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. 2024 Feb 20;14(1):31–40. doi: 10.62347/HUQD8000

Noninvasive PET imaging of tumor PD-L1 expression with 64Cu-labeled Durvalumab

Sara Malih 1, Wilson Lin 2, Zhongmin Tang 3, Molly C DeLuca 2, Jonathan W Engle 2, Behrouz Alirezapour 4, Weibo Cai 2,3, Mohammad J Rasaee 1
PMCID: PMC10944374  PMID: 38500749

Abstract

Breast cancer (BrCa) ranks as the most prevalent malignant neoplasm affecting women worldwide. The expression of programmed death-ligand 1 (PD-L1) in BrCa has recently emerged as a biomarker for immunotherapy response, but traditional immunohistochemistry (IHC)-based methods are hindered by spatial and temporal heterogeneity. Noninvasive and quantitative PD-L1 imaging using appropriate radiotracers can serve to determine PD-L1 expression in tumors. This study aims to demonstrate the viability of PET imaging with 64Cu-labeled Durvalumab (abbreviated as Durva) to assess PD-L1 expression using a murine xenograft model of breast cancer. Durvalumab, a human IgG1 monoclonal antibody against PD-L1, was assessed for specificity in vitro in two cancer cell lines (MDA-MB-231 triple-negative breast cancer cell line and AsPC-1 pancreatic cancer cell line) with positive and negative PD-L1 expression by flow cytometry. Next, we performed the in vivo evaluation of 64Cu-NOTA-Durva in murine models of human breast cancer by PET imaging and ex vivo biodistribution. Additionally, mice bearing AsPC-1 tumors were employed as a negative control. Tumor uptake was quantified based on a 3D region-of-interest (ROI) analysis of the PET images and ex vivo biodistribution measurements, and the results were compared against conventional IHC testing. The radiotracer uptake was evident in MDA-MB-231 tumors and showed minimal nonspecific binding, corroborating IHC-derived results. The results of the biodistribution showed that the MDA-MB-231 tumor uptake of 64Cu-NOTA-Durva was much higher than 64Cu-NOTA-IgG (a nonspecific radiolabeled IgG). In Conclusion, 64Cu-labeled Durvalumab PET/CT imaging offers a promising, noninvasive approach to evaluate tumor PD-L1 expression.

Keywords: Positron emission tomography (PET), programmed death-ligand 1 (PD-L1), breast cancer (BrCa), Durvalumab, 64Cu, immunoPET

Introduction

Breast cancer (BrCa) is the most commonly diagnosed cancer among women worldwide and the leading cause of cancer-related deaths in women [1,2]. The current standard-of-care treatments for BrCa are insufficient, as inherent or drug-induced resistance often underlies metastasis, relapse, and mortality [3,4]. Programmed cell death ligand-1 (PD-L1) is an essential immune checkpoint that is ubiquitously expressed across a spectrum of malignant tumors and involved in the immune evasion process [5]. Immune checkpoint blockade therapy, which targets the programmed death protein 1 (PD-1) and its ligand PD-L1, has shown promising efficacy in treating BrCa and other malignancies [6,7]. However, not all patients respond to this therapy, and there is a need for developing biomarkers to guide patient selection, predict response, and assist with combinatorial therapy [8,9].

PD-L1 expression in tumor cells and tumor-associated immune cells are the most commonly investigated biomarker for immunotherapy, and several companion diagnostic tests for PD-L1 expression, including 22C3 and SP142 assays have been approved by the FDA [10,11]. However, biopsy-based immunohistochemical (IHC) testing is limited by intra-tumoral and inter-lesional heterogeneity, which may lead to a lack of focal PD-L1 expression, dynamic changes in PD-L1 expression after therapeutic intervention, and poor uniformity in the determination of PD-L1 levels by different tests [12,13]. Moreover, the localization of PD-L1 is challenging due to its presence on the surface and in the cytoplasm of tumor cells and immune cells [14,15]. Conventional IHC assays often lack accuracy and reliability due to the interference caused by the staining of cytoplasmic proteins, which hampers accurate measurement of proteins on the cell membrane [16].

