disulphide-bridged cyclic octapeptide which has been refined through numerous structure activity relationship studies for optimal binding towards SSTR2 [20][21][22]. This optimised peptide sequence was selected for conjugation to crown to enable direct comparison to a clinically established radiopharmaceutical.
Crown-TATE was prepared using standardised Fmoc-based solid phase peptide synthesis (SPPS). Briefly, the linear peptide sequence-[DPhe-Cys(Acm)-Tyr(tBu)-DTrp(tBu)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OH]-was synthesised on Wang resin using PyBop/Oxyma Pure coupling methodology, Figure 1. The linear peptide was cyclised between residues Cys 2 and Cys 7 using thallium (III) trifluoroacetate, which simultaneously cleaves the Sacetamidomethyl (Acm) side chain protecting groups and generates the disulphide-bridged cyclic peptide [23,24]. Following Fmoc deprotection, the crown chelator (crown-tris (tBu ester)) [15] was coupled to the free N-terminus using an extended reaction time (21 h) to ensure complete coupling. Global deprotection of the side chain protecting groups and cleavage from the resin was achieved using a standard cleavage cocktail to give the target bioconjugate which was isolated by reverse-phase high performance liquid chromatography (RP-HPLC). Crown-TATE was purified using RP-HPLC and the corresponding mass confirmed by highresolution electrospray ionisation mass spectrometry (HR-ESI-MS). Characterisation data for crown-TATE is provided in the Supporting Information, Figure S1.

Radiochemistry
2.2.1. Radionuclide Production 155 Tb was produced via the proton-induced spallation of tantalum foil targets using the 480 MeV proton beamline at the Isotope Separator and Accelerator facility with Isotope Separation On-Line (ISOL) at TRIUMF (Canada) [25]. The ISAC/ISOL facility permits the production and separation of singly charged isobaric radioactive ion beams and allowed for the isolation of a single beam with mass-to-charge ratio of 155 A/q containing 155 Tb (t1/2 = 5.32 d), 155 Dy (t1/2 = 9.9 h), 155 Ho (t1/2 = 48 min), and 155 Er (t1/2 = 5.3 min). High

Radionuclide Production
155 Tb was produced via the proton-induced spallation of tantalum foil targets using the 480 MeV proton beamline at the Isotope Separator and Accelerator facility with Isotope Separation On-Line (ISOL) at TRIUMF (Canada) [25]. The ISAC/ISOL facility permits the production and separation of singly charged isobaric radioactive ion beams and allowed for the isolation of a single beam with mass-to-charge ratio of 155 A/q containing 155 Tb (t 1/2 = 5.32 d), 155 Dy (t 1/2 = 9.9 h), 155 Ho (t 1/2 = 48 min), and 155 Er (t 1/2 = 5.3 min). High purity 155 Tb was obtained by beam implantation onto an aluminium target coated with an NH 4 Cl salt layer on the surface. After a 5-day cool-down period, which allowed for decay of 155 Dy to 155 Tb, and decay of short-lived 155 Ho and 155 Er, the activity was easily recovered by dissolution of the salt layer with de-ionised H 2 O to give [ 155 Tb]Tb 3+ in a form that was directly suitable for radiolabelling [25]. 161 Tb was produced via the neutron bombardment of enriched [ 160 Gd][Gd 2 O 3 (98.2%) targets using the high thermal neutron flux (3 × 10 14 neutrons/cm 2 /s) BR2 reactor at SCK-CEN (Belgium) [26] and subsequently purified at TRIUMF using a semi-automated TRASIS system with solid phase extraction (SPE) resins (TK211, TK 212, TK221) [27].

