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. 2024 Jan 30;21(3):1515–1525. doi: 10.1021/acs.molpharmaceut.3c01151

PET Imaging of Peptide Probe Al[18F]F-NOTA-PCP1 for Monitoring the Engagement of PD-L1 Antibodies in Tumors

Yang Zhang §,§, Yong Wang §, Yunhao Chen ⊥, Xingchen Ding §, Shijie Wang ∥, Wei Liu §, Man Hu ‡,§,*, Zhiguo Liu #,*
PMCID: PMC10915797  PMID: 38291578

Abstract

graphic file with name mp3c01151_0008.jpg

Immune checkpoint inhibitors (ICIs) are a powerful treatment modality for various types of cancer. The effectiveness of ICIs is intimately connected to the binding status of antibodies to receptors. However, validated means to accurately evaluate target specificity and predict antibody efficacy in vivo are lacking. A novel peptide-based probe called Al[18F]F-NOTA-PCP1 was developed and validated for its specificity to PD-L1 in A549, U87MG, GL261, and GL261-iPDL1 cell lines, as well as in xenograft models. Then the probe was used in PET/CT scans to determine the binding status of PD-L1 antibodies (atezolizumab, avelumab, and durvalumab) in U87MG xenograft model mice. Moreover, Al[18F]F-NOTA-PCP1 was used to evaluate the impact of different treatment times and doses. Al[18F]F-NOTA-PCP1 PET/CT can be used to evaluate the interaction between PD-L1 and antibodies to determine the effectiveness of immunotherapy. By quantifying target engagement, the probe has the potential to predict the efficacy of immunotherapy and optimize the dose and treatment schedules for PD-L1 immunotherapy. This imaging agent could be a valuable tool in guiding personalized treatment strategies and improving cancer patient outcomes.

Keywords: PET/CT imaging, PD-L1 antibody, glioma, peptide tracer, immunotherapy

1. Introduction

Immune checkpoint inhibitors (ICIs) have greatly transformed the treatment of numerous malignancies. The programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis has been a critical focus in cancer treatment due to its significant role in the tumor microenvironment (TME).1 Various immune cell types, such as lymphocytes, monocytes, and natural killer cells, have been found to express PD-1. When PD-1 is overexpressed on effector T cells, it impairs their ability to eliminate tumor cells.2 PD-L1 is overexpressed in numerous solid tumors such as breast cancer, nonsmall cell lung cancer (NSCLC), urothelial cancers, ovarian cancer, and glioblastoma (GBM).3,4 Moreover, the PD-1/PD-L1 pathway is activated when PD-L1 is overexpressed on the surface of cancerous cells, promoting tumor growth.5 Studies have shown that certain factors can induce PD-L1 expression to facilitate immune evasion. For example, β-catenin, a protein involved in cell signaling, has been found to cause an increase in PD-L1 expression levels, facilitating immune evasion.6 Another study showed that NPM1, a protein associated with leukemia, could upregulate PD-L1 to evade host antitumor immunity.7 ICIs have been shown to effectively block the PD-1/PD-L1 pathway in various cancer types. By inhibiting the PD-1/PD-L1 interaction, ICIs help to restore the ability of the immune system to attack and eliminate tumor cells, leading to improved patient outcomes.

Recently, it has become clear that an increasing number of ICIs have been applied in many solid tumors to improve the outcomes of cancer patients since PD-1/PD-L1 inhibitors were approved by the Food and Drug Administration (FDA). Unfortunately, the great benefit from ICIs is only concentrated on a small minority. Approximately 70% of patients do not show a response to ICIs, particularly in some refractory settings. The objective response rate (ORR) of ICIs was found to range from 10% to 30%.8,9 This variability can be attributed to the inadequate and nonuniform distribution of antibodies within tumors.10 Several studies have indicated that antibody treatment is not effective in GBM, which is the most aggressive tumor in the central nervous system (CNS) and has numerous immune evasion mechanisms. It has been reported that using the anti-PD-L1 monoclonal antibody durvalumab (DurMab) against the PD-1/PD-L1 axis has a suitable tolerability profile. However, it failed to improve the outcomes of GBM patients.11 In addition, Rimas et al. enrolled 16 recurrent GBM patients and treated them with atezolizumab (AtzMab), which was shown to be well-tolerated but not clinically effective.12 Notably, the success of cancer immunotherapy is determined by the sufficient and safe accessibility of drugs to tumors, heterogeneity of tumors, endocytosis, integrity of drug targets, and so on.13−16 A better understanding of target engagement will ensure an efficient immune response in immunotherapy. However, due to the poor penetration of ICIs, the degree of target engagement in solid tumors for immunotherapy remains unknown.17 ICIs response variability still remains a major challenge in clinical workflow. Proper patient selection is crucial for ensuring the effectiveness as well as the safety of ICIs in cancer treatment.

