Tumor PD-L1-targeted radioactive diagnosis and treatment medicine as well as labeled precursor, preparation method and application of tumor PD-L1-targeted radioactive diagnosis and treatment medicine

Abstract

The invention discloses a tumor PD-L1-targeted radioactive diagnosis and treatment drug as well as a labeled precursor, a preparation method and application thereof, and belongs to the technical field of biological medicines. Experimental verification finds that the prepared radioactive diagnosis and treatment medicine can be specifically combined with PD-L1, the position of a PD-L1 positive tumor is accurately positioned through PET imaging, and early diagnosis of the PD-L1 positive tumor and patient screening are achieved; moreover, the tumor residence time is long, the therapeutic nuclide can be used for treating PD-L1 positive tumors after being labeled, and the radiopharmaceutical is a diagnosis and treatment integrated radiopharmaceutical with great clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a radioactive diagnostic and therapeutic drug targeting tumor PD-L1 and a labeled precursor thereof, a preparation method and an application thereof. Background Art

[0002] In recent years, the development of PD-1 / PD-L1-based immune checkpoint blockade (ICB) has revolutionized cancer treatment. PD-1 is an immune checkpoint expressed by tumor-infiltrating immune cells, and its expression is upregulated in tumor-infiltrating CD4+, CD8+T cells, and regulatory T cells. PD-L1 is one of the ligands of PD-1, which is expressed to varying degrees in a variety of malignant tumors, including non-small cell lung cancer, ovarian cancer, bladder cancer, nasopharyngeal carcinoma, esophageal cancer, and breast cancer. The interaction between PD-1 and PD-L1 inactivates PD-1, resulting in the inhibition of anti-tumor immune responses. Therefore, blocking the interaction between PD-L1 and PD-1 can enhance the killing effect of immune cells and achieve the purpose of eliminating tumor cells. Currently, immunohistochemistry is the most commonly used method for stratification and screening of ICB patients. However, due to the heterogeneous expression of PD-L1 in tumor cells, the immunohistochemistry results of biopsy often cannot accurately assess the expression of PD-L1 in patients' tumors. Only 20%-40% of patients obtain long-term benefit from ICB, thus, there is an urgent need to develop new approaches to avoid false-negative results caused by heterogeneity in immune checkpoint expression.

[0003] With the development of nuclear medicine, the application of molecular imaging, especially positron emission tomography (PET), has shown great potential and advantages in tumor diagnosis and treatment. Radiolabeled antibodies, peptides and small molecules can all be tracers for monitoring PD-L1 expression in cancer patients. Early clinical trials showed that monoclonal antibody-based immunoPET tracers had poor pharmacokinetic properties and limited clinical application value. Compared with antibodies, peptides exhibit faster blood clearance and are easy to chemically modify. WL12 is reported to be a peptide that can specifically bind to PD-L1. Radiolabeled tracers based on WL12 peptides [ 64 Cu]WL12,[ 68 Ga]WL12 and [ 18 F]FPyWL12 has a high in vivo specific tumor targeting ability and can obtain high-quality images of PD-L1 expression in tumors; however, due to its high hydrophobicity and high liver enrichment, the clinical application of WL12 is limited. Other radiolabeled peptides, such as TPP-1 and SETSKSF, can specifically bind to PD-L1 and have a certain in vivo stability. They can also be used to develop radionuclide diagnostic and therapeutic drugs targeting PD-L1, providing new strategies for tumor immunotherapy patient screening, treatment plan optimization, efficacy testing, and prognosis evaluation. Summary of the invention

[0004] The purpose of the present invention is to provide a diagnostic and therapeutic radiopharmaceutical targeting tumor PD-L1 and its labeled precursor, preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The diagnostic and therapeutic radiopharmaceutical can specifically bind to PD-L1, accurately locate the position of PD-L1 positive tumors through PET imaging, and realize early diagnosis and patient screening of PD-L1 positive tumors; and its tumor retention time is relatively long, and it can be used for the treatment of PD-L1 positive tumors after therapeutic nuclide labeling, and is an integrated diagnostic and therapeutic radiopharmaceutical with great clinical application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a labeled precursor of a diagnostic and therapeutic radioactive drug targeting tumor PD-L1, characterized in that the labeled precursor consists of a polypeptide compound, a connecting arm and a chelating agent, the amino side of the connecting arm is connected to the chelating agent, the carboxyl side of the connecting arm is connected to the N-terminal serine amino group of the polypeptide compound, and the amino acid sequence of the polypeptide compound is SGQYASYHCWCWRDPGRSGGSK.

