This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.
The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.
The discovery of immune checkpoint proteins was a breakthrough in tumor therapy, and has led to major advances in the development of immune checkpoint blockade therapy1. Programmed cell death-protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) are potential drug targets with several antibodies approved by the Food and Drug Administration (FDA). PD-1 is expressed by tumor-infiltrating immune cells, such as CD4+, CD8+ T cells, and regulatory T cells. PD-L1 is one of the PD-1 ligands, which is overexpressed in a variety of tumor cells2,3. The interaction between PD-1 and PD-L1 inactivates PD-1, thus suppressing the antitumor immune response4. These findings suggest that the inhibition of PD-L1 can improve the killing effect of immune cells and eliminate tumor cells5. Currently, chromogenic immunohistochemistry (IHC) is the most commonly used approach to identify patients who are most likely to respond to immune checkpoint therapy6,7. However, due to the heterogeneous expression of PD-L1 in tumor cells, IHC results from biopsies cannot provide accurate information about PD-L1 expression in patients8. Previous studies have reported that only 20%-40% of patients gain long-term benefits from immune checkpoint blockade therapy1,9,10. There is, therefore, an urgent need to develop a new method to circumvent the false-negative results caused by the heterogeneous expression of these immune checkpoint proteins.
Molecular imaging technology, such as positron emission tomography (PET), enables real-time visualization of the whole body in a noninvasive way, and thus can outperform the conventional IHC method11,12,13. Radiolabeled antibodies, peptides, and small molecules are promising tracers for monitoring PD-L1 expression in cancer patients14,15,16,17,18,19,20,21,22,23,24,25. The FDA has approved three PD-L1 therapeutic monoclonal antibodies: avelumab, atezolizumab, and durvalumab26. Immuno-PET tracers based on these antibodies have been well documented27,28,29,30,31,32. Early-phase clinical trials have revealed limited value for clinical application, because of the unfavorable pharmacokinetics30. Compared with antibodies, peptides exhibit faster blood and organ clearance from healthy organs, and can be easily chemically modified33. Multiple peptides with high affinities for PD-1/PD-L1 have been reported2; WL12 is a reported peptide that shows specific binding to PD-L134. Radiolabeled tracers, [64Cu]WL12, [68Ga]WL12, and [18F]FPyWL12, have been reported to show high in vivo specific tumor-targeting ability, which allows for the harvest of high-quality images of PD-L1 expression in tumors26,35,36,37. Moreover, the first in-human evaluation of radiolabeled WL12 has demonstrated that [68Ga]WL12 (chelated by NOTA) has a safe and efficient potential for clinical tumor imaging38. Due to its high hydrophobicity and high uptake in the healthy liver, WL12 has limited clinical use. Other radiolabeling peptides, such as TPP1 and SETSKSF, which specifically bind to PD-L1, have also showed potential stability and specificity to visualize whole-body PD-L1 expression39,40. However, unmodified peptides are easily degraded by proteases, and are rapidly metabolized by the kidney. Dextrorotary(D)-peptides have been widely used as effective mediators, due to the poor stability of left-handed (L)-peptides41,42,43. D-peptides are hyper-resistant to proteolytic degradation and have remarkably prolonged metabolic half-lives. Compared with their L-counterparts, D-peptides mostly show specific binding abilities44,45,46.
This study designed a new method to detect PD-L1 expression, based on PET imaging of a 68Ga-labeled PD-L1-targeted D-peptide, D-dodecapeptide antagonist (DPA), in a tumor-bearing mouse model47. The stability of [68Ga]DPA was first studied in phosphate-buffered saline (PBS) and mouse serum, after which the binding affinity of [68Ga]DPA in PD-L1-overexpressing tumors was tested. Thereafter, PET imaging was performed in glioblastoma xenograft models to confirm whether [68Ga]DPA was an ideal PET tracer to monitor PD-L1 expression in tumors. The combination of PET imaging and DPA not only provides a new approach to overcome challenges associated with the heterogeneous expression of PD-L1, but also lays the basis for the development of D-peptide-based radiotracers.
The animal experimental procedures were approved by the Animal Ethics Committee of Nanjing Medical University or the National Institutes of Quantum Science and Technology. Mice experiments were strictly performed in compliance with the institutional guidelines of the Committee for the Care and Use of Laboratory Animals.
1. Peptide synthesis
2. 68Ga radiolabeling
NOTE 68Ga was generated in-house at Nanjing First Hospital (Nanjing, China) using a 68Ge/68Ga generator.
