Polypeptide positron probe targeting pd-l1 and preparation method and application thereof

Abstract

The application discloses a polypeptide positive probe targeting PD-L1 and a preparation method and application thereof, and provides a polypeptide compound, which is obtained by combining different connecting groups and metal ion chelating agent NOTA on the basis of the structure of a PD-L1 polypeptide CLP002. The polypeptide compound is combined with a nuclide to obtain two PD-L1-targeting polypeptide molecular probes. The two polypeptide probes have high molar activity and good in-vivo and in-vitro stability, can be quickly distributed to the whole body in a model animal, are partially taken up in a PD-L1 positive tumor, are quickly removed from the body, can obtain a PET image with a high target / non-target ratio, and can monitor the expression level and change of PD-L1 in a tumor in real time, dynamically and non-invasively at a molecular level. Therefore, the polypeptide probes have a clinical application prospect in a PD-L1-targeting imaging agent and a tumor imaging drug.

Description

Technical Field

[0001] This invention belongs to the field of peptide drug technology, specifically relating to peptide positron emission tomography probes targeting PD-L1, their preparation methods, and applications. Background Technology

[0002] Positron emission tomography (PET) or PET / CT is currently the best molecular imaging equipment for in vivo monitoring of tumor development and progression, and it is of great value for the early diagnosis, staging, and monitoring of treatment efficacy in solid tumors. Imaging the binding of radionuclide-labeled ligands to their corresponding tumor receptors using PET / CT is one of the important directions for the development of molecular functional imaging in nuclear medicine. However, the number of positron-emitting probes currently available for tumor PET imaging is relatively limited; therefore, the development of novel, specific PET probes is crucial.

[0003] Programmed death receptor 1 (PD-1), also known as CD279, is an important immunosuppressive molecule. PD-1 is a cell membrane surface molecule composed of 288 amino acids, primarily expressed on the surface of activated T cells, B cells, monocytes, and dendritic cells, playing a crucial role in tumor immune escape. Currently identified PD-1 ligands include PD-L1 and PD-L2, with PD-L1 being more important. PD-L1 is a transmembrane protein with a relative molecular mass of 40 kDa, widely expressed in various tumors, including breast cancer, gastric cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, non-small cell lung cancer, melanoma, and glioma, while its expression is low in normal tissues. Therefore, PD-L1 can serve as an important target for PET imaging.

[0004] In recent years, researchers both domestically and internationally have conducted extensive research on PET imaging targeting tumor PD-L1. Imaging agents are mainly divided into two categories: antibodies and peptides. PD-L1-targeting antibody imaging agents are the most studied, with several currently popular agents including 18F-BMS-986192 and 89Zr-Atezolizumab. The advantages of radionuclide-labeled antibodies are their strong affinity for PD-L1 and simple labeling methods. However, their disadvantages include a long biological half-life, difficulty in 18F labeling, the need for longer half-lived radionuclides, long imaging times, poor tumor penetration, and slow in vivo clearance, resulting in a lower target / non-target ratio for monoclonal antibody probes.

[0005] Compared to antibody-based imaging agents, peptide imaging agents offer several advantages, including ease of synthesis, the ability to be modified, radiolabeled, and chelated, high tumor permeability, high receptor affinity, non-immunogenicity, good thermal stability, and fewer side effects. Despite these advantages, there are currently very few reported peptide imaging agents targeting PD-L1. Summary of the Invention

[0006] To overcome the problems existing in the prior art, one objective of the present invention is to provide a polypeptide complex. A second objective of the present invention is to provide a method for preparing the aforementioned polypeptide complex. A third objective of the present invention is to provide applications of the aforementioned polypeptide complex. A fourth objective of the present invention is to provide a polypeptide probe. A fifth objective of the present invention is to provide a method for preparing the aforementioned polypeptide probe. A sixth objective of the present invention is to provide applications of the aforementioned polypeptide probe. A seventh objective of the present invention is to provide applications of the aforementioned polypeptide probe.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a polypeptide complex, wherein the polypeptide complex is a polypeptide CLP002 coupled with a chelating agent; the amino acid sequence of the CLP002 is WHRSYYTWNLNT; the chelating agent is further linked to a linking group; the linking group includes PEG2-Asp2-IPB or PEG2-Asp2.

