Precursor compound of targeted human PD-L1, radioactive marker as well as preparation method and application of precursor compound and radioactive marker

Abstract

The invention provides a cyclic peptide intermediate compound targeting human PD-L1. The structure of the cyclic peptide intermediate compound is shown as a formula (I). The invention further provides a cyclic peptide molecule precursor compound of the targeted human PD-L1, and the structure of the cyclic peptide molecule precursor compound is shown as a formula (II). The invention also provides a radionuclide-labeled compound, which is a coordination compound formed by taking the precursor compound with the structure as shown in the formula (II) as a ligand and radionuclide. The invention also provides methods of making the intermediate compounds, cyclic peptide molecule precursor compounds, and radionuclide-labeled compounds. The invention also provides an application of the cyclic peptide molecule precursor compound as a labeled precursor in preparation of a PET imaging agent, and an application of a radionuclide labeled compound in living body imaging of a non-therapeutic or diagnostic organism. According to the present invention, the cyclic peptide in the radionuclide labeled compound structure can highly target the human source PD-L1, and has dual compatibility with < 18 > F and < 68 > Ga; # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the manufacture of biopharmaceuticals in the biopharmaceutical industry, and particularly to a cyclic polypeptide molecule with targeting properties, specifically to a cyclic polypeptide molecule capable of targeting human PD-L1, its radionuclide label, and its preparation and application in in vivo imaging of organisms. Background Art

[0002] Immune checkpoint blockade therapies targeting PD-1 and its ligand PD-L1 have become standard treatment strategies for various malignancies, especially non-small cell lung cancer (NSCLC). Despite their transformative impact, accurately identifying patients most likely to benefit from these therapies remains a major challenge. Biomarkers such as PD-L1 expression, tumor mutational burden (TMB), and microsatellite instability (dMMR)-high / deficient mismatch repair status (MSI-H) have been validated in specific cancer types through clinical trials. In patients with advanced NSCLC who are negative for epidermal growth factor receptor and / or anaplastic lymphoma kinase mutations, the tumor proportion score (TPS) of PD-L1 has become a key efficacy prediction biomarker. Higher TPS values are associated with improved survival outcomes with immunotherapy, and 1% and 50% are commonly used as thresholds in NSCLC to guide clinical decision-making. However, the relationship between PD-L1 expression and clinical benefit remains unclear. Not all patients with high PD-L1 expression respond to PD-1 / PD-L1 blockade, and some patients with low or negative PD-L1 staining can still benefit from immunotherapy. This discrepancy highlights the limitations of static tissue biomarker assessment, which is often affected by sampling bias, temporal changes, and tumor heterogeneity.

[0003] Positron emission tomography (PET) imaging using radiolabeled anti-PD-L1 antibodies has emerged as a promising method for comprehensively and dynamically assessing PD-L1 expression in systemic lesions. Although antibody-based PET imaging agents have potential, they have practical limitations, including immunogenicity, high production costs, and long imaging times, typically taking more than 72 hours after administration to achieve optimal image contrast. Small molecule peptides have become attractive alternatives due to their rapid clearance, excellent tissue permeability, and ability to provide high-contrast imaging within one hour after administration.

[0004] Cyclic peptide WL12 was the first PD-L1 targeting peptide developed for PET imaging and has undergone early clinical evaluation. However, it exhibits high background activity and relatively low tumor uptake in normal organs, limiting its clinical application. In addition to cyclic peptide WL12, most other PD-L1 targeting probes rely on DOTA chelators and cannot be used with the most commonly used positron radionuclides 18Coordination with F limits its clinical application. With the increasing popularity of medical cyclotrons, 18 F may provide a more suitable option for this new imaging modality to benefit patients.

[0005] Therefore, it is necessary to develop a new PD-L1 targeting peptide to improve its tumor uptake and background activity, and conjugate it with a positron radionuclide based on the NOTA chelator 18 F for better clinical applicability. Summary of the Invention

[0006] An object of the present invention is to provide a new cyclic peptide molecular precursor compound targeting human PD-L1, which has higher tumor uptake and lower background activity than WL12.

[0007] Another object of the present invention is to provide a radionuclide-labeled compound that coordinates the cyclic peptide molecular precursor compound with 18 F, significantly improving its clinical applicability.

[0008] Still another object of the present invention is to provide a method for preparing the cyclic peptide molecular precursor compound and the radionuclide-labeled compound.

[0009] Still another object of the present invention is to provide the application of the cyclic peptide molecular precursor compound and the radionuclide-labeled compound in the preparation of PET imaging agents or in vivo imaging of organisms.

[0010] The above objects of the present invention are achieved by the following technical solutions:

[0011] In a first aspect, the present invention first provides a cyclic peptide intermediate compound targeting human PD-L1, the structure of which is shown in the following formula (I):

[0012]

[0013] The intermediate compound of the present invention is a cyclic polypeptide molecule composed of 15 amino acids (the amino acid sequence is shown in SEQ ID No.1), and has high targeting to human PD-L1.