3. Tracer stability test
4. Analysis of PD-L1 expression by flow cytometry
5. Immunocytochemistry
6. Cellular uptake and inhibition experiment
7. PET imaging
NOTE: Perform small animal PET imaging, using a micro PET scanner that provides 159 transverse axial sections spaced 0.796 mm apart (center-to-center), with a horizontal field of view of 10 cm and an axial field of view of 12.7 cm. All data collected in the list mode are organized into three-dimensional sinograms. The Fourier is then reassembled into two-dimensional sinograms (frame × min: 4 × 1, 8 × 2, 8 × 5).
8. Ex vivo biodistribution
9. Immunohistochemistry
[68Ga]DPA radiolabeling and stability
The model peptide, DPA, is an effective PD-L1 antagonist. DOTA-DPA was obtained with >95% purity and a yield of 68%. The mass of DOTA-DPA is experimentally observed at 1,073.3 ([M+2H]2+). 68Gallium is considered a suitable radionuclide to label peptides for PET imaging, and therefore was chosen for this study. To radiolabel DPA with 68Ga (half-life: 68 min), DOTA-PEG3-DPA was synthesized (Figure 1A). DOTA was used as a chelator for the 68Ga radiolabeling. To space DOTA and DPA, PEG3 was used as a linker. The [68Ga]-DOTA-PEG3-DPA (referred to as [68Ga]DPA in the following text) showed high radiochemical yield (>95%) and radiochemical purity (>95%) (Table 1). A tracer stability test was also performed using HPLC, and the results showed that [68Ga]DPA had great stability both in PBS and mouse serum. The 68Ga decomposition or peptide hydrolysis was not detected after a 4 h incubation at 37 °C (Figure 1B).
Expression of PD-L1 in U87MG cells
A previous study showed that an increased expression of PD-L1 correlated with poor patient survival in glioblastoma tumors, indicating that PD-L1 may be a remarkable prognostic biomarker and therapeutic target in glioblastoma48. Therefore, a human glioblastoma cell line, U87MG, was used to establish a tumor model to determine the efficacy of [68Ga]DPA in PET/CT for PD-L1 tumor imaging. The flow cytometry results suggested that approximately 60% of the U87MG cells were PD-L1 positive (Figure 2A). Moreover, immunofluorescent staining confirmed the strong expression of PD-L1 in the U87MG cells (Figure 2B). Together, these data demonstrated that the U87MG cell line was suitable for this study.
Cellular uptake and specificity of [68Ga]DPA
The uptake of [68Ga]DPA by U87MG cells presented a time-dependent pattern. When a PD-L1 inhibitor, BMS202, was used as a blocking agent, the binding portion and the uptake of [68Ga]DPA were significantly reduced (Figure 3A). A competitive binding assay further examined the binding affinity (Ki) of BMS202 to the U87MG cells. The estimated binding affinity was 43.8 ± 8.6 nmol/L for BMS202 when [68Ga]DPA was used as a competitor (Figure 3B).
[68Ga]DPA PET imaging of tumor models
PET imaging of [68Ga]DPA was performed in U87MG tumor-bearing BALB/C nude mice. [68Ga]DPA was administered through intravenous injection until the U87MG tumor grew to 100 mm3. Whole-body PET images revealed high [68Ga]DPA accumulation in the tumor after 30 and 60 min of injection, and showed the highest accumulation in the kidney and bladder (Figure 4A). To confirm whether [68Ga]DPA specifically accumulated in PD-L1-positive tumors, another mouse model bearing the PanNET cell line Bon-1 was used as a negative control. A parallel experiment demonstrated little [68Ga]DPA accumulation in Bon-1 tumors at 60 min post-injection (Figure 4B).
To clarify this difference, immunohistochemical staining was conducted to analyze PD-L1 expression in tumor tissues. The results revealed that the U87MG cells presented considerable PD-L1 expression (Figure 5A,C), but not the Bon-1 tumor (Figure 5B,C). These data were consistent with the PET results. Therefore, it is possible that the different tumor cell growth statuses resulted in different PD-L1 expression (e.g., tissue necrosis). To verify this, hematoxylin and eosin (H&E) staining was performed. As expected, a similar cell morphology was observed between the two tumor tissues (Figure 5D).