[0009] Preferably, the chelating agent is selected from any one of HYNIC, NOTA, DOTA, and DTPA.

[0010] More preferably, the polypeptide complex has a structure as shown in formula (a):

[0011]

[0012] Where R is H or

[0013] A second aspect of the present invention provides a method for preparing the polypeptide complex described in the first aspect, comprising the following steps:

[0014] S1. Preparation of CLP002 polypeptide coupled with a bifunctional chelating agent;

[0015] S2. The CLP002 polypeptide coupled with the bifunctional chelating agent is sequentially coupled with Fmoc-PEG2-OH, Fmoc-Asp-OtBu and Fmoc-Asp-OtBu to obtain a polypeptide complex with PEG2-Asp2 as the linking group.

[0016] A polypeptide complex with a linker group of PEG2-Asp2 was coupled with 4-iodophenylbutyric acid to prepare a polypeptide complex with a linker group of PEG2-Asp2-IPB.

[0017] Preferably, the preparation steps of CLP002 include: starting from king resin, coupling it with Fmoc-Thr(tBu)-OH to prepare Fmoc-Thr(tBu)-wang resin, and removing the Fmoc protecting group using piperidine solution; then, by solid-phase synthesis, sequentially coupling amino acids Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Trp(Boc) -OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-L-Lys(Dde)-OH, and after each coupling, the Fmoc protecting group was removed with piperidine solution.

[0018] More preferably, the coupled amino acids are prepared using hydroxytriazole and N,N'-diisopropylcarbodiimide as peptide condensing agents.

[0019] Preferably, in step S1, HBTU is used as the peptide coupling reagent and N,N-diisopropylethylamine is used as the base reagent.

[0020] Preferably, step S2 further includes the following step: removing the Dde side chain protecting group of the NOTA-CLP002 peptide with hydrazine hydrate solution.

[0021] Preferably, in step S2, the coupling reaction with Fmoc-PEG2-OH takes place at a temperature of 15-40°C for 20-40 minutes.

[0022] Preferably, in step S2, the coupling reaction with Fmoc-Asp-OtBu takes place at a temperature of 15-40°C for 20-40 minutes.

[0023] Preferably, in step S2, the coupling amino acids are coupled using hydroxybenztriazole and N,N'-diisopropylcarbodiimide as peptide condensing agents.

[0024] Preferably, step S2 further includes the following step: using a cleavage reagent to cut the peptide chain from the resin.

[0025] More preferably, the cleavage reagent is a mixed solution containing trifluoroacetic acid (TFA), ethylene dithiol (EDT), and triisopropylsilane (TIS).

[0026] A third aspect of the present invention provides any of the following applications of the above-described polypeptide complex:

[0027] 1) Used as a molecular probe;

[0028] 2) Used in the preparation of imaging agents.

[0029] A fourth aspect of the present invention provides a polypeptide probe, wherein the polypeptide probe is a polypeptide complex described in the first aspect labeled with a radionuclide.

[0030] Preferably, the radionuclide is selected from... 68 Ga 3+ 、[Al 18 F] 2+ , 177 Lu、 64 Cu 2+ , 111 In 3+ , 89 Zr 4+ , 225 Ac or 212 Pb.

[0031] The fifth aspect of the present invention provides a method for preparing the above-mentioned polypeptide probe, comprising the following steps: reacting the polypeptide complex described in the first aspect with a radioactive metal nuclide in solution to obtain the polypeptide probe.

[0032] A sixth aspect of the present invention provides the use of the above-described peptide probe in the preparation of imaging agents targeting PD-L1.

[0033] A seventh aspect of the present invention provides the use of the above-described polypeptide probe in the preparation of tumor imaging drugs.