[0014] Based on the above cyclic peptide intermediate compound, the present invention further provides a cyclic peptide molecular precursor compound targeting human PD-L1, which is formed by conjugating the cyclic peptide intermediate compound with the structure shown in formula (I) of the present invention with a NOTA chelator, and the structure is shown in the following formula (II):

[0015]

[0016] The precursor compound with the structure shown in formula (II) according to the present invention can be named "NK224", and the cyclic polypeptide in its structure can highly target human PD-L1, and the NOTA chelating group can respectively coordinate with 18 F and 68 Ga.

[0017] In a second aspect, the present invention also provides a radionuclide-labeled compound, which is a coordination compound formed by using the precursor compound with the structure shown in formula (II) according to the first aspect of the present invention as a ligand and a radionuclide.

[0018] In a preferred embodiment of the present invention, the radionuclide may be 18 F or 68 Ga.

[0019] The radionuclide-labeled compound according to the present invention is suitable as a PET imaging agent and has excellent performance in terms of biodistribution and background clearance.

[0020] In a third aspect, the present invention provides methods for preparing the precursor compound according to the first aspect and the radionuclide-labeled compound according to the second aspect.

[0021] The method for preparing the precursor compound according to the first aspect includes:

[0022] 1) Based on Fmoc-protected amino resin, through solid-phase synthesis of polypeptides, the following amino acids are sequentially coupled using a coupling agent: Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-NMe-Nle-OH, Fmoc-NMe-Nle-OH, Fmoc-Trp(N-CH 2 -COOtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-NMeAla-OH, and Fmoc-Tyr(tBu)-OH; then the Fmoc protecting group is removed to obtain a resin peptide with the following structure: H-Tyr(tBu)-NMeAla-Asn(Trt)-Pro-His(Trt)-Glu(OtBu)-Hyp(tBu)-Trp(Boc)-Ser(tBu)-Trp(N-CH 2 -COOtBu)-NMeNle-NMeNle-Lys(Boc)-Cys(Trt)-Gly-Resin;

[0023] 2) Couple chloroacetic anhydride to the tyrosine of the resin peptide obtained in 1), and then cleave the peptide chain from the resin to obtain a crude polypeptide with the following structure:

[0024] Cl-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(N-CH 2 -COOH)-NMeNle-NMeNle-Ly s-Cys-Gly-NH 2 ;

[0025] 3) Purify and cyclize the crude polypeptide obtained in 2) to obtain a cyclic polypeptide with the structure shown in formula (I);

[0026] 4) Couple the NOTA group to the glutamate of the cyclic polypeptide obtained in 3) to obtain a precursor compound with the structure shown in formula (II), denoted as "NK224";

[0027] In a preferred embodiment of the present invention, the amino resin described in 1) is Rink Amide MBHA Resin.

[0028] In a preferred embodiment of the present invention, the coupling agent described in 1) is a mixture of DIC and HOBt.

[0029] In a preferred embodiment of the present invention, the removal of the Fmoc protecting group is carried out by cleavage with a 20% Pip / DMF solution.

[0030] In a preferred embodiment of the present invention, the cleavage in 2) is carried out using a TFA solution as the cleavage solution.

[0031] The method for preparing the radionuclide-labeled compound described in the second aspect includes:

[0032] Use a solution containing radionuclide ions, preferably a solution containing 18 F ions or 68 Ga ions, react with the NK224, carry out radionuclide labeling, to obtain the radionuclide-labeled compound described in the second aspect of the present invention, such as 18 F-NK224 or 68 Ga-NK224.

[0033] Fourthly, the present invention also provides the application of the cyclic peptide molecular precursor compound described in the first aspect as a labeling precursor in the preparation of PET imaging agents; and the application of the radionuclide-labeled compound described in the second aspect as an imaging agent in the in vivo imaging of organisms for non-therapeutic or diagnostic purposes.

[0034] The present invention developed and characterized a novel cyclic peptide molecular precursor compound NK224, which can obtain a novel PET imaging agent after being radiolabeled. The cyclic peptide ligand in its structure can highly target human PD-L1. In addition, NOTA is used as a chelating agent in its structure, which has dual compatibility with 18 F and 68 Ga, expanding its clinical applicability and facilitating its adoption in various clinical settings.

[0035] Compared with the existing WL12, the cyclic peptide molecular precursor compound of the present invention has a different peptide structure. In particular, a carboxyl group is introduced at a specific position of the cyclic peptide molecule, presumably thereby improving the water solubility of the compound, improving its in vivo pharmacokinetic properties, reducing hepatobiliary uptake, and contributing to improving the imaging quality. Preclinical experimental verification shows that after the precursor compound NK224 of the present invention is radiolabeled with 68 radioisotopes such as Ga, it is indeed superior to the existing 68 Ga-NOTA-WL12 in tumor imaging, biodistribution, and background clearance. Description of the Drawings

[0036] Figure 1 It is the liquid chromatography-mass spectrometry analysis chart of Compd1 in Example 1.

[0037] Figure 2 It is the liquid chromatography-mass spectrometry analysis chart of Compd2 in Example 1.

[0038] Figure 3 It is the liquid chromatography-mass spectrometry analysis chart of NK224 in Example 1.

[0039] Figure 4 It is the high performance liquid chromatography (HPLC) chart of NK224 in Example 1.

