Synthesis and Evaluation of ⁶⁴ Cu-Labeled Small-Size Antibody Fragments for Immuno-PET Imaging of Thyroid-Stimulating Hormone Receptor in Thyroid Cancer

The thyroid-stimulating hormone receptor (TSHR) is a promising molecular target for thyroid cancer imaging and therapy. Our previous work has demonstrated that PET imaging with the radiolabeled anti-TSHR human monoclonal antibody K1-70 enables assessment of TSHR expression in thyroid cancer. However, full-length antibody-based radiopharmaceuticals exhibit delayed systemic clearance and increased off-target radiation burden, resulting in suboptimal pharmacokinetics for immuno-PET imaging. Herein, we report the synthesis and evaluation of ⁶⁴Cu-labeled anti-TSHR K1-70 antibody fragment antigen-binding (Fab) and single-chain variable fragment (scFv), for immuno-PET imaging of TSHR in thyroid cancer mouse models. These smaller formats enabled rapid tumor targeting and favorable pharmacokinetics with high tumor-to-background contrast. Two radiotracers, named ⁶⁴Cu-NOTA-TSHR-Fab and ⁶⁴Cu-NOTA-TSHR-scFv, were prepared by conjugating TSHR-Fab or TSHR-scFv to <i>p</i>-SCN-Bn-NOTA, followed by radiolabeling with ⁶⁴Cu, achieving high radiochemical purity (>99%). The specificity and binding affinity of each radiotracer were determined by cellular uptake and binding assays using TSHR-positive THJ529T cells and corresponding wild-type controls. Both radiotracers exhibited specific, nanomolar binding affinity to TSHR-positive cells. Immuno-PET imaging, ex vivo biodistribution, and blocking studies of each radiotracer were performed in NSG mice bearing subcutaneous TSHR-positive THJ529T tumor xenografts at various time points (1, 4, 18, and 24 h postinjection). In comparative in vivo evaluations, ⁶⁴Cu-NOTA-TSHR-Fab showed rapid and superior TSHR-specific tumor accumulation compared with ⁶⁴Cu-NOTA-TSHR-scFv, evident as early as 1 h postinjection. Both radiotracers demonstrated rapid pharmacokinetics and low background signal, but with high renal uptake. This head-to-head comparison of small-size antibody fragment-based radiotracers for TSHR-targeted immuno-PET imaging identifies ⁶⁴Cu-NOTA-TSHR-Fab as a promising immuno-PET radiotracer for in vivo detection of TSHR expression in thyroid cancer and for guiding TSHR-targeted therapy.