To the best of our understanding, PD-L1 immunohistochemistry (IHC) staining is routinely utilized as a simple and direct method in PD-1/PD-L1 immunotherapy treatment, which requires invasive methods to obtain tumor tissue samples.18 However, it may not completely reflect the real-time changes in the expression of PD-L1 and the degree of drug-target engagement in solid tumors. Furthermore, it is significant to noninvasively keep track of the real-time fluctuations in drug-target interactions. Positron emission tomography (PET) allows processes noninvasively to monitor different PD-L1 status in both tumor and metastatic foci.19−21 More importantly, PET can visualize intratumoral and intercellular heterogeneity.22 Peptides are regarded as promising candidates for PET imaging because of their simple synthesis and favorable physicochemical characteristics, including their small molecular size and effective tissue permeability.23−25 For instance, WL12 has been used for PET/CT imaging to detect the PD-L1 status of tumors. However, due to incomplete modification of the structure of WL12, the tracer uptake in liver and the tumor background are not satisfactory.26,27

In this study, we optimized the chemical structure of NOTA-WL12 and synthesized NOTA-PCP1 to target PD-L1, and it is radiolabeled with 18F for PET imaging. PET/CT scans using this probe have demonstrated high affinity for PD-L1 and have shown strong contrast images at 120 min postadministration. One of the main advantages of Al[18F]F-NOTA-PCP1 PET/CT is the measurement of the engagement of PD-L1 after antibody treatment in patients undergoing ICI therapy. This method can help determine the effectiveness of the treatment and guide subsequent therapeutic decisions. Additionally, Al[18F]F-NOTA-PCP1 PET/CT may help elucidate mechanisms governing GBM progression, thereby improving the GBM diagnosis and treatment. By performing PET/CT on GBM patients, we can further understand of the important function of PD-L1 in the illness and potentially identify novel treatment options. Furthermore, Al[18F]F-NOTA-PCP1 PET/CT may also help identify patients who may benefit from immunotherapy and spare nonresponsive patients from unnecessary treatment. In conclusion, our proposed noninvasive, rapid, and accurate approach using Al[18F]F-NOTA-PCP1 PET/CT holds promise for determining the expression of PD-L1, guiding immune checkpoint therapy, exploring GBM mechanisms, and identifying patients who may be suitable candidates for immunotherapy.

2. Materials and Methods

2.1. Binding Affinity Assays (Surface Plasmon Resonance Analysis)

The Biacore X100 instrument, equipped with a CM5 chip (catalog BR100012, Cytiva), was used to perform surface plasmon resonance (SPR) analysis at a temperature of 25 °C. Recombinant PD-L1 protein from human and mouse sources was immobilized on the CM5 chip, and any excess carboxyl groups on the chip surface were blocked using ethanol hydrochloride (pH 8.5). The peptides, NOTA-PCP1 and NOTA-WL12, were diluted in running buffer called PBS-P (0.01 M HEPES pH 7.2–7.4, 0.15 M NaCl, and 0.005% (v/v) surfactant P20). Peptides were prepared at seven distinct concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 nM) and applied to a CM5 Sensor chip loaded with the recombinant human PD-L1 protein. In a parallel setup, the same peptides were diluted to equivalent concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM) and introduced into another CM5 Sensor chip featuring the recombinant murine PD-L1 protein. The peptides were introduced at a flow rate of 10 μL/min, and the binding signals were continuously recorded as response units. The contact time (time of peptide injection) was set at 120 s, and the dissociation time (time without peptide flow) was set at 360 s. To regenerate the chip surface, glycine pH 2.0 was injected for 15 s. The affinity of the peptide–protein interaction, represented as the KD value, was calculated by using the association rate constant (Ka) and dissociation rate constant (Kd). The peptide binding curve was fitted onto the sensorgrams after the blank values were subtracted, and this analysis was prepared by Biacore X100 Evaluation Software (Cytiva, Marlborough, MA).

2.2. Synthesis of Al[18F]F-NOTA-PCP1

The synthesis module (ChelationLab@Al18F) was used for Al[18F]F-NOTA-PCP1 automatic radiosynthesis, as reported in our previous publication.28 In brief, 100 μg of NOTA-PCP1 in 20 μL of deionized (DI) water was added into a 2 mL Eppendorf (EP) tube containing 10 mM AlCl3 (5 μL), 0.1 M NaOAc-HOAc buffer (50 μL), and acetonitrile (300 μL). The EP tube was placed in the heating block and sealed with the PEEK cap as the reactor. The automated synthesis procedures were performed as described previously.29

2.3. Cell Culture

The A549 NSCLC cell line derived from humans, U87MG glioma cell lines derived from humans, and GL261 glioma cell line derived from mice were purchased from Cas9X (Suzhou, China). GL261 cells were infected with a lentivirus vector containing the mouse-PD-L1 gene (Cd274, 45904–1) to create a cell line stably expressing high PD-L1 (GL261-iPDL1). Following infection, the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The cells were placed in a humidified environment at 37 °C with 5% CO2.

2.4. Cell-Binding Studies

The expression of PD-L1 on A549, U87MG, GL261, and GL261-iPDL1 cells was confirmed using Western blot analysis with an antihuman PD-L1 antibody (ab205921, Abcam, UK) or antimouse PD-L1 antibody (ab213480, Abcam, UK) at a dilution of 1:1000. To measure the cell-bound activity, A549, U87MG, GL261, and GL261-iPDL1 cells (n = 3) were plated and the cell count was 1 × 106 for each type of cell. Then, cells were incubated with 0.1 μCi of Al[18F]F-NOTA-PCP1 at 4 °C for 30 min. Afterward, the cells were rinsed three times with a PBS solution containing Tween-20 (0.1%). The radioactivity in cells was quantified with an automated γ-counter (2470 Wizard2, PerkinElmer, USA). To evaluate the binding specificity of the antibodies, U87MG cells were incubated with PBS or 50 nM PD-L1 monoclonal antibodies at 4 °C for 30 min. Then, the cells were incubated with Al[18F]F-NOTA-PCP1 and washed, and the cell-bound activity was calculated. Finally, the uptake of the tracer by the cells was described as a percentage of the injected dose (%ID).