This dual localization impacts the detection and quantification of PD-L1, as surface PD-L1 plays a pivotal role in immune response modulation, while cytoplasmic PD-L1 may be involved in different intracellular processes. Accurately identifying and quantifying PD-L1 in these distinct cellular locations is crucial for understanding its role in tumor biology and for the effective application of targeted therapies [17,18].

In contrast, in vivo techniques to assess PD-L1 expression via molecular imaging can be used to monitor disease progression, localize metastatic sites, and generate personalized therapies [19-23]. PET imaging with a positron-emitting radionuclide is a popular choice due to its high sensitivity, superb tissue penetration, reproducible quantification, and potential for clinical translation, among others [24-30]. U.S. Food and Drug Administration (FDA) has approved three anti-PD-L1 antibodies for the treatment of various types of cancer: Atezolizumab (Tecentriq), Durvalumab (Imfinzi), and Avelumab (Bavencio) [31]. Several immunoPET tracers targeting PD-L1 have been reported in pre-clinical and clinical studies [32-34]. Of these, Atezolizumab (Atz, MPDL3280A) is a practical option given its clinical usage and reliability in assessing PD-L1 expression in BrCa [35-37] and when labeled with zirconium-89 (89Zr), it has been used in PET imaging to visualize PD-L1 expression in tumors. This tracer is beneficial due to the long half-life of 89Zr, allowing for delayed imaging which matches the pharmacokinetics of antibodies. However, the long half-life may also increase the radiation dose to the patient [38,39].

Similar to Atezolizumab, Durvalumab is another anti-PD-L1 antibody. Durvalumab, a fully human IgG1 monoclonal antibody targeting PD-L1, has a high affinity for PD-L1. Early human studies employing Durvalumab labeled with 89Zr for evaluation of PD-L1 expression in squamous cell carcinoma of head and neck and non-small cell lung cancer (NSLC) patients have yielded promising results [40-42]. 124I-labeled F(ab’)2 fragments of Durvalumab and 124I-labeled Durvalumab have been used for PD-L1 expression in human NSLC tumors in vivo, as well [43]. FDA has approved Durvalumab for the treatment of urothelial carcinoma and other malignancies, and it is currently under phase I and II clinical trials for the combination therapy of BrCa [44-46]. These advancements have enhanced our confidence in the safety and feasibility of immunoPET imaging based on the tracer derived from Durvalumab. Furthermore, recent studies have used different radiotracers such as 89Zr-Atezolizumab and 89Zr-labeled Avelumab for assessing the PD-L1 expression using immune-PET imaging [47-49]. 89Zr, with a half-life of about 78.4 hours, offers a longer imaging window, which can be beneficial for slower pharmacokinetics of antibodies but increases radiation exposure [50]. 64Cu has a half-life of about 12.7 hours, which can be a good balance between providing enough time for the antibody to localize to the tumor and limiting radiation exposure [51]. Previous studies have demonstrated the feasibility of antibody labeling with 64Cu [52] and its successful application in detecting PD-L1 expression patterns using PET/CT [32]. The in vivo findings indicated that 64Cu-Atezolizumab has the specificity for detecting different levels of PD-L1 expression in Triple Negative Breast Cancers (TNBCs) using PET/CT, validating its use in varied PD-L1 detection [35].

In this study, we evaluated the efficacy of immunoPET for noninvasive monitoring of PD-L1 expression in BrCa using 64Cu-NOTA-Durva. We confirmed the high expression of PD-L1 in MDA-MB-231 human BrCa cell line in murine xenograft models via biopsy IHC and validated the efficacy of this tracer for PET imaging. In our control experiments, we utilized the PD-L1 negative AsPC-1 tumor cell line with the 64Cu-NOTA-Durva, and the PD-L1 positive MDA-MB-231 tumor cell line with the 64Cu-NOTA-Rituximab, a nonspecific radiolabeled IgG.

