Skip to main content
Download PDF
Download ePub

Preclinical evaluation and preliminary clinical study of 68Ga-NODAGA-NM-01 for PET imaging of PD-L1 expression

Cancer Imaging volume 25, Article number: 6 (2025) Cite this article

Abstract

Background

Programmed cell death 1/programmed death ligand-1 (PD-L1)-based immune checkpoint blockade is an effective treatment approach for non-small-cell lung cancer (NSCLC). However, immunohistochemistry does not accurately or dynamically reflect PD-L1 expression owing to its spatiotemporal heterogeneity. Herein, we assessed the feasibility of using a 68Ga-labeled anti-PD-L1 nanobody, 68Ga-NODAGA-NM-01, for PET imaging of PD-L1.

Methods

Micro-PET/CT and biodistribution studies were performed on PD-L1-positive and -negative tumor-bearing mice. Additionally, a preliminary clinical study was performed on two patients with NSCLC. NM-01 was radiolabeled with 68Ga without further purification under mild conditions.

Results

68Ga-NODAGA-NM-01 exhibited radiochemical purity (> 98%), high stability in vitro, and rapid blood clearance in vivo. Specific accumulation of 68Ga-NODAGA-NM-01 was observed in PD-L1-positive tumor-bearing mice, with a good tumor-to-background ratio 0.5h post-injection. Furthermore, 68Ga-NODAGA-NM-01 PET/CT imaging was found to be safe with no adverse events and distinct uptake in primary and metastatic lesions of the PD-L1-positive patient, with a higher maximal standardized uptake value than that in lesions of the PD-L1-negative patient 1h post-injection.

Conclusions

68Ga-NODAGA-NM-01 can be prepared using a simple method under mild conditions and reflect PD-L1 expression in primary and metastatic lesions. However, our findings need to be confirmed in a large cohort.

Trial registration

NCT02978196. Registered February 15, 2018.

Background

Lung cancer is the leading cause of cancer-related mortality, with approximately 2.5 million new cases and 1.8 million deaths worldwide in 2022 [1,2,3]. In 2022, the National Cancer Center of China released annual cancer registration data, reporting 828,100 new cases and 657,000 deaths attributed to lung cancer nationwide in 2016. Non-small-cell lung cancer (NSCLC) constituted nearly 80–85% of all lung cancer cases [4]. Recently, programmed cell death 1 (PD-1)/programmed death ligand-1 (PD-L1) blockade immunotherapy has emerged as a highly promising approach for the comprehensive treatment of NSCLC. This strategy improves the durable response rate, providing long-lasting benefits for advanced NSCLC compared to conventional chemotherapy or radiotherapy [4,5,6,7].

PD-1 expression is upregulated in activated T cells, whereas its dominant ligand PD-L1 is primarily expressed on tumor cells following exposure to pro-inflammatory cytokines in the tumor microenvironment, resulting in adaptive immune resistance [6, 8]. High PD-L1 expression in patients with NSCLC correlates with shorter survival time and poorer prognosis [9]. To block tumor immune escape, immune checkpoint inhibitors are used to relieve the inhibitory effect of tumors on T lymphocytes and enhance the immune response. Consequently, they have become effective and safe therapeutic options for advanced NSCLC. Several PD-1/PD-L1 inhibitors, such as nivolumab, pembrolizumab, and atezolizumab have received clinical approval [10, 11]. Immunohistochemistry (IHC) is recommended for assessing PD-L1 expression in NSCLC. Considering the invasiveness of the procedures required for IHC (tissue collection), spatial and temporal heterogeneity, the impossibility of repeated and dynamic evaluation, and a higher risk of false-negative results, it is difficult for pathologists to provide precise measurements [12, 13]. Hence, there is an urgent need to design and develop a non-invasive and accurate imaging method to monitor PD-L1 expression for optimal personalized treatment.

Radiolabeled PD-L1-binding molecules, capable of capturing the entire extent of tumor heterogeneity, have been introduced as tracers to visualize PD-L1 expression in tumor-bearing animals and patients with NSCLC. Recent preclinical and clinical studies have demonstrated that these tracers can quantify PD-L1 expression in tumor cells in real-time, encouraging further development of molecular imaging for immunotherapy [14,15,16,17,18,19]. Owing to their relatively large size (150 kDa), antibody-based PD-L1-targeted tracers pose certain disadvantages, such as prolonged blood circulation, low tumor uptake, and limited tumor penetration. Nanobodies, single-domain antibodies derived from camels, offer a promising alternative owing to their lower molecular weight (12–15 kDa), facilitating deep tumor penetration, fast blood clearance, and easy modification and processing, increasing the potential for their use in constructing tracers for molecular imaging [10, 20,21,22,23]. In a previous study, we screened a PD-L1-targeted nanobody (NM-01) radiolabeled with 99mTc as a single-photon emission computed tomography (SPECT)/CT tracer for the assessment of PD-L1 expression in NSCLC [10]. 99mTc-labeled NM-01 exhibited an excellent safety profile, favorable imaging characteristics, and a significant correlation with PD-L1 IHC results in patients with NSCLC. In the present study, NM-01 was further conjugated with the chelator NODAGA and radiolabeled with 68Ga as a positron emission tomography (PET) tracer. Preclinical evaluation of 68Ga-labeled NM-01 (68Ga-NODAGA-NM-01) was conducted to validate its specific targeting ability in vivo in PD-L1-positive tumor-bearing mice. Furthermore, the safety, biodistribution, and tumor-targeting potential of 68Ga-NODAGA-NM-01 were assessed in a preliminary clinical study involving two patients with NSCLC.

