PD-L1 targeting polypeptide positron probe as well as preparation method and application thereof

Abstract

The invention discloses a PD-L1-targeted polypeptide positron probe as well as a preparation method and application thereof, and provides a polypeptide compound which is obtained by adding different linking groups on the basis of the structure of PD-L1-targeted polypeptide CLP002 and connecting and combining the PD-L1-targeted polypeptide CLP002 with a metal ion chelating agent NOTA. The polypeptide compound is combined with nuclide to obtain two PD-L1 targeted polypeptide molecular probes, and the two polypeptide probes have high molar activity and good in-vivo and in-vitro stability, can be rapidly distributed to the whole body in a model animal body, can be taken in a PD-L1 positive tumor part, and can be rapidly removed from the body, so that the PD-L1 targeted polypeptide molecular probes can be used for detecting the PD-L1 positive tumor. In addition, the two probes can dynamically and noninvasively monitor the expression level and change condition of the PD-L1 in the tumor in real time at the molecular level, so that the two probes have clinical application prospects in imaging agents of the targeted PD-L1 and tumor imaging drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide drugs, and particularly relates to a polypeptide-based positron probe targeting PD-L1, a preparation method thereof, and an application thereof. Background Art

[0002] A positron emission tomograph (PET) or PET / CT is currently the best molecular imaging device for monitoring the occurrence and development of tumors in vivo, and it has important value for the early diagnosis, tumor staging, and efficacy monitoring of solid tumors. Imaging the binding of a radiolabeled ligand to a corresponding tumor receptor by PET / CT is one of the important development directions of nuclear medicine molecular functional imaging. However, the current positron probes applied to tumor PET imaging are relatively limited, so it is very important to develop new specific PET probes.

[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, mainly expressed on the surface of activated T cells, B cells, monocytes, and dendritic cells, and plays an important role in the process of tumor immune escape. The currently discovered PD-1 ligands include PD-L1 and PD-L2, among which PD-L1 is more important. PD-L1 is a transmembrane protein with a relative molecular mass of 40 kDa, which is widely expressed in a variety of tumors, including breast cancer, gastric cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, non-small cell lung cancer, melanoma, and glioblastoma, etc., while normal tissues have low expression. Therefore, PD-L1 can be used as an important target for PET imaging.

[0004] In recent years, domestic and foreign research scholars have done a lot of work in the research of PET imaging targeting tumor PD-L1. The main imaging agents are divided into two categories, namely antibody-based and polypeptide-based. Antibody-based imaging agents targeting PD-L1 are a type that has been studied more. Several currently studied popular imaging agents are 18F-BMS-986192 and 89Zr-Atezolizumab. The advantage of radionuclide-labeled antibodies is their strong binding affinity to PD-L1 and simple labeling methods. The disadvantages are their relatively long biological half-life, difficult 18F labeling, the need to use radionuclides with longer half-lives for labeling, long imaging time required, poor tumor penetrability, and slow in vivo clearance, which result in a low target / non-target ratio of monoclonal antibody-based probes.

[0005] Compared with antibody-based imaging agents, polypeptide-based imaging agents have the advantages of being easy to synthesize, amenable to modification, radiolabeling, and chelation, high tumor permeability, high receptor affinity, non-immunogenicity, good thermal stability, and low side effects. Despite these many advantages of polypeptides, there are few reported polypeptide imaging agents targeting PD-L1 to date. Summary of the Invention

[0006] To overcome the problems existing in the above-mentioned prior art, one of the objectives of the present invention is to provide a polypeptide complex. Another objective of the present invention is to provide a method for preparing the above-mentioned polypeptide complex. A third objective of the present invention is to provide the application of the above-mentioned 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 above-mentioned polypeptide probe. A sixth objective of the present invention is to provide the application of the above-mentioned polypeptide probe. A seventh objective of the present invention is to provide the application of the above-mentioned polypeptide probe.

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

[0008] In the first aspect of the present invention, a polypeptide complex is provided, and the polypeptide complex is a polypeptide CLP002 conjugated with a chelating agent; the amino acid sequence of CLP002 is WHRSYYTWNLNT; the chelating agent is also connected to a linking group; the linking group includes PEG 2 -Asp 2 -IPB or PEG 2 -Asp 2 .