Radiolabelling Studies
The radiolabelling properties of crown-TATE with [ 155/161 Tb]Tb 3+ were evaluated under mild conditions (ambient temperature, 10 min, pH 6.0) and showed quantitative radiolabelling with both [ 155 Tb]Tb 3+ and [ 161 Tb]Tb 3+ , achieving molar activities of 19.4 MBq/nmol and 11.4 MBq/nmol, respectively. Quality control studies were undertaken using radio-HPLC which showed a single sharp peak for the [ 155 Tb]Tb-crown-TATE product (T R = 8.7 min) and no free [ 155 Tb]Tb 3+ activity (T R = 1.0 min) in the final product (>99% radiochemical purity (RCP)), Figure 2A. MBq/nmol and 11.4 MBq/nmol, respectively. Quality control studies were undertaken using radio-HPLC which showed a single sharp peak for the [ 155 Tb]Tb-crown-TATE product (TR = 8.7 min) and no free [ 155 Tb]Tb 3+ activity (TR = 1.0 min) in the final product (>99% radiochemical purity (RCP)), Figure 2A. Human serum contains numerous endogenous proteins (e.g., albumin, transferrin) which can compete with the chelate for the binding of different metal ions. Prior to undertaking any preclinical studies, the kinetic inertness of [ 161 Tb]Tb-crown-TATE was evaluated using a human serum stability challenge assay, Figure 2B. No evidence of transmetalation of bound activity to serum proteins was observed over the course of 7 days (>99.5% RCP); implying excellent overall stability.

SPECT/CT Studies
Preclinical SPECT/CT studies were undertaken to evaluate the potential of [ 155 Tb]Tb-crown-TATE and [ 161 Tb]Tb-crown-TATE for imaging pancreatic exocrine tumours. The 155 Tb/ 161 Tb-labelled radiotracers were each administered to male NRG mice bearing AR42J tumour xenografts (left-shoulder) and a series of dynamic quantitative SPECT/CT scans were acquired from 0 to 2.5 h post-administration, Figures 3 and 4. From the quantitative SPECT/CT scans, mean standardised uptake values (SUVmean) were extracted for regions of interest (ROIs) in tumour, kidneys, and bladder. The quantitative imaging studies enabled direct measurement of the activity concentrations (%ID/g) in ROIs in the SPECT/CT scans over time which can be compared to the ex vivo biodistribution results obtained after euthanising the mice following acquisition of the final SPECT/CT scan (2.5 h p.i.). Full biodistribution results for the imaging studies are provided in the supplementary materials, Figure S3 and Table S1. Human serum contains numerous endogenous proteins (e.g., albumin, transferrin) which can compete with the chelate for the binding of different metal ions. Prior to undertaking any preclinical studies, the kinetic inertness of [ 161 Tb]Tb-crown-TATE was evaluated using a human serum stability challenge assay, Figure 2B. No evidence of transmetalation of bound activity to serum proteins was observed over the course of 7 days (>99.5% RCP); implying excellent overall stability.