Methods

Trial registration

99mTc/68Ga Labeled Anti-PD-L1 sdAb in Assessment of PD-L1 Expression in NSCLC (NCT02978196; Registered February 15, 2018; https://www.clinicaltrials.gov/study/NCT02978196).

Materials

The preparation and characterization of NM-01 are reported in our previous study [10]. NODAGA-NM-01 was synthesized by conjugating maleimide-NODAGA as described previously [24] and was kindly supplied by NanoMab (Shanghai, China). 68Ga was eluted from a 68Ge/68Ga generator (ITG, Baden-Württemberg, Germany) with 4 mL 0.05 M HCl solution. Phosphate-buffered saline (PBS), fetal bovine serum (FBS), and Dulbecco’s modified Eagle medium were purchased from Beijing BioDee Biotechnology Co., Ltd. (Beijing, China). All other chemicals and solvents were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Preparation and quality control of 68Ga-NODAGA-NM-01

NODAGA-NM-01 was labeled with 68Ga as described previously [25, 26]. In brief, a mixture of 150 μg NODAGA-NM-01, 1 mL 68GaCl3 (555–666 MBq), and 100 μL sodium acetate (1.0 M) was incubated at room temperature (20–25°C) for 10 min. The final product was analyzed using instant radio-layer chromatography (radio-TLC) and radio-high-performance liquid chromatography (radio-HPLC) according to the procedure described in our previous study [10]. Other quality control tests including visual checks, pH measurements, and endotoxin tests were performed. In vitro stabilities were confirmed by measuring the radiochemical purities (RCPs) of 68Ga-NODAGA-NM-01 in PBS at room temperature and in FBS at 37°C within 3 h.

Cells and animal models

HCC827 and A549 cells were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured as recommended by the supplier. Female BALB/c nude mice (4-week-old, 16–18 g) were sourced from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). Tumor-bearing mouse models were established according to previously published protocols [27], wherein mice were subcutaneously inoculated with 100 μL cell suspension in their right-side flanks; the suspension contained 5 × 106 cells in a 1:1 PBS and Matrigel mixture. Animal experiments were commenced when the tumor diameter reached 0.8–1.2 cm.

Western blot analysis and saturation binding assays

PD-L1 expression levels in the A549 and HCC829 cells were verified by Western blotting. Total protein of tumor cells was extracted and quantified using a BCA protein assay kit according to a previously published procedure [27]. The blots were quantified using ImageJ software. HCC827 cells were inoculated into 24-well plates at a density of 2 × 105 cells per well and incubated overnight. Different concentrations of 68Ga-NODAGA-NM-01 (0.01–70 nM) were added into the wells (n = 3) and incubated for 2 h at 37°C. The cells were washed twice with PBS and then lysed with pre-cooled NaOH (0.1M, 1 mL). Finally, the radioactivity was measured by a γ-counter (CAPINTEC, USA). Kd value was calculated using Graph pad Prism 8.0.

Pharmacokinetics studies

Five 6-week-old healthy ICR mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences for evaluating the pharmacokinetic profile of 68Ga-NODAGA-NM-01. Each mouse was intravenously injected with 68Ga-NODAGA-NM-01 at a dose of 740 KBq in 200 μL saline solution. Blood samples (10 μL) were immediately collected and weighed after 1, 2, 5, 15, 30, 60, and 120 min, and the radioactivity was measured using a γ counter to calculate the percentage of injected dose per gram (%ID/g). Additionally, pharmacokinetic data were analyzed by DAS 2.0 (Shanghai, China) using a two-compartment model to calculate the half-life of 68Ga-NODAGA-NM-01 in blood.

Micro-PET imaging study

PET imaging was performed using an Inveon small-animal PET scanner and the acquired images were reconstructed using Inveon Research software (Siemens Medical Solutions, Erlangen, Germany). Tumor-bearing mice were injected with 3.7–5.55 MBq 68Ga-NODAGA-NM-01 via the tail vein, equivalent to 10 µg NM-01 in 200 μL saline solution. For the blocking group, mice bearing HCC827 tumor xenografts were co-injected with 400 μg NM-01 and the same dose of 68Ga-NODAGA-NM-01. PET images were acquired 30 and 90 min after injection.