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

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

[0011]

[0012] wherein, R is H or

[0013] In the second aspect of the present invention, a method for preparing the polypeptide complex according to the first aspect is provided, including the following steps:

[0014] S1. Prepare a CLP002 polypeptide conjugated with a bifunctional chelating agent;

[0015] S2. Sequentially react the CLP002 polypeptide conjugated with the bifunctional chelating agent with Fmoc-PEG 2-OH, Fmoc-Asp-OtBu, and Fmoc-Asp-OtBu are subjected to a coupling reaction to obtain a polypeptide complex with a linking group of PEG2-Asp2;

[0016] The polypeptide complex with a linking group of PEG2-Asp2 is subjected to a coupling reaction with 4-iodophenylbutyric acid to obtain a polypeptide complex with a linking group of PEG2-Asp2-IPB.

[0017] Preferably, the preparation steps of CLP002 include: starting from Wang resin, coupling with Fmoc-Thr(tBu)-OH to prepare Fmoc-Thr(tBu)-wang resin, and deprotecting the Fmoc protecting group using a piperidine solution; by solid-phase synthesis method, successively 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, and deprotecting the Fmoc protecting group with a piperidine solution after each coupling.

[0018] More preferably, hydroxybenzotriazole and N,N'-diisopropylcarbodiimide are used as polypeptide condensing agents for coupling amino acids.

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

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

[0021] Preferably, in step S2, the reaction temperature in the coupling reaction with Fmoc-PEG 2 -OH is 15 - 40 °C and the reaction time is 20 - 40 min.

[0022] Preferably, in step S2, the reaction temperature in the coupling reaction with Fmoc-Asp-OtBu is 15 - 40 °C and the reaction time is 20 - 40 min.

[0023] Preferably, hydroxybenzotriazole and N,N'-diisopropylcarbodiimide are used as polypeptide condensing agents for coupling amino acids in step S2.

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

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

[0026] The third aspect of the present invention provides any one of the following applications of the above polypeptide complex:

[0027] 1) Used as a molecular probe;

[0028] 2) Used for preparing an imaging agent.

[0029] The fourth aspect of the present invention provides a polypeptide probe, which is the 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 preparation method of the above polypeptide probe, including the following steps: reacting the polypeptide complex described in the first aspect with a radioactive metal nuclide in a solution to obtain the polypeptide probe.

[0032] The sixth aspect of the present invention provides an application of the above polypeptide probe in preparing an imaging agent targeting PD-L1.

[0033] The seventh aspect of the present invention provides an application of the above polypeptide probe in preparing a tumor imaging drug.

[0034] The beneficial effects of the present invention are:

[0035] The present invention provides a polypeptide complex, which is obtained by adding different linking groups on the basis of the structure of the PD-L1-targeting polypeptide CLP002 (sequence: WHRSYYTWNLNT, abbreviated as WT12) and linking and combining with the metal ion chelator NOTA. The present invention combines the above polypeptide complex with a radionuclide to obtain two PD-L1-targeting polypeptide molecular probes, which can be applied to the preparation of imaging agents and tumor imaging drugs targeting PD-L1. These two polypeptide probes have high molar activity, good in vitro and in vivo stability, can be rapidly distributed throughout the body in model animals, are taken up in PD-L1-positive tumor parts, and are rapidly cleared from the body, and can obtain PET images with a high target / non-target ratio. Therefore, they have good clinical application prospects. Compared with existing techniques such as pathological immunohistochemistry, these two probes can achieve real-time, dynamic and non-invasive monitoring of the PD-L1 expression level and changes in tumors at the molecular level, and provide help for guiding immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0041] Figure 6 is 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 radioactive high performance liquid chromatography analysis and its stability;

[0042] Figure 7 is 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 radioactive high performance liquid chromatography analysis and its stability