SPECT/CT Studies
Preclinical SPECT/CT studies were undertaken to evaluate the potential of [ 155 Tb]Tbcrown-TATE and [ 161 Tb]Tb-crown-TATE for imaging pancreatic exocrine tumours. The 155 Tb/ 161 Tb-labelled radiotracers were each administered to male NRG mice bearing AR42J tumour xenografts (left-shoulder) and a series of dynamic quantitative SPECT/CT scans were acquired from 0 to 2.5 h post-administration, Figures 3 and 4. From the quantitative SPECT/CT scans, mean standardised uptake values (SUV mean ) were extracted for regions of interest (ROIs) in tumour, kidneys, and bladder. The quantitative imaging studies enabled direct measurement of the activity concentrations (%ID/g) in ROIs in the SPECT/CT scans over time which can be compared to the ex vivo biodistribution results obtained after euthanising the mice following acquisition of the final SPECT/CT scan (2.5 h p.i.). Full biodistribution results for the imaging studies are provided in the Supplementary Materials, Figure S3 and Table S1.  Table 1. Quantification of the SPECT/CT scans revealed a tumour uptake of 32.6 %ID/g 2.5 h p.i., which was consistent with the ex vivo biodistribution measurements (3 %ID/g). As anticipated, the SPECT/CT imaging results for [ 161 Tb]Tb-crown-TATE w  Both radiotracers showed fast clearance from the bloodstream, with rapid elimination through the renal pathway, as evidenced by activity in the kidneys and bladder, giving high contrast images. This is further supported by the time-activity curves, Figure  5, where most of each radiotracer was cleared from circulation and tumour uptake reached a plateau within 0.5 h p.i., as shown in Table 1. Comparatively, the preclinical SPECT studies with 155 Tb yielded superior imaging quality over 161 Tb, with higher image contrast and lower signal noise. However, these studies clearly show the suitability of 161 Tb for performing dosimetry and patient monitoring studies following administration of 161 Tb therapy treatments, as is standard practice for 177 Lu therapies.   Table 1. Quantification of the SPECT/CT scans revealed a tumour uptake of 32.6 %ID/g at 2.5 h p.i., which was consistent with the ex vivo biodistribution measurements (31.5 %ID/g). As anticipated, the SPECT/CT imaging results for [ 161 Tb]Tb-crown-TATE were highly comparable to those of the 155 Tb-labelled radiotracer, exhibiting a similar pharmacokinetic profile and high tumour uptake (30.0 %ID/g at 2.5 h p.i.), which was further confirmed in the biodistribution studies (28.6 %ID/g). Both radiotracers showed fast clearance from the bloodstream, with rapid elimination through the renal pathway, as evidenced by activity in the kidneys and bladder, giving high contrast images. This is further supported by the time-activity curves, Figure 5, where most of each radiotracer was cleared from circulation and tumour uptake reached a plateau within 0.5 h p.i., as shown in Table 1. Comparatively, the preclinical SPECT studies with 155 Tb yielded superior imaging quality over 161 Tb, with higher image contrast and lower signal noise. However, these studies clearly show the suitability of 161 Tb for performing dosimetry and patient monitoring studies following administration of 161 Tb therapy treatments, as is standard practice for 177 Lu therapies. Both radiotracers showed fast clearance from the bloodstream, with rapid elimination through the renal pathway, as evidenced by activity in the kidneys and bladder, giving high contrast images. This is further supported by the time-activity curves, Figure  5, where most of each radiotracer was cleared from circulation and tumour uptake reached a plateau within 0.5 h p.i., as shown in Table 1. Comparatively, the preclinical SPECT studies with 155 Tb yielded superior imaging quality over 161 Tb, with higher image contrast and lower signal noise. However, these studies clearly show the suitability of 161 Tb for performing dosimetry and patient monitoring studies following administration of 161 Tb therapy treatments, as is standard practice for 177 Lu therapies.

Biodistribution Studies
Full biodistribution studies were performed for the 155 Tb-and 161 Tb-radiolabelled bioconjugates in tumour-bearing mice at 2 h post-administration, Figure 6. Mice were administered with either [ 155 Tb]Tb-crown-TATE (~175 kBq, 93 pmol per animal) or [ 161 Tb]Tbcrown-TATE (~850 kBq, 76 pmol per animal). Additional blocking control studies were undertaken to confirm SSTR2 specific uptake by co-administration of excess unlabelled DOTA-TOC (23 nmol per animal) with each radiotracer. Full biodistribution results for each radiotracer are provided in the Supporting Information, Figure S4 and Table S2. The ex vivo biodistribution results show good consistency with the SPECT/CT studies, with high tumour uptake for both [ 155 Tb]Tb-crown-TATE (38.7 ± 8.0 %ID/g) and [ 161 Tb]Tb-crown-TATE (38.5 ± 3.5 %ID/g), and elimination primarily through the renal pathway. Blocking control studies confirmed tumour uptake of each radiotracer was SSTR2 specific (p < 0.001 and p < 0.0001, respectively). Additionally, statistically significant decreases in radiotracer uptake by non-target organs expressing SSTR2 (lungs, stomach, pancreas, adrenals glands, and intestines) are observed in the blocking studies, further confirming SSTR2 specific uptake. Although [ 155 Tb]Tb-crown-TATE showed an 86% reduction in tumour uptake with co-administration of the blocking agent, and a comparable decrease of 81% was demonstrated for [ 161 Tb]Tb-crown-TATE, the blocking The ex vivo biodistribution results show good consistency with the SPECT/CT studies, with high tumour uptake for both [ 155 Tb]Tb-crown-TATE (38.7 ± 8.0 %ID/g) and [ 161 Tb]Tbcrown-TATE (38.5 ± 3.5 %ID/g), and elimination primarily through the renal pathway. Blocking control studies confirmed tumour uptake of each radiotracer was SSTR2 specific (p < 0.001 and p < 0.0001, respectively). Additionally, statistically significant decreases in radiotracer uptake by non-target organs expressing SSTR2 (lungs, stomach, pancreas, adrenals glands, and intestines) are observed in the blocking studies, further confirming SSTR2 specific uptake. Although [ 155 Tb]Tb-crown-TATE showed an 86% reduction in tumour uptake with co-administration of the blocking agent, and a comparable decrease of 81% was demonstrated for [ 161 Tb]Tb-crown-TATE, the blocking groups did not demon-strate complete knock-down of tumour uptake for [ 155 Tb]Tb-crown-TATE (5.51 %ID/g) or [ 161 Tb]Tb-crown-TATE (7.50 %ID/g); this result was anticipated since the blocking agent was co-administered with each radiotracer injection, rather than using a pre-blocking approach. This is comparable to previous reports which demonstrated that co-injection of unlabelled TATE decreased tumour uptake of [ 64 Cu]Cu-DOTATATE by 83% in mice at 4 h post-injection [28].