Biodistribution

Mice with subcutaneous HCC827 or A549 xenografts were divided into two groups (five mice per group) to evaluate the biodistribution of 68Ga-NODAGA-NM-01 in the main organs and tumors. Each mouse was intravenously injected with 68Ga-NODAGA-NM-01 at a dose of 740 KBq in 200 µL saline solution. The mice were sacrificed 30 and 90 min after injection, and tumors and main organs, including the kidneys, liver, spleen, lungs, heart, and muscles, were collected. Each sample was immediately weighed and its radioactivity counted using a γ counter. The results are expressed as %ID/g.

Preliminary clinical study

Two newly diagnosed patients with NSCLC between January and November 2019 were recruited and underwent PET/CT imaging and a follow-up after 1week. Patient inclusion criteria were age between 18 and 80 years with histopathologically diagnosed NSCLC, no prior lung cancer-related treatment, and an Eastern Cooperative Oncology Group Performance Score of 1 or less. Exclusion criteria included pregnancy or lactation, severe liver or kidney dysfunction, and previous chemotherapy, radiotherapy, or targeted therapy before PET/CT scans. PD-L1 expression in the available lesions was assessed through IHC, as described previously [10].

Patients were intravenously injected with 68Ga-NODAGA-NM-01 and asked to drink 300–500 mL water and empty their bladders before PET/CT imaging. 68Ga-NODAGA-NM-01 PET/CT scans were conducted at 1 h post-injection using a GE Discovery STE PET/CT scanner (GE Healthcare, Buckinghamshire, UK). The CT scans used a voltage of 140 kV, current of 150 mA, scanning layer thickness of 3 mm, and pitch of 1.2 mm. PET collections, lasting 3 min for each bed, were performed in two-dimensional mode. The ordered subset expectation maximization method was used for image reconstruction, and attenuation correction of the PET images was performed using CT projection scanning data processing to obtain whole-body fusion images of PET, CT, and PET/CT. Two experienced nuclear medicine physicians analyzed the images, delineated the regions of interest for the lesions, and calculated the maximum standardized uptake value (SUVmax). In addition to the physiological uptake of the liver, spleen, kidney, and other parts of the image, when there was an abnormal concentration of local radioactivity or the uptake level was significantly higher than that of the surrounding normal tissues, the diagnosis was one of a primary tumor or metastasis after excluding the possibility of benign lesions, such as inflammation.

Statistics

Data are presented as mean ± standard deviation. Statistical significance was evaluated using a one-way analysis of variance. A p-value of 0.05 was selected as the threshold of significance: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Results

Preparation, quality control, and stability of 68Ga-NODAGA-NM-01

The NODAGA modified NM-01 was characterized by mass spectrometry and HPLC (Figs. S1 and 1A). The binding affinity of NM-01 to human PD-L1 (Kd = 0.8 nM) has been reported, which was not repeated in this study [28]. 68Ga-NODAGA-NM-01 could be readily prepared with high radiochemical yield (99.3 ± 0.5%) and specific activity (> 45Ci/g). 68Ga-NODAGA-NM-01 was characterized using radio-HPLC and radio-TLC. 68Ga-NODAGA-NM-01 displayed a single radioactive peak at 12.43 min (Fig. 1B), which is consistent with the retention time of NODAGA-NM-01 (11.95 min). The RCP of 68Ga-NODAGA-NM-01 was calculated to be greater than 98% without further purification, which could also be rapidly estimated using radio-TLC. 68Ga-NODAGA-NM-01 exhibited a retention factor of 0–0.2 (Fig. 1C), while 68GaCl3 had a retention factor of 0.8–1.0. Furthermore, the stability of 68Ga-NODAGA-NM-01 was analyzed in PBS at room temperature and in FBS at 37°C using radio-TLC. 68Ga-NODAGA-NM-01 was stable in vitro, with an RCP greater than 98% after 3 h (Fig. 1D).

Fig. 1
figure 1

Characterization and stability of 68Ga-NODAGA-NM-01. A, B Radio-HPLC and (C) radio-TLC results of 68Ga-NODAGA-NM-01. D In vitro stability of 68Ga-NODAGA-NM-01 in phosphate-buffered saline (PBS) at room temperature and fetal bovine serum (FBS) at 37°C within 3 h

Full size image

Western blot and binding affinity

PD-L1 expression levels in the selected tumor cells were examined using western blotting. As presented in Fig. 2A, HCC827 cells showed high PD-L1 expression levels, whereas low plectin expression were found in A549 cells. Quantitative analysis revealed a higher PD-L1 expression level in HCC827 cells than in A549 cells, further confirming the significance between these cells (Fig. 2B). To demonstrate the affinity of 68Ga-NODAGA-NM-01 to PD-L1, saturation binding assays were conducted in Hcc827 cells (Fig. 2C). With the increase of radioactivity concentration, the cell uptake of 68Ga-NODAGA-NM-01 gradually increased, and the dissociation constant (Kd) was calculated to be 1.5 ± 0.1 nM.