2.5. Animal Models

BALB/c nude mice and C57/BL6 mice were obtained from Hua Fukang Biological Technology Co., Ltd. Female mice aged 6 to 8 weeks and weighing between 18.0 and 23.0 g were used in the study. The mice were subcutaneously inoculated with U87MG (6 × 106) or A549 (1 × 107) cells in the right flanks for BALB/c nude mice and with GL261 (3 × 106) or GL261-iPDL1 (3 × 106) cells in the right flanks for C57/BL6 mice using a hypodermic needle. The tumors were permitted to proliferate until their volume reached 100–300 mm3 before PET/CT imaging or biodistribution studies. This animal study protocol was approved by the Animal Ethics Committee of Shandong Cancer Hospital affiliated with Shandong First Medical University.

2.6. Micro-PET/CT Imaging In Vivo

The specificity of Al[18F]F-NOTA-PCP1 binding with PD-L1 protein was determined by measuring tracer uptake in tumors derived from U87MG, A549, GL261, and GL261-iPDL1 cells on PET/CT. To visualize the target engagement of PD-L1 antibodies, U87MG tumor-bearing nude mice (n = 3) were administered AtzMab (20 mg/kg dose), Avelumab (AveMab, 10 mg/kg dose), or DurMab (10 mg/kg dose). Al[18F]F-NOTA-PCP1 PET/CT was performed to quantify the effect of the PD-L1 antibody therapeutic dose at 24 and 120 h. In addition, U87MG tumor-bearing mice were administered different doses of AtzMab (including 0.05, 0.5, 5, and 20 mg/kg) to assess the impact of the antibody treatment dose on PD-L1 target engagement at 120 h (n = 3). The impact of time on PD-L1 target engagement was further measured at 24 and 120 h for the 5 mg/kg and 20 mg/kg groups (n = 3). First, 6.0 ± 0.91 MBq of Al[18F]F-NOTA-PCP1 was injected into mice from the tail vein. Afterward, the mice were single-housed in an induction chamber supplied with 2% isoflurane gas for 10 min. Subsequently, the mice were placed on the imaging stage to receive PET/CT scanning 2 h postinjection of Al[18F]F-NOTA-PCP1 noninvasively. Next, tumor-bearing mice were anesthetized by 1.5%–2% isoflurane vaporized in oxygen at a flow of 0.5 L/min. Micro-PET/CT scans were administered on a small-animal IRIS PET/CT scanner (Inviscan Strasbourg, France), and image reconstruction was performed using a 3D-OSEM/OP-MAP algorithm with attenuation correction. Regions of interest (ROIs) were drawn manually to delineate the tumors using OsiriX MD 11 software. The percentage of injected dose per cubic centimeter (%ID/cc) was determined as the tracer uptake of tumors.

2.7. Biodistribution Studies

The U87MG-bearing nude mice treated with antibodies (n = 3) were euthanized by decapitation 2 h postinjection of Al[18F]F-NOTA-PCP1. Tissues including tumor, heart, liver, spleen, lung, kidneys, bone, brain, blood, muscle, and bowel were extracted from each mouse, washed, weighed and evaluated for radiotracer accumulation. The accumulation of radiotracer in organs and tumors was determined using a γ counter and calculated as the percentage of injected dose per gram (%ID/g).

2.8. Immunohistochemistry

U87MG, A549, GL261, and GL261-iPDL1 tumor-bearing mice were sacrificed after Al[18F]F-NOTA-PCP1 PET/CT imaging, and tumors were immediately removed and placed in 4% neutral buffered formalin for 48 h. Subsequently, tumors were dehydrated, embedded in paraffin, and then sections were baked for 60 min at 65 °C before dewaxing and hydration. Subsequently, antigens were repaired via antigen repair buffer (K8004 EnVisionTM FLEX Target Retrieval Solution, High PHX50) for 50 min. The sections were incubated with antihuman PD-L1 antibody (M3653, Dako, Agilent, USA) or antimouse PD-L1 antibody (ab238697, Abcam, UK). Afterward, the samples were incubated with FLEX/HRP (Agilent, SM802) for 20 min and then with Flex (SM803) DAB+Sub chromo (diaminobenzidine-peroxidase substrate, Dako) for 10 min. Finally, the sections were scanned via a ZEISS Automatic Digital Slide Scanner (Axio Scan. Z1, German), and ZEN 2012 software (blue edition) was utilized to analyze the data.

2.9. Statistical Analysis

This analysis was conducted with GraphPad Prism 8.0.2 software. The uptake of tumor cells and xenografts after administering Al[18F]F-NOTA-PCP1 and antibodies was compared using an unpaired two-tailed t test. A p value of less than 0.05 was considered statistically significant.

3. Results

3.1. NOTA-PCP1 Binding to Human PD-L1 with High Affinity

We measured the binding affinity of NOTA-PCP1 for both human PD-L1 and mouse PD-L1. SPR (surface plasmon resonance) showed that the Ka value of NOTA-PCP1 to human and mouse PD-L1 were 1.474 × 106 Ms1– and 1.741 × 104 Ms1–, respectively, and the Kd value were 5.925 × 10–4 1/s and 0.5202 1/s. The KD of NOTA-PCP1 for human and mouse PD-L1 was 4.019 × 10–10 M and 2.998 × 10–5 M, respectively (Figure 1A, B). Furthermore, a comparison of the affinity of NOTA-PCP1 and the leading compound for PD-L1 is shown in Figure S1 and Tables S1 and S2. Overall, these results indicated that the NOTA-PCP1 had a higher binding affinity for human PD-L1 than mouse PD-L1.