Our results provide insights into the potential of immunoPET imaging with 64Cu-NOTA-Durva as a noninvasive approach for monitoring of PD-L1 expression in BrCa.

Materials and methods

Chemicals

Durvalumab (anti-PD-L1) was obtained from Selleck Chemicals, Inc. (Houston, TX). 1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was purchased from Macrocyclics, Inc. (Dallas, TX). 64CuCl2 in 0.05 M HCl was obtained from UW-Madison’s cyclotron research group. PD-L1 (E1L3N) XP Rabbit monoclonal antibody was purchased from Cell Signaling, Inc. (Danvers, MA). PE anti-human CD274 (B7-H1, PD-L1) antibody was purchased from BioLegend, Inc. (San Diego, CA). APC Conjugation Kit - Lightning-Link was purchased from Abcam, Inc. (Boston, MA).

Rituximab (monoclonal anti-CD20 antibody) was purchased from Selleck Chemicals, Inc. (Houston, TX). PD-10 desalting columns were supplied by GE Healthcare, Inc. (Piscataway, NJ). 4’,6-diamidino2-phenylindole (DAPI) is the product of ThermoFisher, Inc. (Burlingame, CA). Dulbecco Modified Eagle Medium (DMEM) was obtained from Gibco, Inc. (Grand Island, NY). Fetal bovine serum (FBS) was provided by Gibco, Inc. (Grand Island, NY). All other reagents were from ThermoFisher, Inc.

Cell culture

Human BrCa cell line MDA-MB-231 and human pancreas cancer cell line AsPC-1 were provided by the American Type Culture Collection (ATCC; Manassas, VA). The cells were cultured in DMEM and RPMI-1640 medium, respectively, with high glucose, FBS (10%), and Penicillin/streptomycin (1%). The T75 flasks containing the cells were placed in a humidified constant thermoincubator at 37°C with CO2 (5%). Cells were trypsinized and harvested for tumor inoculation and in vitro experiments after reaching ~70% confluence.

Subcutaneous xenograft model

Female athymic nude mice aged 4 to 7 weeks were purchased from Envigo (Cambridge Shire, UK). Approximately 5×106 MDA-MB-231 cells and 4×106 AsPC-1 cells, suspended in 150 μL of medium were implanted subcutaneously on the right shoulder of each mouse. The injected mixture can be adsorbed in 1-2 days. At ~5 weeks post-inoculation of MDA-MB-231 cells and 10 days post-inoculation of AsPC-1 cells, tumors with a diameter of ~10 mm were accepted for in vivo experiments. 12-h light-dark cycle was maintained, and food and water were continuously available. All experiments complied with current regulations of the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin-Madison (UW-Madison).

Identification of PD-L1 expression

Immunofluorescent staining and confocal imaging were conducted to validate the expression of PD-L1 on the MDA-MB-231 and AsPC-1 cell lines. The cells were cultured in glass-bottom dishes (50 mm, ~2×105 cells/dish) and incubated at 37°C in CO2 (5%) overnight. Then cells were incubated with PE-anti-PD-L1, APC-Durvalumab (10 μg/mL), and DAPI (5 μg/mL) at 4°C overnight in the dark and imaged on an A1R confocal microscope (Nikon, Inc.; Melville, NY).

PD-L1 expression on the tumor cell surface was verified in the MDA-MB-231 and AsPC-1 cell lines by flow cytometry. The cells were suspended in a staining buffer (4°C) and split into aliquots of 1×106 cells/tube. Then the cells were incubated with PE-anti-PD-L1, APC-Durvalumab (10 μg/mL) for 1 h on ice in darkness. Finally, all cells were re-suspended in 500 μL of staining buffer (4°C) and DAPI was added 10 min before the flow cytometry analysis on a 5-Laser LSR Fortessa cytometer (Becton-Dickinson, Inc.; San Jose, CA). Cell counts were recorded and analyzed using FlowJo (ver. X.0.9; Tree Star, Inc.; Ashland, OR) software.

