Programmed cell death receptor 1 (PD-1) and its ligand PD-L1 are particularly interesting immune checkpoint proteins for human cancer treatment. Positron emission tomography (PET) imaging allows for the dynamic monitoring of PD-L1 status during tumor progression, thus informing patients' response index. Herein, we report the synthesis of two linear peptide-based radiotracers, [64Cu]/[68Ga]HKP2201 and [64Cu]/[68Ga]HKP2202, and validate their utility for PD-L1 visualization in preclinical models. The precursor peptide HKP2201 was derived from a linear peptide ligand, CLP002, which was previously identified by phage display and showed nanomolar affinity toward PD-L1. Appropriate modification of CLP002 via PEGylation and DOTA conjugation yielded HKP2201. The dimerization of HKP2201 generated HKP2202. The 64Cu and 68Ga radiolabeling of both precursors was studied and optimized. PD-L1 expression in mouse melanoma cell line B16F10, mouse colon cancer cell line MC38, and their allografts were assayed by immunofluorescence and immunohistochemistry staining. Cellular uptake and binding assays were conducted in both cell lines. PET imaging and ex vivo biodistribution studies were employed in tumor mouse models bearing B16F10 and MC38 allografts. [64Cu]/[68Ga]HKP2201 and [64Cu]/[68Ga]HKP2202 were obtained with satisfactory radiocharacteristics. They all showed lower liver accumulation compared to [64Cu]/[68Ga]WL12. B16F10 and MC38 cells and their tumor allografts sections were verified to express PD-L1. These tracers demonstrated a concentration-dependent cell affinity and a comparable half-maximal effect concentration (EC50) with radiolabeled WL12. Competitive binding and blocking studies demonstrated the specific target of these tracers to PD-L1. PET imaging and ex vivo biodistribution studies revealed notable tumor uptake in tumor-bearing mice and rapid clearance from blood and major organs. Importantly, [64Cu]/[68Ga]HKP2202 showed higher tumor uptake compared to [64Cu]/[68Ga]HKP2201. Of note, [64Cu] labeled tracers showed longer retention in tumors than [68Ga] labeled traces, indicating advantages in the long-term tracking of PD-L1 dynamics. In comparison, [68Ga]HKP2201 and [68Ga]HKP2202 showed lower liver accumulation, enabling its great potential in the fast detection of both primary and metastatic tumors, including hepatic carcinoma. [64Cu]/[68Ga]HKP2201 and [64Cu]/[68Ga]HKP2202 are promising PET tracers for visualizing PD-L1 status. Notably, their combination would cooperate in rapid diagnosis and subsequent treatment guidance. Future assessment of the radiotracers in patients is needed to fully evaluate their clinical value.
Keywords: PD-L1; PET imaging; [64Cu]/[68Ga]HKP2201; [64Cu]/[68Ga]HKP2202; immune checkpoint block therapy; linear peptides.