The field of oncology has witnessed remarkable advancements in recent years, particularly within the domain of immunotherapy. Notably, immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and its ligand PD-L1 have reshaped cancer treatment paradigms [1,2,3]. However, the efficacy of these therapies relies heavily on precise patient stratification, meticulous monitoring of treatment response, and early detection of potential adverse events [4, 5]. Positron emission tomography (PET) imaging has emerged as a formidable tool for the non-invasive visualization and quantification of molecular processes within the body [6, 7]. While PET imaging with radiolabeled antibodies against PD-L1 has shown promise, the inherent limitations associated with antibodies, such as immunogenicity, short half-life, and high cost, have motivated the development of small molecular tracers for PET imaging of PD-L1 [8,9,10,11].

Small molecules offer several advantages over antibodies, including rapid clearance, improved tissue penetration, and the potential for radiolabeling with longer-lived isotopes [8]. Leveraging these properties, researchers have embarked on the development of PD-L1 specific small molecule PET tracers, aiming to enable real-time assessment of PD-L1 expression in tumors and immune cells. This pursuit represents a multifaceted approach, encompassing the design, synthesis, and evaluation of compounds with high specificity and affinity, as well as desired pharmacokinetic properties. Key challenges in this endeavor include optimizing tracer specificity for PD-L1, achieving sufficient tumor uptake and retention, and ensuring compatibility with clinical PET imaging systems. Despite these hurdles, recent preclinical studies have demonstrated the feasibility and potential clinical utility of PD-L1 small molecule PET imaging across various cancer models [12,13,14].

WL12 is the first high-affinity cyclic peptide with PD-L1 binding, which can be used as a PET imaging agent for detecting the expression level of PD-L1 in tumors and other tissues. WL12 has been labeled with 18F, 64Cu, and 68Ga radionuclides thus far [14,15,16]. In the process of fluorination to synthesize [18F]F-FPy-WL12, the hydrophilic carboxylates are replaced with a lipophilic nicotinic group, thus setting it apart from [64Cu]Cu-WL12 and [68Ga]Ga-WL12. This alteration in molecular structure significantly impacts the behavior and properties of [18F]F-FPy-WL12 [15, 17]. Carboxylates, owing to their hydrophilic nature, are known to enhance water solubility and facilitate rapid clearance from the body, making them favorable for applications that require renal clearance and minimal non-specific binding. Conversely, compounds that are highly lipophilic in nature demonstrate enhanced membrane permeability, which can lead to increased uptake in lipid-rich tissues, such as the liver and kidneys, along with non-specific binding to other tissues [15].

The PEGylation of peptides, a common strategy for improving pharmacokinetics, involves conjugating polyethylene glycol (PEG) chains to the peptide, thereby enhancing its circulation time and reducing nonspecific interactions [18]. The synthesis of PEGylated TPP-1 (PEG-TPP-1) as a star tetramer represents an innovative approach to enhance the pharmacokinetics and biodistribution of the imaging agent [19]. However, previous studies investigating [18F]F-PEG-TPP-1 and [64Cu]Cu-PEG-TPP-1 have found that the star tetrameric structure increases molecular weight and size, potentially resulting in prolonged circulation time, reduced renal clearance, and enhanced off-target accumulation. Therefore, the translation potential and clinical applicability of this approach may be limited [20, 21].

Nanobodies offer several advantages compared to conventional antibodies or small molecules, including small size (typically 15 kDa), high stability, low immunogenicity, and rapid tissue penetration [22]. Liu et al. have reported that the 68Ga-labeled nanobody Nb109 offers high sensitivity and spatial resolution for detecting PD-L1 expression in lung cancer PDX models via PET imaging [23]. This allows for accurate localization and characterization of PD-L1-positive tumors within the xenografts, aiding in the evaluation of tumor heterogeneity and the identification of potential therapeutic targets. However, they observed differences in background uptake of the imaging agent, particularly in the liver and kidneys, between the two PDX models (ADC and SCC). These differences likely stem from inherent biological variations and characteristics of the tumor models [24].

When comparing the biodistribution characteristics of [18F]F-NOTA-IPB-PDL1P with [18F]F-FPy-WL12 and [64Cu]Cu-PEG-TPP-1, Sun et al. found that all three tracers exhibit high uptake in the kidneys and moderate uptake in tumors. In addition, [18F]F-NOTA-IPB-PDL1P exhibits higher uptake in muscle tissue compared to [68Ga]Ga-WL12 [25]. This finding prompts further improvements of molecular imaging agent [18F]F-NOTA-IPB-PDL1P in future studies.

[18F]F-BMS-986229 is a macrocyclic peptide ligand specifically designed to target PD-L1 [26]. Macrocyclic peptides offer improved stability and binding affinity compared to linear peptides, thereby highlighting their suitability as PET imaging agents [27, 28]. Although [18F]BMS-986192 proves valuable in clinical settings, its synthesis remains challenging, resulting in the isolation of the ligand in modest yields [28]. Technical hurdles stemming from the complex synthesis process and the requirement for high radiochemical purity constrain the widespread adoption and clinical utility of [18F]BMS-986192.