Chelator conjugation and radiolabeling

p-SCN-Bn-NOTA was dissolved in 10 μL of dimethyl-sulfoxide (DMSO) and Durvalumab was conjugated to NOTA at a molar ratio of 1:5 at pH 8.4. The mixture was incubated at room temperature (RT) for 2 h with constant shaking and later purified by size-exclusion columns using metal-free phosphate buffer solution (PBS) as the mobile phase to remove unbound NOTA. Fractions containing NOTA-conjugated Durvalumab were collected, and the peak fraction of solute was confirmed on the NanoDrop One (ThermoFisher; Waltham, MA). For 64Cu radiolabeling, Durvalumab/IgG was incubated with 64CuCl2 (111 MBq/3 mCi) in 300 μL of sodium acetate buffer (0.1 mol/L, pH 5) for 60 min at 37°C with constant shaking. The reaction solution was purified by PD-10 (PBS as the eluent). Eluted 64Cu-NOTA-Durvalumab (64Cu-NOTA-Durva) fractions were combined and used for mouse studies.

Serum stability testing of 64Cu-NOTA-Durvalumab

The in vitro serum stability of 64Cu-NOTA-Durva was assessed using instant thin-layer chromatography. 100 μCi (3.7 MBq) of 64Cu-NOTA-Durva was incubated in 200 μL of human serum at 37°C for 4, 24, and 48 h.

PET imaging and biodistribution studies

In vivo PET imaging was performed using an Inveon Micropet/CT scanner (Siemens Medical Solutions USA, Inc.). Tumor xenograft mice (n = 3/group) were injected with 3.7-7.4 MBq (0.10-0.20 mCi) of 64Cu-NOTA-Durvalumab/NOTA-IgG via the lateral tail vein. PET/CT scans were acquired at 4, 24, and 48 h post-injection (p.i.). The Inveon Research Workplace (IRW) software (Siemens, Inc.) was used to quantify the mean uptake of the region of interest (ROI) in major organs in terms of the percentage of injected dose per gram (%ID/g, decay-corrected). The %ID/g value was calculated by dividing tissue activity in MBq/g (converted from ROI uptake) by the total radioactive dose injected.

Ex vivo biodistribution studies were carried out after the last PET scan at 48 h p.i. and all mice were euthanized by CO2 asphyxiation. The tumor, blood, brain, heart, lung, liver, spleen, kidneys, stomach, intestine, muscle, bone, and pancreas were harvested and wet weighed. The radioactivity of major organs was determined using a Wizard 2480 automatic γ-counter (PerkinElmer, Inc.; Waltham, MA), and the results are presented as %ID/g, mean ± standard deviation.

Immunohistochemistry

Tumors were excised from mice, fixed overnight in 4% paraformaldehyde, and subsequently transferred to 70% ethanol for preservation. Immunohistochemical analysis was performed at the University of Wisconsin-Madison’s Translational Research Initiatives in Pathology (TRIP) facility. The samples were embedded in paraffin for tissue processing. Deparaffinization was achieved using standard protocols, followed by heat-induced epitope retrieval using an EDTA-based buffer (Ventana #950-500) at 95°C for 32 minutes. The primary antibody (E1L3N®) XP® Rabbit mAb #13684 was applied at a dilution of 1:100 and incubated at 37°C for 32 minutes. The secondary antibody, Discovery OmniMap anti-Rabbit HRP (Ventana #760-4311), was applied and incubated at 37°C for 16 minutes. Counterstaining was performed with Harris hematoxylin at a 1:5 dilution for 30 seconds, and the slides were then covered with xylene. Finally, the slides were imaged using ImageScope.

