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Immune checkpoint inhibitor therapy targeting the PD-1/PD-L1 axis in cancer patients, is a promising oncological treatment. However, the number of non-responders remains high, causing a burden for the patient and the healthcare system. Consequently, a diagnostic tool to predict treatment outcomes would help with patient stratification. Molecular imaging provides said diagnostic tool by offering a whole-body quantitative assessment of PD-L1 expression, hence supporting therapy decisions. Four PD-L1 radioligand candidates containing a linker-chelator system for radiometalation, along with three hydrophilizing units—one sulfonic and two phosphonic acids—were synthesized. After labeling with 64Cu, log D7.4 values of less than −3.03 were determined and proteolytic stability confirmed over 94% intact compound after 48 h. Binding affinity was determined using two different assays, revealing high affinities up to 13 nM. µPET/CT imaging was performed in tumor-bearing mice to investigate PD-L1-specific tumor uptake and the pharmacokinetic profile of radioligands. These results yielded an unexpected in vivo distribution, such as low tumor uptake in PD-L1 positive tumors, high liver uptake, and accumulation in bone/bone marrow and potentially synovial spaces. These effects are likely caused by Ca2+-affinity and/or binding to macrophages. Despite phosphonic acids providing high water solubility, their incorporation must be carefully considered to avoid compromising the pharmacokinetic behavior of radioligands. [ABSTRACT FROM AUTHOR]

Simple Summary: PD-L1 plays a crucial role in the immune responses against cancer. Only around 30% of cancer patients respond to an anti-PD-L1 immune checkpoint inhibitor therapy. Noninvasive molecular imaging techniques such as positron emission tomography (PET) would allow identification of patients likely to respond. Here we report on the synthesis of nine PET radioligands targeting PD-L1, based on small-molecule inhibitors. We introduced a chelator for radiolabeling and water-soluble groups to aim for clearance through the kidneys. The compounds showed binding affinities toward PD-L1 in the lower nanomolar range and stability in vitro. Mouse experiments showed moderate accumulation in tumor tissue but mainly clearance through the liver. Additionally, the compounds showed unexpected long circulation times due to strong binding to albumin in blood. Nevertheless, our compounds are a starting point for further development of PD-L1 small molecule PET radiotracer to support therapy decisions. Noninvasive molecular imaging of the PD-1/PD-L1 immune checkpoint is of high clinical relevance for patient stratification and therapy monitoring in cancer patients. Here we report nine small-molecule PD-L1 radiotracers with solubilizing sulfonic acids and a linker–chelator system, designed by molecular docking experiments and synthesized according to a new, convergent synthetic strategy. Binding affinities were determined both in cellular saturation and real-time binding assay (LigandTracer), revealing dissociation constants in the single digit nanomolar range. Incubation in human serum and liver microsomes proved in vitro stability of these compounds. Small animal PET/CT imaging, in mice bearing PD-L1 overexpressing and PD-L1 negative tumors, showed moderate to low uptake. All compounds were cleared primarily through the hepatobiliary excretion route and showed a long circulation time. The latter was attributed to strong blood albumin binding effects, discovered during our binding experiments. Taken together, these compounds are a promising starting point for further development of a new class of PD-L1 targeting radiotracers. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitor (ICI) therapy has emerged as a major treatment option for a variety of cancers. Among the immune checkpoints addressed, the programmed death receptor 1 (PD-1) and its ligand PD-L1 are the key targets for an ICI. PD-L1 has especially been proven to be a reproducible biomarker allowing for therapy decisions and monitoring therapy success. However, the expression of PD-L1 is not only heterogeneous among and within tumor lesions, but the expression is very dynamic and changes over time. Immunohistochemistry, which is the standard diagnostic tool, can only inadequately address these challenges. On the other hand, molecular imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) provide the advantage of a whole-body scan and therefore fully address the issue of the heterogeneous expression of checkpoints over time. Here, we provide an overview of existing PET, SPECT, and optical imaging (OI) (radio)tracers for the imaging of the upregulation levels of PD-1 and PD-L1. We summarize the preclinical and clinical data of the different molecule classes of radiotracers and discuss their respective advantages and disadvantages. At the end, we show possible future directions for developing new radiotracers for the imaging of PD-1/PD-L1 status in cancer patients. [ABSTRACT FROM AUTHOR]