[0007] Preferably, the linker arm is selected from one of the following compounds:

[0008]

[0009] Wherein, n is an integer in the range of 1-20.

[0010] Preferably, the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane, 1,4,7,10-tetraazacyclododecane-1-pentanedioic acid-4,7,10-triacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine, 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine, diethylenetriamine pentaacetate or deferoxamine.

[0011] The present invention also provides a method for preparing the labeled precursor, characterized in that it comprises the following steps:

[0012] synthesizing the polypeptide compound;

[0013] Carrying out coupling reaction between the polypeptide compound, the connecting arm and the chelating agent to obtain a crude product of the labeling precursor;

[0014] The crude product of the labeling precursor is purified to obtain the labeling precursor.

[0015] Preferably, the molar ratio of the polypeptide compound, the linker arm and the chelating agent is 1:(1-5):(1-5);

[0016] The purification reagent is a mixture of diethyl ether and n-hexane, and the volume ratio of the diethyl ether to the n-hexane is 1:1.

[0017] The present invention also provides a radioactive diagnostic and therapeutic drug targeting tumor PD-L1, characterized in that the radioactive diagnostic and therapeutic drug is obtained by labeling the labeled precursor with a radionuclide, and the radionuclide is selected from 18 F. 64 Cu, 67 Cu, 68 Ga, 89 Zr, 99m Tc, 111 In, 177 Lu, 186 Re, 188 Re, 203 Pb, 212 Pb, 213 Bi, 225 Ac, 227 Th.

[0018] The present invention also provides a method for preparing a radioactive diagnostic and therapeutic drug targeting tumor PD-L1, characterized in that the labeled precursor is mixed and incubated with a radionuclide solution to prepare the radioactive diagnostic and therapeutic drug targeting tumor PD-L1;

[0019] The incubation condition is 20-110° C. for 5-60 min.

[0020] Before the labeling precursor is mixed with the nuclide solution, the labeling precursor is first dissolved in a buffer solution, wherein the buffer solution is a mixture of one or more of sodium acetate, water, ethanol, phosphate buffer solution or dimethyl sulfoxide, and the pH value is 4.0-10.0;

[0021] The present invention also provides a pegylated radioactive diagnostic and therapeutic drug targeting tumor PD-L1, characterized in that the polypeptide compound according to claim 1 is connected with polyethylene glycol to form a pegylated radioactive diagnostic and therapeutic drug targeting tumor PD-L1.

[0022] Preferably, the polyethylene glycol molecule is selected from one of the following compounds:

[0023]

[0024] Wherein, n is an integer in the range of 1-20;

[0025] R is a common chemical group such as H, hydroxyl, amino, azido, alkynyl, NHS, etc.

[0026] The present invention also provides a method for preparing a pegylated tumor-targeting PD-L1 radioactive diagnostic and therapeutic drug, characterized in that the labeling precursor, the polyethylene glycol and the radionuclide solution are mixed and incubated to prepare the pegylated tumor-targeting PD-L1 radioactive diagnostic and therapeutic drug;

[0027] It is characterized in that before the labeling precursor is mixed with the nuclide solution, the labeling precursor is first dissolved in a buffer solution, wherein the buffer solution is a mixture of one or more of sodium acetate, water, ethanol, phosphoric acid, saline buffer solution or dimethyl sulfoxide, and the pH value is 4.0-10.0;

[0028] The incubation condition is 20-110° C. for 5-60 min.

[0029] The present invention also provides the use of the labeled precursor, the radioactive therapeutic drug targeting tumor PD-L1, or the pegylated radioactive therapeutic drug targeting tumor PD-L1 in the preparation of reagents, drugs, and / or drug combinations for diagnosing and / or treating one or more tumors or cells expressing PD-L1.

