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.