A Novel Small Cyclic Peptide-Based 68Ga-Radiotracer for Positron Emission Tomography Imaging of PD-L1 Expression in Tumors

In the tumor microenvironment, programmed death protein 1 and programmed death protein ligand 1 (PD-L1) signaling pathways help tumors escape the immune system. We designed a gallium-68 (68Ga)-labeled small-molecule peptide-targeting PD-L1 and used positron emission tomography/computed tomography (PET/CT) to detect and dynamically monitor the expression level of PD-L1 in tumors. S-Cyclo(ETSK)-SF-NH2 (SETSKSF) is a cyclic peptide inhibitor comprising seven amino-acid residues. We connected it with the chelating agent DOTA, labeled DOTA-SETSKSF, with the short half-life nuclide Ga-68, and measured the stability of Ga-68-2,2',2 ''-(10-(2-((S)-1-((3S,6S,9S,18S)-18-((S)- 1-((S)-1-amino-1-oxo- 3- henylpropan-2-ylamino)-3-hydroxy-1-oxopropan-2-ylcarbamoyl)-6-((R)-1-hydrox-yethyl)-3-(hydroxymethyl)- 2,5,8,12-tetraoxo-1,4,7,13-tetraazacyclooctadecan-9-ylamino)-3-ydroxy-1-oxopropan-2-ylamino)-2-oxoethyl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid (Ga-68-DOTA-SETSKSF) in normal saline (NS), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) in vitro. We conducted the Ga-68-DOTA-SETSKSF affinity test, cell-specific uptake experiments, time-combined experiments, western blotting, and laser confocal experiments to confirm the expression and localization of PD-L1 at the cell level and determine the uptake. Biodistribution and imaging experiments were performed using the H1975, B16F10, and A549 tumor models. Ga-68-DOTA-SETSKSF was successfully synthesized, and the radiochemical purity was >99% after purification. The in vitro stability of Ga-68-DOTA-SETSKSF was >95% in NS, PBS, and FBS at 37 degrees C after 4 h of incubation. Cell-binding experiments confirmed that Ga-68-DOTA-SETSKSF exhibited high uptake in H1975 tumors with high PD-L1 expression and low uptake in A549 tumors with low PD-L1 expression. The clear half-life (T-1/2) of Ga-68-DOTA-SETSKSF from the blood was 14.48 +/- 3.26 min. The percentages of the injected dose per gram of tissue (%ID/g) for H1975 and A549 tumors were 5.29 +/- 0.21 and 0.89 +/- 0.10 at 1 h after injection, respectively. The H1975 tumor-to-muscle and tumor-to-blood ratios were 41.79 +/- 5.81 and 4.75 +/- 0.19 at 4 h, respectively. Apart from the H1975 tumor, the kidney and the bladder showed high accumulation because Ga-68-DOTA-SETSKSF was excreted through the urinary system. PET/CT images showed high accumulation of Ga-68-DOTA-SETSKSF in H1975 tumors and low uptake in A549 tumors, which was consistent with the results of biodistribution experiments. Ga-68-DOTA-SETSKSF is convenient to prepare, has high stability, can be used to monitor the expression of PD-L1, and has an extremely high clinical application value.

Automated summary

In this study, a novel PET/CT detection method targeting PD-L1 was designed and validated through experiments, demonstrating its feasibility and accuracy in dynamically monitoring the expression of PD-L1 in tumors.