[0030] Wherein, the one or more tumors or cells expressing PD-L1 are selected from the group consisting of non-small cell lung cancer and cells, ovarian cancer and cells, osteosarcoma and cells, nasopharyngeal cancer and cells, liver cancer and cells, kidney cancer and cells, bladder cancer and cells, colon cancer and cells, breast cancer and cells, esophageal cancer and cells, liver cancer and cells;

[0031] The one or more tumors or cells expressing PD-L1 are in vitro, in vivo or ex vivo.

[0032] The present invention discloses the following technical effects:

[0033] The present invention first connects the chelator to the polypeptide compound targeting PD-L1 through a connecting arm to synthesize a radioactive diagnostic and therapeutic drug labeled precursor QM-2308 with a sequence of SGQYASYHCWCWRDPGRSGGSK. 64 Cu labeling to obtain high-purity radioactive therapeutic drugs[ 64 Cu]QM-2308. At the same time, through the modification method of PEGylation and 64 Cu labeling, synthesis of PEGylated radioactive therapeutic drugs [ 64 Cu]QM-2309, 64 Cu]QM-2310 and [ 64Cu] QM-2311. Experiments have shown that the radioactive diagnostic and therapeutic drug prepared by the present invention has good binding specificity with tumor PD-L1 and a long tumor retention time. The radioactive diagnostic and therapeutic drug prepared by the present invention is an integrated diagnostic and therapeutic radioactive drug with great clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 HPLC and mass spectrometry analysis results of peptide compounds that are precursors of radioactive diagnostic and therapeutic drugs, QM-2308;

[0036] Figure 2 for[ 64 Cu]QM-2308(A),[ 64 Cu]QM-2309(B),[ 64 Cu]QM-2310(C) and [ 64 Radioactive high performance liquid chromatography (HPLC) analysis results of Cu]QM-2311(D);

[0037] Figure 3 for[ 64 Cu]QM-2308(A),[ 64 Cu]QM-2309(B),[ 64 Cu]QM-2310(C) and [ 64 In vivo PET / CT imaging study of Cu]QM-2311(D) in tumor-bearing mice;

[0038] Figure 4 for[ 64 Cu]QM-2308, 64 Cu]QM-2309, 64 Cu]QM-2310 and [ 64 Cu]QM-2311 accumulation in different organs of tumor-bearing mice; A: accumulation of four PET tracers in tumors; B: accumulation of four PET tracers in kidneys; C: accumulation of four PET tracers in livers; D: accumulation of four PET tracers in spleens; E: accumulation of four PET tracers in hearts;

[0039] Figure 5 for[ 64Cu]QM-2308, 64 Cu]QM-2309, 64 Cu]QM-2310 and [ 64 Evaluation of the therapeutic effect of QM-2311 on tumor-bearing mice; A: [ 64 Cu] QM-2308 treatment group mice tumor volume change curve; B: [ 64 Cu] QM-2309 treatment group mice tumor volume change curve; C: [ 64 Cu] QM-2310 treatment group mice tumor volume change curve; D: [ 64 Cu] QM-2311 treatment group mouse tumor volume change curve; E: four radioactive diagnostic and therapeutic drug treatment group mouse survival rate statistical chart; F: four radioactive diagnostic and therapeutic drug treatment group mouse body weight change statistical chart; G: four radioactive diagnostic and therapeutic drug treatment group mouse tumor volume change statistical chart;

[0040] Figure 6 for[ 64 Cu]QM-2311 and [ 64 Comparative study on the toxic effects of Cu]WL-12; A: statistical graph of changes in mouse body weight; B: statistical graph of changes in white blood cell count in mouse blood; C: statistical graph of changes in red blood cell count in mouse blood; D: statistical graph of changes in platelet count in mouse blood. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] In the present invention 64 Cu was purchased from commercial sources with a radionuclide purity of 98%. Radioactive high performance liquid chromatography (HPLC) analysis of all radiotracers was performed using an HPLC system (Shimadzu, Japan).

[0047] The present invention uses mouse colon cancer MC38 cell line as a cell model, and its culture medium is: DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin / streptomycin, and the incubator is set at: 37°C, 5% CO 2 . Subculture every three days. Change the medium every two days.

[0048] All animal experiments used in this invention were approved by the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences. All animal experiments followed the 3R principle and were conducted under the guidance of the Experimental Animal Care and Ethics Committee. The tumor-bearing C57BL / 6J mouse model was established by subcutaneous injection of MC38 cells. Each mouse was injected unilaterally in the axilla with 1×10 6 cells, and one tumor was established in each mouse. After 2 weeks, when the tumor tissue grew to about 100-300mm 3 At 4 pm, mice were dosed and subjected to PET / CT imaging studies.