[0040] Figure 5 It reflects the 68 in vitro evaluation results of Ga-NK224 and 18 F-NK224 in Experimental Example 1, the affinity and specificity test results in Experimental Example 2, and the expression results of PD-L1 in different tumor cells. Among them, (A) reflects the chemical structure of NK224 and the radiolabeling mechanism of binding to 68 Ga and 18 F. (B-C) reflect the in vitro stability analysis results of 68 Ga-NK224 and 18 F-NK224 in PBS and FBS. (D) reflects the results of analyzing the binding affinity of NK224 to PD-L1 by IC50. (E) reflects the 68Cell uptake assay results of Ga-NK224, including blocking studies using unlabeled precursor and hPD-L1 antibody to confirm specificity (n = 3 / group). (F-G) show the results of evaluating PD-L1 expression in A549, C666-1, and U87-MG cells by Western blot and flow cytometry.

[0041] Figure 6 show the results in Experimental Example 3 68 Ga-NK224 and 18 in vivo evaluation results of F-NK224. Among them, (A) shows the representative PET imaging results using 68 Ga-WL12 and 68 Ga-NK224 in the U87-MG tumor model, and the blocking study results using 68 Ga-NK224. (B) shows the quantification results of the biodistribution study of 68 Ga-WL12 and 68 Ga-NK224 in the U87-MG tumor model, including the blocking experiment results using 68 Ga-NK224. (C) shows the representative PET imaging results using 18 F-NK224 in the U87-MG tumor model. (D) shows the representative PET imaging results of A549, C666-1, and U87-MG tumor-bearing mice after 1 hour, showing PD-L1-specific tumor uptake. (G) shows the IHC staining and H&E staining of PD-L1 in the corresponding tumors.

[0042] Figure 7 show the results in Experimental Example 4 68 Distribution of Ga-NK224 in three healthy volunteers. (A) shows the representative PET imaging results at 15, 30, 60, and 120 minutes after injection, showing the biodistribution in normal organs. (B) shows the semi-quantitative analysis (SUVmean) results of the main organs (including liver, spleen, lung, kidney) at 15, 30, 60, and 120 minutes after injection.

[0043] Figure 8 show the results in Experimental Example 4 68 Correlation between Ga-NK224 tumor uptake and PD-L1 expression level. (A) shows the results of the Kruskal-Wallis test showing 18 no significant correlation between F-FDG SUVmax and PD-L1 expression (P = 0.138), while 68There was a significant correlation between Ga-NK224 SUVmax and PD-L1 expression (H = 24.994, P < 0.001). (B) shows representative 18 F-FDG and 68 Ga-NK224 PET / CT images and immunohistochemically stained tissue samples from lesions with different PD-L1 expressions (red circles indicate CT-guided biopsy sites); among them, the upper row shows respectively: 18 F-FDG SUVmax 15.3, 68 Ga-NK224 SUVmax 8.2, PD-L1 TPS 80%; the middle row shows respectively: 18 F-FDG SUVmax 10.1, 68 Ga-NK224 SUVmax 4.8, PD-L1 TPS 2; the lower row shows respectively: 18 F-FDG SUVmax 10.4, 68 Ga-NK224 SUVmax 2.2, PD-L1 TPS < 1%. Bar = 50 μm.

[0044] Figure 9 shows representatives of patients with different PD-L1 expression levels in Experimental Example 4 18 F-FDG and 68 PET / CT maximum intensity projection maps of Ga-NK224: Patient A: 18 F-FDG SUVmax 15.3, 68 Ga-NK224 SUVmax 8.2, PD-L1 TPS 80%; Patient B: 18 F-FDG SUVmax 10.1, 68 Ga-NK224 SUVmax 4.8, PD-L1 TPS 2; Patient C: 18 F-FDG SUVmax 10.4, 68 Ga-NK224 SUVmax 2.2, PD-L1 TPS < 1%.

[0045] Figure 10 shows the results of visualizing PD-L1 expression and inter-tumor heterogeneity by 68 Ga-NK224 PET / CT in Experimental Example 4. (A) shows representative PET / CT images of metastatic NSCLC patients showing the 18 similar F-FDG uptake between the primary tumor (red arrow) and bone metastasis (blue arrow) (primary tumor SUVmax 7.8; bone metastasis SUVmax 8.6). In contrast, 68Ga-NK224 uptake varied among the lesions (SUVmax of primary tumor was 5.0; SUVmax of bone metastasis was 11.8). CT-guided biopsy and IHC staining confirmed weak PD-L1 expression in the lung lesions and strong PD-L1 expression in the bone metastases. (B) shows the overview of 68 Ga-NK224 uptake (SUVmax) in the lesions of 24 patients, including 24 primary lesions and 215 metastatic lesions. This figure shows the SUVmax values of individual lesions in different patients, as well as tumor size, biopsy site, and lesion location.

