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Background: Programmed cell death ligand 1 (PD-L1) plays a critical role in the tumor microenvironment and overexpression in several solid cancers has been reported. This was associated with a downregulation of the local immune response, specifically of T-cells. Immune checkpoint inhibitors have the potential to reactivate the immune system, but only 30% of patients are considered responders. New diagnostic approaches re therefore needed to determine patient eligibility. Small molecule radiotracers targeting PD-L1 may serve as such diagnostic tools, addressing the heterogeneous PD-L1 expression between and within tumor lesions, thus aiding in therapy decisions. Results: Four small-molecule biphenyl-based PD-L1 ligands were synthesized using a convergent synthetic route with a linear sequence of up to eleven steps. Three different chelators (NODA-GA, CB-TE2A, DiAmSar) were employed to efficiently radiolabel these compounds with copper-64, and a dimeric structure was also synthesized. All radioligands exhibited high proteolytic stability (>95%) for 48 hours post-radiolabeling. Saturation binding yielded moderate affinities ranging from 100 to 265 nM. Conversely, real-time radioligand binding revealed more promising KD values of about 20 nM for [64Cu]Cu-14 and [64Cu]Cu-15. In vivo PET imaging in mice bearing PC3 PD-L1 overexpressing and PD-L1-negative tumors was performed at 0–2, 4–5 and 24–25 h post injection (p.i.). This revealed considerably different pharmacokinetic profiles, depending on the substituted chelator. [64Cu]Cu-14, substituted with NODA-GA, showed renal clearance with low liver uptake, whereas substitution with the cross-bridged cyclam chelator CB-TE2A resulted in a primarily hepatobiliary clearance. Notably, the monomeric DiAmSar radioligand [64Cu]Cu-16 demonstrated a higher liver uptake than [64Cu]Cu-15, but was still renally cleared as evidenced by the lack uptake in gall bladder and intestines. The dimeric structure [64Cu]Cu-17 showed extensive accu

Der Programmed Death-Ligand 1 (PD-L1) wird von verschiedenen Tumorentitäten überexprimiert und hemmt durch die Bindung an das auf T-Zellen vorkommende Programmed Cell Death Protein (PD-1) die Immunantwort. Immuncheckpoint-Inhibitoren sind in der Lage, diese Blockade aufzubrechen und die Immunantwort zu reaktivieren. Auf eine solche Monotherapie sprechen aber nur etwa 30 % der Patienten an, sodass klinischer Bedarf an nichtinvasiven, bildgebenden Diagnosemethoden besteht. Eine Möglichkeit stellen Radioliganden für die nichtinvasive Bildgebung mittels Positronen-Emissions-Tomographie (PET) oder Einzelphotonen-Emissionscomputertomographie (SPECT) dar, welche einen Therapieerfolg prognostizieren könnten. Die vorliegende Dissertationsschrift beschäftigt sich mit der Entwicklung und Synthese einer neuen Klasse von PD-L1-adressierenden Radioliganden sowie deren biologischer Evaluierung in Zellen (in vitro) und in Kleintieren (in vivo). Nach der Identifikation von drei hochaffinen PD-L1-Inhibitoren in der Patentliteratur, wurden zunächst diese als Referenzverbindungen in einer neuen, konvergenten Synthesestrategie dargestellt. Diese ermöglichte nicht nur eine höhere Gesamtausbeute der Substanzen, sondern erlaubte auch dank des modularen Aufbaus den einfachen Zugang zu einer großen Substanzbibliothek an PD-L1-Radioliganden. Mithilfe einer Kokristallstruktur aus PD-L1 und einem Inhibitor wurden Modifikationen des Inhibitors für eine Transformation in einen Radioliganden geplant. Zur Erhöhung der Hydrophilie der Substanzen wurden löslichkeitsvermittelnde Gruppen (Sulfon-/Phosphonsäuren) in dem Molekülteil eingeführt, welcher in der „Solvent-Exposed-Region“ des Proteins lokalisiert ist. Funktionalisierung des Bindungsmotivs mit einem Alkin erlaubte die Einführung eines Linker-Chelator-Konstruktes mittels kupferkatalysierter Azid-Alkin-Cycloaddition (CuAAC) zur Komplexierung des Radiometalls. In der ersten Generation an PD-L1-Radioliganden wurden zwei Bindungsmotive und drei ver

