First-Generation Radiolabeled Cyclic Peptides for Molecular Imaging of Platelet-Derived Growth Factor Receptor α

Occult nodal spread and metastatic disease require longstanding imaging and biochemical assessments for thyroid cancer, a disease that has a propensity for diffuse, small-volume disease. We have developed a ⁶⁴Cu-labeled platelet-derived growth factor receptor α (PDGFRA) antibody for immuno-PET of PDGFRA in metastatic papillary thyroid cancer (PTC). The present work describes the discovery of small cyclic PDGFRA-targeting peptides, their binding features, and radiolabeling with positron emitter gallium-68 (⁶⁸Ga) for in vitro and in vivo characterization in thyroid cancer models. Phage-display technology with two separate libraries and seven different cell lines was used through three rounds of biopanning as well as flow cytometry and comparative analysis with recombinant protein to select specific peptide sequences. Phenotypic binding analysis was completed by using phosphorylation and cell migration assays. In vitro protein binding was analyzed with thermophoresis and flow cytometry using the fluorescent-labeled PDGFRA peptide. Peptide candidates were modified with the NOTA chelator for radiolabeling with ⁶⁸Ga. In vitro cell uptake was studied in various thyroid cancer cell lines. In vivo studies of ⁶⁸Ga-labeled peptides included metabolic stability and PET imaging. From the original library (10¹³ compounds), five different peptide groups were identified based on biopanning experiments with and without the α subunit of PDGFR, leading to ∼50 peptides. Subsequent phenotypic screening revealed two core peptide sequences (CP16 and CP18) that demonstrated significant changes in the level of PDGFRA phosphorylation and cell migration. Alanine scan sublibraries were created from these two lead peptide sequences, and peptides were radiolabeled using ⁶⁸Ga-GaCl₃ at pH 4.5, resulting in RCP > 95% within 34-40 min, including SPE purification. Cyclic peptide CP18.5 showed the strongest effects on cell migration, flow cytometry, and binding by visual interference color assay. ⁶⁸Ga-labeled PDGFRA-targeting peptides showed elevated cell and tumor uptake in models of thyroid cancer, with ⁶⁸Ga-NOTA-CP18.5 being the lead candidate. However, metabolic stability in vivo was compromised for ⁶⁸Ga-NOTA-CP18.5 vs ⁶⁸Ga-NOTA-CP18 but without impacting tumor uptake or clearance profiles. First-generation radiolabeled cyclic peptides have been developed as novel radiotracers, particularly ⁶⁸Ga-NOTA-CP18.5, for the molecular imaging of PDGFRA in thyroid cancer.