Figure 1.

Figure 1

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Affinity of NOTA-PCP1 binding to human as well as mouse PD-L1 protein detected by SPR (surface plasmon resonance). (A) Representative sensograms of NOTA-PCP1 binding to the human PD-L1 protein. (B) Representative sensograms of NOTA-PCP1 binding to the mouse PD-L1 protein.

3.2. Al[18F]F-NOTA-PCP1 Synthesis, Stability, and In Vitro Characteristics

PCP1 is a cyclic peptide composed of 12 amino acids that specifically targets PD-L1. It was derived from WL1226 and modified with a polyethylene glycol linker and NOTA as a chelator for Al18F radiofluorination. (Figure 2A). Al[18F]F-NOTA-PCP1 was radiosynthesized for less than 25 min by using a custom-made automated radiosynthesizer (ChelationLab@Al18F) (Figure S2). The isolated radiochemical yield was 37–45% with nondecay correction (n = 20) within 25 min, and the radiochemical purity exceeded 95% (n = 20) (Figure 2B). The molar activity of the radiotracer was 37–55 GBq/μmol at the end of radiosynthesis. The radiopharmaceutical solution exhibited a transparent and colorless appearance with a pH of 5.5–6.0. The attributes of the injectable tracer solution are listed in Table S3. Moreover, the stability of Al[18F]F-NOTA-PCP1 was also tested in saline and 5% HSA in vitro. It was demonstrated that the radiochemical purity of the tracer remained greater than 95% for 4 h based on radio-HPLC analysis (Figure S3).

Figure 2.

Figure 2

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Preparation of the Al[18F]F-NOTA-PCP1 probe and in vitro assays. (A) Schema and structure for the production of the Al[18F]F-NOTA-PCP1. (B) Radio-HPLC profile of the purified probe Al[18F]F-NOTA-PCP1. (C) PD-L1 expression in the A549, U87MG, GL261, and GL261-iPDL1 cell lines. Loading control: GAPDH. (D) Al[18F]F-NOTA-PCP1 uptake by the A549, U87MG, GL261, and GL261-iPDL1 cell lines. (E) Tracer uptake of the PBS group, AtzMab group, AveMab group, and DurMab group in the U87MG cell line. *** p < 0.001, **** p < 0.0001. ns, not significant.

3.3. Binding of Al[18F]F-NOTA-PCP1 to Tumors In Vitro

The PD-L1 levels of A549 and U87MG (derived from human), as well as GL261 and GL261-iPDL1 (derived from mice), cell lines were evaluated by Western blotting. It had been confirmed the human PD-L1 expression of A549 was lower than U87MG. Meanwhile, it illustrated that the mouse PD-L1 expression of GL261-iPDL1 was higher than GL261 (Figure 2C).

To substantiate the binding specificity of Al[18F]F-NOTA-PCP1 with PD-L1 in vitro, cellular binding assays were performed on A549, U87MG, GL261, and GL261-iPDL1 cells. After 30 min of incubation, significantly higher uptake of Al[18F]F-NOTA-PCP1 was observed in the U87MG cell compared with the A549 cell, with %ID values of 22.19 ± 0.18 and 1.31 ± 0.10, respectively (p < 0.001). However, there was almost no uptake of Al[18F]F-NOTA-PCP1 in GL261 and the GL261-iPDL1 cell (1.38 ± 0.14, and 1.15 ± 0.24%ID, p = 0.24; Figure 2D). These results indicated that Al[18F]F-NOTA-PCP1 uptake in tumor cells was correlated to the presence of human PD-L1, while it is not associated with the expression of mouse PD-L1.

3.4. The Interaction of PD-L1 Antibodies with Tumor Cells In Vitro

To measure the target engagement of PD-L1 antibodies in vitro, U87MG cells were treated with PD-L1 antibodies (50 nM; AtzMab, AveMab, and DurMab). The treated cells were compared to PBS-treated controls and were incubated with Al[18F]F-NOTA-PCP1 for 30 min. The %ID values for the cells were 1.26 ± 0.05, 1.40 ± 0.05, 1.34 ± 0.09, and 22.37 ± 0.85 (p < 0.0001; Figure 2E), which indicated that PD-L1 antibodies effectively blocked the PD-1/PD-L1 axis and that Al[18F]F-NOTA-PCP1 can visualize the PD-L1/tumor cell interactions, which can be quantified by determined the tracer uptake by the cells.

3.5. Validation for the Specificity of Al[18F]F-NOTA-PCP1 in Different Xenograft Models

To confirm the specificity of Al[18F]F-NOTA-PCP1 to tumors, PET-CT was performed on the U87MG, A549, GL261, and GL261-iPDL1-bearing models. The U87MG and A549 tumors showed significantly different uptake of Al[18F]F-NOTA-PCP1, with values of 11.72 ± 0.61 and 2.12 ± 0.36%ID/cc, respectively (p < 0.0001; Figure 3A-B). In addition, the GL261 and GL261-iPDL1 tumors had similar uptake values, with %ID/cc values of 2.57 ± 0.83 and 2.84 ± 0.37, respectively (p = 0.63; Figure 4A, B). These findings suggest that Al[18F]F-NOTA-PCP1 could bind to human PD-L1 specifically and could be applied to measure different binding statuses of PD-L1 in tumors. However, Al[18F]F-NOTA-PCP1 PET-CT could not detect mouse PD-L1 expression.