[0046] Figure 11 Shows the consecutive 68 Ga-NK224 PET / CT scan results before and after PD-L1 antibody treatment in Experimental Example 4. (A) are consecutive 68 Ga-NK224 PET / CT images of NSCLC patients before treatment and on days 1, 7, and 21 after administration of the PD-L1 antibody. (B) shows the bar graph depicting the changes in SUVmax of major organs (including tumor, liver, spleen, bone marrow, lung, and kidney) before and after PD-L1 antibody treatment. Detailed implementation manners

[0047] The technical solutions and technical effects of the present invention will be further described below by listing examples and experimental examples. Example 1. Synthesis method of PD-L1 targeting probe

[0048] 1. Synthesis of Compound 1 (Compd1):

[0049] First, Fmoc-Gly-OH was coupled to Rink Amide MBHA Resin, and then Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-NMe-Nle-OH, Fmoc-NMe-Nle-OH, Fmoc-Trp(N-CH 2-COOtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-NMeAla-OH, and Fmoc-Tyr(tBu)-OH were coupled successively. The coupling of all amino acids was carried out in DMF using DIC (3 eq) and HOBt (3 eq) as coupling reagents. The Fmoc protecting group was cleaved with 20% Pip / DMF solution. Then chloroacetic anhydride was coupled with the previous resin peptide under the conditions of DIPEA (3 eq) and DMF. Finally, the crude peptide compound 1 (Compd1) was cleaved from the resin with TFA solution at room temperature for 2 h. The crude peptide was purified by HPLC on a C18 preparative column to obtain the pure compound 1 (Compd1) (Yield: 20.5%). The results of its liquid chromatography-mass spectrometry analysis are shown in Figure 1 。

[0050] 2. Synthesis of compound 2 (Compd2):

[0051] Compd1 (1 eq) obtained in the above step 1 was dissolved in 10 mL of DMF, DIEA (5 eq) was added, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by LC-MS until completion. The solvent was evaporated, and the product was purified by reverse-phase preparative liquid chromatography to obtain compound 2 (Copmd2) (Yield: 15.8%). The results of its liquid chromatography-mass spectrometry analysis are shown in Figure 2 。

[0052] 3. Synthesis of NK224:

[0053] Compd2 (1 eq) obtained in the above step 2 and NOTA-succinimide ester (compd3) (1 eq) were dissolved in 10 mL of DMF, DIEA (3 eq) was added, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by LC-MS until completion. The solvent was evaporated, and the product was purified by reverse-phase preparative liquid chromatography to obtain NK224 (Yield: 35%). The results of its liquid chromatography-mass spectrometry analysis are shown in Figure 3 , and the high-performance liquid chromatography (HPLC) diagram is shown in Figure 4 , and its amino acid sequence is shown in SEQ ID No.1.

[0054] The synthetic route of the above NK22 is as follows:

[0055]

[0056]

[0057] The present invention conducted a preliminary clinical evaluation on Ga-NK224 prepared in Example 1 through the following Experimental Examples 1-4, focusing on its safety, dosimetry, and consistency with PD-L1 immunohistochemistry (IHC). In addition, we evaluated its ability to accurately visualize the heterogeneity of PD-L1 expression in systemic lesions. 68 Ga-NK224 was subjected to a preliminary clinical evaluation, with a focus on its safety, dosimetry, and concordance with PD-L1 immunohistochemistry (IHC). Additionally, we evaluated its ability to accurately visualize the heterogeneity of PD-L1 expression in systemic lesions.

[0058] Experimental Example 1. Radioactive Labeling and Stability Testing of the Probe

[0059] Ga-NK224 and 68 F-NK224 were prepared according to conventional methods: 18 As shown in A of

[0060] 1 mL of NK224 (50 μg dissolved in 0.25 M sodium acetate) was added to the Figure 5 GaCl 68 solution (4 mL, 925 - 1110 MBq, pH 5 - 6) obtained from a germanium-gallium generator, and the mixture was incubated at 80 °C for 10 minutes. For 3 F-NK224, 200 μL of 18 F ions (1850 - 2035 MBq) were added to 20 μL of 20 mM AlCl 18 (dissolved in 0.5 M acetic acid-sodium acetate solution, pH 4.0), then 50 μg of the precursor NK224, 100 μL of 0.5 M acetic acid-sodium acetate solution, and 300 μL of acetonitrile were added. The mixture was reacted at 100 °C for 15 minutes. 3 The specific activities of 68 Ga-NK224 and 18 F-NK224 after radioactive labeling were 30.6 - 36.7 GBq / μmol and 33.2 - 36.5 GBq / μmol, respectively. 68 Both the 18 Ga- and 68 F-labeled products were purified using a C18 column (WAT020515, Waters Corporation). The radiochemical purity and stability of 18 Ga-NK224 and 18 F-NK224 were evaluated by radio-high performance liquid chromatography (HPLC). The in vitro stability of the 68 F-NK224 and 18 Ga-NK224 injection solutions was evaluated in phosphate-buffered saline (pH = 7.4) and fetal bovine serum (FBS). Specifically, 0.5 MBq (50 μL) of 68Ga-NK224 was mixed with 200 μL of 5% FBS or 200 μL of PBS buffer (0.01 M, pH 7.4) and incubated at room temperature for 2 hours. The mixture was analyzed by radio-HPLC to evaluate in vitro stability. The analytical method was as follows: 85% A (aqueous solution of 0.1% TFA) and 30% B (acetonitrile solution of 0.1% TFA), at a flow rate of 1 mL / min for 20 minutes, increased to 95% B. The results of HPLC analysis showed (as shown in B and C of Figure 5 ) that both radiotracers showed high radiochemical purity after purification and remained stable for up to 2 hours, and no obvious demetallization was observed in phosphate-buffered saline (PBS) and fetal bovine serum (FBS) (≥99%). For clinical translational research, the sterility test of the probe was carried out inside the radiochemistry facility of the First Affiliated Hospital of Xiamen University. Before being approved for clinical use and for human administration, 68 Ga-NK224 had met all established criteria.