D-peptides are mirror images of naturally occurring L-peptides and are resistant to enzymatic degradation, making them attractive candidates for therapeutic and diagnostic applications [29]. PET imaging with 18F-labeled D-peptide antagonist enables the assessment of PD-L1 status in tumors, providing valuable information about the immune microenvironment and potential response to immunotherapy [12]. However, [68Ga]Ga-AUNP-12 offers several advantages over 18F-labeled D-peptide imaging agents, making it an attractive alternative for PET imaging in certain contexts [30]. One the one hand, the synthesis of [68Ga]Ga-AUNP-12 requires less precursor materials, thus reducing the production costs and resource requirements [30]. Additionally, the radiolabeling process of [68Ga]Ga-AUNP-12 is notably straightforward and convenient, allowing for streamlined production workflows and increased efficiency [30]. [68Ga]Ga-AUNP-12 can be produced using a generator system, thereby eliminating the need for expensive cyclotron facilities and infrastructure. Both of these different tracers have undergone clinical verification, which is significant for their promotion, transformation, and clinical application.

In this issue of the European Journal of Nuclear Medicine and Molecular Imaging, Yang et al. developed a novel small-molecule radiotracer named [68Ga]Ga-D-PMED [31]. This tracer was designed based on a 2-methyl-3-biphenyl methanol scaffold, significantly increasing the lipophilicity of the probe. Building upon this design, they synthesized two additional variants. One variant, [68Ga]Ga-D-PEG-PMED, incorporates a PEG linker. This addition aims to enhance the water solubility and reduce the lipophilicity of the radiotracer, potentially improving its pharmacokinetic profile and biodistribution in vivo. The other variant, [68Ga]Ga-D-pep-PMED, incorporates a hydrophilic peptide sequence by introducing a hydrophilic peptide moiety into the radiotracer. This modification aims to further increase water solubility and reduce lipophilicity. The hydrophilic peptide sequence also offers notable advantages, such as improved biocompatibility and reduced non-specific binding. High radiochemical yields were successfully obtained for all three radiotracers. Specifically, the yields were reported as 87 ± 6 % for [68Ga]Ga-D-PMED, 82 ± 4 % for [68Ga]Ga-D-PEG-PMED, and 79 ± 9 % for [68Ga]Ga-D-pep-PMED. As is commonly understood, lower IC50 (half maximal inhibitory concentration) values indicate a higher affinity of the radioligand for the target. In this study, in vitro competition assays demonstrated the high affinities of the radioligands, where the IC50 values for [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]Ga-D-pep-PMED were reported as 90.66 ± 1.24 nM, 160.8 ± 1.35 nM, and 51.6 ± 1.32 nM, respectively. Due to its hydrophilic modification, [68Ga]Ga-D-pep-PMED showed the highest target-to-nontarget (T/NT) ratio among the different radioligand variants, with a value of approximately 6.2 ± 1.2. A higher T/NT ratio indicates better specificity and lower off-target binding, which is desirable for PET imaging agents. Lastly, this study evaluated the effects of dose and duration of treatment using anti-mouse PD-L1 antibody on PD-L1 levels in the tumor via PET imaging with [68Ga]Ga-D-pep-PMED. The authors found that the tumor uptake of [68Ga]Ga-D-pep-PMED significantly decreased with increasing doses of PD-L1 mAb treatment. After 8 days of treatment with a single antibody dose, the uptake of [68Ga]Ga-D-pep-PMED in the tumor significantly increased compared to baseline levels. However, despite this increase, the uptake remained lower than that in the saline (control) group. The findings suggest that the efficacy of anti-PD-L1 antibody in modulating PD-L1 levels in the tumor is dose-dependent and influenced by the duration of treatment [31].

In conclusion, the hydrophilic property (log P =  − 2.31 ± 0.09) and pharmacokinetics of [68Ga]Ga-D-pep-PMED greatly improved due to the peptide sequence. This resulted in better T/NT ratios as evidenced by PET imaging. Additionally, BMS202-derived [68Ga]Ga-D-pep-PMED failed to block the tumor uptake of radioligands. This failure could be attributed to the high expression of PD-L1 in the liver. As the dose of BMS202 increased, radioligand uptake in the liver decreased, while uptake in the tumor was not significantly inhibited, consistent with previous findings [31]. Conversely, tumor uptake dramatically decreased in in vivo competitive inhibition assays with PD-L1 mAbs, demonstrating that [68Ga]Ga-D-pep-PMED specifically binds to PD-L1 [31]. Overall, these results suggest that [68Ga]Ga-D-pep-PMED is a promising PET tracer for visualizing PD-L1 expression in vivo. At present, there are several 68Ga-labeled PD-L1 specific tracers, such as [68Ga]Ga-NOTA-WL12 and [68Ga]Ga-NOTA-Nb109. These tracers have also shown high specific affinity for PD-L1 and favorable pharmacokinetics, indicating great potential for noninvasive PET imaging of PD-L1 expression in tumors [13, 23, 32]. Through direct comparison of these tracers, researchers can gain insights into their respective strengths and limitations, facilitating informed decisions regarding their optimal application in specific clinical and research settings. Additionally, such comparative studies may reveal potential synergies or complementary functions among these tracers, thereby fostering the development of improved PET imaging strategies for assessing PD-L1 expression and guiding personalized immunotherapy in cancer patients.