Fig. 2
figure 2

A Western blot results and (B) quantitative analysis of plectin expression in HCC827 and A549. C Saturation binding curve of 68Ga-NODAGA-NM-01 in HCC827 cells. D Blood clearance of 68Ga-NODAGA-NM-01 in normal ICR mice

Full size image

Pharmacokinetics of 68Ga-NODAGA-NM-01

The radioactivity–time curve of 68Ga-NODAGA-NM-01 between 1 and 120 min post-injection is shown in Fig. 2D. At 1 min post-injection, blood radioactivity was 24.1 ± 3.9 %ID/g, which rapidly decreased to 7.8 ± 1.5 %ID/g 5 min post-injection. By 120 min post-injection, less than 2 %ID/g could be recovered from the blood pool. A two-phase exponential model was used to determine the biological blood half-life of 68Ga-NODAGA-NM-01. The elimination distribution phase half-life (t1/2α) and clear phase half-life (t1/2β) of 68Ga-NODAGA-NM-01 were approximately 1.02 and 35.66 min, respectively.

Micro-PET/CT imaging and biodistribution

Micro-PET/CT imaging was performed on PD-L1-positive HCC827 and PD-L1-negative A549 tumor-bearing mice. HCC827 tumors showed distinct high uptake of 68Ga-NODAGA-NM-01 30 and 90 min post-injection, whereas tumor uptake of 68Ga-NODAGA-NM-01 in the HCC827 blocking group and A549 tumors was negligible at both time points (Fig. 3A). High accumulation of 68Ga-NODAGA-NM-01 was observed in the kidneys and bladder, with low uptake in other major organs, such as the liver, spleen, heart, lung, and muscle. After micro-PET/CT imaging, IHC was performed to confirm PD-L1 levels in the tumors and analyze the correlation with tumor uptake of this probe; muscle tissues served as the negative control. IHC staining for PD-L1 in the same xenografts supported the PET/CT findings, revealing a higher uptake in tumors with higher PD-L1 levels (Fig. 3B).

Fig. 3
figure 3

Uptake of 68Ga-NODAGA-NM-01 in tumors. A Micro-PET/CT images of 68Ga-NODAGA-NM-01 in PD-L1 positive HCC827 and PD-L1 negative A549 tumor-bearing mice. B Immunohistochemistry for PD-L1 in muscle, A549 tumors, and HCC827 tumors. Scale bar, 200 μm

Full size image

To quantitatively analyze the radioactivity in the main organs and tumors in tumor-bearing mice, biodistribution experiments were performed at 30 and 90 min post-injection. The kidneys in the HCC827 tumor-bearing, HCC827 blocking, and A549 tumor-bearing mice had the highest uptake (113.11 ± 25.94 %ID/g, 106.38 ± 19.04 %ID/g, and 130.56 ± 35.06 %ID/g, respectively) 30 min post-injection, while the livers and other organs displayed low uptake, less than 2 %ID/g (Fig. 4A). Tumor uptake in HCC827 tumor-bearing mice was higher (2.59 ± 0.65 %ID/g) than in HCC827 blocking (0.93 ± 0.11 %ID/g) and A549 tumor-bearing mice (0.62 ± 0.11 %ID/g). The reduced tumor uptake in PD-L1-positive HCC827 after NM-01 blocking indicated the specificity of 68Ga-NODAGA-NM-01 in vivo. A slight decrease in uptake in the major organs and tumors was observed 90 min post-injection (Fig. 4B). Tumor uptake in HCC827 tumor-bearing, HCC827 blocking, and A549 tumor-bearing mice was 2.33 ± 0.48, 0.66 ± 0.18, and 0.57 ± 0.10 %ID/g, respectively. Accordingly, the tumor-to-muscle (T/M) ratio in HCC827 tumor-bearing mice (5.61 ± 1.68) was higher than that in HCC827 blocking (1.78 ± 0.25) and A549 tumor-bearing mice (1.15 ± 0.38) 30 min post-injection (Fig. 4C), and T/M ratios 90 min post-injection increased to 5.96 ± 0.74, 1.67 ± 0.70, and 1.51 ± 0.44, respectively.

Fig. 4
figure 4

Biodistribution of 68Ga-NODAGA-NM-01 in tumors. Biodistribution in HCC827 and A549 tumor-bearing mice at (A) 30 and (B) 90 min post-injection, and (C) their tumor-to-muscle (T/M) ratios

Full size image

Preliminary clinical study of 68Ga-NODAGA-NM-01 PET/CT imaging

Two male patients (aged 77 and 69 years) with histopathologically confirmed NSCLC (one squamous cell carcinoma and one adenocarcinoma) were enrolled for a preliminary clinical study. One patient tested positive for PD-L1 expression, whereas the other tested negative. The administered doses of 68Ga-NODAGA-NM-01 were 82 and 107 MBq, respectively, corresponding to 75 μg of NODAGA-NM-01. No drug-related adverse effects were observed during the 7-day follow-up period. There were no significant changes in vital clinical signs, blood and urine laboratory test results, and other clinical parameters such as heart rate, respiratory rate, body temperature, and blood pressure.