Small molecules offer some advantages for developing PET tracers and are therefore a promising approach for imaging and therapy monitoring of PD-L1 positive tumors. Here, we report six biphenyl PD-L1 radioligands, using the NODA-GA-chelator for efficient copper-64 complexation. These radioligands contain varying numbers of sulfonic and/or phosphon-ic acid groups, serving as hydrophilizing units to lower the log D7.4 value down to –4.28. Binding affinities of compounds were evaluated using saturation binding and a real-time binding assay, with lowest binding affinity of 21 nM. Small ani-mal PET imaging revealed vastly different pharmacokinetic profiles, depending on the quantity and type of hydrophiliz-ing units. Of the investigated radioligands, [64Cu]Cu-3 showed the most favorable kinetics in vitro. This was also found in vivo, with a predominantly renal clearance and a specific uptake in the PD-L1-positive tumor. With further modifications, this compound could be a promising candidate for imaging of PD-L1 in the clinical setting.

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.

Immune checkpoint inhibitor therapy targeting the PD-1/PD-L1 axis in cancer patients holds promise as an oncological treatment. However, the number of non-responders remains high. Consequently, clinicians need a diagnostic tool to predict treatment outcomes. PET imaging can play an important role in supporting therapy decisions by offering whole-body scan while quantitatively assessing PD-L1 expression. In multi-step organic synthesis, four PD-L1 radiolig-ands containing a linker-chelator system for radiometallation, along with three hydrophilizing units – one sulfonic acid and two phosphonic acids – were synthesized. After labeling with 64Cu, log D7.4 values of below –3.03 were determined and proteolytic stability studies were conducted confirming stabilities over 94% after 48 hours. Binding affinities studies were conducted using two different binding assays revealing high affinities up 13 nM. µPET/CT imaging was performed in tumor-bearing mice to investigate PD-L1 specific tumor uptake and the pharmacokinetic profile. The µPET images revealed an unexpected in vivo behavior, including low tumor uptake in PD-L1 positive tumors, high liver uptake, and accumulation in bone/bone marrow/joints. These effects were attributed to Ca2+-affinity and/or binding to macrophages. Despite phosphonic acids offering a high degree of water-solubility, their incorporation must be carefully considered to avoid ex-acerbating the radioligands’ pharmacokinetic behaviors.

Bei diesem Datensatz handelt es sich um die chemische Charakterisierung der Verbindungen, die in-vitro-, in-vivo- und ex-vivo-Daten.

Bei diesem Datensatz handelt es sich um die chemische Charakterisierung der Verbindungen, die in-vitro- und in-vivo-Daten.

Bei diesem Datensatz handelt es sich um die chemische Charakterisierung der Verbindungen, die in-vitro- und in-vivo-Daten.

Bei diesem Datensatz handelt es sich um die chemische Charakterisierung der Verbindungen, die in-vitro-, in-vivo- und ex-vivo-Daten.

Aim: The programmed cell death ligand 1 (PD-L1) is overexpressed by various cancers, resulting in a downregulation of the local immune response and therefore enabling further tumor growth.[1] Immune checkpoint inhibitors (ICIs) can reactivate the immune system, however, only 30% of the patients respond to an ICI monotherapy.[2] Since PD-L1 is heterogeneously expressed within and across tumor sites, there is an urgent clinical need for non-invasive diagnostic tools to support the therapeutic decision process. Small molecule-based radiotracers for noninvasive molecular PD-L1 imaging offer improved tissue penetration, fast blood clearance and low immunogenicity over radiolabeled antibodies. Methods: Based on a published small molecule PD-L1 inhibitor, 10 different radioligands were synthesized and radiolabeled with copper-64 (HZDR, 30 MeV TR-FLEX cyclotron). Binding affinities were determined on PC3 cells stably overexpressing human PD-L1. For in vivo evaluation, qualitative PET/CT imaging (nanoSCAN PET/CT, Mediso) was performed in NMRI-FoxN1-nude mice bearing PC3-hPD-L1 xenografted tumors. Results: Modification of the PD-L1 binding motif with strongly water-solubilizing sulfonate and phosphonate groups, different linker units and a NODAGA-chelator in 21-25 organic synthesis steps (12-13 longest linear sequence) yielded 10 different ligands [3]. The 64Cu-labelled radiotracers exhibited logD values between -3.17 and -4.15 for six ligands of the first series, with dissociation constants (Kd) between 80.5 and 532.8 nM, as determined by saturation binding assays. Depending on the number and pattern of sulfonate and phosphonate groups, the in vivo experiments showed drastically different pharmacokinetic profiles: Compounds bearing a less hydrophilic linker showed improved tumor uptake. Three sulfonates resulted in increased blood circulation times of up to 24 h due to albumin binding increased renal clearance but also low tumor uptake (SUVmax = 1.4). Substitution of one sul