Statistical analysis

Quantitative data were analyzed using GraphPad PRISM (version 10.1.1) and presented as mean ± standard deviation (SD).

Results

PD-L1 expression

As depicted in Figure 1A, the nuclei of MBA-MB-231 cells were stained by DAPI (blue channel). The PD-L1 on the cells was heavily stained by APC-Durvalumab and exhibited strong fluorescence (red channel), the cells engaging with PE-anti-PD-L1 antibody showed an intense red fluorescent signal as well. This verifies the high expression of PD-L1 on the MDA-MB-231 cell line. AsPC-1 PD-L1 negative cell lines (Figure 1B) were also stained to confirm the specificity of APC-Durvalumab binding. The nuclei of AsPC-1 cells were stained by DAPI (blue channel) and there was no significant PD-L1 expression on these cells after staining with APC-Durvalumab/PE-anti-PD-L1 antibody.

Figure 1.

Figure 1

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Characterization of in vitro PD-L1 expression in human BrCa cell line. A, B. The confocal imaging of MDA-MB-231 and AsPC-1 cells after immuno-fluorescent staining, Scale bar: 10 μm. A. MDA-MB-231 cells stained by APC-Durva and PE-anti-PD-L1. B. AsPC-1 cells stained by APC-Durva and PE-anti-PD-L1. Sample groups: DAPI, the nucleus stained by DAPI; APC, the PD-L1 expression stained by APC-Durvalumab; PE, the PD-L1 expression stained by PE-anti-PD-L1. C, D. The PD-L1 expression on the cell membrane of the MDA-MB-231 and AsPC-1 cell lines were evaluated using flow cytometry. C. The cell lines were stained by a commercialized PE-anti-PD-L1 antibody. D. The cell lines were stained by APC-Durva antibody. The blue area represents cells without PD-L1 expression, and the red area represents cells with PD-L1 expression. There is a significant shift in both Anti-PD-L1 antibody and Durvalumab treated group in MDA-MB-231 PD-L1 positive cell line compared to AsPC-1 PD-L1 negative cell line.

In the results of flow cytometry, cells stained with PE-anti-PD-L1 (Figure 1C) and APC-Durva (Figure 1D) shared notable positive shifts compared with control groups, confirming the high expression of PD-L1 on the MDA-MB-231 cell surface. The shift in the PD-L1 negative group of flow cytometry and the negative signal in the confocal images of the PD-L1 negative group is circumstantial evidence of the specificity APC-Durva. The in vitro results demonstrate that the MDA-MB-231 cell line is PD-L1 positive and Durvalumab shows specificity for PD-L1.

Radiochemistry

Durvalumab was successfully conjugated with NOTA and radiolabeled with 64Cu. The 64Cu-labeling achieved a radiochemical yield of >80% 64Cu-NOTA-Durva. RTLC analysis results showed that 64Cu-NOTA-Durva has high stability in serum, with >74% radiochemical purity at 48 h (Figure 2).

Figure 2.

Figure 2

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RTLC analysis of 64Cu-NOTA-Durva. The radiochemical purity of 64Cu-NOTA-Durva was ≥74% after 48 h incubation in serum. n = 3.

PET/CT imaging and biodistribution of 64Cu-NOTA-Durvalumab

Figure 3 shows the PET/CT images and biodistribution results of 64Cu-NOTA-Durva. 64Cu-NOTA-Durva showed noticeable tumor uptake differences between the targeting and non-targeting groups at all timepoints from 4 to 48 h p.i. 64Cu-NOTA-Durva showed longer blood retention in the non-targeting group. We further performed a biodistribution study at 48 h p.i.

Figure 3.

Figure 3

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Representative PET/CT images and biodistribution of MDA-MB-231 and AsPC-1 tumor-bearing mice over time. Representative PET/CT images (A) and biodistribution (B) of MDA-MB-231 and AsPC-1 tumor-bearing mice injected with 64Cu-NOTA-Durva/64Cu-NOTA-IgG. n = 3.