[0049] The Chinese meanings of the English and abbreviations involved in the present invention are as follows:

[0050] Fmoc: 9-methoxycarbonyl;

[0051] Rink Amide-MBHA Resin: 4-Methylbenzhydrylamine resin hydrochloride;

[0052] DMF: N,N,-dimethylformamide;

[0053] DCM: dichloromethane;

[0054] HCTU: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate;

[0055] TFA: trifluoroacetic acid;

[0056] DIPEA: N,N-diisopropylethylamine;

[0057] TIS: triisopropylsilane;

[0058] Trt: trityl;

[0059] tBu: tert-butyl;

[0060] Pbf: 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl;

[0061] PEG: polyethylene glycol;

[0062] NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid;

[0063] DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0064] Example 1 Labeled precursor QM-2308 of a diagnostic and therapeutic radiopharmaceutical targeting tumor PD-L1 and a diagnostic and therapeutic radiopharmaceutical [ 64 Synthesis of Cu]QM-2308

[0065] Using the Fmoc-based solid phase peptide synthesis method, a peptide with the sequence of SGQYASYHCWCWRDPGRSGGSK was synthesized, and then coupled with a linker and a chelating agent, followed by deprotection and shearing of the crude product, and purified by HPLC to obtain pure QM-2308. The synthetic route is as follows:

[0066]

[0067] The specific synthesis steps are:

[0068] (1) Resin swelling: 100 mg of Rink Amide-MBHA Resin with a degree of substitution of 0.32 mmol / g was added to a reaction tube, followed by 3 mL of DMF, and the mixture was reacted for 30 minutes under nitrogen blowing.

[0069] (2) Removal of Fmoc protecting group from resin: discard the solvent, add 3 mL of 20% piperidine / DMF solution (piperidine:DMF=1:4, v:v), react for 5 minutes, discard the solvent, add 3 mL of 20% piperidine / DMF solution (piperidine:DMF=1:4, v:v), react for 15 minutes.

[0070] (3) Resin washing: Wash with DMF, DCM, and DMF in sequence, twice each.

[0071] (4) First amino acid connection: A condensation system was prepared in a centrifuge tube, to which Fmoc-Lys(Boc)-OH (4 eq, 59.97 mg) and HCTU (4 eq, 52.96 mg) were added, followed by DMF for dissolution, and finally DIPEA (8 eq, 44.6 μL) was added; the solvent in the reaction tube was discarded, the condensation system was added thereto, and nitrogen was blown for reaction for 60 minutes.

[0072] (5) Resin washing: Wash with DMF, DCM, and DMF in sequence, twice each.

[0073] (6) Removal of amino acid protecting group Fmoc: discard the solvent, add 3 mL of 20% piperidine / DMF solution (piperidine:DMF=1:4, v:v), react for 5 minutes, discard the solvent, add 3 mL of 20% piperidine / DMF solution (piperidine:DMF=1:4, v:v), react for 15 minutes.

[0074] (7) Resin washing: Wash with DMF, DCM, and DMF in sequence, twice each.

[0075] (8) Second amino acid connection: A condensation system was prepared in a centrifuge tube, and Fmoc-Ser(tBu)-OH (49.08 mg) and HCTU (4 eq, 52.96 mg) were added thereto. DMF was then added to dissolve the mixture, and finally DIPEA (8 eq, 44.6 μL) was added. The solvent in the reaction tube was discarded, and the condensation system was added thereto. The mixture was reacted for 60 minutes under nitrogen blowing.

