Fabian Krutzek

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 D_7.4 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.

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.

Efficient ¹⁸F-fluorination procedures for the production of radiopharmaceuticals are urgently needed to satisfy the increasing demand for clinical diagnostics using positron emission tomography (PET). However, the development of PET tracers is often a time-consuming and challenging process. This work examines the applicability of the recently described sulfur [¹⁸F]fluoride exchange ([¹⁸F]SuFEx) chemistry as a fast screening approach towards a number of clinically relevant PET tracer preparations. The preparation of a number of ¹⁸F-labeled compounds commenced with [¹⁸F]fluoride loading onto a quarternary methylammonium (QMA) cartridge, which was eluted with a methanolic solution containing a base, followed by solvent removal under reduced pressure. Thereafter, the radiolabeling precursors in MeCN were added to the reaction vessels, and allowed to react via [¹⁸F]SuFEx at room temperature for 5 min. The radiofluorination reactions were quenched by water dilution followed by C18 cartridge purification. The ¹⁸F-labeled products were isolated by elution from the cartridge with EtOH and the identities of the products were confirmed by radio-ultra high performance liquid chromatography (UHPLC). The optimized preparations of ¹⁸F-labeled prostate-specific membrane antigen (PSMA) inhibitor, Programmed death-ligand 1 (PD-L1) ligand, cyclooxygenase-2 inhibitor (COXIB), and Fibroblast activation protein alpha inhibitor (FAPI) were achieved with high non-decay corrected isolated activity yields (AY) of 33–57% (n = 12) and >95% radiochemical purity (RCP) in 25 min. The automated radiosynthesis procedures afforded the radiolabeled products in an unoptimized 8–15% AY (n = 5), with >95% RCP in 40 min. The ultra-fast [¹⁸F]SuFEx reaction permitted several structurally diverse ¹⁸F-labeled compounds for potential imaging to be rapidly achieved in excellent isolated AYs and high RCP. Presently, optimization of the automated radiosynthesis and biological assessment of the ¹⁸F-labeled products is underway.

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.