[0034] The beneficial effects of this invention are:

[0035] This invention provides a polypeptide complex, based on the structure of the PD-L1-targeting polypeptide CLP002 (sequence WHRSYYTWNLNT, abbreviated as WT12), with the addition of different linking groups, and combined with the metal ion chelating agent NOA. This invention combines the above polypeptide complex with radionuclides to obtain two PD-L1-targeting polypeptide molecular probes, which can be used to prepare PD-L1-targeting imaging agents and tumor imaging drugs. These two polypeptide probes have high molar activity, good in vitro and in vivo stability, can rapidly distribute throughout the body in model animals, are taken up in PD-L1-positive tumors, and are rapidly cleared from the body, enabling the acquisition of PET images with high target / non-target ratios, thus showing good clinical application prospects. Compared with existing pathological histochemistry and other techniques, these two probes can achieve real-time, dynamic, and non-invasive monitoring of PD-L1 expression levels and changes in tumors at the molecular level, providing assistance in guiding immunotherapy. Attached Figure Description

[0036] Figure 1 This is the preparation process of polypeptide complexes;

[0037] Figure 2 This is the HPLC chromatogram of purified Nota-PEG2-Asp2-WT12;

[0038] Figure 3 This is the mass spectrum of purified Nota-PEG2-Asp2-WT12;

[0039] Figure 4 This is the HPLC chromatogram of purified Nota-PEG2-Asp2-IPB-WT12;

[0040] Figure 5 This is the mass spectrum of purified Nota-PEG2-Asp2-IPB-WT12;

[0041] Figure 6 yes[ 18 Radioactive high-performance liquid chromatography analysis and stability of F]AlF-NOTA-PEG2-Asp2-IPB-WT12;

[0042] Figure 7 yes[ 18 Radioactive high-performance liquid chromatography analysis and stability of F]AlF-NOTA-PEG2-Asp2-WT12

[0043] Figure 8 It is a cell experiment study; where A is [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 cellular uptake; B is [ 18F] Blocking study of AlF-NOTA-PEG2-Asp2-WT12; C is [ 18 F]AlF-NOTA-PEG2-Asp2-IPB-WT12 cellular uptake; D is [ 18 Blocking study of F]AlF-NOTA-PEG2-Asp2-IPB-WT12;

[0044] Figure 9 yes[ 18 Dynamic Micro PET / CT imaging of [F]AlF-NOTA-PEG2-Asp2-WT12; where A is the intravenous injection of [F]AlF-NOTA-PEG2-Asp2-WT12 in nude mice carrying 4T1-hPD-L1 tumors. 18 F] Representative maximum intensity projection (MIP) images at different time points after AlF-NOTA-PEG2-Asp2-WT12; B is the intravenous injection [ 18 F] Time-activity curves of AlF-NOTA-PEG2-Asp2-WT12 followed by 4T1-hPD-L1 tumors and major organs; C is the intravenous injection in nude mice carrying A549-hPD-L1 tumors [ 18 F] Representative maximum intensity projection (MIP) images at different time points after AlF-NOTA-PEG2-Asp2-WT12; D is the intravenous injection [ 18 F]Time-activity curves of A549-hPD-L1 tumors and major organs after AlF-NOTA-PEG2-Asp2-WT12; E is the time-activity curve of 4T1-hPD-L1 tumors and A549-hPD-L1 tumors; F is [ 18 Time-activity ratio curves of F]AlF-NOTA-PEG2-Asp2-WT12 against muscle in 4T1-hPD-L1 and A549-hPD-L1 tumors; (red circles indicate tumors);