Ex vivo biodistribution of [68Ga]DPA in U87MG tumors
The ex vivo biodistribution study was also conducted using U87MG-bearing mice (Table 2). The results showed a rapid clearance in blood and most analyzed organs, including the heart, liver, lung, and muscle. The kidney accumulated the highest amount of radioactivity and showed a clearance rate of 0.12% ID/(g∙min) from 5 min to 120 min. The tumor exhibited uptake of the second-highest tracer uptake at all time points. In addition, from 5 min to 60 min post-injection, the tumor presented a lower tracer clearance rate of 0.027% ID/(g∙min). For blood, the clearance rate was 0.069% ID/(g∙min), while for muscle, the clearance rate was 0.037% ID/(g∙min).
Figure 1: [68Ga]DPA radiolabeling and stability. (A) The chemical structure of [68Ga]DPA and the schematic representation of its binding to PD-L1 expressing tumor cells. (B) HPLC curves showing the radioactivity of [68Ga]DPA after incubation with PBS or mouse serum for 0.5, 2, and 4 h. This figure was modified from Hu et al.47. Abbreviations: DPA = dodecapeptide antagonist; PD-L1 = programmed death-ligand 1; PBS = phosphate-buffered saline; HPLC = high-performance liquid chromatography. Please click here to view a larger version of this figure.
Figure 2: Expression of PD-L1 in the U87MG cell line. (A) The expression of PD-L1 in the U87MG cell line was measured through flow cytometry analysis. (B) The expression of PD-L1 in U87MG cells was measured by immunofluorescence staining assay. Scale bar = 100 µm. This figure was modified from Hu et al.47. Abbreviations: PD-L1 = programmed death-ligand 1; SSC = side scatter; FITC = fluorescein isothiocyanate. Please click here to view a larger version of this figure.
Figure 3: Cellular uptake and inhibition of [68Ga]DPA. (A) The uptake of U87MG cells when incubated with [68Ga]DPA (0.74 MBq/mL) or [68Ga]DPA (0.74 MBq /mL) + BMS202 (100 µmol/L) for different durations. (B) Competitive binding of [68Ga]DPA (0.74 MBq /mL) to the U87MG cells following incubation with BMS202. The Ki value is shown in the panel. This figure was modified from Hu et al.47. Abbreviations: DPA = dodecapeptide antagonist; %AD = administered dose (with respect to the binding portion). Please click here to view a larger version of this figure.
Figure 4: PET imaging of [68Ga]DPA in PD-L1-overexpressing U87MG tumors. (A,B) PET-CT images showing the distribution of [68Ga]DPA in U87MG-bearing mice (A) and Bon-1-bearing mice (negative control, B) after intravenous injection (~18.5 MBq) for 30 min and 60 min. Representative maxiumum-intensity projection (MIP) (top panel) and transverse PET-CT images (bottom panel) are presented. The tumor positions are marked by white dashed circles. This figure was modified from Hu et al.47. Abbreviations: DPA = dodecapeptide antagonist; PD-L1 = programmed death-ligand 1; PET-CT = positron emission tomography-computed tomography; MIP = maxiumum-intensity projection; p.i. = post-injection. Please click here to view a larger version of this figure.
Figure 5: Immunohistochemical analysis of [68Ga]DPA-treated tumors. (A,B) Whole-section immunohistochemical images of PD-L1 in (A) U87MG and (B) Bon-1 tumors. (C) Enlarged pictures of the marked areas in A and B. (D) H&E staining of U87MG and Bon-1 tumor. Scale bars = 100 µm (C,D). This figure was modified from Hu et al.47. Abbreviations: DPA = dodecapeptide antagonist; PD-L1 = programmed death-ligand 1; H&E = hematoxylin and eosin. Please click here to view a larger version of this figure.
Tracers | [68Ga]DPA |
Radiochemical yield (%) | >95 |
Molar activity (GBq µmol-1) | 37 ± 8 |
Radiochemical puritya (%) | >95 |
Table 1: Radiolabeling and quality control of [68Ga]DPA. Radiochemical yield, molar activity, and radiochemical purity of [68Ga]DPA. Data are represented as the mean ± SD (n = 7). This table was modified from Hu et al.47. Abbreviation: DPA = dodecapeptide antagonist. aRadiochemical purity of [68Ga]DPA was analyzed by reverse-phase HPLC under an optimized condition: 1) column: YMC-Triat-C18 (4.6 mm i.d., 150 mm, 5 mm); 2) solvent gradient: solvent A-deionized water; solvent B-acetonitrile (0.1% trifluoroacetic acid); flow time: 20 min, with acetonitrile from 10% to 90%; flow rate of 1 mL/min.