Figure 3.

Figure 3

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In vivo studies on U87MG and A549 models and the IHC assay. (A) Al[18F]F-NOTA-PCP1 PET/CT images of U87MG (high human PD-L1 expression) as well as A549 (low human PD-L1 expression) xenograft models. Tumors are indicated by red arrows with red dashed circles. (B) Al[18F]F-NOTA-PCP1 uptake of U87MG and A549 tumors. (C) PD-L1 expression of U87MG and A549 tumors by IHC analysis. **** p < 0.0001.

Figure 4.

Figure 4

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In vivo studies on GL261 and GL261-iPDL1 models and the IHC assay. (A) Al[18F]F-NOTA-PCP1 PET/CT images of GL261 (low mouse PD-L1 expression) and GL261-iPDL1 (high mouse PD-L1 expression) models. Tumors are indicated by red arrows with red dashed circles. (B) Al[18F]F-NOTA-PCP1 uptake of GL261 and GL261-iPDL1 tumors. (C) IHC analysis of the PD-L1 expression of GL261 and GL261-iPDL1 tumors. ns, not significant

3.6. Immunohistochemistry

The immunohistochemical staining results showed that the U87MG tumor exhibited the highest level of PD-L1 expression among all tumors analyzed. In contrast, the A549 tumors displayed the lowest level of PD-L1 expression (Figure 3C). Furthermore, the GL261-iPDL1 tumors exhibited PD-L1 expression higher than that of the regular GL261 tumors (Figure 4C).

3.7. Quantification of Target Engagement in GBM with PD-L1 Antibodies

To visualize the binding status of PD-L1 antibodies to tumors, U87MG bearing nude mice were administered saline or different PD-L1 antibodies. Compared with the saline-treated group, the uptake of Al[18F]F-NOTA-PCP1 in tumors was lower in the AtzMab-, AveMab-, and DurMab-treated groups. The uptake in tumors was measured at 11.56 ± 0.36, 1.58 ± 0.02, 1.80 ± 0.31, and 1.53 ± 0.10%ID/cc, respectively (p < 0.0001; Figure 5A, B). Similar results were obtained from PET/CT at 120 h (Figure S4 and S5). Overall, the results demonstrated no significant difference in the Al[18F]F-NOTA-PCP1 uptake among the different monoclonal antibodies tested. All PD-L1 antibodies demonstrated effective blockade of PD-L1 in tumors as observed through PET/CT imaging.

Figure 5.

Figure 5

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In vivo and ex vivo studies on tumor models after treatment with different PD-L1 antibodies. (A) Al[18F]F-NOTA-PCP1 PET/CT images of the saline-treated group, AtzMab-treated group, AveMab-treated group, and DurMab-treated group in U87MG models at 24 h. Tumors are indicated by red arrows with red dashed circles. (B) Tumor Al[18F]F-NOTA-PCP1 uptake in the saline-treated group, AtzMab-treated group, AveMab-treated group, and DurMab-treated group at 24 h. (C) Biodistribution study of Al[18F]F-NOTA-PCP1 in nude mice bearing U87MG xenografts and treated with different antibodies for 24 h. **** p < 0.0001. ns, not significant

The biodistribution of Al[18F]F-NOTA-PCP1 2 h postinjection was determined in the saline-, AtzMab-, AveMab-, and DurMab-treated groups. The results presented in Figure 5C with a γ counter show the %ID/g in various organs. In U87MG tumors, the %ID/g was 11.24 ± 0.62 in the control group, 3.44 ± 0.70 in the AtzMab group, 2.60 ± 0.52 in the AveMab group, and 2.89 ± 0.62 in the DurMab group (p < 0.0001). The tracer accumulation in heart, liver, spleen, lung, kidneys, bone, brain, blood, muscle, and bowels showed no significant difference among the control group, AtzMab group, AveMab group, and DurMab group (Figure 5C, Table 1).

Table 1. Biodistribution Study of Control Group and mAb Treated Groups (%ID/g)a.

  Control AtzMab AveMab DurMab
Tumor 11.24 ± 0.62 3.44 ± 0.70 2.60 ± 0.52 2.89 ± 0.62
Heart 2.43 ± 0.41 2.39 ± 0.16 2.08 ± 0.62 2.24 ± 0.35
Liver 12.44 ± 1.12 11.37 ± 0.88 10.95 ± 1.20 10.99 ± 0.34
Spleen 2.09 ± 0.60 1.64 ± 0.11 2.15 ± 0.57 1.78 ± 0.24
Lung 3.10 ± 0.39 2.85 ± 0.73 2.37 ± 0.13 2.49 ± 0.40
Kidneys 130.78 ± 9.70 126.72 ± 15.77 121.29 ± 11.63 110.72 ± 6.26
Bone 4.13 ± 0.92 4.48 ± 0.51 3.46 ± 2.21 1.84 ± 0.55
Brain 0.10 ± 0.02 0.09 ± 0.01 0.08 ± 0.01 0.08 ± 0.02
Blood 0.54 ± 0.06 0.59 ± 0.07 0.55 ± 0.10 0.63 ± 0.08
Muscle 1.12 ± 0.31 1.54 ± 1.22 1.48 ± 0.85 0.93 ± 0.20
Bowel 1.90 ± 0.29 1.61 ± 0.19 1.68 ± 0.20 1.69 ± 0.13
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a

%ID/g: the percentage of injected dose per gram; AtzMab: Atezolizumab; AveMab: Avelumab; DurMab: Durvalumab.

