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Purpose: 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. Methods: Radiotracers of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]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 [68Ga]Ga-D-pep-PMED was performed to noninvasively quantify PD-L1 blocking using an anti-mouse PD-L1 antibody (PD-L1 mAb). Results: The radiosyntheses of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]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 IC50 values of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]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, [68Ga]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 [68Ga]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 [68Ga]Ga-D-pep-PMED in the tumor significantly increased but remained lower than that in the saline group. Conclusion: PET imaging with [68Ga]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. [ABSTRACT FROM AUTHOR]

Purpose: Evans blue as an albumin binder has been widely used to improve pharmacokinetics and enhance tumor uptake of radioligands, including prostate-specific membrane antigen (PSMA) targeting agents. The goal of this study is to develop an optimal Evans blue-modified radiotherapeutic agent that could maximize the absolute tumor uptake and tumor absorbed dose thus the therapeutic efficacy to allow treatment of tumors even with moderate level of PSMA expression. Methods: [177Lu]Lu-LNC1003 was synthesized based on PSMA-targeting agent and Evans blue. Binding affinity and PSMA targeting specificity were verified through cell uptake and competition binding assay in 22Rv1 tumor model that has moderate level of PSMA expression. SPECT/CT imaging and biodistribution studies in 22Rv1 tumor-bearing mice were performed to evaluate the preclinical pharmacokinetics. Radioligand therapy studies were conducted to systematically assess the therapeutic effect of [177Lu]Lu-LNC1003. Results: LNC1003 showed high binding affinity (IC50 = 10.77 nM) to PSMA in vitro, which was comparable with that of PSMA-617 (IC50 = 27.49 nM) and EB-PSMA-617 (IC50 = 7.91 nM). SPECT imaging of [177Lu]Lu-LNC1003 demonstrated significantly improved tumor uptake and retention as compared with [177Lu]Lu-EB-PSMA and [177Lu]Lu-PSMA-617, making it suitable for prostate cancer therapy. Biodistribution studies further confirmed the remarkably higher tumor uptake of [177Lu]Lu-LNC1003 (138.87 ± 26.53%ID/g) over [177Lu]Lu-EB-PSMA-617 (29.89 ± 8.86%ID/g) and [177Lu]Lu-PSMA-617 (4.28 ± 0.25%ID/g) at 24 h post-injection. Targeted radioligand therapy results showed noteworthy inhibition of 22Rv1 tumor growth after administration of a single dose of 18.5 MBq [177Lu]Lu-LNC1003. There was no obvious antitumor effect after [177Lu]Lu-PSMA-617 treatment under the same condition. Conclusion: In this study, [177Lu]Lu-LNC1003 was successfully synthesized with high radiochemical purity and stability. High binding affinity and PSMA targeting specificity were identified in vitro and in vivo. With greatly enhanced tumor uptake and retention, [177Lu]Lu-LNC1003 has the potential to improve therapeutic efficacy using significantly lower dosages and less cycles of 177Lu that promises clinical translation to treat prostate cancer with various levels of PSMA expression. [ABSTRACT FROM AUTHOR]