[68Ga]DPA | ||||||||
5 min | 30 min | 60 min | 120 min | |||||
blood | 3.89 | ±0.43 | 1.55 | ±1.07 | 0.11 | ±0.02 | 0.05 | ±0.01 |
heart | 1.19 | ±0.39 | 0.52 | ±0.33 | 0.06 | ±0.02 | 0.05 | ±0.02 |
liver | 1.06 | ±0.26 | 0.59 | ±0.43 | 0.19 | ±0.02 | 0.16 | ±0.04 |
spleen | 0.98 | ±0.14 | 0.68 | ±0.67 | 0.14 | ±0.06 | 0.09 | ±0.03 |
lung | 1.64 | ±0.42 | 1.03 | ±0.9 | 0.13 | ±0.05 | 0.09 | ±0.02 |
kidney | 19.23 | ±1.95 | 16.13 | ±1.51 | 11.5 | ±0.44 | 5.2 | ±0.31 |
stomach | 1.54 | ±0.1 | 0.61 | ±0.35 | 0.08 | ±0.01 | 0.08 | ±0.03 |
intestinal | 0.72 | ±0.27 | 0.47 | ±0.35 | 0.08 | ±0.02 | 0.06 | ±0.03 |
pancreas | 1.88 | ±0.28 | 0.77 | ±0.75 | 0.16 | ±0.03 | 0.13 | ±0.03 |
muscle | 2.21 | ±0.27 | 0.71 | ±0.37 | 0.18 | ±0.02 | 0.14 | ±0.04 |
bone | 2.18 | ±0.11 | 0.85 | ±0.51 | 0.26 | ±0.09 | 0.14 | ±0.06 |
brain | 0.19 | ±0.04 | 0.11 | ±0.08 | 0.03 | ±0.01 | 0.02 | ±0.01 |
tumor | 4.5 | ±0.32 | 3.77 | ±0.27 | 2.99 | ±0.03 | 0.89 | ±0.19 |
fat | 2.09 | ±0.49 | 0.81 | ±0.12 | 0.27 | ±0.07 | 0.1 | ±0.07 |
Table 2: Biodistribution of [68Ga]DPA in U87MG tumor-bearing mice after administration for different durations (n = 3 per time point). This table is modified from Hu et al.47. Abbreviation: DPA = dodecapeptide antagonist.
The critical steps described in this method include the efficient labeling of 68Ga to DPA and choosing a suitable time window for PET imaging, which must perfectly match the pharmacodynamic pattern of DPA in the tumor.
In contrast to IHC, PET imaging enables real-time detection of whole-body PD-L1 expression in a noninvasive manner, allowing the visualization of each positive area in a heterogeneous tumor6,7. Peptides were chosen as ligands to avoid the disadvantages of antibodies and small molecules. Antibodies with large molecular weights generally have long circulating half-lives, which causes higher toxicity to healthy organs. The clearance of small molecules is usually too rapid to attain the required tumor retention. The molecular weight of peptides ranges between that of antibodies and small molecules. This enables peptide-based radiotracers to achieve both long-term tumor retention and good tissue penetration with minimal toxicity13,49,50,51,52,53. Importantly, the utility of the D-peptide DPA, rather than the commonly reported L-peptides, confer the [68Ga]DPA with a remarkably prolonged metabolic half-life. Moreover, DPA is positively charged and hydrophilic in vivo, and hence has high solubility and can avoid nonspecific targeting in blood, facilitating the generation of PET images with high imaging quality.
Notably, successful 68Ga radiolabeling requires a specific pH and no interference from transition metal ions, such as Cu (II) and Fe (III) cations. In some cases, the Cu2+ contamination leads to low radiochemical yield. Therefore, it is critical to ensure that all the containers and pipette tips are not contaminated. In addition, in this method, U87MG was used for tumor inoculation. Although PD-L1 expression in U87MG xenografts was verified in previous studies, its expression varies across individual animals. Therefore, the absolute uptake of the tracer in the U87MG tumors varied across individual mice. To ensure effective tracer uptake in the tumors, animals with an appropriate tumor size (500 mm3 < volume < 100 mm3) must be selected for PET scanning.
One of the limitations of [68Ga]DPA is that the binding affinity of DPA to PD-L1 is relatively low compared to several other PD-L1 targeting peptides, such as WL12, which makes it unsuitable for tumors with relatively low PD-L1 expression26,47. Further modification of the D-peptide will improve its specific binding capacity. In addition, to enhance the imaging effect of [68Ga]DPA, the formulation of the injection strategy can be optimized, for example, by concurrently injecting unlabeled DPA before [68Ga]DPA to block the nonspecific binding sites54,55,56.