[0061] Experimental Example 2. Probe Specificity and Affinity Test

[0062] The cell lines used in the experiment included A549 (non-small cell lung cancer), C666-1 (EBV-positive nasopharyngeal carcinoma), and U87-MG (glioma). The A549 and U87-MG cell lines were from the National Experimental Cell Resource Sharing Platform of China, and the C666-1 was from the research group of Professor Zhao Chong of the Sun Yat-sen University Cancer Center. The binding specificity was further evaluated by cell binding and blocking studies on the U87-MG, C666-1, and A549 cell lines.

[0063] To determine 68 the binding affinity of Ga-NK224 for PD-L1, the half-maximal inhibitory concentration (IC50) was determined. For the IC50 determination, U87-MG cells with high expression of human PD-L1 were used for evaluation. When the cell growth reached about 80% of the well plate, 68 Ga-NK224 was used as a radioligand, and U87-MG cells were treated with different concentrations of unlabeled NK224 (4.5×10-5 to 10-14 M; n = 3). After incubation for 60 minutes, the cells were washed with cold PBS supplemented with 2% FBS, harvested, and the radioactivity was measured using a gamma counter. The IC50 values were analyzed using Prism v7.0 (GraphPad Software Inc., San Diego, Canada). Each experiment was repeated three times for each condition. The experimental results were as shown in D of Figure 5 , and NK224 showed high affinity, with a 50% inhibitory concentration (IC50) of 2.45 nM.

[0064] To evaluate its specificity for PD-L1, cell binding and blocking assays were performed using glioma cell line U87-MG expressing hPD-L1, nasopharyngeal carcinoma cell line C666-1, and hPD-L1-negative lung cancer cell line A549. The tumor cells were cultured in 24-well plates until they reached 80% of the well. Subsequently, the regular medium was replaced with serum-free medium for subsequent experiments. The cell uptake assay involved treating the three cell lines (A549, C666-1, and U87-MG) with 68 68Ga-NK224 alone or in combination with 10 nmol of blocking agent (unlabeled NK224) or hPD-L1 antibody (Adebrelimab, Jiangsu Hengrui Pharmaceutical Co., Ltd., China) for 60 minutes to evaluate 68 the specificity of 68Ga-NK224 uptake.

[0065] For western blotting analysis, cell proteins were extracted with lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 1 mM EDTA, and 1% protease inhibitor and phosphatase inhibitor). Approximately 20 μg of total protein from each sample was separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). The membrane was pre-incubated with 5% non-fat milk in TBST for 1 hour and then incubated with human PD-L1 antibody (abcam, catalog number ab205921). The membrane was washed three times with TBST and incubated with horseradish peroxidase-labeled secondary antibody (Abclonal, catalog number AS039), and detected using an enhanced chemiluminescence detection system (CLINX, ChemiScope 6200).

[0066] For flow cytometry, viable cells were counted and suspended in cell staining buffer at a concentration of 5 - 10×10 6 cells / mL. Next, 100 μL / tube of cell suspension (5 - 10×10 5 cells / tube) was dispensed into 2 mL plastic tubes. Then, 2.5 μg of purified anti-human CD16 / CD32 mAb (BD PharmingenTM, catalog number 564219) was pre-incubated with every 10 6 cells at 100 μL for 10 minutes at room temperature to block Fc receptors. Then, 100 μL of cells per tube was incubated with a fluorescent full-length mAb against human PD-L1 (BioLegend, catalog number 563738) at 2 - 8 °C for 30 minutes. Then the tubes were washed once in 2 mL of staining buffer and centrifuged at 350×g for 5 minutes. Cells were analyzed using a BD FACSCanto II flow cytometer and data were acquired.

[0067] For histological analysis, tissue specimens were subjected to a series of preparations. They were fixed in 10% buffered formalin, dehydrated by a series of ethanol washes, embedded in paraffin, and subsequently stained using immunohistochemistry (IHC). Paraffin-embedded samples were deparaffinized in ethanol and antigen retrieval was performed using 0.01 mol / L sodium citrate containing 0.05% Tween. IHC was performed on paraffin-embedded mouse tissues using a primary antibody targeting the PD-L1 antibody (Agilent, catalog number 22C3) and a secondary antibody (Yuanxi Biotech, catalog number H-D110041-100T). Photographs were taken using an Aperio Versa 8 tissue imaging system (3D HISTECH).

