Evaluation of 11C-NR2B-SMe and its Enantiomers as PET Radioligands for Imaging the NR2B Subunit within the NMDA Receptor Complex in Rats

1212 Objectives: The pretargeting strategy, which has been proposed as an alternative to radioimmunotherapy, increases the absorbed dose to the tumor while reducing the side effects of normal tissues, resulting in a high therapeutic effect. From the early development stage, the combination of streptavidin and biotin for in vivo binding, which controls the pharmacokinetics and therapeutic effects, has been expected to have clinical application owing to its high affinity for the recognition pair. However, due to the strong immunogenicity of streptavidin in vivo, the streptavidin/biotin pair has not been studied extensively. Hence, we designed Cupid, which is a mutated streptavidin with low immunogenicity and can fuse to four single-chain Fv fragments (scFv), and synthesized Psyche, which is a corresponding biotin variant with a specific affinity to Cupid (Sugiyama et al., Prc. Jpn. Acad. Ser. B 95:602, 2019). In this study, in addition to using these combinations, we labeled Psyche with an alpha-emitting radionuclide 211At, which is expected to have high therapeutic effects in the tumor and fewer side effects on the surrounding tissue in vivo. The aim of this study was to evaluate the labeling, stability, and pharmacokinetics of 211At labeled Psyche and scFV-Cupid in mice. Methods: 211At was produced using 209Bi(α, 2n)211At reaction and isolated through dry distillation. 80 MBq of 211At was labeled to Psyche derivatives (Psyche-B) and purified as a no-carrier-added product using Reversed-Phase HPLC. For the stability evaluation, 211At-Psyche-B was incubated with PBS and serum for 24 h. In total, 1×107 human gastric cancer cells (MKN-45) with a high expression of CEA were xenografted subcutaneously in male, 9-week-old BALB/c nu/nu mice. Thirteen days after the transplantation, 100 pmol of anti-CEACAM5 scFv-Cupid was intravenously administered to mice. After an interval of 24 h, 250 kBq of 211At labeled Psyche-B was intravenously administered to mice. The mice were sacrificed 1 min, 1 h, 6 h, and 24 h after the 211At-Psyche-B injection (n = 3 in each group). Samples were collected from the blood, brain, thyroid, heart, lung, liver, spleen, stomach, small intestine, pancreas, kidney, adrenal gland, muscle, bone, urine, and feces. Subsequently, they were weighed and measured for radioactivity using a gamma counter. Results: The radiochemical yield of 211At-Psyche-B was 96%, as determined by a radio-HPLC analysis of the crude product. 211At-Psyche-B showed high stability in PBS after 24 h but dissociated in serum within 1 h. In the biodistribution study, the clearance of 211At-Psyche-B from blood was 46.4 ± 1.4%ID/g after 1 min, 40.6 ± 3.2 %ID/g after 1 h, 29.4 ± 0.9 %ID/g after 6 h, and 6.2 ± 1.3 %ID/g after 24 h. The accumulation of 211At-Psyche-B in blood decreased with time, and approximately 94% of the injected dose was excreted within 24 h. The accumulation of 211At-Psyche-B in the tumor was 0.6 ± 0.1 %ID/g in 1 min, 3.2 ± 0.4 %ID/g in 1 h, 11.8 ± 1.5 %ID/g in 6 h, and 16.3 ± 4.1 %ID/g in 24 h. The tumor-to-blood ratios were 0.01 ± 0.00 after 1 min, 0.08 ± 0.01 after 1 h, 0.40 ± 0.05 after 6 h, and 2.63 ± 0.10 after 24 h. 211At-Psyche-B showed high liver uptakes in the experiment period: 17.4 ± 2.3 %ID/g in 1 min, 13.6 ± 1.4 %ID/g in 1 h, 16.0 ± 1.4 %ID/g in 6 h, and 15.5 ± 0.1 %ID/g in 24 h. 211At-Psyche-B uptakes in the spleen were 4.9 ± 1.7 %ID/g in 1 min, 7.9 ± 1.1 %ID/g in 1 h, 10.1 ± 0.3 %ID/g in 6 h, and 9.6 ± 2.4 %ID/g in 24 h. 211At-Psyche-B uptakes in the kidney were 29.3 ± 1.5 %ID/g in 1 min, 14.2 ± 1.3 %ID/g in 1 h, 9.8 ± 0.7 %ID/g in 6 h, and 4.7 ± 0.8 %ID/g in 24 hours. Conclusion: Although 211At-Psyche-B showed low serum stability in vitro, tumor uptake was confirmed and accumulation increased over time in vivo. Thus, this study suggested that our pre-targeting strategy of low-immunogenic CEA scFv-Cupid and 211At labeled Psyche might work for advanced cancer treatment. Acknowledgments: This work was financially supported by AMED under Grant Number JP19ck0106414.