Histology

Tumor samples were fixed in PFA 4% overnight and then transferred to cold Ethanol 70%. Slide scans of these tissues after immunofluorescent staining are shown in Figure 4. The fluorescence from PD-L1 of MDA-MB-231 (in brown) is visible and overlays with the cell nuclei (in blue) in tumor tissue.

Figure 4.

Figure 4

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Assessment of PD-L1 expression pattern in MDA-MB-231 and AsPC-1 tumor samples using immunohistochemistry, Scale bar: 200 µm. Strong PD-L1 staining is observed in MDA-MB-231, indicating high expression levels, and very little staining in AsPC-1 tumor sample indicating low expression level.

Discussion

The level of PD-L1 expression has been shown to greatly influence the patient’s response to therapy [53,54] and could be a superior predictor of patient response to immune checkpoint blockade compared to biopsy IHC analysis, which is the current clinical standard [10]. However, PD-L1 expression is not consistently stable on the cell surface and can be influenced by various signaling pathways, post-translational modifications, and the release of inflammatory factors during treatments like radiotherapy and chemotherapy [40-43,55].

In light of these challenges, the development of advanced imaging techniques such as PD-L1 PET imaging becomes imperative. By allowing noninvasive and real-time monitoring of PD-L1 expression, PD-L1 PET imaging offers a valuable tool for accurately assessing PD-L1 status and guiding treatment decisions. This imaging modality overcomes the limitations of traditional methods and provides timely information on PD-L1 expression dynamics, ensuring optimal patient selection and treatment efficacy.

Several studies have investigated tumor detection using molecular imaging based on PD-L1 expression, often using murine models [32-37,56,57]. This study differs from previous ones in a few key aspects. Firstly, human cell lines were used to induce the tumor models, enhancing clinical translatability compared to studies using murine cell lines such as 4T1 [58-60]. Secondly, the positive cell line used in this study (MDA-MB-231) expresses native levels of PD-L1 without artificial overexpression, unlike some previous studies that have used human PD-L1 gene-transfected A375 cells and human PD-L1-gene transfected B16-F10 [61,62]. Lastly, the study avoided predosing/blocking strategies employed in other studies [48,61,63] to minimize off-target lymphatic uptake. Only the radiolabeled protein was administered at a low dose to avoid potential pharmacologic responses while achieving sufficient imaging signal. Predosing involves administering an excess of unlabeled antibody before introducing the radiolabeled counterpart, aiming to saturate target binding sites in normal tissues, thus reducing background signal. Some studies have highlighted the advantages of predosing in improving image quality and target-to-background ratios, emphasizing its potential clinical relevance [64]. Predosing or blocking strategies, while effective in minimizing non-specific binding, can introduce complexities such as altering the pharmacokinetics of the radiotracer, potentially influencing its distribution and clearance patterns [48,65,66]. The choice to avoid these strategies in the current study aimed to maintain simplicity in the experimental design and enhance the reliability of the obtained imaging data.

PD-L1 imaging presents challenges compared to traditional tumor markers. The widespread expression of PD-L1 reduces the imaging signal in tumors, and conventional control experiments cannot be applied. As the injection of a blocking dose would saturate PD-L1 in the spleen and lymph nodes, this method might lead to increased tracer accumulation in the tumor, challenging its specificity for cancer cells, contrary to conventional studies. Using a nonspecific, isotype-matched antibody as a control also presents issues, as it would not bind to ubiquitous PD-L1 throughout the body and may accumulate to higher levels in tumor tissue that were elaborated in previous studies [47,67]. In our study, as a negative control, mice with AsPC-1 tumors were used to assess the 64Cu-NOTA-Durva accumulation in tumor tissues not attributed to PD-L1 expression by tumor cells. Furthermore, nonspecific uptake was assessed by a radiolabeled control IgG antibody, 64Cu-NOTA-IgG, in MDA-MB-231 mice models.