[0045] Figure 10 yes[ 18 Dynamic Micro PET / CT imaging of [F]AlF-NOTA-PEG2-Asp2-IPB-WT12; where A is the intravenous injection of [F]AlF-NOTA-PEG2-Asp2-IPB-WT12 in nude mice carrying 4T1-hPD-L1 tumors. 18 F] Representative maximum intensity projection (MIP) images at different time points after AlF-NOTA-PEG2-Asp2-IPB-WT12; B is the intravenous injection [ 18 F] Time-activity curves of AlF-NOTA-PEG2-Asp2-IPB-WT12 followed by 4T1-hPD-L1 tumors and major organs; C is the intravenous injection in nude mice carrying A549-hPD-L1 tumors. 18F] Representative maximum intensity projection (MIP) images at different time points after AlF-NOTA-PEG2-Asp2-IPB-WT12; D is the intravenous injection [ 18 F]Time-activity curves of A549-hPD-L1 tumors and major organs after AlF-NOTA-PEG2-Asp2-IPB-WT12; E is the time-activity curve of 4T1-hPD-L1 tumors and A549-hPD-L1 tumors; F is [ 18 Time-activity ratio curves of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 against muscle in 4T1-hPD-L1 and A549-hPD-L1 tumors; (red circles indicate tumors);

[0046] Figure 11 This is a static Micro PET / CT image of nude mice carrying 4T1-hPD-L1 and A549-hPD-L1; where A is the result of intravenous injection of 4T1-hPD-L1 and A549-hPD-L1 in nude mice. 18 F] MIP images 60 minutes after AlF-NOTA-PEG2-Asp2-WT12; B is the intravenous injection of 4T1-hPD-L1 and A549-hPD-L1 in nude mice. 18 F] MIP images at 60 minutes after AlF-NOTA-PEG2-Asp2-IPB-WT12; C is the MIP image at 60 minutes of 4T1-hPD-L1 and A549-hPD-L1 tumors. 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 F] Quantitative uptake of AlF-NOTA-PEG2-Asp2-IPB-WT12; D is the tumor-to-muscle ratio at 60 minutes;

[0047] Figure 12 yes[ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 [F] Biodistribution of AlF-NOTA-PEG2-Asp2-IPB-WT12 in tumor-bearing mice; (A) 30 and 60 minutes after injection, [ 18 Biodistribution of AlF-NOTA-PEG2-Asp2-WT12 in mice carrying 4T1-hPD-L1, A549-hPD-L1, 4T1, and CHO tumors (n=3); (B) 18 F]AlF-NOTA-PEG2-Asp2-WT12 tumor to muscle ratio at 30 and 60 minutes in different tumors; (C)[ 18Biodistribution of AlF-NOTA-PEG2-Asp2-IPB-WT12 in mice carrying 4T1-hPD-L1, A549-hPD-L1, and CHO tumors at 30 and 60 min post-injection (n=3); (D) 18 Tumor-to-muscle ratio in different tumors at 30 and 60 minutes using F]AlF-NOTA-PEG2-Asp2-IPB-WT12;

[0048] Figure 13 Immunohistochemical staining of PD-L1 expression in tumors: (A) 4T1-hPD-L1 (B) A549-hPD-L1 (C) 4T1 (D) CHO; Scale bar: 100 μm. Detailed Implementation

[0049] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0050] The terminology involved in this invention:

[0051] HOBt: Hydroxybenzotriazole, a polypeptide condensing agent;

[0052] DIC: N,N'-Diisopropylcarbodiimide;

[0053] HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate, peptide coupling reagent;

[0054] DIPEA: N,N-diisopropylethylamine;

[0055] NOTA-COOH: A bifunctional chelating agent for NOTA macrocyclic compounds modified with carboxylic acid.

[0056] Example 1

[0057] This embodiment provides a polypeptide complex targeting PD-L1, the synthesis method of which is as follows: Figure 1 As shown, Nota-PEG2-Asp2-IPB-WT12 and Nota-PEG2-Asp2-WT12 were synthesized via solid-phase peptide synthesis (SPPS). The specific preparation method is as follows:

[0058] S1. Resin swelling: First, place the resin into a reaction tube and treat it with dichloromethane (DCM, 15 mL / g) for 30 minutes.

[0059] S2, Introducing the first amino acid: Add a solution containing Fmoc-Thr(tBu)-OH (413 mg), HOBt (121 mg), DIC (464 μL), and DMF (20 mL), and stir at room temperature for 1 hour. Then filter off the solvent and remove the Fmoc protecting group using a 20% piperidine DMF solution (20 mL). Subsequent washing steps include DMF (wash once), DCM (wash twice), and DMF (wash twice).