Following PET/CT imaging, two experienced nuclear medicine physicians independently conducted visual interpretation and quantitative analysis of all lesions. The PET/CT images of the two patients with NSCLC are shown in Figs. 5 and 6. Biodistribution of 68Ga-NODAGA-NM-01 was as expected with main radioactivity in the liver, spleen, and kidneys. Low accumulation was found in the lungs, bone marrow and muscle. In terms of tumor uptake, patient 1 displayed moderate uptake of 68Ga-NODAGA-NM-01 in the primary lesion and metastases. The SUVmax of the right lower lobe primary lesion and right hilar lymph node were 3.3 and 4.4, respectively (Fig. 5A), and the SUVmax of the mediastinal lymph nodes were 3.5 and 3.6 (Fig. 5B). IHC staining confirmed PD-L1 positivity in the primary tumor. Physiologically, 68Ga-NODAGA-NM-01 mainly accumulated in the kidneys, liver, and spleen, with a relatively low uptake in the muscle and bone marrow (Fig. 5C). In patient 2, the primary lesion exhibited low uptake of 68Ga-NODAGA-NM-01 with an SUVmax of 1.7 (Fig. 6A), consistent with negative PD-L1 expression as confirmed by IHC. The left hilar lymph node and bone metastases had mild levels of tracer uptake with SUVmax of 2.4 and 2.8 (Fig. 6B), while a low SUVmax of 1.5 in the occipital metastasis (Fig. 6C) and background levels in the bone marrow and muscle were observed (Fig. 6D). This suggests varying PD-L1 expression between primary lesions and metastases.

Fig. 5
figure 5

68Ga-NODAGA-NM-01 PET/CT of patient 1. Different uptake in the (A) right lung lower lobe primary lesion (green arrow), right hilar lymph node (red arrow), and (B) mediastinal lymph metastases. The SUVmax of primary lesion and metastases were 3.3, 4.4, and 3.6, respectively. C Representative maximum-intensity-projection image of patient 1

Full size image
Fig. 6
figure 6

68Ga-NODAGA-NM-01 PET/CT of patient 2. Mild uptake in the (A) left hilum primary lesion with SUVmax of 1.7, while heterogeneous uptake in the (B) mediastinal lymph node with SUVmax of 2.4 (green arrow), sternum metastasis with SUVmax of 2.8 (red arrow), and (C) occipital metastasis with SUVmax of 1.5, respectively. D Representative maximum-intensity-projection image of patient 2

Full size image

Discussion

PD-1/PD-L1 blockade therapy has emerged as a promising approach for managing NSCLC, although its effectiveness is limited to a subset of patients. Many radiolabeled PD-L1-targeted imaging ligands, such as antibodies, nanobodies and peptides, have been evaluated in preclinical and clinical studies to dynamically and non-invasively assess PD-L1 status, potentially extending the benefits of PD-1/PD-L1 blockade therapy to a larger population [14, 17,18,19]. Despite the successful use of monoclonal antibodies, such as 89Zr-atezolizumab and 89Zr-durvalumab, for in vivo tumor imaging, their applications are still restricted by their large molecular size, which prevent tumor penetration and plasma clearance. By contrast, nanobody- and peptide-based tracers have gained significance due to their smaller molecular size and excellent targeting properties, allowing rapid blood clearance and a high tumor-to-background ratio. In our previous study, we developed a nanobody, NM-01, labeled with 99mTc as a SPECT probe for non-invasive assessment of PD-L1 expression [10]. We demonstrated its safety, tolerable radiation dose, favorable biodistribution, and image characteristics correlating with PD-L1 IHC results. Based on these findings, in the present study, we synthesized 68Ga-NODAGA-NM-01 and assessed its potential for PET imaging of PD-L1 expression in NSCLC.

The radiolabeling strategy for NM-01 is site-specific and simple. 68Ga-NODAGA-NM-01 was prepared within 30 min with a high RCP and good stability in vitro. Prior to the preliminary clinical study, we validated the imaging performance of 68Ga-NODAGA-NM-01 in vivo using tumor-bearing mice. High uptake of 68Ga-NODAGA-NM-01 in PD-L1-positive tumors (HCC827), but low accumulation in the blocking group and PD-L1-negative tumors (A549), confirmed the specificity of the tracer for PD-L1. The tumor retention of radioactivity remained stable but was rapidly cleared from the blood, resulting in good-quality PET images with high T/M ratios as early as 0.5 h post-injection. This was attributed to its hydrophilicity and small molecular size. Similar to other radiolabeled PD-L1-targeted nanobodies, the radioactivity of 68Ga-NODAGA-NM-01 mainly in the liver, spleen, and kidneys with low background levels in the lungs, bone marrow and muscle. The tumor uptake of 68Ga-NODAGA-NM-01 was 2.59 ± 0.65 %ID/g at 0.5 h post-injection, which was close to other reported nanobody-based tracers, such as 68Ga-NOTA-Nb109 and 68Ga-NOTA-RW102 [17, 29]. Notably, 68Ga-NODAGA-NM-01 showed lower tumor uptake than some reported nanobodies and peptides, such as 68Ga-THP-APN09 and 68Ga-NOTA-WL12 [30, 31]. This was probably related to the high PD-L1 levels in the used mouse xenograft models constructed by gene-transfected tumor cells. Meanwhile, several peptide-based agents also demonstrated high tumor uptake in natural high PD-L1-expressing tumor models, providing support for clinical studies and future development of immunoimaging technology [18, 32, 33].