[0043] Figure 8 is cell experiment research; where A is 18 F]AlF-NOTA-PEG2 -Asp 2 - Cellular uptake of WT12; B is 18 F]AlF-NOTA-PEG 2 -Asp 2 - Blocking study of WT12; C is 18 F]AlF-NOTA-PEG 2 -Asp 2 - Cellular uptake of IPB-WT12; D is 18 F]AlF-NOTA-PEG 2 -Asp 2 - Blocking study of IPB-WT12;

[0044] Figure 9 is 18 F]AlF-NOTA-PEG 2 -Asp 2 - Dynamic Micro PET / CT imaging of WT12; where A is intravenous injection in nude mice bearing 4T1-hPD-L1 tumors 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 at different time points of representative maximum intensity projection (MIP) images; B is intravenous injection 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 of 4T1-hPD-L1 tumors and major organs of the time-activity curve; C is intravenous injection in nude mice bearing A549-hPD-L1 tumors 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 at different time points of representative maximum intensity projection (MIP) images; D is intravenous injection 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 of A549-hPD-L1 tumors and major organs of the time-activity curve; E is the time-activity curve of 4T1-hPD-L1 tumors and A549-hPD-L1 tumors; F is 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 in 4T1-hPD-L1 and A549-hPD-L1 tumors of the time-activity ratio curve of muscle; (red circles represent tumors);

[0045] Figure 10 is18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 dynamic Micro PET / CT imaging; where A is intravenous injection in nude mice bearing 4T1-hPD-L1 tumors 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 at different time points of representative maximum intensity projection (MIP) images; B is intravenous injection 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 after 4T1-hPD-L1 tumor and major organ time-activity curves; C is intravenous injection in nude mice bearing A549-hPD-L1 tumors 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 at different time points of representative maximum intensity projection (MIP) images; D is intravenous injection 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 after A549-hPD-L1 tumor and major organ time-activity curves; E is 4T1-hPD-L1 tumor and A549-hPD-L1 tumor time-activity curves; F is 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 in 4T1-hPD-L1 and A549-hPD-L1 tumors to muscle time-activity ratio curves; (red circles represent tumors);

[0046] Figure 11 Static Micro PET / CT imaging of nude mice bearing 4T1-hPD-L1 and A549-hPD-L1; where A is intravenous injection in 4T1-hPD-L1 and A549-hPD-L1 nude mice 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 60 minutes after MIP images; B is intravenous injection in 4T1-hPD-L1 and A549-hPD-L1 nude mice 18 F]AlF-NOTA-PEG 2 -Asp 2-MIP images of IPB-WT12 at 60 minutes post-injection; C shows, at 60 minutes, in 4T1-hPD-L1 and A549-hPD-L1 tumors 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -Quantitative uptake of IPB-WT12; D shows the tumor-to-muscle ratio at 60 minutes;

[0047] Figure 12 is 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -Biodistribution of IPB-WT12 in tumor-bearing mice; (A) at 30 and 60 minutes post-injection, 18 F]AlF-NOTA-PEG 2 -Asp 2 -Biodistribution of WT12 in mice bearing 4T1-hPD-L1, A549-hPD-L1, 4T1, and CHO tumors (n = 3); (B) 18 F]AlF-NOTA-PEG 2 -Asp 2 -Tumor-to-muscle ratio of WT12 in different tumors at 30 and 60 minutes; (C) 18 F]AlF-NOTA-PEG 2 -Asp 2 -Biodistribution of IPB-WT12 in mice bearing 4T1-hPD-L1, A549-hPD-L1, and CHO tumors at 30 and 60 min post-injection (n = 3); (D) 18 F]AlF-NOTA-PEG 2 -Asp 2 -Tumor-to-muscle ratio of IPB-WT12 in different tumors at 30 and 60 minutes;

[0048] Figure 13 is 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 methods

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

[0050] Terms related to the present invention:

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

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

[0053] HBTU: O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate, a peptide coupling reagent;

[0054] DIPEA: N,N-Diisopropylethylamine;

[0055] NOTA-COOH: A carboxylic acid-modified NOTA macrocyclic compound, a bifunctional chelating agent.