The choice of PD-L1-targeted radiotracer for immunoPET imaging depends on the specific requirements of the study, including the desired imaging window, the balance between resolution and radiation dose, and the pharmacokinetic properties of the tracer in relation to the tumor characteristics. 64Cu has a half-life of approximately 12.7 hours. Our study benefits from the relatively short half-life of the 64Cu, enabling multiple imaging sessions in a day and capturing changes in PD-L1 expression over shorter time spans. Furthermore, shorter-lived radionuclides like 64Cu typically result in lower radiation exposure for patients and medical personnel, enhancing safety in clinical settings. 64Cu is more accessible and easier to handle in many clinical settings, which can streamline the logistics of our study as well.

On the other hand, 89Zr forms stable chelates with antibodies, ensuring long-lasting imaging signals. However, for our specific study, the shorter half-life of 64Cu may still provide sufficient stability while offering more flexibility in imaging scheduling. In summary, based on the title and focus of our study on breast cancer and PD-L1 expression using 64Cu-labeled Durvalumab, 64Cu appears to be a suitable choice. Its shorter half-life, rapid imaging capabilities, and lower radiation exposure align well with the objectives of noninvasively evaluating PD-L1 expression dynamics in breast cancer tumors.

The results presented in this study demonstrate the successful conjugation of NOTA with Durvalumab and its radiolabeling with 64Cu, resulting in a stable and pure 64Cu-NOTA-Durva tracer. The in vitro experiments also showed high expression of PD-L1 on the MDA-MB-231 cell line, which indicates that Durvalumab can be a promising tracer for PD-L1 imaging in breast cancer.

The in vivo PET/CT imaging and biodistribution studies of 64Cu-NOTA-Durva showed noticeable tumor uptake differences between the targeting and non-targeting groups, confirming the specificity of the tracer for PD-L1 positive tumors (14.47 ± 3.70 %ID/g for 64Cu-NOTA-Durva vs 5.55 ± 2.5 %ID/g for 64Cu-NOTA-IgG; n = 3). A five-fold higher uptake of radioactivity in MDA-M231 cells than in AsPC-1 cells further confirmed the specificity of 64Cu-NOTA-Durva for PD-L1. The biodistribution study at 48 h p.i. further supported the selective accumulation of the tracer in the tumor, as evidenced by the higher uptake of the targeting group compared to the non-targeting group. The immunofluorescent staining of tumor tissue and flow cytometry results also confirmed the expression of PD-L1 in the MDA-MB-231 tumors. The findings of this study are consistent with previous studies that have used Durvalumab as a tracer for PD-L1 imaging in various types of cancer [42,43].

Conclusion

Our study demonstrates the potential of PD-L1 PET/CT imaging with 64Cu-labeled Durvalumab for noninvasive evaluation of PD-L1 expression in tumors. The radiolabeling of Durvalumab with 64Cu was straightforward, and the radiolabeled compound showed high in vitro serum stability and specificity for PD-L1-expressing cells. The PET/CT imaging studies showed that 64Cu-labeled Durvalumab can accumulate in PD-L1-expressing tumors and provide high-contrast images. The quantitative measurement of PD-L1 expression using PET/CT imaging with 64Cu-labeled Durvalumab showed a significant correlation with PD-L1 expression levels in tumors, and the specificity of 64Cu-labeled Durvalumab binding to PD-L1 was confirmed by non-targeting models. The results from this work can help guide the development of new PD-L1 inhibitors and improve our understanding of the role of PD-L1 in cancer biology.

Acknowledgements

This work was supported by the University of Wisconsin-Madison, the National Institutes of Health (P30 CA014520), and the Tarbiat Modares University.

Disclosure of conflict of interest

Weibo Cai declares conflict of interest with the following corporations: Actithera, Inc., Portrai, Inc., rTR Technovation Corporation, Four Health Global Pharmaceuticals Inc., and POP Biotechnologies, Inc.

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