General
All solvents and reagents were purchased from commercial suppliers (Sigma-Aldrich (Markham, ON, Canada), (Alfa Aesar, Tewksbury, MA, USA) and used directly without further purification. Crown-tris(tBu ester) was prepared as previously described [15]. Radiolabelling studies were monitored using instant thin-layer chromatography (iTLC) with silica gel (SG)-impregnated paper TLC plates (Agilent technologies, Santa Clara, CA, USA). TLC imaging was performed using an AR-2000 imaging scanner (Eckert & Ziegler, Berlin, Germany) equipped with P-10 gas, and radiochemical yields (RCYs) analysed using WinScan V3_14 software. Radio-HPLC was carried out using an Agilent 1200 instrument equipped with a Phenomenex Luna C18 reverse phase column (100 × 46 mm, 5 µm) and a GABI star radioactive HPLC flow monitor (Elysia-raytest GmbH, Germany). Radioactivity was quantified using a calibrated high-purity germanium (HPGe) detector (Mirion Technologies (Canberra) Inc., Meriden, CT, USA) with Genie 2000 software. All work with radionuclides at TRIUMF was undertaken in shielded fume hoods to minimise the dose to experimenters (and special precautions were used to prevent contamination) under nuclear energy worker (NEW) status earned by attending TRIUMF's Advanced Radiation Protection course and passing the final exam. Peptides were prepared using a Focus Xi semi-automated solid phase peptide synthesiser (AAPPTec, Louisville, KY, USA). SPECT/CT studies were performed using a multimodal VECTor/CT system (MILabs, Houten, Netherlands) in combination with an extra ultra-high sensitivity (XUHS) pinhole collimator (2-mm). Image analysis was performed using AMIDE (v. 1.0.5) software [30].

[ 155 Tb]Tb 3+
155 Tb was produced via proton-induced spallation of tantalum foil targets using the 480 MeV proton beamline at the Isotope Separator and Accelerator facility with Isotope Separation On-Line (ISOL) at TRIUMF (Canada) [25]. Radioisotopes produced via the ISAC/ISOL technique are liberated from the target material by diffusion and effusion processes under ultra-high vacuum at high temperature (2300 • C) [31]. Released radioisotopes are then ionised in a combined surface and laser ion source and accelerated under high voltage to produce a heterogenous radioactive ion beam which is passed through a pre-separator magnet, followed by a high-resolution mass separator magnet to give a singly charged, isobaric ion beam. For 155 Tb, a radioactive ion beam with mass-to-charge ratio of 155 A/q is separated. The ion beam was implanted onto aluminium targets with an NH 4 Cl salt layer deposited on the surface and retrieved following a 5-day cool-down period after implantation which allows for decay of short-lived radionuclides ( 155 Ho, 155 Er) and ingrowth of 155 Tb from 155 Dy decay. The activity is then easily recovered by dissolution of the NH 4 Cl salt layer using de-ionised H 2 O and yields high-purity [ 155 Tb]Tb 3+ at high specific activity (~175 MBq/184 µL) which can be used directly without additional purification [25,32]. Typical production yields of 155 Tb were~300 MBq, with no detectable isotopic impurities as determined by gamma spectroscopy.