A preliminary clinical study of 68Ga-NODAGA-NM-01 was conducted in patients with NSCLC with varying PD-L1 expression status (PD-L1 positive and negative). We demonstrated that 68Ga-NODAGA-NM-01 PET/CT is a safe procedure with no adverse events. The PET/CT images of the primary lesions were carefully evaluated and compared with PD-L1 IHC results. The primary lesion of the PD-L1-positive patient was clearly visualized with a low background signal 1 h post-injection, whereas the primary lesion of the PD-L1-negative patient showed low uptake, consistent with their IHC results and SUVmax. However, probably owing to the heterogeneity of PD-L1 expression, metastases of both PD-L1-positive and -negative patients showed distinct accumulation of radioactivity. This hypothesis requires further investigation. Similar to 99mTc-NM-01 and other nanobody-based PD-L1 tracers, 68Ga-NODAGA-NM-01 exhibited the highest radioactivity in the kidneys [34,35,36]. Therefore, efforts must be made to reduce high kidney retention [37].

Although our data demonstrated that the ability of 68Ga-NODAGA-NM-01 to reflect PD-L1 expression in primary and metastatic lesions of patients with NSCLC, the present study has potential limitations. To further verify the safety and efficiency of this tracer, a relatively large cohort is needed. Bone and lymph node metastases that were positive for PD-L1 expression on 68Ga-NODAGA-NM-01 PET/CT were not examined pathologically, opening the possibility of false-positive results. In addition, without tracking and follow-up, we were unable to investigate the potential of 68Ga-NODAGA-NM-01 PET/CT imaging for assessing the prognosis of patients undergoing or not having received immunotherapy. Future studies with 68Ga-NODAGA-NM-01 PET/CT imaging may address these issues, improving our understanding of its potential application for guiding NSCLC treatment.

Conclusions

A 68Ga-labeled PD-L1 targeted nanobody was successfully prepared using a simple method under mild conditions. The prepared 68Ga-NODAGA-NM-01 had a high RCP, good stability in vitro, rapid blood clearance, and specific accumulation in PD-L1-positive tumors in vivo. A preliminary clinical study showed that 68Ga-NODAGA-NM-01 PET/CT imaging was a safe procedure with no adverse events in two patients with NSCLC that could reflect PD-L1 expression in primary and metastatic lesions. However, further validation in a larger patient cohort is warranted to substantiate these findings.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

FBS:

Fetal bovine serum

IHC:

Immunohistochemistry

NSCLC:

Non-small-cell lung cancer

PBS:

Phosphate-buffered saline

PD-1:

Programmed cell death 1

PD-L1:

Programmed death ligand-1

PET:

Positron emission tomography

Radio-HPLC:

Radio-high-performance liquid chromatography

Radio-TLC:

Radio-layer chromatography

RCP:

Radiochemical purity

SPECT:

Single-photon emission computed tomography

SUVmax :

Maximum standardized uptake value

T/M:

Tumor-to-muscle

t1/2α :

Elimination distribution phase half-life

t1/2β :

Clear phase half-life

68Ga-NODAGA-NM-01:

68Ga-labeled NM-01

%ID/g:

Percentage of injected dose per gram

References

  1. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–63.

    Article  PubMed  Google Scholar 

  2. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA-Cancer J Clin. 2023;73:17–48.

    Article  PubMed  Google Scholar 

  3. Cao W, Chen HD, Yu YW, Li N, Chen WQ. Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020. Chin Med J. 2021;134(7):783–91.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Chen P, Liu Y, Wen Y, Zhou C. Non-small cell lung cancer in China. Cancer Commun. 2022;42(10):937–70.

    Article  Google Scholar 

  5. Gandhi L, Rodríguez-Abreu D, Gadgeel S, Esteban E, Felip E, De Angelis F, et al. Pembrolizumab plus chemotherapy in metastatic non-small-cell lung cancer. N Engl J Med. 2018;378(22):2078–92.

    Article  CAS  PubMed  Google Scholar 

  6. Xia LL, Liu YY, Wang Y. PD-1/PD-L1 blockade therapy in advanced non-small-cell lung cancer: current status and future directions. Oncologist. 2019;24(Suppl 1):S31–41.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Duma N, Santana-Davila R, Molina JR. Non-small cell lung cancer: epidemiology, screening, diagnosis, and treatment. Mayo Clin Proc. 2019;94(98):1623–40.

    Article  CAS  PubMed  Google Scholar 

  8. Patel SA, Weiss J. Advances in the treatment of non-small cell lung cancer: immunotherapy. Clin Chest Med. 2020;41(2):237–47.