[0056] Example 1

[0057] This example provides a polypeptide complex targeting PD-L1, and its synthesis method is as Figure 1 shown. The synthesis of NOTA-PEG 2 -Asp 2 -IPB-WT12 and NOTA-PEG 2 -Asp 2 -WT12 was achieved through solid-phase peptide synthesis (SPPS), and its preparation method is as follows:

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

[0059] S2. Incorporation of 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 use a 20% piperidine in DMF solution (20 mL) to remove the Fmoc protecting group. The subsequent washing steps include DMF (washing 1 time), DCM (washing 2 times), and DMF (washing 2 times).

[0060] S3. Incorporate the second amino acid: Subsequently, Fmoc-Asn(Trt)-OH (537 mg), HOBt (121 mg), and DIC (464 μL) in 20 mL of DMF were added to the resin and stirred at room temperature for 30 minutes. After filtering the solvent, the Fmoc group was deprotected using a 20% piperidine in DMF solution (20 mL), followed by additional washes with DMF (1 wash), DCM (2 washes), and DMF (2 washes).

[0061] S4. Repeat S3 and sequentially couple the 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. The procedure was the same: Fmoc-amino acid (0.9 mmol × molecular weight), HOBt (121 mg), and DIC (464 μL) in 20 mL of DMF were added to the resin and stirred at room temperature for 30 minutes. After filtering the solvent, the Fmoc group was deprotected using a 20% piperidine in DMF solution (20 mL), followed by additional washes with DMF (1 wash), DCM (2 washes), and DMF (2 washes). The amino acid sequence was LNWTYYSRHW, and finally the last amino acid Fmoc-L-Lys(Dde)-OH was attached.

[0062] S5. Add NOTA-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 for 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 of DMF; (2) Fmoc-Asp-OtBu (370 mg), HOBt (121 mg) and DIC (464 μL) were reacted in 10 mL of DMF; (3) Fmoc-Asp-OtBu (370 mg), HOBt (121 mg) and DIC (464 μL) were reacted in 10 mL of 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 2 times), MeOH (washed 2 times), DMF (washed 2 times) and DCM (washed 2 times). Finally, compound F was obtained. Subsequently, the Fmoc protecting group in compound F was removed with a DMF solution of 20% piperidine (20 mL).

[0064] S7. It was coupled with 4-iodophenylbutyric acid by adding HBTU (341 mg) and DIPEA (522 μL) to 10 mL of DMF and stirring at room temperature for 30 minutes. Then, the resin was washed again with DMF (washed 2 times), MeOH (washed 2 times), DMF (washed 2 times) and DCM (washed 2 times) to obtain compound G.

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

[0066] The obtained product was analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), Figure 2 is the HPLC chromatogram of purified NOTA-PEG 2 -Asp 2 -WT12; Figure 3 is the mass spectrum of purified NOTA-PEG 2 -Asp 2 -WT12; Figure 4 is purified NOTA-PEG2 -Asp 2 HPLC chromatogram of -IPB-WT12; Figure 5 is purified NOTA-PEG 2 -Asp 2 -Mass spectrum of IPB-WT12; from Figures 2 to 5 it can be shown that the synthesis of NOTA-PEG in Example 1 was successful 2 -Asp 2 -IPB-WT12 and NOTA-PEG 2 -Asp 2 -WT12.

[0067] Example 2

[0068] This example provides a polypeptide-based positron probe targeting PD-L1, and its preparation method is as follows:

[0069] Using the radionuclide 18 F to label the polypeptide precursor of Example 1 with aluminum fluoride (Al 18 F). The labeling process is as follows: Add 9 μL / AlCl 3 solution (2 mM), 6 μL glacial acetic acid, 280 μL acetonitrile, and 50 μL deionized water containing 100 μg NOTA-PEG 2 -Asp 2 -WT12 to the reaction vial, then add 80 μL of fluorine-18 (3.7 GBq). The pH of the reaction mixture is 4. React the reaction mixture at 100 °C for 15 minutes. Subsequently, dilute the reaction mixture with 6 mL of water and cool to room temperature. After cooling, pass the mixture liquid through a Waters C18 column, hang the reaction product on the C18 column, and wash the impurities with 50 mL of water. Then elute the radionuclide-labeled product with 1.5 mL of ethanol-water mixture (50% each), and then dilute with saline and perform radiochemical purity analysis.

