Development of small-molecular-based radiotracers for PET imaging of PD-L1 expression and guiding the PD-L1 therapeutics

Programmed cell death protein ligand 1 (PD-L1) is a crucial biomarker for immunotherapy. However, nearly 70% of patients do not respond to PD-L1 immune checkpoint therapy. Accurate monitoring of PD-L1 expression and quantification of target binding during treatment are essential. In this study, a series of small-molecule radiotracers were developed to assess PD-L1 expression and direct immunotherapy. Radiotracers of [${}^{68}$Ga]Ga-D-PMED, [${}^{68}$Ga]Ga-D-PEG-PMED, and [${}^{68}$Ga]Ga-D-pep-PMED were designed based on a 2-methyl-3-biphenyl methanol scaffold and successfully synthesized. Cellular experiments and molecular docking assays were performed to determine their specificity for PD-L1. PD-L1 status was investigated via positron emission tomography (PET) imaging in MC38 tumor models. PET imaging of [${}^{68}$Ga]Ga-D-pep-PMED was performed to noninvasively quantify PD-L1 blocking using an anti-mouse PD-L1 antibody (PD-L1 mAb). The radiosyntheses of [${}^{68}$Ga]Ga-D-PMED, [${}^{68}$Ga]Ga-D-PEG-PMED, and [${}^{68}$Ga]Ga-D-pep-PMED were achieved with radiochemical yields of 87 ± 6%, 82 ± 4%, and 79 ± 9%, respectively. In vitro competition assays demonstrated their high affinities (the IC${}_{50}$ values of [${}^{68}$Ga]Ga-D-PMED, [${}^{68}$Ga]Ga-D-PEG-PMED, and [${}^{68}$Ga]Ga-D-pep-PMED were 90.66 ± 1.24, 160.8 ± 1.35, and 51.6 ± 1.32 nM, respectively). At 120 min postinjection (p.i.) of the radiotracers, MC38 tumors displayed optimized tumor-to-muscle ratios for all radioligands. Owing to its hydrophilic modification, [${}^{68}$Ga]Ga-D-pep-PMED had the highest target-to-nontarget (T/NT) ratio of approximately 6.2 ± 1.2. Interestingly, the tumor/liver ratio was hardly affected by different concentrations of the inhibitor BMS202. We then evaluated the impacts of dose and time on accessible PD-L1 levels in the tumor during anti-mouse PD-L1 antibody treatment. The tumor uptake of [${}^{68}$Ga]Ga-D-pep-PMED significantly decreased with increasing PD-L1 mAb dose. Moreover, after 8 days of treatment with a single antibody, the uptake of [${}^{68}$Ga]Ga-D-pep-PMED in the tumor significantly increased but remained lower than that in the saline group. PET imaging with [${}^{68}$Ga]Ga-D-pep-PMED, a small-molecule radiotracer, is a promising tool for evaluating PD-L1 expression and quantifying the target blockade of PD-L1 to assist in the development of effective therapeutic regimens.