[0060] S3. Adding the second amino acid: Subsequently, 537 mg of Fmoc-Asn(Trt)-OH, 121 mg of HOBt, and 464 μL of DMF from 20 mL of DMF were added to the resin, and the mixture was stirred at room temperature for 30 minutes. After filtering the solvent, the Fmoc group was deprotected using a 20 mL solution of 20% piperidine in DMF, followed by additional washing with DMF (wash once), DCM (wash twice), and DMF (wash twice).

[0061] S4. Repeat S3, and then sequentially couple amino acids Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-Trp(Boc)-OH, and Fmoc-L-Lys(Dde)-OH. The steps are the same: add 20 mL of Fmoc-amino acids (0.9 mmol × molecular weight), HOBt (121 mg), and DIC (464 μL) from DMF to the resin and stir at room temperature for 30 minutes. After filtering the solvent, the Fmoc group was deprotected using a 20 mL solution of 20% piperidine in DMF, followed by additional washing with DMF (wash once), DCM (wash twice), and DMF (wash twice). The amino acid sequence was LNWTYYSRHW, with the last amino acid, Fmoc-L-Lys(Dde)-OH, attached.

[0062] S5. Add NOA-NHS (50 mg), DMF (30 mL), HBTU (1 g), and DIPEA (20 mL) to the product obtained in S4, and stir at room temperature for 30 minutes to perform coupling.

[0063] S6. After removing the Dde side chain protecting group with 4% hydrazine hydrate, the peptide chain was further extended through several coupling steps: (1) Fmoc-PEG2-OH (346 mg), HOBt (121 mg), and DIC (464 μL) were reacted in 10 mL DMF; (2) Fmoc-Asp-OtBu (370 mg), HOBt (121 mg), and DIC (464 μL) were reacted in 10 mL DMF; (3) Fmoc-Asp-OtBu (370 mg), HOBt (121 mg), and DIC (464 μL) were reacted in 10 mL DMF. Each coupling reaction was stirred at room temperature for 30 minutes. After the coupling reaction was completed, the resin was washed with DMF (washed twice), MeOH (washed twice), DMF (washed twice), and DCM (washed twice). Finally, compound F was obtained. The Fmoc protecting group in compound F was then removed with a 20 mL solution of 20% piperidine in DMF.

[0064] S7. HBTU (341 mg) and DIPEA (522 μ L) were added to 10 mL of DMF and stirred at room temperature for 30 minutes to couple the resin with 4-iodophenylbutyric acid. The resin was then washed again with DMF (washed twice), MeOH (washed twice), DMF (washed twice), and DCM (washed twice) to obtain compound G.

[0065] S7. The two products (F and G) were dried under high vacuum and dissolved in 10 mL of a mixed solution of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS), and ultrapure water at a volume ratio of 95:2:2:1 (v / v), and the reaction was carried out over 2 hours. After filtration of both solutions, the filtrate was precipitated in 100 mL of ice-cold ether to obtain two solid products. Finally, the two solids were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain high-purity peptides NOA-PEG2-Asp2-IPB-WT12 and NOA-PEG2-Asp2-WT12.

[0066] The obtained products were analyzed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Figure 2 This is the HPLC chromatogram of purified Nota-PEG2-Asp2-WT12; Figure 3 This is the mass spectrum of purified Nota-PEG2-Asp2-WT12; Figure 4 This is the HPLC chromatogram of purified Nota-PEG2-Asp2-IPB-WT12; Figure 5 This is the mass spectrum of purified Nota-PEG2-Asp2-IPB-WT12; from Figures 2 to 5This demonstrates that Example 1 successfully synthesized NOA-PEG2-Asp2-IPB-WT12 and NOA-PEG2-Asp2-WT12.