    Article  PubMed  Google Scholar 

  9. Sun C, Mezzadra R, Schumacher TN. Regulation and function of the PD-L1 checkpoint. Immunity. 2018;48(3):434–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Xing Y, Chand G, Liu C, Cook GJR, O’Doherty J, Zhao L, et al. Early phase I study of a 99mTc labeled anti-PD-L1 sdAb in SPECT/CT assessment of programmed death ligand-1 expression in non-small cell lung cancer. J Nucl Med. 2019;60(9):1213–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tang S, Qin C, Hu H, Liu T, He Y, Guo H, et al. Immune checkpoint inhibitors in non-small cell lung cancer: progress, challenges, and prospects. Cells. 2022;11(3):320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. McLaughlin J, Han G, Schalper KA, Carvajal-Hausdorf D, Pelekanou V, Rehman J, et al. Quantitative assessment of the heterogeneity of PD-L1 expression in non-small-cell lung cancer. JAMA Oncol. 2016;2(1):46–54.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Doroshow DB, Bhalla S, Beasley MB, Sholl LM, Kerr KM, Gnjatic S, et al. PD-L1 as a biomarker of response to immune-checkpoint inhibitors. Nat Rev Clin Oncol. 2021;18(6):345–62.

    Article  CAS  PubMed  Google Scholar 

  14. Bensch F, van der Veen EL, Lub-de Hooge MN, Jorritsma-Smit A, Boellaard R, Kok IC, et al. 89Zr-atezolizumab imaging as a non-invasive approach to assess clinical response to PD-L1 blockade in cancer. Nat Med. 2018;24(12):1852–8.

    Article  CAS  PubMed  Google Scholar 

  15. Nimmagadda S. Imaging PD-L1 expression in melanoma brain metastases. J Nucl Med. 2022;63(6):897–8.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Smit J, Borm FJ, Niemeijer AN, Huisman MC, Hoekstra OS, Boellaard R, et al. PD-L1 PET/CT imaging with radiolabeled durvalumab in patients with advanced-stage non-small cell lung cancer. J Nucl Med. 2022;63(5):686–93.

    CAS  PubMed  Google Scholar 

  17. Liu Q, Jiang L, Li K, Li H, Lv G, Lin J, et al. Immuno-PET imaging of 68Ga-labeled nanobody Nb109 for dynamic monitoring the PD-L1 expression in cancers. Cancer Immunol Immunother. 2021;70(6):1721–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Mishra A, Kumar D, Gupta K, Lofland G, Sharma AK, Banka DS, et al. Gallium-68-labeled peptide PET quantifies tumor exposure of PD-L1 therapeutics. Clin Cancer Res. 2023;29(3):581–91.

    Article  CAS  PubMed  Google Scholar 

  19. Zhu S, Liang B, Zhou Y, Chen Y, Fu J, Qiu L, et al. Development of novel peptide-based radiotracers for detecting PD-L1 expression and guiding cancer immunotherapy. Eur J Nucl Med Mol Imaging. 2024;51(3):625–40.

    Article  CAS  PubMed  Google Scholar 

  20. Sun S, Ding Z, Yang X, Zhao X, Zhao M, Gao L, et al. Nanobody: a small antibody with big implications for tumor therapeutic strategy. Int J Nanomed. 2021;16:2337–56.

    Article  Google Scholar 

  21. Lecocq Q, De Vlaeminck Y, Hanssens H, D’Huyvetter M, Raes G, Goyvaerts C, et al. Theranostics in immuno-oncology using nanobody derivatives. Theranostics. 2019;9(25):7772–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu M, Li L, Jin D, Liu Y. Nanobody-A versatile tool for cancer diagnosis and therapeutics. Wiley Interdiscip Rev-Nanomed Nanobiotechnol. 2021;13(4):e1697.

    Article  CAS  PubMed  Google Scholar 

  23. Su J, Liu X, Guo S, Zhang J, Wei X, Li X. Nanobodies: a new potential for prostate cancer treatment. J Cancer Res Clin Oncol. 2023;149(9):6703–10.

    Article  PubMed  Google Scholar 

  24. Mitran B, Güler R, Roche FP, Lindström E, Selvaraju RK, Fleetwood F, et al. Radionuclide imaging of VEGFR2 in glioma vasculature using biparatopic affibody conjugate: proof-of-principle in a murine model. Theranostics. 2018;8(16):4462–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang Y, Wang C, Huang M, Qin S, Zhao J, Sang S, et al. Pilot study of a novel nanobody 68Ga-NODAGA-SNA006 for instant PET imaging of CD8+ T cells. Eur J Nucl Med Mol Imaging. 2022;49(13):4394–405.

    Article  CAS  PubMed  Google Scholar 

  26. Ge S, Wang C, You X, He H, Zhang B, Jia T, et al. Imaging and monitoring HER2 expression in tumors during HER2 antibody-drug conjugate therapy utilizing a radiolabeled site-specific single-domain antibody probe: 68Ga-NODAGA-SNA004-GSC. J Med Chem. 2024;67(15):12855–67.