[0068] The experimental results are as shown in Figure 5 E-G below, 68 The radioactive uptake of Ga-NK224 was correlated with the PD-L1 expression levels observed by Western blotting and flow cytometry, in the order of U87-MG > C666-1 > A549. Binding blockade studies were performed using 10 μM excess non-radioactive NK224 peptide or hPD-L1 antibody, reducing the uptake of 68 Ga-NK224 in PD-L1-positive cells by more than 90%, confirming receptor targeting specificity (P < 0.001; Figure 5 E). Specifically, in U87-MG cells, NK224 or hPD-L1 antibody significantly reduced the binding of 68 Ga-NK224 to hPD-L1 from 28.08 ± 1.27% to 0.81 ± 0.19% or 0.56 ± 0.29%, with P values all < 0.001; in C666-1 cells, from 4.71 ± 0.25% to 0.37 ± 0.20% or 0.44 ± 0.30%, with P values all < 0.001).

[0069] Experimental Example 3. 68 Ga / 18 In vivo evaluation of F-NK224 in an animal model

[0070] Six-week-old BALB / c nude mice were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and were housed in a specific pathogen-free (SPF) facility at the Experimental Animal Center of Xiamen University. Tumor models were established by subcutaneous injection of 5 × 10 6 tumor cells (U87-MG, C666-1, or A549) into the right shoulder of each mouse. Once the tumor diameter reached 6 - 10 mm, PET imaging and biodistribution studies were performed.

[0071] For small animal PET imaging, mice bearing U87-MG xenograft tumors (n = 3 per group) were intravenously injected with 7.4 MBq 68Ga-NOTA-WL12, 68 Ga-NK224 or 18 F-NK224. Static PET scans were performed using an Inveon small animal PET scanner (Siemens, Germany) at 1 hour and 2 hours post-injection. Blocking studies included co-injecting 25 nmol of unlabeled NK224 or 10 mg / kg of humanized PD-L1 antibody (Adebrelimab, Jiangsu Hengrui Medicine Co., Ltd., China) with 68 Ga-NK224, followed by PET imaging at 1 hour post-injection. Additional imaging was performed to compare tumor uptake in three tumor models (n = 3 per group). Images were reconstructed using 3D OPMAP 256 (pPetRcn; Siemens Medical Solutions, Erlangen, Germany) and analyzed by region of interest (ROI) segmentation. Tumor uptake was quantified as the percentage of injected dose per gram of tissue (%ID / g).

[0072] For biodistribution studies, different groups of mice bearing U87-MG xenograft tumors (n = 3 per group) were injected with 1.48 MBq 68 Ga-NK224 (with or without NK224 or Adebrelimab), or 1.48 MBq 68 Ga-NOTA-WL12. Mice were euthanized at predetermined time points post-injection, and major organs and tumors were harvested, weighed, and analyzed for radioactivity. Tumor tissues from all three tumor types were fixed in formalin, embedded in paraffin, and sectioned into 4-μm slices for H&E staining and PD-L1 analysis.

[0073] As shown by the above experimental results, Figure 6 small animal PET imaging showed that at 1 hour and 2 hours post-injection, 68 Ga-NK224 and 68 Ga-WL12 had comparable tumor uptake ( Figure 6 A). However, 68 Ga-NK224 showed lower physiological uptake in the liver, kidneys, and lungs, resulting in a higher tumor-to-background ratio than 68 Ga-WL12 ( Figure 6 A). After co-administering excess unlabeled NK224 or hPD-L1 antibody, the tumor uptake of 68 Ga-NK224 was significantly reduced, confirming the PD-L1 targeting specificity of 68 Ga-NK224. The results of the biodistribution experiment were consistent with the small animal PET results ( Figure 6 B).

[0074] In U87-MG tumor xenografts 18The results of small animal PET imaging with F-NK224 were similar to those of 68 Ga-NK224, and the tumor-to-background ratios at 1 hour and 2 hours after injection were high ( Figure 6 of C). In addition, to verify the ability of 68 Ga-NK224 to detect different PD-L1 expression levels, human xenograft models with different PD-L1 expression levels were used. The order of tumor uptake was U87-MG > C666-1 > A549 ( Figure 6 of D), which was consistent with the PD-L1 expression levels detected by Western blotting and flow cytometry in Experimental Example 2 ( Figure 5 of F-G). PD-L1 IHC staining of the corresponding tumor xenografts further confirmed the PET imaging results ( Figure 6 of E).

[0075] Experimental Example 4. 68 Clinical translation of

[0076] This single-center, prospective, investigator-initiated trial evaluated the 68 clinical utility of

[0077] Ga-NK224 PET / CT in patients with NSCLC. Inclusion criteria included: (1) adults (>18 years old), suspected, newly diagnosed, or treated NSCLC; (2) no prior anti-cancer treatment within 4 weeks before PET / CT scan; (3) biopsy or surgery to confirm pathology and PD-L1 immunohistochemical staining; and (4) informed consent according to the guidelines of the clinical research ethics committee. Exclusion criteria included pregnant women, patients who were unwilling to provide informed consent, and patients with non-malignant diseases or other lung malignancies.

