Preclinical ImmunoPET Imaging of Thyroid-Stimulating Hormone Receptor Expression in Thyroid Cancer using [⁶⁴Cu]Cu-NOTA-TSHR-Ab

Advanced thyroid cancers are aggressive and often refractory to the current standard of care. The thyroid-stimulating hormone receptor (TSHR) is highly expressed in thyroid cancers and rarely expressed outside the thyroid, making it a viable target for developing radiotheranostics for imaging and therapy of advanced thyroid cancer. This study reports the radiosynthesis and preclinical evaluation of a ⁶⁴Cu-labeled human antibody for positron emission tomography (PET) imaging of TSHR expression in advanced thyroid cancer mouse models. Human anti-TSHR recombinant antibody K1-70 (TSHR-Ab) was labeled with copper-64, yielding [⁶⁴Cu]Cu-NOTA-TSHR-Ab with a radiochemical yield of 46.89 ± 3.74%, radiochemical purity of 98.77 ± 0.89%, and specific activity >212 GBq/μmol (<i>n</i> = 5). <i>In vitro</i> studies on TSHR-positive (THJ529T<i>^TSHR+</i>) and wild-type (THJ529T<i>^WT</i>) cells demonstrated the radiotracer's high specificity and nanomolar binding affinity for THJ529T<i>^TSHR+</i> cells, with a dissociation constant (<i>K</i>_d) of 4.74 nM and an inhibition constant (<i>K</i>_i) of 0.92 nM. ImmunoPET imaging in mice bearing dual-flank tumors (THJ529T<i>^WT</i> and THJ529T<i>^TSHR+</i>) at multiple time points (1, 2, 4, 18, 24, and 48 h) postinjection (p.i.) revealed rapid tumor targeting and 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 studies with varying doses of unlabeled TSHR-Ab (0, 25, 50, 100 μg) demonstrated that the coinjection significantly reduced background signals, especially in the spleen, liver, and bone, with a dose of 25 μg effectively reducing off-target signals without affecting tumor uptake. Biodistribution and immunohistochemistry analyses supported these immunoPET imaging results. Furthermore, a comparison study with traditional [¹⁸F]FDG PET imaging showed that [⁶⁴Cu]Cu-NOTA-TSHR outperformed [¹⁸F]FDG in tumor detection. In conclusion, [⁶⁴Cu]Cu-NOTA-TSHR-Ab is a promising radiotracer for PET imaging of TSHR-positive advanced thyroid cancers, with the potential to guide and monitor TSHR-targeted therapies. Further clinical evaluation of [⁶⁴Cu]Cu-NOTA-TSHR-Ab could provide valuable insights for patient stratification and optimization of anti-TSHR treatments.