Objective: The programmed cell death-ligand 1 (PD-L1) is upregulated on many different cancers and allows the tumor cells to evade immune response through binding to the PD-1 receptor.[1] Monoclonal antibodies, i.e. checkpoint inhibitors, are able to break this blockade and thus reactivate the immune system.[2] However, only 30% of the patients respond to antibody-based immunotherapy. Because PD-L1 is heterogeneously expressed within and across tumor sites, there is an urgent clinical need for a non-invasive, diagnostic imaging approach helping for therapy decision. Radiotracers for PET and SPECT imaging are able to meet these requirements. Especially small molecules are favourable, because of their short clearance times and for providing high imaging contrast.[3] Methods: Modification of two literature known small molecule PD-L1 inhibitors with water-solubilizing groups, different linkers and a DOTA chelator resulted in six different radioligands. Labeling was performed with Cu-64 (HZDR, 30 MeV TR-FLEX cyclotron) and binding affinities to PD-L1 were determined in vitro on transduced PC3 cells stably overexpressing human PD-L1. Qualitative PET scans (nanoSCAN PET/CT scanner, Mediso) were performed in NMRI-FoxN1-nude mice bearing PC3-hPD-L1 xenografted tumors. Results: Organic synthesis started from biaryl building blocks (R1 = H, R2 = Br and R1 = R2 = Me), which underwent a Mitsunobu reaction with the central chloroaryl moiety. The bis(sulfonic acid) group was attached via a sarcosine spacer. Three different linker structures were synthesized and attached by Cu(I)-catalyzed click reaction. Synthesis was finished with DOTA conjugation and subsequent quantitative labeling with Cu-64 under standard labeling conditions was achieved. Using the shake flask method, log(D) values ranging from –1.5 to –2.5 were obtained. Saturation binding assays revealed that biphenyl compounds with R1 = R2 = Me showed promising binding affinities to PD-L1 (KD between 60 and 123 nM). In mic

Ziel: Der Programmed Cell Death Ligand 1 (PD-L1) ist auf verschiedenen Tumorentitäten überexprimiert und hemmt die Immunantwort durch Bindung an PD-1 auf T-Zellen. Immuncheckpoint-Inhibitoren können diese Blockade aufbrechen und die Immunantwort reaktivieren. Da nur ca. 30% der Patienten auf eine solche Therapie ansprechen, besteht klinisch ein dringender Bedarf an einem nicht-invasiven PET/SPECT-Radioliganden, um die Ansprechrate der Patienten auf diese Therapie abzuschätzen.[1] Methoden: Basierend auf den Strukturen von nicht-peptidischen PD-L1-Inhibitoren, wurden sechs verschiedene Radioliganden synthetisiert und mit [64Cu]Cu2+ markiert (HZDR, 30 MeV TR-FLEX-Zyklotron). Bindungsaffinitäten wurden auf stabil hPD-L1 überexprimierenden PC3 Zellen bestimmt. In vivo wurden qualitative PET/CT-Bilder (nanoSCAN PET/CT, Mediso) an NMRI-FoxN1-Nacktmäusen mit PC3-hPD-L1- und mock-Tumoren aufgenommen. Ergebnisse: Zwei PD-L1-Inhibitoren wurden synthetisch mit stark wasserlöslichen Säuregruppen, hydrophilen Linkern und NODAGA modifiziert (Abb. 1a).[2] Die LogD7.4-Werte der [64Cu]Cu-Radioliganden lagen zwischen –3,17 und –4,15 und die Bindungsaffinitäten (KD) zwischen 80,5 und 532,8 nM. Abhängig von der Zahl und dem Substitutionsmuster der Sulfon- und Phosphonsäuregruppen, zeigte sich in vivo eine stark unterschiedliche Pharmakokinetik. Der Radioligand mit R1 = SO2Me, R2 = PO3H2 und n = 0 zeigte in vivo eine kurze Zirkulationszeit, renale Ausscheidung, gute Tumoraufnahme (SUVmax = 3.5) und einen deutlichen Kontrast zwischen hPD-L1- und mock-Tumor (Abb. 1b). Schlussfolgerungen: Der PD-L1-Radioligand mit je einer Sulfon- und Phosphonsäuregruppe wies das beste pharmakokinetische Profil auf. Um die Bindungsaffinität und die Tumoraufnahme zu verbessern, wird das Leitmotiv derzeit weiter modifiziert. Referenzen: [1] Postow M. A., Callahan M. K., Wolchok J. D. J. Clin. Oncol. 2015, 33, 1974-1982. [2] Stadlbauer S., Krutzek F., Kopka K. EP21212444.0, 2021.

