PET Imaging of Tumor PD-L1 Expression with a 18F-labeled Bicyclic Peptide

Introduction: Bicyclic peptides have been one of the most promising platforms for diagnostic and therapeutic drug development, owing to their chemical diversity, access to chemical synthesis, biocompatibility and antibody-like affinity and selectivity. BCY10959 is a bicyclic peptide scaffold with nanomolar affinity towards PD-L1. Using radioconjugates thereof, noninvasive and quantitative imaging of PD-L1 expression with PET was evaluated in a U87MG-bearing mice model.

Methods: The bicyclic peptide BCY10959 and its precursor NOTA-BCY10959 were custom synthesized. The radiosynthesis of [¹⁸F]AlF-BCY10959 was performed in the ChelationLab@Al¹⁸F automatic platform. The radiochemical yield, radiochemical purity, molar activity and partition coefficient of [¹⁸F]AlF-BCY10959 were determined. The binding model of [¹⁸F]AlF-BCY10959 to PD-L1 protein was studied by molecular docking in Maestro12.0. Cell uptake and binding assays were completed in U87MG and U87MG-PDL1-/- cells. The PD-L1 PET imaging and biodistribution were investigated in U87MG and U87MG-PDL1-/- bearing tumor models. The PD-L1 expression was further verified by western blotting, flow cytometry and immunohistochemistry.

Results: [¹⁸F]AlF-BCY10959 was automatically produced with a decay-corrected radiochemical yield of 71.6 ± 8.8 % , radiochemical purity of > 99 % and molar activity of 52.4 ± 3.2 GBq/μmol (n = 3). The partition coefficient (Log P) was calculated as 0.83 ± 0.24, indicating its lipophilicity (n = 6). The optimized binding conformation of [¹⁹F]AlF-BCY10959 towards PD-L1 was formed with a binding energy of - 8.456 kcal/mol. In vitro assays revealed that [¹⁸F]AlF-BCY10959 has high affinity and specificity with an IC₅₀ of 8.73 nM and K_D of 6.25 nM. PET imaging demonstrated the specific accumulation of [¹⁸F]AlF-BCY10959 in U87MG tumor with an uptake value of 8.05 ± 0.89 % ID/g and a tumor-to-muscle ratio of 12.80 ± 2.7 at 60 min after injection. The biodistribution of [¹⁸F]AlF-BCY10959 was consistent with the observations from PET imaging. The primary distribution of radioactivity was observed in kidneys and liver, indicating its metabolic pathway.

Conclusions: [¹⁸F]AlF-BCY10959 is a promising bicyclic peptide-based PET ligand with clinical translation potentials for noninvasive imaging of tumor PD-L1 expression.