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.

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.

Early detection and treatment of cancers can significantly increase patient prognosis and enhance the quality of life of affected patients. The emerging significance of the tumor microenvironment (TME) as a new frontier for cancer diagnosis and therapy may be exploited by radiolabeled tracers for diagnostic imaging techniques such as positron emission tomography (PET). Cancer-associated fibroblasts (CAFs) within the TME are identified by biomarkers such as fibroblast activation protein alpha (FAPα), which are expressed on their surfaces. Targeting FAPα using small-molecule ¹⁸F-labeled inhibitors (FAPIs) has recently garnered significant attention for non-invasive tumor visualization using PET. Herein, two potent aryl-fluorosulfate-based FAPIs, 12 and 13, were synthetically prepared, and their inhibition potency was determined using a fluorimetric FAP assay to be IC₅₀ 9.63 and 4.17 nM, respectively. Radiofluorination was performed via the sulfur [¹⁸F]fluoride exchange ([¹⁸F]SuFEx) reaction to furnish [¹⁸F]12 and [¹⁸F]13 in high activity yields (AY) of 39–56% and molar activities (A_m) between 20–55 GBq/µmol. In vitro experiments focused on the stability of the radiolabeled FAPIs after incubation with human serum, liver microsomes and liver cytosol. Preliminary PET studies of the radioligands were performed in healthy mice to investigate the in vivo biodistribution and ¹⁸F defluorination rate. Fast pharmacokinetics for the FAP-targeting tracers were retained and considerable bone uptake, caused by either ¹⁸F defluorination or radioligand accumulation, was observed. In summary, our findings demonstrate the efficiency of [¹⁸F]SuFEx as a radiolabeling method as well as its advantages and limitations with respect to PET tracer development.

Small molecules offer some advantages for developing positron emission tomography (PET) tracers and are therefore a promising approach for imaging and therapy monitoring of programmed death ligand 1 (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 phosphonic acid groups, serving as hydrophilizing units to lower the log D_7.4 value down to -4.28. The binding affinities of compounds were evaluated using saturation binding and a real-time binding assay, with a highest binding affinity of 21 nM. Small-animal PET imaging revealed vastly different pharmacokinetic profiles depending on the quantity and type of hydrophilizing 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-overexpressing tumor. With further modifications, this compound could be a promising candidate for the imaging of PD-L1 in the clinical setting.

Background: Targeted alpha therapy represents an advanced and rapidly evolving form of precision cancer treatment with increasing importance in recent years. The alpha-emitter ²²⁵ Ac plays a key role in this clinical development due to its attractive physical and chemical properties. In this context, the macropa chelator has favorable characteristics in terms of labeling conditions and complex stability, making its derivatives exceptionally appealing for ²²⁵ Ac-labeling of heat-sensitive biomolecules. However, preclinical evaluation of such ²²⁵ Ac-containing molecules and comprehensive assessment of their pharmacokinetics, dosimetry and radiobiology necessitate a suitable diagnostic counterpart. Due to its attractive radiation properties, ¹³³ La represents an adequate positron-emitting radionuclide to form a matched pair with ²²⁵ Ac for macropa-based radiopharmaceuticals. Herein, we describe the preparation and radiopharmacological characterization of macropa-functionalized, ¹³³ La/ ²²⁵ Ac-labeled single-domain antibodies (sdAbs) targeting the epidermal growth factor receptor (EGFR) to demonstrate the general suitability of this theranostic pair of radionuclides. Results: The synthesis of a clickable, bicyclononyne-modified macropa chelator and its site-specific conjugation to azide-modified, monovalent and biparatopic sdAbs is presented. Subsequent labeling at room temperature (rt) for 15 min resulted in molar activities of 30 MBq/nmol for ¹³³ La and 0.5 MBq/nmol for ²²⁵ Ac, respectively. In vitro studies using the ¹³³ La-labeled sdAbs revealed comparable binding characteristics, but an enhanced cellular internalization of the biparatopic variant compared to its monovalent counterparts. This increased uptake consequently resulted in higher cytotoxicity of the ²²⁵ Ac-labeled biparatopic conjugate. In vivo PET imaging of the ¹³³ La-labeled conjugates indicated comparable uptake and retention of the mono- and biparatopic variants in liver and kidneys, with the former showing slightly higher tumor accumulation. Ex vivo biodistribution studies conducted with ²²⁵ Ac-labeled conjugates largely confirmed the findings obtained by PET imaging, albeit with a marginally higher tumor accumulation of the biparatopic ²²⁵ Ac-radioimmunoconjugate. Final histological examinations of tumor and kidney tissues showed DNA damage in the renal cortex of the ²²⁵ Ac-radioimmunoconjugate-treated mice, but no differences in the number of γ-H2AX-positive cells in the corresponding tumor tissues could be detected. Conclusions: We present a comprehensive study on the theranostic application of ¹³³ La and ²²⁵ Ac for antibody-based biomolecules and lay the foundation for the future application of this matched pair of radionuclides towards labeling of heat-sensitive, macropa-functionalized radiopharmaceuticals in general.

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 showed a potential to break this localized immune paralysis, but only 30% of patients are considered responders. New diagnostic approaches are 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 biphenyl-based small-molecule PD-L1 ligands were synthesized using a convergent synthetic route with a linear sequence of up to eleven steps. As a chelator NODA-GA, CB-TE2A or DiAmSar was used to allow radiolabeling with copper-64 ([⁶⁴Cu]Cu-14–[⁶⁴Cu]Cu-16). In addition, a dimeric structure based on DiAmSar was synthesized ([⁶⁴Cu]Cu-17). All four radioligands exhibited high proteolytic stability (> 95%) up to 48 h post-radiolabeling. Saturation binding yielded moderate affinities toward PD-L1, ranging from 100 to 265 nM. Real-time radioligand binding provided more promising K_D values around 20 nM for [⁶⁴Cu]Cu-14 and [⁶⁴Cu]Cu-15. In vivo PET imaging in mice bearing both PC3 PD-L1 overexpressing and PD-L1-mock 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 of uptake in gall bladder and intestines. The dimeric structure [⁶⁴Cu]Cu-17 showed extensive accumulation and trapping in the liver but was also cleared via the renal pathway. Of all tracer candidates and across all timepoints, [⁶⁴Cu]Cu-17 showed the highest accumulation at 24 h p.i. in the PD-L1-overexpressing tumor of all timepoints and all radiotracers, indicating drastically increased circulation time upon dimerization of two PD-L1 binding motifs. Conclusions: This study shows that chelator choice significantly influences the pharmacokinetic profile of biphenyl-based small molecule PD-L1 radioligands. The NODA-GA-conjugated radioligand [⁶⁴Cu]Cu-14 exhibited favorable renal clearance; however, the limited uptake in tumors suggests the need for structural modifications to the binding motif for future PD-L1 radiotracers.