[0067] Example 2

[0068] This embodiment provides a peptide-based positron emission tomography probe targeting PD-L1, and its preparation method is as follows:

[0069] Use of nuclides 18 F is used to make aluminum fluoride (Al) 18 F) Labeling the polypeptide precursor of Example 1. The labeling procedure was as follows: 9 μL / AlCl3 solution (2 mM), 6 μL glacial acetic acid, 280 μL acetonitrile, and 50 μL deionized water containing 100 μg NOTA-PEG2-Asp2-WT12 were added to a reaction vial, followed by 80 μL of fluorine-18 (3.7 GBq). The pH of the reaction mixture was 4. The reaction mixture was reacted at 100 °C for 15 min. The reaction mixture was then diluted with 6 mL of water and cooled to room temperature. After cooling, the liquid mixture was passed through a Waters C18 column, the reaction product was attached to the C18 column, and impurities were washed with 50 mL of water. The radiolabeled product was then eluted with 1.5 mL of an ethanol-water mixture (50% each), followed by dilution with brine and radiochemical purity analysis.

[0070] The same method was used to label Nota-PEG2-Asp2-IPB-WT12.

[0071]

[0072] [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 The undecay-corrected radiochemical yields of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 were 15-25% and 25-35% (n>5), respectively. The molar activities of both were calculated using the formula: molar activity (Am) = product radioactivity / M (molar mass of the precursor). 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 The molar activities of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 were 17.97-30.11 GBq / μmol and 18.09-26.46 GBq / μmol, respectively.

[0073] Experimental Analysis

[0074] 1. Study on lipid-water partition coefficient and in vitro-in vivo stability

[0075] Will[ 18F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 The two tracers, F]AlF-NOTA-PEG2-Asp2-IPB-WT12, were mixed with 2.0 mL of PBS and 2.0 mL of n-octanol in a 15 mL centrifuge tube. The solution was then centrifuged at 10,000 rpm for 3 minutes to separate the n-octanol and PBS layers. After centrifugation, 100 μL was collected from each layer and analyzed using a gamma counter to determine the logP value (n = 4).

[0076] Will[ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 F]AlF-NOTA-PEG2-Asp2-IPB-WT12 was incubated in PBS (200 μL) or FBS (200 μL) at 37°C for 2 hours (n=3) to evaluate [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 In vitro stability of F]AlF-NOTA-PEG2-Asp2-IPB-WT12. For in vivo metabolic analysis, 0.5 mL mouse serum samples were collected 60 min (17.5 MBq) after tracer injection. Blood samples were collected from nude mice (n=3), centrifuged at 10,000 rpm for 5 min, and the supernatant was analyzed by radioactive HPLC. HPLC mobile phase was collected for 0.5 min intervals over 14 min, followed by measurements using a gamma counter.

[0077] [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 The lipid-water partition coefficients (logP) of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 were -2.23±0.06 and -1.84±0.02 (n>3), respectively.

[0078] Results of in vitro and in vivo stability studies, such as Figure 6 and Figure 7 As shown, in [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 After incubation in PBS and FBS for 120 min, F]AlF-NOTA-PEG2-Asp2-IPB-WT12 showed only a single main peak, and the radiochemical purity of the probes was >90%, indicating that the probes have good in vitro stability. No defluorination or decomposition occurred in serum within 60 min, indicating that the probes are relatively stable in serum.

[0079] 2. Cellular experimental research

[0080] 4T1-hPD-L1 and A549-hPD-L1 cells were seeded in 12-well plates and cultured for 24 hours until they reached confluence (over 90%). For cell uptake assays, cells were incubated with 0.5 mL of serum-free medium at 37°C for different time points (5, 15, 30, 60, and 120 minutes). This medium contained […]. 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 [F]AlF-NOTA-PEG2-Asp2-IPB-WT12 (0.37 MBq). After incubation, cells were washed twice with PBS and lysed with NaOH-SDS buffer (0.2 M NaOH, 1% SDS). The resulting cell lysates were then collected and analyzed using a gamma counter. In the blocking assay, BMS-1 ​​(10 μg) was used as the blocking agent to inhibit PD-L1.

