Therapeutic Potential of 47Sc in Comparison to 177Lu and 90Y: Preclinical Investigations

Targeted radionuclide therapy with 177Lu- and 90Y-labeled radioconjugates is a clinically-established treatment modality for metastasized cancer. 47Sc is a therapeutic radionuclide that decays with a half-life of 3.35 days and emits medium-energy β−-particles. In this study, 47Sc was investigated, in combination with a DOTA-folate conjugate, and compared to the therapeutic properties of 177Lu-folate and 90Y-folate, respectively. In vitro, 47Sc-folate demonstrated effective reduction of folate receptor-positive ovarian tumor cell viability similar to 177Lu-folate, but 90Y-folate was more potent at equal activities due to the higher energy of emitted β−-particles. Comparable tumor growth inhibition was observed in mice that obtained the same estimated absorbed tumor dose (~21 Gy) when treated with 47Sc-folate (12.5 MBq), 177Lu-folate (10 MBq), and 90Y-folate (5 MBq), respectively. The treatment resulted in increased median survival of 39, 43, and 41 days, respectively, as compared to 26 days in untreated controls. There were no statistically significant differences among the therapeutic effects observed in treated groups. Histological assessment revealed no severe side effects two weeks after application of the radiofolates, even at double the activity used for therapy. Based on the decay properties and our results, 47Sc is likely to be comparable to 177Lu when employed for targeted radionuclide therapy. It may, therefore, have potential for clinical translation and be of particular interest in tandem with 44Sc or 43Sc as a diagnostic match, enabling the realization of radiotheragnostics in future.

TFA in MilliQ water (A) and ACN (B) using a linear gradient of solvent A (95-20% over 15 min) in solvent B at a flow rate of 1 mL/min. An aliquot (~0.3 MBq) of the radiolabeling solution was diluted in 100 µL MilliQ water containing sodium diethylenetriamine pentaacetic acid (Na-DTPA; 50 µM) for analysis.
Results: Representative chromatograms of 47 Sc-folate, 177 Lu-folate and 90 Y-folate are shown in Figure S2. No difference in the retention time (tR = 11.4 ± 0.1 min) was observed among the radiofolates. All radiofolates were obtained at a radiochemical purity ≥97% and were used for in vitro and in vivo evaluation without further purification.

Cell Internalization Experiments
Purpose: Cell experiments were performed to demonstrate FR-specific uptake and internalization of the folate conjugate labeled with the radionuclides in question ( 47 Sc, 177 Lu and 90 Y) using FR-positive IGROV-1 ovarian cancer cells.
Methods: IGROV-1 tumor cells were seeded in 12-well plates (0.5 × 10 6 cells in 2 mL FFRPMI medium/well) allowing cell adhesion and growth overnight at 37 °C, 5% CO2. After removal of the supernatant, the cells were washed once with PBS prior to the addition of FFRPMI medium without supplements (975 µL/well). 47 Sc-folate, 177 Lu-folate or 90 Y-folate were labeled at a molar activity of 10 MBq/nmol. The radiofolates (25 µL, ~38 kBq, ~3.75 pmol) were added to each well. In some cases, cells were incubated with excess folic acid (100 µM) to block FRs on the surface of the cancer cells. After incubation of the well plates for 4 h at 37 °C, the cells were washed with ice-cold PBS to determine total uptake of 47 Sc-folate, 177 Lu-folate and 90 Y-folate. In order to assess the fraction of internalized radiolabeled folates, the cells were additionally washed with a stripping buffer (aqueous solution of 0.1 M acetic acid and 0.15 M NaCl, pH 3 [3]) to release FR-bound radiofolates from the cell surface. The tumor cells were lysed by addition of NaOH (1.0 M, 1 mL) to each well, followed by transfer of the cell suspensions to tubes for measuring in a γ-counter (Perkin Elmer, Wallac Wizard 1480). The results were expressed as percentage of total added activity and graphs were prepared using GraphPad Prism software (version 7.0).

Results:
The results are reported in the main article ( Figure 1).