3.8. Quantifying the Effect of Dose and Time on the Occupancy of PD-L1 in GBM by Atezolizumab

The imaging results of mice indicated that the tumor uptake of Al[18F]F-NOTA-PCP1, a marker for PD-L1, decreased as the dose of AtzMab increased (Figure 6A). The %ID/cc values of Al[18F]F-NOTA-PCP1 uptake were 11.64 ± 1.13, 6.17 ± 0.35, 2.11 ± 0.24, and 2.02 ± 0.14 for doses of 0.05, 0.5, 5, and 20 mg/kg AtzMab at 120h, respectively (Figure 6B). Further study is needed to confirm the temporal changes in the unbound PD-L1 levels. Al[18F]F-NOTA-PCP1 PET/CT imaging was conducted on U87MG tumor-bearing mice after administering a fixed dose (5 mg/kg or 20 mg/kg) of AtzMab at 24 and 120 h (Figure 7A). The %ID/cc values of tumors at 24 h were 2.16 ± 0.20 and 2.19 ± 0.13 for the 5 mg/kg dose group and 20 mg/kg dose group, respectively (Figure 7B). At 120 h, the %ID/cc values increased to 6.91 ± 0.18 for the 5 mg/kg dose group (p < 0.0001), while no difference was observed (2.29 ± 0.04, p = 0.27) for the 20 mg/kg dose group. The results of the biodistribution study are shown in Figures 6C and 7C and Tables 2 and 3. These findings suggest that Al[18F]F-NOTA-PCP1 PET/CT imaging could be utilized to assess dose-dependent changes in the binding of PD-L1 to antibodies and to measure alterations in target interactions over time and in response to varying doses.

Figure 6.

Figure 6

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In vivo and ex vivo studies on tumor models at different doses and time points of PD-L1 antibody treatment. (A) Al[18F]F-NOTA-PCP1 PET/CT images of 0.05 0.5, 5, and 20 mg/kg AtzMab-treated groups in U87MG models at 120 h. Tumors are indicated by red arrows with red dashed circles. (B) Al[18F]F-NOTA-PCP1 uptake of 0.05 mg/kg, 0.5 mg/kg, 5 mg/kg, and 20 mg/kg AtzMab-treated groups in U87MG models at 120 h. (C) Biodistribution study of Al[18F]F-NOTA-PCP1 in nude mice bearing U87MG xenografts and treated with 0.05 mg/kg, 0.5 mg/kg, 5 mg/kg, and 20 mg/kg doses of AtzMab for 24 h. ** p < 0.01, **** p < 0.0001. ns, not significant.

Figure 7.

Figure 7

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In vivo and ex vivo studies on tumor models at different doses and time points of PD-L1 antibody treatment. (A) Al[18F]F-NOTA-PCP1 PET/CT images of the 5 and 20 mg/kg AtzMab-treated groups at 24 and 120 h. Tumors are indicated by red arrows with red dashed circles. (B) Al[18F]F-NOTA-PCP1 uptake of the 5 mg/kg and 20 mg/kg AtzMab-treated groups in U87MG models at 24 and 120 h. (C) Biodistribution study of Al[18F]F-NOTA-PCP1 in nude mice bearing U87MG xenografts treated with 5 mg/kg and 20 mg/kg doses of AtzMab for 24 and 120 h. **** p < 0.0001. ns, not significant.

Table 2. Effect of Different Doses of Antibody on Al[18F]F-NOTA-PCP1 Uptake (%ID/g) by Biodistribution Studya.

  0.05 mg/kg 0.5 mg/kg 5 mg/kg 20 mg/kg
Tumor 11.47 ± 0.51 5.86 ± 0.62 2.41 ± 0.21 2.49 ± 0.10
Heart 2.26 ± 0.08 2.21 ± 0.08 2.20 ± 0.19 2.16 ± 0.25
Liver 12.36 ± 0.62 11.71 ± 0.40 13.69 ± 2.44 11.79 ± 0.39
Spleen 2.00 ± 0.19 1.46 ± 0.01 1.48 ± 0.15 1.41 ± 0.12
Lung 2.80 ± 0.16 2.55 ± 0.23 2.75 ± 0.28 2.53 ± 0.15
Kidneys 138.18 ± 9.88 136.08 ± 15.34 123.82 ± 7.19 138.12 ± 15.43
Bone 4.05 ± 0.55 4.01 ± 0.42 1.96 ± 0.24 2.28 ± 0.10
Brain 0.10 ± 0.01 0.09 ± 0.01 0.10 ± 0.01 0.09 ± 0.01
Blood 0.52 ± 0.09 0.44 ± 0.07 0.51 ± 0.08 0.52 ± 0.06
Muscle 1.18 ± 0.34 0.94 ± 0.43 0.95 ± 0.16 1.25 ± 0.76
Bowel 1.64 ± 0.09 1.58 ± 0.08 1.62 ± 0.06 1.60 ± 0.10
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a

%ID/g: the percentage of injected dose per gram.

Table 3. Biodistribution Study of Different Groups Treated with Antibody on Different Time Points (%ID/g)a.