The programmed cell death ligand (PD-L1) is expressed on a number of different tumor entities and inhibits the immune response through binding to PD-1 on T-cells. Immune checkpoint inhibitors (ICI) prevent this blockade and thus can reactivate an immune response. However, only about 30% of the patients respond to an ICI monotherapy. Therefore, clinicians are in need for a non-invasive PET/SPECT radioligand for patient stratification and therapy monitoring. Based on the structures of non-peptidic PD-L1 inhibitors, six different radiotracers were synthesized and radiolabelled with [64Cu]Cu2+ (HZDR, 30 MeV TR-FLEX cyclotron). Binding affinities were determined on PC3 cells stably overexpressing hPD-L1. For in vivo studies, qualitative PET/CT imaging experiments (nanoSCAN PET/CT, Mediso) were performed in NMRI-FoxN1-nude mice bearing PC3-hPD-L1 and mock xenograted tumors. Two PD-L1 inhibitors were modified with strongly water-soluble acid groups, hydrophilic linker units and a NODAGA-chelator resulting in six different radioligands. The log(D) values of the copper-64 labelled radiotracers were between –3.17 and –4.15 and binding affinities ranged between 80.5 and 533 nM. Depending on the number and the pattern of sulfonate and phosphonate groups, in vivo experiments showed drastically different pharmacokinetic profiles. The radiotracer with one sulfonate and phosphonate group and the most hydrophobic linker exhibited a short circulation time, renal clearance, good tumor uptake (SUVmax = 3.5) and a distinct contrast between the hPD-L1 and the mock tumor. In conclusion one PD-L1 radiotracer showed a promising pharmacokinetic profile, which is currently further modified to improve the binding affinity and tumor uptake.

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.

Aim/Introduction The programmed cell death ligand 1 (PD-L1) is expressed by several cancer types and leads to a downregulation of the local immune response, therefore enabling tumour cells to evade the immune response.[1] So-called immune checkpoint inhibitors (ICI) are able to reactivate the immune system, however, only 30% of the patients respond to an ICI monotherapy.[2] Since PD-L1 is heterogeneously expressed within and across tumour sites, there is an urgent clinical need for a diagnostic, non-invasive imaging probe to support therapy decision. Small molecule-based radiotracers for PD-L1 PET or SPECT imaging fulfil these requirements due to their fast clearance, low risk of side effects and highly sensitive imaging at the molecular level.[3] Materials & Methods Based on a published small molecule PD-L1 inhibitor, six different radioligands were synthesized and radiolabelled with copper-64 (HZDR, 30 MeV TR-FLEX cyclotron). Binding affinities were determined on PC3 cells stably overexpressing human PD-L1 which were kindly provided by the Department of Radioimmunology. For in vivo evaluation, qualitative PET/CT imaging experiments (nanoSCAN PET/CT scanner, Mediso) were performed in NMRI-FoxN1-nude mice bearing PC3-hPD-L1 xenografted tumours. Results We designed six radioligands by modifying the PD-L1 binding motif with strongly water-solubilizing sulfonate and phosphonate groups, hydrophilic linker units and a NODAGA-chelator in 21 – 25 organic synthesis steps (12-13 longest linear sequence).[4] The copper-64 labelled radiotracers exhibited log(D) values between –3.17 and –4.15. Binding affinities (Kd) were between 80.5 and 532.8 nmol/L. Depending on the number and pattern of sulfonate and phosphonate groups, the in vivo experiments showed drastically different pharmacokinetic profiles: The radiotracer containing three sulfonates showed long circulation times of 24 h due to albumin binding, renal clearance but low tumour uptake (SUVmax = 1.4). Substitution of one