Radiolabelling Studies
Initial radiolabelling studies of crown-TATE with [ 155 Tb]Tb 3+ and [ 161 Tb]Tb 3+ were performed to establish suitable labelling conditions and quality control methods.

Human Serum Stability
For human serum stability challenge studies, a high activity stock solution of [ 161 Tb]Tbcrown-TATE (~1 MBq, 100 µL) was prepared using a similar procedure as outlined above. Aliquots of [ 161 Tb]Tb-crown-TATE (~300 kBq, 30 µL) were diluted into pooled human serum (270 µL) and incubated at 37 • C for 7 days with gentle mechanical shaking. All studies were performed in triplicate (n = 3). The radiochemical purity was determined using radio-TLC measurements under the same conditions as outlined above.

Tumour Inoculation
Male NRG mice (24-weeks old) were inoculated with AR42J exocrine pancreatic tumour cells (3 × 10 6 ) on the left shoulder and waited for tumours to grow to reach 8-10 mm in diameter for subsequent experiments.

Radiotracer Preparation
All sample preparations were performed using low retention pipette tips and lowprotein binding Eppendorf tubes to minimise losses of the radiolabelled bioconjugates through surface adsorption. [ 155 Tb]Tb-crown-TATE was prepared by addition of [ 155 Tb]Tb 3+ (19.5 MBq, 35 µL) to a solution containing NH 4 OAc (0.5 M, pH 6.0, 10 µL) and ultra-pure deionised H 2 O (10 µL). Crown-TATE (10 −3 M, 1 µL, 1 nmol) was added and the reaction mixture agitated at 40 • C for 30 min to ensure quantitative radiolabelling. Reaction completion was confirmed using radio-TLC. To minimise the presence of any free metal ions and other impurities in the final preparation, the entire solution of quantitatively radiolabelled [ 155 Tb]Tb-crown-TATE (60 µL) was loaded onto a Sep-Pak ® C18 Plus Light Cartridge (Waters TM ) (pre-conditioned with EtOH (5 × 1 mL), then saline (5 × 1 mL)). The reaction vessel was rinsed with saline (2 × 200 µL) and loaded onto the Sep-Pak cartridge to ensure quantitative transfer. The Sep-Pak ® cartridge was washed with saline (5 mL) and the purified product eluted with an EtOH/saline solution (9:1; 2 × 500 µL).  [33,34]. After the first scan, mice were allowed to roam freely in their cages, and further static SPECT/CT scans were acquired at 2.5 h p.i. SPECT data were reconstructed with the pixel-based ordered subset expectation maximisation (POSEM) algorithm, with a voxel size of 0.4 mm 3 , 16 subsets, and 6 iterations. For 155 Tb, three photopeaks were detected: 44, 85, and 106 keV, while for 161 Tb, the photopeaks were 25, 47, and 75 keV. Reconstructions were carried out using photopeaks of 44 and 47 keV, respectively, for 155 Tb and 161 Tb, with a spectral width of 50%. Each imaging scan was decay corrected to the time of injection and recorded counts were adjusted using attenuation factors determined from CT scans at each time point. After acquisition of the final SPECT/CT scans, mice were kept under isoflurane and promptly euthanised by CO 2 asphyxiation, followed by cardiac puncture to recover blood activity, and relevant organs were harvested for biodistribution studies. Point source measurements of known samples of 155 Tb and 161 Tb were used to determine calibration factors relating counts/voxel to activity concentration prior to performing the preclinical studies. From the quantitative SPECT/CT scans, mean standardised uptake values (SUV mean ) were extracted for volumes of interest (VOIs) in the tumour, kidneys, and bladder, using AMIDE software (v. 1.0.5) [30]. SUV mean is defined as follows: SUV mean (g/mL) = [radioactivity concentration (MBq/mL)]/[injected dose (MBq)/animal weight (g)]. The %ID/g (injected dose/gram body tissue) in relevant organs was calculated by assuming a tissue density of 1 g/mL directly from the quantitative SPECT/CT scans using the following formula: %ID/g = [radioactivity concentration (MBq/mL) in VOIs]/[injected dose (MBq)] × 100. Maximum intensity projections (MIPs) for each time-point were generated using AMIDE software [30], where a Gaussian filtering of FWHM = 2 mm was used for image rendering.