In conclusion, this study developed a noninvasive and real-time method to track PD-L1 distribution in the whole body of living animals using [68Ga]DPA as a radiotracer. The results revealed a relatively high, in vivo specifical binding affinity, favorable stability, and excellent imaging capacity of [68Ga]DPA, suggesting that [68Ga]DPA-PET is a promising approach for visualizing PD-L1-overexpressing tumors. Furthermore, this technique can also be applied to the treatment of PD-L1 positive tumors when labeling DPA with other radionuclides, such as 177Lu and 225Ac. Therefore, the DPA radiolabeling technique not only overcomes the limitation of IHC dependent diagnosis, but also provides a new option for treatment.
The authors have nothing to disclose.
This study was supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (no. 2022-RC350-04) and the CAMS Innovation Fund for Medical Sciences (nos. 2021-I2M-1-026, 2022-I2M-1-026-1, 02120101, 02130101, and 2022-I2M-2-002).
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) | Merck | 60239-18-1 | 68Ga chelation |
3,3-diaminobenzidine (DAB) Kit | Sigma-Aldrich | D7304-1SET | Immunohistochemistry |
anti-PD-L1 monoclonal antibody | Wuhan Proteintech | 17952-1-ap | Immunohistochemistry: primary antibody |
BMS202 | Selleck | 1675203-84-5 | Competitive binding assay: inhibitor |
BSA | Merck | V900933 | Immunofluorescent : blocking |
DAPI | Merck | D9542 | Immunofluorescent: staining of nucleus |
Dichloromethane (DCM) | Merck | 34856 | Solvent |
DIPEA | Merck | 3439 | Peptide coupling |
EDC·HCl | Merck | E6383 | Activation of DOTA |
FBS | Gibco | 10099 | Cell culture: supplement |
FITC-conjugated anti-human IgG Fc Antibody | Biolegend | 409310 | Immunofluorescent: secondary antibody |
FITC-conjugated anti PD-L1 antibody | Biolegend | 393606 | Flow cytometry: direct antibody |
HCTU | Energy Chemical | E070004-25g | Peptide coupling |
HRP labeled goat anti-rabbit antibody | Servicebio | GB23303 | Immunohistochemistry: secondary antibody |
Hydroxysuccinimide (NHS) | Merck | 130672 | Activation of DOTA |
MeCN | Merck | PHR1551 | Solvent |
Morpholine | Merck | 8.06127 | Fmoc- deprotection |
NMP | Merck | 8.06072 | Solevent |
Paraformaldehyde | Merck | 30525-89-4 | Fixation of tissues |
PBS | Gibco | 10010023 | Cell culture: buffer |
Penicillin-streptomycin | Gibco | 10378016 | Cell culture: supplement |
RIA tube | PolyLab | P10301A | As tissue sample container |
RPMI-1640 medium | Gibco | 11875093 | Cell culture: basic medium |
Sodium acetate | Merck | 1.06264 | Salt for buffer |
Trypsin-EDTA | Gibco | 25200056 | Cell culture: dissociation agent |
U87MG cell line | Procell Life Science & Technology Co | CL-0238 | Cell model |
Equipment | |||
68Ge/68Ga generator | Isotope Technologies Munich, ITM | Not applicable | Generation of [68Ga] |
Autogamma counter | Perkin Elmer | Wizard2 | Detection of radioactivity |
Confocal fluorescent microscopy | Keyence | Observation of immunofluorescent results | |
Flow cytometer | Becton Dickinson, BD | LSRII | Monitoring the PD-L1 positive cells |
High-performance liquid chromatography (HPLC) | SHIMAZU | LC-20AT | Purification of DPA peptide |
PET scanner | Siemens Medical Solutions | Inveon MultiModality System | PET imaging |
Optical microscopy | Nikon | Eclipse E100 | Observation of immunohistochemistry results |
Solid phase peptide synthesizer | Promega Vac-Man Laboratory Vacuum Manifold | LOT#11101 | Synthesis of DPA-DOTA peptide |
Software | |||
ASIPro | Siemens Medical Solutions | Not applicable | Analysis of PET-CT results |
FlowJo | Becton Dickinson, BD | FlowJo 7.6.1 | Analysis of the flow cytometer results |
Inveon Acquisition Workplace (IAW) | Siemens Medical Solutions | Not applicable | Management of PET mechine |
Prism | Graphpad | Prism 8.0 | Analysis of the data |