[0081] [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 The uptake values ​​of the two probes, F]AlF-NOTA-PEG2-Asp2-IPB-WT12, slowly increased over time in 4T1-hPD-L1 and A549-hPD-L1 cells, reaching their respective maximum uptake values ​​at 120 minutes (e.g., F1F-NOTA-PEG2-Asp2-IPB-WT12). Figure 8 (As shown in A and C). At 120 minutes, [ 18 Cellular uptake of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 was significantly higher than [ 18 [F]AlF-NOTA-PEG2-Asp2-WT12, this may be due to the introduction of the IPB group increasing its lipophilicity and internalization in cells. Furthermore, the addition of BMS-1 ​​significantly reduced [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 The uptake of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 in 4T1-hPD-L1 and A549-hPD-L1 cells indicates that both tracers can specifically target PD-L1 on tumors (e.g., ...). Figure 8 (As shown in B and D in the diagram).

[0082] 3. Micro PET-CT Imaging Research

[0083] Nude mice bearing 4T1-hPD-L1, A549-hPD-L1, 4T1, or CHO tumors were intravenously injected via the tail vein. 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18F]AlF-NOTA-PEG2-Asp2-IPB-WT12 (5.55-7.40 MBq) (n=3 per group), followed by 120 minutes of dynamic PET imaging. In the blockade experiment, 4T1-hPD-L1 tumor-bearing mice and [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 [F]AlF-NOTA-PEG2-Asp2-IPB-WT12 (5.55-7.40 MBq) and WT12 (200 μg / mouse) were co-injected, and static micro-PET scans were performed at 30 and 60 minutes post-injection. PET images were reconstructed using the 3D Ordered Subset Expectation-Maximization (OSEM) algorithm (Siemens, Germany). For data analysis, regions of interest (ROIs) were manually plotted on tumors and major organs using Inveon ResearchWorkplace 4.1, and the results were expressed as the percentage of injected dose per gram of tissue or organ (%ID / g).

[0084] Figure 9 yes[ 18 Dynamic Micro PET / CT imaging of F]AlF-NOTA-PEG2-Asp2-WT12, as can be seen from the PET images, [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 is mainly excreted through the kidneys, with high uptake in tumors and low uptake in major organs such as the heart, liver, lungs, brain, and muscles. Figure 10 yes[ 18 Dynamic Micro PET / CT imaging of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 In addition, [ 18 F]AlF-NOTA-PEG2-Asp2-IPB-WT12 is also mainly excreted through the kidneys, exhibiting high uptake in tumors, but higher uptake in the heart, lungs, and muscles, and lower uptake in other organs. This probe shows […]. 18 [F]AlF-NOTA-PEG2-Asp2-WT12 showed higher tumor uptake and higher background. However, the tumor / muscle ratio was still [ 18 F]AlF-NOTA-PEG2-Asp2-IPB-WT12 is better than [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 is higher (e.g.) Figure 11 (As shown). Therefore, these two probes have good targeting properties for tumors with high PD-L1 expression and can monitor the expression of PD-L1 in tumor sites of tumor-bearing mice.

[0085] 4. Biological distribution research

[0086] Mice bearing 4T1-hPD-L1 and A549-hPD-L1 tumors were injected intravenously via the tail vein. 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 [F]AlF-NOTA-PEG2-Asp2-IPB-WT12 (1.11-1.85 MBq, n=3), and mice were sacrificed at 30 and 60 minutes after injection. Mice bearing 4T1 and CHO tumors were also injected with the same doses of both tracers. In the blockade experiment, 4T1-hPD-L1 tumor-bearing mice and [ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 [F]AlF-NOTA-PEG2-Asp2-IPB-WT12 (1.11-1.85 MBq) and competitor WT12 (200 μg / mouse) were co-injected. Mice were sacrificed at the corresponding time points after injection, and relevant organs, tumors, and blood were collected and weighed. Radioactivity was measured using a gamma counter. Results are expressed as a percentage of the injected dose per gram of tissue (%ID / g).