[0077] Participants received an intravenous injection of 3.0 - 3.7 MBq / kg 68 Ga-NK224. PET / CT imaging was performed using a hybrid PET / CT scanner (Discovery MI, GE Healthcare) 1 hour after injection. In addition, one patient received serial scans before and after PD-L1 antibody treatment (Atezolizumab, Roche Registration GmbH, Germany). The intravenous injection doses of 18 F-FDG or 68 Ga-NK224 were calculated based on the patient's body weight (3.7 MBq [0.1 mCi] / kg for FDG; 68The dose of Ga-NK224 was 1.8 - 2.2 MBq [0.05 - 0.06 mCi] / kg). One hour after intravenous administration, data were acquired using a hybrid PET / CT scanner (Discovery MI, GE Healthcare, Milwaukee, WI, USA). PET scans were performed in 3D acquisition mode immediately after CT scanning, with 6 - 8 bed positions and 2.0 - 2.5 minutes per position. All acquired data were transferred to Advantage Workstation (version AW 4.7, GE Healthcare, Milwaukee, WI, USA); the data were reconstructed using the Bayesian penalized likelihood (BPL) reconstruction algorithm (Q.clear, GE Healthcare, Milwaukee, WI, USA), with a penalty factor (beta) of 500. The reconstructed images were then registered and displayed.

[0078] All participants underwent 18 F-FDG PET / CT as a routine imaging examination for tumor detection. For quantitative assessment of radiopharmaceutical uptake, both SUVmax and SUVmean were used to evaluate the distribution in normal organs and tumor tissues. PET / CT scans were evaluated by two board-certified nuclear medicine physicians with expertise in PET / CT data interpretation. Uptake was considered positive if the visually detected area of local tracer uptake exceeded the background level after excluding physiological uptake, trauma, infection, and inflammatory diseases. Any differences in interpretation were resolved by discussion to reach a consensus.

[0079] To elucidate 68 the biodistribution of Ga-NK224, PET imaging was performed on three healthy volunteers at 15, 30, 60, and 120 minutes to provide a comprehensive overview of the in vivo behavior of the radiotracer. The OLINDA / EXM software (version 1.1) was used to calculate the critical dose for evaluating the safety and efficacy of the administered radiopharmaceutical. For quantitative assessment of radiopharmaceutical uptake, both SUVmax and SUVmean were used to evaluate its distribution in normal organs and tumor tissues. The PD-L1 IHC staining procedure for biopsy tissues was described in the supplementary material. Since the PD-L1 expression thresholds of 1% and 50% are clinically relevant for guiding immunotherapy decisions in NSCLC, we divided PD-L1 TPS into three groups based on these thresholds in the analysis: negative expression (<1%), moderate expression (1 - 49%), and high expression (≥50%).

[0080] Dose analysis of three healthy volunteers showed that the effective dose calculated using OLINDA software was 2.06E-02 mSv / MBq (Table 1; Figure 7 ). The in vivo distribution was mainly observed in the spleen and liver (Figure 7 B).

[0081] Table 1. Dosimetry data for the effective dose of 68 Ga-NK224

[0082]

[0083]

[0084] A preliminary clinical study conducted between June 2023 and November 2024 enrolled 37 lung cancer patients who underwent 68 Ga-NK224 PET / CT imaging. Six patients were excluded due to the absence of PD-L1 IHC staining, and a total of 31 eligible lung cancer subjects were finally enrolled. Both healthy volunteers and lung cancer patients showed good tolerance to 68 Ga-NK224, and no adverse events were reported during the injection or the 4-hour follow-up period.

[0085] The study results suggest that PD-L1 expression in NSCLC is highly correlated with 68 Ga-NK224 uptake, but not with 18 F-FDG uptake.

[0086] A total of 31 lung cancer patients were included in this study. Among these patients, 28 were newly diagnosed patients and underwent PET / CT imaging for initial staging, while the remaining 3 were lung cancer patients with post-treatment recurrence / metastasis and underwent PET / CT for tumor recurrence and metastasis assessment. Tumor biopsy samples from 26 patients were from lung cancer, and those from 6 patients were from other organ metastases (one patient received multi-part biopsies of synchronous lung and bone metastasis lesions). According to the PD-L1 immunohistochemistry results, 11 specimens showed negative expression (<1%), 14 specimens showed moderate expression (1-49%), and 7 specimens showed high expression (≥50%). 68 The median and interquartile range (IQR) SUVmax values for the negative, intermediate, and high PD-L1 expression groups obtained with 18 Ga-NK224 were 2.6 (IQR, 2.0 - 3.3), 4.3 (IQR, 3.7 - 4.9), and 7.8 (IQR, 7.3 - 11.8), respectively. Figure 8 A). Conversely, 68 there was a statistically significant positive correlation between Figure 8 Ga-NK224 uptake and PD-L1 expression ( Figure 8 B, p < 0.001). Figure C shows representative68 Ga-NK224 PET / CT images and corresponding immunohistochemical staining biopsy results. Figure 9 Showed those of three representative patients 68 Ga-NK224 and 18 F-FDG PET / CT maximum intensity projection (MIP) images.