  5 mg/kg
 20 mg/kg
  24 h 120 h 24 h 120 h
Tumor 2.30 ± 0.16 7.58 ± 0.41 2.55 ± 0.60 2.54 ± 0.14
Heart 2.12 ± 0.06 2.15 ± 0.15 2.24 ± 0.18 2.19 ± 0.11
Liver 12.61 ± 0.69 11.31 ± 0.33 12.61 ± 1.16 12.16 ± 1.32
Spleen 1.68 ± 0.13 1.34 ± 0.06 1.36 ± 0.16 1.71 ± 0.51
Lung 2.74 ± 0.15 2.57 ± 0.09 2.77 ± 0.14 2.67 ± 0.38
Kidneys 136.93 ± 12.75 139.25 ± 18.57 130.10 ± 16.64 127.68 ± 6.22
Bone 3.76 ± 1.18 3.57 ± 0.62 2.20 ± 0.18 3.06 ± 1.49
Brain 0.09 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 0.09 ± 0.01
Blood 0.53 ± 0.04 0.46 ± 0.04 0.56 ± 0.06 0.53 ± 0.05
Muscle 1.22 ± 0.39 1.34 ± 0.79 1.32 ± 0.67 0.90 ± 0.10
Bowel 1.67 ± 0.05 1.59 ± 0.07 1.62 ± 0.10 1.60 ± 0.08
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a

%ID/g: the percentage of injected dose per gram.

4. Discussion

Several PD-1/PD-L1 inhibitors have been approved by the FDA for clinical trials. However, the objective response rate of ICIs varies in different subgroups of patients. Numerous clinical trials have confirmed that PD-L1 antibody monotherapy including AtzMab and DurMab is not effective in GBM patients.11,12 Therefore, it is urgent to predict the therapeutic efficacy and improve the regimens of the drugs. It has been confirmed that the response rate of ICIs is related to PD-L1 expression in tumors.30−32 Additionally, the binding status of receptors and ICIs may influence the pharmacokinetic properties of drugs. Moreover, the target engagement of the drugs may change during the period of treatment, which is difficult to detect. Previous reports have also highlighted the potential of 89Zr-atezolizumab PET/CT evaluating efficacy of ICIs in multiple types of cancer patients before initiating ICI treatment.20 However, multiple studies have shown inconsistent results in terms of predicting treatment response and overall survival using this imaging modality.33,34 Furthermore, the limited availability and long biological half-life of 89Zr-durvalumab PET/CT imaging pose additional challenges for its widespread use in clinical practice. Therefore, alternative imaging modalities or biomarkers may be more suitable for the treatment of cancer patients. Other probes such as antibody Fab fragments and nanobody show rapid absorption by the tumor and efficient clearance from the bloodstream and nontarget organs.35,36 Additionally, peptides have a lower molecular weight with the advantage of more easily penetrating tumor tissues, allowing for rapid and accurate monitoring of the dynamic changes in PD-L1 expression during treatment. Thus, exploring the use of peptide-based PET/CT imaging may provide a valuable alternative for monitoring the binding status of antibodies in patients receiving immune checkpoint inhibitor treatment.

We prepared Al[18F]F-radiolabeled NOTA-PCP1 with steady physical characteristics for PET/CT immunoimaging. To enhance receptor targeting imaging, we developed a specialized automated radiosynthesizer (ChelationLab@Al18F) with optimized conditions28 for Al[18F]F labeling of NOTA-PCP1. Our study demonstrated that Al[18F]F-NOTA-PCP1 production could be completed within 25 min at the end of synthesis, yielding products with a molar activity of 37–55 GBq/μmol and high radiochemical purity (>95%). These results demonstrate that Al[18F]F-NOTA-PCP1 has several advantages, including favorable physical characteristics and the ability to be synthesized on demand, making it highly promising for PD-L1-targeting PET/CT imaging.

In this research, a novel compound, NOTA-PEG3-WL12 (NOTA-PCP1), was created through the strategic assembly of three functional elements: a cyclic peptide derived from WL12 for the specific targeting of PD-L1, a chelator (NOTA group) enabling radionuclide labeling for PET imaging (Al18F), and a PEG linker utilized to enhance pharmacokinetic (PK) and pharmacodynamic (PD) characteristics. Novel peptide probe Al[18F]F-NOTA-PCP1 based PET/CT immunoimaging was conducted to measure the expression of human PD-L1 with high sensitivity and specificity and to visualize the binding status of tumor cells to several PD-L1 monoclonal antibodies. The probe showed higher uptake in the U87MG cell line than in the A549 cell line, which had lower human PD-L1 levels. However, in regard to mouse PD-L1, the binding of Al[18F]F-NOTA-PCP1 in the GL261 (low expression of mouse PD-L1) and GL261-iPDL1 (high expression of mouse PD-L1) cell lines was not significant. Similarly, significant Al[18F]F-NOTA-PCP1 uptake was observed in U87MG xenografts from PET/CT imaging, while no significant difference in tracer uptake in GL261 and GL261-iPDL1 tumors with mouse PD-L1 expression was observed. These results of the in vitro and in vivo studies could be explained by the SPR results, which showed that the KD value of NOTA-PCP1 to human PD-L1 and mouse PD-L1 were 4.019 × 10–10 M, and 2.998 × 10–5 M, respectively. These results indicated that NOTA-PCP1 binds to human PD-L1 with a higher affinity than mouse PD-L1. Thus, Al[18F]F-NOTA-PCP1 could detect the human PD-L1 expression in cells or tumors but could not detect mouse PD-L1 status secondary to the intrinsic properties of the probe, which demonstrated that the probe binds to human PD-L1 specifically rather than mouse PD-L1. Overall, Al[18F]F-NOTA-PCP1 PET/CT is capable of visually detecting tumor lesions derived from humans with high specificity and sensitivity. This finding suggests that the technique has substantial potential for future clinical translation.