    Article  CAS  PubMed  Google Scholar 

  27. Gong J, Zhao L, Yang J, Zhu M, Zhao J. [99mTc]Tc-labeled plectin-targeting peptide as a novel SPECT probe for tumor imaging. Pharmaceutics. 2022;14(5):996.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wong NC, Cai Y, Meszaros LK, Biersack HJ, Cook GJ, Ting HH, et al. Preclinical development and characterisation of 99mTc-NM-01 for SPECT/CT imaging of human PD-L1. Am J Nucl Med Mol Imaging. 2021;11(3):154–66.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhang Y, Cao M, Wu Y, Malih S, Xu D, Yang E, et al. Preclinical development of novel PD-L1 tracers and first-in-human study of [68Ga]Ga-NOTA-RW102 in patients with lung cancers. J Immunother Cancer. 2024;12(4):e008794.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Ma X, Zhou X, Hu B, Li X, Yao M, Li L, et al. Preclinical evaluation and pilot clinical study of [68Ga]Ga-THP-APN09, a novel PD-L1 targeted nanobody radiotracer for rapid one-step radiolabeling and PET imaging. Eur J Nucl Med Mol Imaging. 2023;50(13):3838–50.

    Article  CAS  PubMed  Google Scholar 

  31. Zhou X, Jiang J, Yang X, Liu T, Ding J, Nimmagadda S, et al. First-in-humans evaluation of a PD-L1-binding peptide PET radiotracer in non-small cell lung cancer patients. J Nucl Med. 2022;63(4):536–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhou M, Xiang S, Zhao Y, Tang Y, Yang J, Yin X, et al. [68Ga]Ga-AUNP-12 PET imaging to assess the PD-L1 status in preclinical and first-in-human study. Eur J Nucl Med Mol Imaging. 2024;51(2):369–79.

    Article  CAS  PubMed  Google Scholar 

  33. Zhang L, Zhang S, Wu J, Wang Y, Wu Y, Sun X, et al. Linear peptide-based PET tracers for imaging PD-L1 in tumors. Mol Pharm. 2023;20(8):4256–67.

    Article  CAS  PubMed  Google Scholar 

  34. Qin S, Yu Y, Guan H, Yang Y, Sun F, Sun Y, et al. A preclinical study: correlation between PD-L1 PET imaging and the prediction of therapy efficacy of MC38 tumor with 68Ga-labeled PD-L1 targeted nanobody. Aging. 2021;13(9):13006–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhu M, Zhang J, Yang M, Zhang H, Xu T, Kan F, et al. In vitro and in vivo study on the treatment of non-small cell lung cancer with radionuclide labeled PD-L1 nanobody. J Cancer Res Clin Oncol. 2023;149(11):8429–42.

    Article  CAS  PubMed  Google Scholar 

  36. Lv G, Sun X, Qiu L, Sun Y, Li K, Liu Q, et al. PET imaging of tumor PD-L1 expression with a highly specific nonblocking single-domain antibody. J Nucl Med. 2020;61(1):117–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hu B, Ma X, Shi L, Liu T, Li L, Yao M, et al. Noninvasive evaluation of tumoral PD-L1 using a novel 99mTc-labeled nanobody tracer with rapid renal clearance. Mol Pharm. 2024;21(4):1977–86.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This research was funded by the National Natural Science Foundation of China, grant numbers 82171978 and 81971647.

Author information

Author notes

  1. Lingzhou Zhao, Jiali Gong and Sisi Liao contributed equally to this work.

Authors and Affiliations

  1. Department of Nuclear Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Hongkou District, No. 100, Haining Road, Shanghai, 200080, China

    Lingzhou Zhao, Jiali Gong, Sisi Liao, Jinhua Zhao & Yan Xing

  2. Nanomab Technology Limited, No. 333, North Chengdu Road, Jingan District, Shanghai, 200041, China

    Wenhua Huang

Authors
  1. Lingzhou Zhao
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Jiali Gong
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Sisi Liao
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Wenhua Huang
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Jinhua Zhao
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Yan Xing
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Conceptualization, LZ and JG; methodology, LZ, JG, SL, and WH; validation, YX and JZ; formal analysis, LZ and JG; investigation, LZ; resources, YX and JZ; writing—original draft preparation, LZ and SL; writing—review and editing, YX and JZ; supervision, YX; project administration, YX; funding acquisition, YX and JZ. All authors have read and agreed to the published version of the manuscript. LZ, JG, and SL contributed to this work equally.

Corresponding authors

Correspondence to Jinhua Zhao or Yan Xing.

Ethics declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Shanghai General Hospital (protocol code [2019]310). Informed consent was obtained from all subjects involved in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, L., Gong, J., Liao, S. et al. Preclinical evaluation and preliminary clinical study of 68Ga-NODAGA-NM-01 for PET imaging of PD-L1 expression. Cancer Imaging 25, 6 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40644-025-00826-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40644-025-00826-8

Keywords

Cancer Imaging

ISSN: 1470-7330

Contact us