[0087] Among 31 patients, a total of 31 primary lung lesions and 215 metastatic tumors were found. The median SUVmax of primary lung cancer lesions was 4.1 (IQR, 3.3 - 4.9), while that of metastatic lesions was also 4.1 (IQR, 2.6 - 5.8). There was no significant difference in 68 Ga-NK224 uptake between lung cancer and metastatic lesions (p = 0.709). Spearman correlation analysis showed that among 246 lung cancer lesions, 68 there was no significant association between Ga-NK224 uptake and lesion size (p = 0.187). To evaluate whether 68 Ga-NK224 PET / CT could visualize the heterogeneity of PD-L1 expression within tumors, the uptake of 68 Ga-NK224 in different tumor lesions of the same patient was analyzed. Figure 10 A of 18 showed a representative case, demonstrating the heterogeneity of PD-L1 expression in different lesions within the patient: Although the 68 F-FDG uptake levels were similar (SUVmax 7.8 in the primary tumor, SUVmax 8.6 in the bone metastasis), the Figure 10 Ga-NK224 uptake between the primary tumor (weak PD-L1 positive, SUVmax 5.0) and the bone metastasis (strong PD-L1 positive, SUVmax 11.8) was different. Among 31 patients, 24 had multiple metastases. The median coefficient of variation (CV) of SUVmax in these patients was 27.5% (range 5.7% - 53.2%). Further data visualization showed heterogeneous lesion characteristics, including changes in lesion SUVmax, lesion size, biopsy site, and lesion type (

[0088] B). 68 In addition, the present invention also demonstrated the specificity of the probe by continuously

[0089] monitoring the occupancy of the PD-L1 target by Ga-NK224 PET / CT. 68Ga-NK224 PET / CT scan. As Figure 11 shown, the primary lung tumor showed significant 68 Ga-NK224 uptake at baseline, which significantly decreased at 1 and 7 days after injection, but slightly increased at day 21. This indicates that even 21 days after injection, the anti-PD-L1 antibody can still effectively occupy the PD-L1 target. In addition, after anti-PD-L1 treatment, the 68 uptake of Ga-NK224 in the liver, spleen, and bone marrow significantly decreased, while the uptake in the lung showed slight changes.

Claims

1. A cyclic peptide intermediate compound targeting human PD-L1, whose structure is shown in the following formula (I):

2. A cyclic peptide molecular precursor compound targeting human PD-L1, which is formed by conjugating the cyclic peptide intermediate compound of the structure shown in (I) of claim 1 with a NOTA chelating agent, and its structure is shown in the following formula (II):

3. A radionuclide-labeled compound, which is a coordination compound formed by a precursor compound having a structure represented by formula (II) as claimed in claim 2 and a radionuclide.

4. The radionuclide-labeled compound according to claim 3, characterized in that The radionuclide is 18 F or 68 Ga.

5. A method for preparing the intermediate compound according to claim 1, comprising: 1) Based on the Fmoc-protected amino resin, the following amino acids were coupled in sequence by solid phase synthesis of peptides using a coupling agent: Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-NMe-Nle-OH, Fmoc-NMe-Nle-OH, Fmoc-Trp(N-CH2-COOtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-NMeAla-OH and Fmoc-Tyr(tBu)-OH; then the Fmoc protecting group is removed to obtain a resin peptide with the following structure: H-Tyr(tBu)-NMeAla-Asn(Trt)-Pro-His(Trt)-Glu(OtBu)-Hyp(tBu)-Trp(Boc)-Ser(tBu)-Trp(N-CH2-COOtBu)-NMeNle-NMeNle-Lys(Boc)-Cys(Trt)-Gly-Resin; the amino resin is preferably Rink Amide MBHAResin; the coupling agent is preferably a mixture of DIC and HOBt; the removal of the Fmoc protecting group is preferably cleaved by 20% Pip / DMF solution; 2) coupling chloroacetic anhydride to the tyrosine of the resin peptide obtained in 1), and then cleaving the peptide chain from the resin to obtain a crude polypeptide with the following structure: Cl-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(N-CH2-COOH)-NMeNle-NMeNle-Lys-Cys-Gly-NH2; the cleavage is preferably performed using a TFA solution as a cleavage solution; 3) Purifying the crude polypeptide obtained in 2) and then cyclizing it to obtain a cyclic polypeptide with a structure as shown in formula (I):

6. A method for preparing the cyclic peptide molecule precursor compound according to claim 2, comprising: The NOTA group is coupled to the glutamic acid of the cyclic polypeptide obtained by the method of claim 5 to obtain a precursor compound having a structure as shown in formula (II):

7. A method for preparing the radionuclide-labeled compound of claim 3, comprising: The radionuclide-labeled compound according to claim 3 is obtained by reacting a solution containing radionuclide ions with the cyclic peptide molecule precursor compound according to claim 2 to carry out radionuclide labeling.

8. The method according to claim 7, characterized in that: The radionuclide ions are 18 F ions or 68 Ga ions.

9. Use of the cyclic peptide molecular precursor compound according to claim 2 as a labeling precursor in the preparation of a PET imaging agent.

10. Use of the radionuclide-labeled compound according to claim 3 as an imaging agent in in vivo imaging of a living organism for non-therapeutic or diagnostic purposes.

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