Biodistribution Studies
For biodistribution studies, experiments were performed using four replicates (n = 4) for each unblocked group and three replicates (n = 3) in the blocked groups. [ 155 Tb]Tbcrown-TATE (~175 kBq, 93 pmol, 100 µL) or [ 161 Tb]Tb-crown-TATE (~850 kBq, 76 pmol, 100 µL) was administered intravenously via the lateral tail using a Tailveiner restrainer. After administration, mice were allowed to roam freely in their cages. Biodistribution studies were performed at 2 h post-administration of [ 155 Tb]Tb-crown-TATE or [ 161 Tb]Tbcrown-TATE. Mice were euthanised by CO 2 asphyxiation under 2% isoflurane anaesthesia, followed by cardiac puncture to recover activity in the blood pool. Organs of interest were completely harvested, weighed, and the activity measured using a gamma counter (Packard Cobra II, Perkin Elmer, Waltham, MA, USA). To account for organs that could not be fully extracted (such as blood, muscle, and bone), standardised organ weights for ageand sex-matched mice were used to correct the data [35,36]. The results are reported as the percentage of the injected dose per gram (%ID/g) and per organ (%ID/organ).

Statistical Analysis
Statistical analysis was performed in Microsoft Excel and presented as average ± standard deviation. p-values were calculated using Welch's t-test for two-samples with unequal variances.

Conclusions
While it may seem reasonable to expect that the biodistribution and pharmacokinetic profiles of the same bioconjugate labelled with either [ 155 Tb]Tb 3+ or [ 161 Tb]Tb 3+ would be identical, it is important to validate this assumption, since the production methods used for each radionuclide involve different nuclear reactions (proton spallation vs. neutron irradiation) and purification strategies (ISOL vs. SPE/HPIC), which result in products of differing radionuclidic purity and specific activities.
In this proof-of-principle study, crown-TATE was synthesised for the first time and evaluated as a new bioconjugate targeting SSTR2-positive NETs. The results showed that crown-TATE can be efficiently radiolabelled with both [ 155 Tb]Tb 3+ and [ 161 Tb]Tb 3+ under mild conditions, and in vivo assessment demonstrated excellent tumour imaging contrast with minimal background organ uptake. Our investigation demonstrates the capability of crown-TATE to provide two new element-matched radiotracers which exhibit equivalent biodistribution and pharmacokinetic properties in vivo when radiolabelled with either 155 Tb or 161 Tb. The interchangeable metal complexation properties of the crown chelator provide a suitable platform for developing 155 Tb as a diagnostic companion for 225 Ac targeted alpha therapy. In addition, the favourable imaging properties of both terbium radionuclides will enable accurate staging of disease progression and assessment of patient suitability for treatment using 155 Tb, as well as post-therapy scintigraphy following 161 Tb administration. Further work focused on 225 Ac-labelled crown-TATE is of high interest and will expand the applicability of this bioconjugate for more effective and personalised treatments of NETs. Additional therapy studies involving [ 161 Tb]Tb-crown-TATE are the subject of ongoing research, which will allow direct comparison of the therapeutic efficacy of Meitner-Auger electron emitters versus alpha emitters.