Biodistribution Study
Purpose: Time-dependent biodistribution experiments were performed with 177 Lu-folate in IGROV-1 tumor-bearing mice in order to be able to estimate the mean absorbed dose. It was assumed that the distribution profile of 47 Sc-folate and 90 Y-folate would be equal to the distribution of 177 Lufolate.
Methods: Biodistribution studies were performed as previously reported by Siwowska et al. [4]. Results: Biodistribution data obtained at 1 h, 4 h, 24 h, 48 h, 4 d, 5 d, 7 d and 10 d after administration of 177 Lu-folate (as a representative for all radioconjugates), using IGROV-1 tumorbearing mice, are listed in Tables S1/S2. The tumor uptake increased from 29 ± 3% IA/g at 1 h p.i. to 33 ± 9% IA/g at 4 h p.i. and reached the highest value 24 h after injection of the radiofolate (39 ± 8% IA/g) ( Table S1). The accumulation of the activity in the tumor began to drop at 48 h p.i. and decreased to 3.5 ± 1.8% IA/g at the last investigated time point, 10 days after injection (Table S2). Kidney accumulation was as prominent as for the tumor and increased up to 24 h after injection, when it reached 35 ± 10% IA/g. Nevertheless, the highest tumor-to-kidney ratio of accumulated activity was observed after 4 h (1.2 ± 0.5). In the blood, radioactivity was present at significant amounts until 4 h p.i. and dropped to 0.82 ± 0.16% IA/g at 24 h p.i.. Radioactivity was also detected in salivary glands, lung and liver, however, it decreased rapidly over time. Values shown represent the mean ± S.D. of data from three animals (n = 3). Values shown represent the mean ± S.D. of data from three animals (n = 3).

Dosimetry Estimation
Purpose: Dosimetry calculations were performed in order to estimate the quantity of activity of 47 Sc-folate, 177 Lu-folate or 90 Y-folate, respectively, which had to be injected to obtain the same mean absorbed dose to tumors and kidneys.
Methods: The dosimetry method is described in the main article. Results: A mono-exponential fit was applied to the biokinetic data. The time integrated activity concentration for 47 Sc, 177 Lu and 90 Y in the tumor were equal to 7.3 × 10 4 , 9.4 × 10 4 and 6.5 × 10 4 decay/g, respectively. The time-integrated activity concentration for 47 Sc, 177 Lu and 90 Y in the kidneys were equal to 7.6 × 10 4 , 10 × 10 4 and 6.6 × 10 4 decay/g, respectively. The absorbed electron energy fractions for the tumors were 0.89, 0.93 and 0.44 for 47 Sc, 177 Lu and 90 Y, respectively. The absorbed electron energy fractions for the kidneys were 0.91, 0.95 and 0.45 for 47 Sc, 177 Lu and 90 Y, respectively. The specific mean absorbed doses are presented in the main article.
To obtain the dose of ~21 Gy in the tumors, mice had to be treated with 12.5 MBq, 10 MBq and 5 MBq of 47 Sc-folate, 177 Lu-folate and 90 Y-folate, respectively (Table S3). This quantity of injected activity resulted in a mean absorbed dose of ~22−23 Gy to the kidneys, which should -according to previously published data [5]-not cause radionephrotoxicity.

Therapy Experiments
Purpose: Therapy experiments were performed to investigate the tumor growth delay in each of the treated groups of mice and compare it with untreated controls. Moreover, the body weight was monitored and blood plasma parameters assessed for each mouse after euthanasia.
Methods: The detailed methods of monitoring mice regarding the tumor growth and body weights are described in the main article. When an endpoint criterion was reached, blood was sampled from the retrobulbar vein immediately before euthanasia and plasma parameters indicative of kidney or liver damage were measured.
Results: The tumor growth curves of each group, presented as the average tumor volume of all mice per group as well as for each single mouse, are shown in Figure S3. The relative body weights (RBW) of each group, presented as the average RBW of all mice per group as well as for each single mouse, are shown in Figure S4. Creatinine (CRE) levels were below the detection limit in all investigated animals (Table S4). Blood urea nitrogen (BUN) levels were comparable among all mice (5.2-7.2 mmol/L). The alkaline phosphatase (ALP) levels were in the same range (40 ± 6 to 57 ± 10 U/L) for all mice and the same held true for total bilirubin (TBIL) levels (3.3 ± 0.8 to 5.5 ± 1.1 µmol/L) (Table S4). Table S4. Plasma parameters measured in the blood plasma of mice at the endpoint.

Toxicity Assessment
Purpose: Non-tumor-bearing mice were euthanized 2 weeks after radiofolate injection at two different activity levels, followed by assessment of the histopathology. The aim was to determine potential short-term histological changes in the kidneys, bone marrow and spleen.
Methods: Non-tumor-bearing mice were injected intravenously with saline or with different activities of 47 Sc-folate, 177 Lu-folate and 90 Y-folate, respectively (Table S5). Body weights were measured twice a week. All mice were euthanized two weeks after the injection of the respective radiofolate. Detailed methods of the pathohistological assessment are reported in the main article.

Results:
The results of the scoring as a measure of potential side effects are listed in Table S6 and the results are reported in the main article. Representative H/E stained kidney sections of each group are shown in Figure S5. 0 0 (9/9) 0 (9/9) 0 (9/9) 1 (1/9) 2 (6/9) 3 (