[0087] Figure 12 yes[ 18 F]AlF-NOTA-PEG2-Asp2-WT12 and [ 18 Biodistribution of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 in tumor-bearing mice; the biodistribution results show that, 18 [F]AlF-NOTA-PEG2-Asp2-WT12 is primarily excreted through the kidneys. The probe shows high uptake in tumors at 30 and 60 minutes, but lower uptake in major organs such as the heart, liver, brain, and muscles. Furthermore, [ 18 F]AlF-NOTA-PEG2-Asp2-IPB-WT12 is also mainly excreted through the kidneys. At 30 and 60 minutes, the probe showed high uptake in tumors, but higher uptake in the heart, lungs, gallbladder, and blood, and lower uptake in other organs. Therefore, the biodistribution results of these two probes are largely consistent with the results of PET imaging.

[0088] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A polypeptide complex, characterized in that, The polypeptide complex is a polypeptide CLP002 coupled with a chelating agent; the amino acid sequence of the CLP002 is WHRSYYTWNLNT; the chelating agent is further connected with a linking group; the linking group is PEG2-Asp2-IPB; The preparation method of the polypeptide complex comprises the following steps: S1, preparing the CLP002 polypeptide coupled with a chelating agent; S2, coupling the CLP002 polypeptide coupled with a chelating agent with Fmoc-PEG2-OH, Fmoc-Asp-OtBu and Fmoc-Asp-OtBu in sequence to prepare a polypeptide complex with a linking group of PEG2-Asp2; The polypeptide complex with a linking group of PEG2-Asp2 is coupled with 4-iodophenyl butyric acid to prepare a polypeptide complex with a linking group of PEG2-Asp2-IPB. The preparation step of S1 comprises: taking Wang resin as a starting end, coupling with Fmoc-Thr(tBu)-OH to prepare Fmoc-Thr(tBu)-Wang resin, and using a piperidine solution to remove the Fmoc protecting group; and then through solid-phase synthesis, coupling amino acids Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-L-Lys(Dde)-OH in sequence, and then coupling with a chelating agent to prepare the polypeptide CLP002 coupled with a chelating agent. The chelating agent is selected from any one of HYNIC, NOTA, DOTA and DTPA.

2. The polypeptide complex of claim 1, wherein, The polypeptide complex has a structure as shown in formula (a): Formula (a); wherein R is .

3. A method of producing the polypeptide complex of any one of claims 1 to 2, characterized in that, Comprising the following steps: S1, preparing the CLP002 polypeptide coupled with a chelating agent; S2, coupling the CLP002 polypeptide coupled with a chelating agent with Fmoc-PEG2-OH, Fmoc-Asp-OtBu and Fmoc-Asp-OtBu in sequence to prepare a polypeptide complex with a linking group of PEG2-Asp2; The polypeptide complex with a linking group of PEG2-Asp2 is coupled with 4-iodophenyl butyric acid to prepare a polypeptide complex with a linking group of PEG2-Asp2-IPB.

4. Any one of the following applications of the polypeptide complex according to any one of claims 1 to 2: 1) used for preparing a molecular probe; 2) used for preparing an imaging agent; 3) used for preparing a tumor detection reagent.

5. A polypeptide probe, characterized in that, The polypeptide probe is the polypeptide complex according to any one of claims 1 to 2 labeled with a radionuclide.

6. The polypeptide probe of claim 5, wherein, The radionuclide is selected from the group consisting of 68 Ga 3+ , [Al 18 F] 2+ , 177 Lu 64 , Cu 2+ , 111 In 3+ , 89 Zr 4+ , 225 Ac or 212 Pb.

7. A method of preparing a polypeptide probe according to claim 5 or 6, characterised in that, Comprising the following steps: reacting the polypeptide complex according to claim 1 with a radioactive metal nuclide in a solution to obtain the polypeptide probe.

8. Use of the polypeptide probe of claim 5 or 6 in the preparation of a tumor imaging agent targeting PD-L1.

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