[0070] Label NOTA-PEG 2 -Asp 2 -IPB-WT12 using the same method.

[0071]

[0072] 18 F]AlF-NOTA-PEG2-Asp2-WT12 and 18 The undecay-corrected radiochemical yields of F]AlF-NOTA-PEG2-Asp2-IPB-WT12 are 15 - 25% and 25 - 35% (n > 5) respectively. Calculate the molar activities of both by 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 the partition coefficient between octanol and water and in vitro and in vivo stability

[0075] Mix 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12, these two tracers, with 2.0 mL of PBS and 2.0 mL of n-octanol in a 15 mL centrifuge tube. Then centrifuge the solution at 10000 rpm for 3 minutes to separate the two layers of n-octanol and PBS. After centrifugation, collect 100 μL from each layer and analyze using a γ counter to determine the logP value (n = 4).

[0076] Mix 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 in PBS (200 μL) or FBS (200 μL) and incubate at 37 °C for 2 hours (n = 3) to evaluate 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 for in vitro stability. For in vivo metabolism analysis, collect 0.5 mL of mouse serum samples 60 minutes after tracer injection (17.5 MBq). Collect blood samples from nude mice (n = 3), centrifuge at 10000 rpm for 5 minutes, and then analyze the supernatant by radioactive HPLC. Collect the HPLC mobile phase every 0.5 minutes within 14 minutes and then measure using a γ counter.

[0077] 18 F]AlF-NOTA-PEG 2 ​-Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 The logP values of -IPB-WT12 and -Asp were -2.23 ± 0.06 and -1.84 ± 0.02 (n > 3), respectively.

[0078] The results of in vitro and in vivo stability studies are as Figure 6 and Figure 7 shown. In 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12, after incubation in PBS and FBS for 120 min, only a single main peak appeared. The radiochemical purity of the above probes was >90%. This result indicates that the probes have good in vitro stability; there was no defluorination and decomposition within 60 min in serum, and they were relatively stable in serum.

[0079] 2. Cell experiment research

[0080] Seed 4T1-hPD-L1 and A549-hPD-L1 cells in a 12-well plate and culture for 24 hours until they reach confluence of more than 90%. For the cell uptake experiment, incubate the cells with 0.5 mL of serum-free medium at 37 °C for different time points (5, 15, 30, 60, and 120 minutes). The medium contains 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 (0.37 MBq). After incubation, wash the cells twice with PBS and lyse them with NaOH-SDS buffer (0.2 M NaOH, 1% SDS). Then collect the resulting cell lysates and analyze them using a gamma counter. In the blocking experiment, use BMS-1 (10 μg) as a blocker to inhibit PD-L1.

[0081] 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 ​- The uptake values of two probes, [F]AlF-NOTA-PEG-Asp-IPB-WT12, slowly increased over time in 4T1-hPD-L1 and A549-hPD-L1 cells and reached their respective maximum uptake values at 120 minutes (as shown by A and C in Figure 8 ). At 120 minutes, 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 showed significantly higher cellular uptake than 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12, which might be due to the introduction of the IPB group increasing its lipophilicity and internalization in cells. Additionally, the addition of BMS-1 significantly reduced the 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 uptake in 4T1-hPD-L1 and A549-hPD-L1 cells, indicating that both tracers could specifically target PD-L1 on tumors (as shown by B and D in Figure 8 ).

[0082] 3. Micro PET-CT imaging study

[0083] Nude mice bearing 4T1-hPD-L1, A549-hPD-L1, 4T1, or CHO tumors were intravenously injected via the tail vein with 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 (5.55 - 7.40 MBq) (n = 3 per group), followed by 120-minute dynamic PET imaging. In the blocking experiment, 4T1-hPD-L1 tumor-bearing mice were co-injected with 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2-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 after 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 drawn on tumors and major organs using Inveon Research Workplace 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 F]AlF-NOTA-PEG 2 -Asp 2 Dynamic Micro PET / CT imaging of [F]AlF-NOTA-PEG 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 was 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 F]AlF-NOTA-PEG 2 -Asp 2 Dynamic Micro PET / CT imaging of [F]AlF-NOTA-PEG 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 was also mainly excreted through the kidneys, with high uptake in tumors. However, its uptake in the heart, lungs, and muscles was relatively high, and its uptake in other organs was low. This probe showed higher tumor uptake and higher background than 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12. However, the tumor / muscle ratio of [F]AlF-NOTA-PEG 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 was higher than that of 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 (as shown in Figure 11 )). Therefore, these two probes had good targeting properties for tumors with high PD-L1 expression and could monitor the expression of PD-L1 in the tumor sites of tumor-bearing mice.

