Preclinical immunoPET imaging of thyroid-stimulating hormone receptor expression in thyroid cancer using [64Cu]Cu-NOTA-TSHR-Ab

Purpose The thyroid-stimulating hormone receptor (TSHR) is a cell-surface receptor predominantly expressed on benign and malignant thyroid cells, making it a viable target for developing radiotheranostics for metastatic thyroid cancer imaging and therapy. This study reports the radiosynthesis and preclinical evaluation of a ⁶⁴ Cu-labelled human antibody for positron emission tomography (PET) imaging of TSHR expression in thyroid cancer mouse models. Methods Human anti-TSHR recombinant antibody K1-70™ (TSHR-Ab) was conjugated with a bifunctional chelator, 2- S -(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid ( p -SCN-Bn-NOTA), and then labelled with [ ⁶⁴ Cu]Cu ²⁺ , yielding radiotracer [ ⁶⁴ Cu]Cu-NOTA-TSHR-Ab. The specificity and binding affinity of the radiotracer to TSHR-positive (THJ529T ^TSHR(+) ) or wild-type (THJ529T ^WT ) cells were assessed in vitro . A series of immnuoPET imaging with [ ⁶⁴ Cu]Cu-NOTA-TSHR-Ab were conducted in mice bearing dual-flank tumors (THJ529T ^WT on the left and THJ529T ^TSHR(+) on the right) at multiple time points (1, 2, 4, 18, 24, and 48 h) post-injection (p.i.). Radiotracer co-injection studies with varying doses of unlabelled (cold) TSHR-Ab (100, 50, 25, 0 µg) were studied to assess its effect on radiotracer uptake in tumors and off-target organs. Biodistribution and immunohistochemistry (IHC) were performed to verify the immnuoPET imaging results. Results [ ⁶⁴ Cu]Cu-NOTA-TSHR-Ab was synthesised with a radiochemical yield of 46.89 ± 3.74%, radiochemical purity of 98.77 ± 0.89%, and specific activity >1.42 GBq/mg (n = 5). In vitro cell studies demonstrated [ ⁶⁴ Cu]Cu-NOTA-TSHR-Ab had high specificity and nanomolar affinity for THJ529T ^TSHR(+) cells, with a dissociation constant (K _d ) of 4.74 nM and an inhibition constant (K _i ) of 0.92 nM. The immnuoPET imaging studies showed that the radiotracer could reach tumor quickly and showed high uptake in TSHR-positive thyroid tumors (SUV _max : 3.63 ± 0.42, 3.82 ± 0.44, and 4.09 ± 0.56 at 18, 24, and 48 h p.i., respectively). Co-injection with cold TSHR-Ab at doses ranging from 25 to 100 µg effectively reduced background signals, especially in spleen, liver, and bone. Notably, a dose of 25 µg cold TSHR-Ab reduced off-target signals without affecting tumor uptake. The biodistribution and IHC analyses further supported PET imaging findings. Conclusion TSHR-targeted radiotracer [64Cu]Cu-NOTA-TSHR-Ab was synthesized with high radiochemical purity. Our preclinical evaluation results demonstrate it is a promising radiotracer for PET imaging of TSHR-positive thyroid cancer, which may guide and monitor TSHR-targeted therapies. High radiotracer uptake in tumor, prolonged retention time, and favourable pharmacokinetics with the support of cold antibody co-injection, pave the way for the development of paired radiotherapeutics to treat TSHR-positive thyroid cancers.