The results suggest that the uptake of the Al[18F]F-NOTA-PCP1 tracer in tumors can provide information about the binding status of the PD-L1 receptors and different antibodies. When U87MG tumors were treated with different antibodies, the tracer uptake was significantly lower than the control treated group, both in vitro as well as in vivo. This decrease in tracer uptake could be explained by the binding of the antibodies to the PD-L1 receptors. In essence, the Al[18F]F-NOTA-PCP1 PET/CT imaging technique could provide a visualization of the degree of unbound PD-L1 receptors on tumors. An ex vivo biodistribution study further supported these findings, as the tumor tracer accumulations of the AtzMab-, AveMab-, and DurMab-administered groups were significantly lower than the saline-administered group. Nevertheless, there was no notable difference in the accumulation of the Al[18F]F-NOTA-PCP1 tracer in normal organs such as the lung, liver, as well as heart. However, further research ise required to determine the feasibility and efficacy of this novel tool in clinical settings.

This study demonstrates the ability of the Al[18F]F-NOTA-PCP1 PET/CT tracer to detect the binding of antibodies to PD-L1 receptors at different doses and time points. Moreover, the findings indicate that at the 0.05 mg/kg dose, the tracer uptake in tumors was higher than that at the 0.5 5, and 20 mg/kg dose. This result suggests that at lower doses there was partial binding of PD-L1 receptors and Atzmab, whereas at higher doses there was complete binding. Furthermore, the study found that tracer uptake in tumors increased significantly from 24 to 120 h at the 5 mg/kg dose, while it remained steady at the 20 mg/kg dose. This finding indicates that target engagement was reduced at the 5 mg/kg dose but maintained at a relatively high level at the 20 mg/kg dose for 120 h. These results are consistent with previous research using the [64Cu]WL12 PET/CT tracer in lung cancer models, which also demonstrated that the unoccupied tumor PD-L1 could be quantified based on dose and time.37 Compared with 64Cu, 18F is more suitable for peptide radiolabeling because of its shorter half-life. 18F has a half-life of approximately 110 min, while 64Cu has a longer half-life of around 12.7 h.19 Collectively, Al[18F]F-NOTA-PCP1 PET/CT imaging was served as a tool to visualize the binding status of tumor cells and antibodies and further monitor changes in binding status during antibody treatment, which could improve immunotherapy regimens, optimize immunotherapy modes, reverse immune resistance, and even guide combination therapy.

However, the sample size used in our study was limited, and larger sample sizes should be included in future studies to increase the statistical power and provide more robust results. Furthermore, the imaging technique used in our study may have limitations in terms of spatial resolution and sensitivity, which could affect the accuracy of determining PD-L1 levels and the binding status. Improvements in imaging technology and protocols are necessary to overcome these limitations. Last, although our study focused on the valuation of PD-L1 expression and binding status, the clinical significance and potential therapeutic implications of these findings need to be further investigated. Evaluating the correlation between imaging results and patient outcomes, such as the response to immunotherapy or overall survival, will be important in determining the utility of this imaging tool in a clinical setting.

5. Conclusions

The present study demonstrates that Al[18F]F-NOTA-PCP1 is able to specifically target and visualize human PD-L1 status in various tumors. In addition, this probe shows high affinity and sensitivity for human PD-L1. Furthermore, it could be used to quantify the degree of PD-L1 antibody binding to tumor cells. The study also utilized PET/CT imaging at different doses and time points to observe the pharmacokinetics of antibodies. In a word, the findings of this study demonstrate that Al[18F]F-NOTA-PCP1 PET/CT imaging can play a crucial role in personalized immunotherapy by providing valuable information about target management, which could help determine patients who will respond to immunotherapy and tailor PD-L1 antibody therapy to individual patients.

Acknowledgments

The study was supported by the foundation of National Natural Science Foundation of China (82202958, 82272751), the foundation of Natural Science Foundation of Shandong (ZR2021LSW002), Science Technology Program of Jinan (No. 202225013, 202225019), and Shandong provincial colleges and universities youth innovation technology support program (2023KJL004).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.3c01151.

  • PR measurements of NOTA-WL12 or NOTA-PCP1 versus human and mouse PD-L1 (Figure S1); ChelationLab@Al18F system for probe automatic radiosynthesis (Figure S2); in vitro stability test of Al[18F]F-NOTA-PCP1 (Figure S3); Al[18F]F-NOTA-PCP1 PET/CT images of control group and antibody-treated groups on U87MG models (Figure S4); tracer uptake in tumor of control group and antibody-treated groups (Figure S5); Ka, Kd, and KD value of NOTA-WL12 and NOTA-PCP1 to human (Table S1) or mouse (Table S2) PD-L1 protein; quality control test data for Al[18F]F-NOTA-PCP1 radiosynthesis (Table S3) (PDF)

Author Contributions

† Authors Y.Z. and Y.W. contributed equally. M.H. and Z.L. were responsible for the conception and design of the study. X.D. and W.L. contributed scientific insights. Y.Z., Z.L., Y.W., and Y.C. conducted the bioinformatic analysis and prepared the figures and tables. Y.Z., Y.C., and S.W. were involved in the experimental operations. Y.Z. and Y.W. contributed significantly to data interpretation, statistical analysis, and manuscript writing. All authors contributed to the article and approved the submitted version.

The authors declare no competing financial interest.

Supplementary Material

mp3c01151_si_001.pdf (470KB, pdf)

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Supplementary Materials

mp3c01151_si_001.pdf (470KB, pdf)

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