[0085] 4. Biodistribution study

[0086] Mice bearing 4T1-hPD-L1 and A549-hPD-L1 tumors were intravenously injected via the tail vein 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 (1.11 - 1.85 MBq, n = 3), and the mice were sacrificed at 30 and 60 minutes after injection. Mice bearing 4T1 and CHO tumors were also injected with the same doses of the two tracers. In the blocking experiment, 4T1-hPD-L1 tumor-bearing mice were co-injected with 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 (1.11 - 1.85 MBq) and the competitor WT12 (200 μg / mouse). The mice were sacrificed at the corresponding time points after injection, and the relevant organs, tumors, and blood were obtained and weighed, and the radioactivity was measured using a gamma counter. The results were expressed as the percentage of the injected dose per gram of tissue (%ID / g).

[0087] Figure 12 is 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 and 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 in tumor-bearing mice; from the biodistribution results, it can be seen that 18 F]AlF-NOTA-PEG 2 -Asp 2 -WT12 was mainly excreted through the kidneys, and the probe had a relatively high uptake in the tumor at 30 and 60 minutes, and a relatively low uptake in the major organs such as the heart, liver, brain, and muscle. In addition, 18 F]AlF-NOTA-PEG 2 -Asp 2 -IPB-WT12 was also mainly excreted through the kidneys, and the probe had a relatively high uptake in the tumor at 30 and 60 minutes, but a relatively high uptake in the heart, lung, gallbladder, and blood, and a relatively low uptake in other organs. Therefore, the biodistribution results of these two probes were basically consistent with the results of PET imaging.

[0088] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also regarded as the protection scope 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 also connected to a connecting group; the connecting group includes PEG2-Asp2-IPB or PEG2-Asp2.

2. The polypeptide complex according to claim 1, characterized in that The chelating agent is selected from any one of HYNIC, NOTA, DOTA and DTPA.

3. The polypeptide complex according to claim 1, characterized in that The polypeptide complex has a structure as shown in formula (a): Where R is H or 4. The method for preparing the polypeptide complex according to any one of claims 1 to 3, characterized in that: The steps include: S1. preparing a CLP002 polypeptide coupled with a chelating agent; S2, sequentially coupling the CLP002 polypeptide coupled with a chelating agent with Fmoc-PEG2-OH, Fmoc-Asp-OtBu, and Fmoc-Asp-OtBu to obtain a polypeptide complex with a connecting group of PEG2-Asp2; Preferably, the polypeptide complex whose linking group is PEG2-Asp2 is coupled with 4-iodophenylbutyric acid to obtain the polypeptide complex whose linking group is PEG2-Asp2-IPB.

5. Any of the following uses of the polypeptide complex according to any one of claims 1 to 3: 1) Used as a molecular probe; 2) Used for preparing imaging agents; 3) Used for preparing tumor detection reagents.

6. A polypeptide probe, characterized in that: The polypeptide probe is the polypeptide complex according to any one of claims 1 to 3 labeled with radionuclides.

7. The polypeptide probe according to claim 6, characterized in that 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.

8. The method for preparing the polypeptide probe according to claim 6 or 7, characterized in that: The method comprises the following steps: reacting the polypeptide complex according to claim 1 with a radioactive metal nuclide in a solution to obtain the polypeptide probe.

9. Use of the polypeptide probe according to claim 6 or 7 in the preparation of an imaging agent targeting PD-L1.

10. Use of the polypeptide probe according to claim 6 or 7 in the preparation of tumor imaging drugs.

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