[0076] Repeat the above steps and connect in sequence: Fmoc-Gly-OH (38.05 mg), Fmoc-Gly-OH (38.05 mg), Fmoc-Ser(tBu)-OH (49.08 mg), Fmoc-Arg(Pbf)-OH (83.04 mg), Fmoc-Gly-OH (38.05 mg), Fmoc-Pro-OH (43.18 mg), Fmoc-Asp(otBu)-OH (52.67 mg), Fmoc-Arg(Pbf)-OH (83.04 mg), Fmoc-Trp(Boc)-OH (67.40 mg), Fmoc-Cys(Trt)-OH (74.97 mg), Fmoc-Trp(Boc)-OH (67.40 mg), Fmoc-Cys (Trt)-OH(74.97mg), Fmoc-His(Trt)-OH(79.32mg), Fmoc-Tyr(tBu)-OH(58.82mg), Fmoc-Ser(tBu)-OH(49.08mg), Fmoc-Ala-OH(39.85mg), Fmoc-Tyr(tBu)-OH(58.82mg), Fmoc-Gln(Trt)-OH(78.17mg), Fmoc-Gly-OH(38.05mg), Fmoc-Ser(tBu)-OH(49.08mg), 4-[2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]ethyl]-1-piperazineacetic acid (52.41 mg), NOTA-tris (t-Bu ester) (69.85 mg), the amount of the above substances used is 4 eq.

[0077] (9) Drain the solution and wash the resin: wash with DMF, DCM, and DMF three times each.

[0078] (10) Resin dehydration: Add 1 mL of methanol, dehydrate for 1 minute, remove the methanol, and blow dry the resin with nitrogen.

[0079] (11) Peptide shearing: Add shearing solution (TFA:TIS:H 2 O=95:2.5:2.5, v:v:v), and place on a shaker for 2 hours;

[0080] (12) Filtration: Plug cotton into a 1 mL pipette tip, pour the shear system into the filter, rinse the resin with an appropriate amount of TFA after filtration, collect the filtrate in a centrifuge tube, and then blow dry the filtrate with nitrogen to obtain a crude peptide solid;

[0081] (13) Peptide purification: A cold ether + hexane mixture (ether: hexane = 1:1, v:v) was added to the crude peptide solid to generate a white precipitate, which was then sonicated for 30 seconds and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the precipitate was blown dry with nitrogen. The resulting precipitate was dissolved in a 20% acetonitrile / water solution, purified by HPLC, and freeze-dried to obtain a white powder of the polypeptide precursor QM-2308.

[0082] HPLC detection of peptide compounds Figure 1 As shown in A, the mass spectrometry detection diagram is as follows Figure 1 As shown in B. The structure of the synthesized labeled precursor of the diagnostic radiopharmaceutical (QM-2308) is shown below:

[0083]

[0084] (14) Radionuclide labeling: 20 μg of QM-2308 was dissolved in sodium acetate solution (0.1 mol / L, 100 mL, pH 4.5) and then added 64 CuCl 2 (10 mCi) solution, incubate at 80°C for 10 minutes. Dilute with physiological saline and filter sterilize using a 0.22 μm filter. Radio-HPLC analysis using a YMC-tria-C18 filter column 64 The radiochemical purity of QM-2308 was determined by the following analysis conditions:

[0085] Solvent gradient: solvent A, deionized water; solvent B, acetonitrile (0.1% TFA); flow time: 20 min, acetonitrile from 10% to 90%; flow rate: 1.0 mL / min.

[0086] The diagnostic radiopharmaceuticals prepared in this example [ 64 The results of Radio-HPLC test of QM-2308 were as follows: 64 The radiochemical yield of Cu]QM-2308 was greater than 98% (Table 1) and the retention time was approximately 7.360 min (e.g. Figure 2 The molar activity of the therapeutic radiopharmaceutical was greater than 74 GBq / μmol (Table 1).

[0087] Table 1. 64 Cu]QM-2308, 64 Cu]QM-2309, 64 Cu]QM-2310 and [ 64 Quality Control Results of Cu]QM-2311

[0088]

[0089] Example 2 Diagnostic and therapeutic radiopharmaceuticals targeting tumor PD-L1 64 Synthesis of Cu]QM-2309

[0090] 20 μg of QM-2308 was dissolved in sodium acetate solution (0.1 mol / L, 100 mL, pH 4.5), and then a linear PEG molecule with a molecular weight of 5000 Da (the molecular structure of PEG is shown in the figure below) was added. 64 CuCl2 (10 mCi) solution, incubated at 80°C for 6 hours. Diluted with physiological saline and sterilized by filtration using a 0.22 μm filter. Radio-HPLC analysis using a YMC-tria-C18 filter column 64 The radiochemical purity of QM-2309 was determined by the following analysis conditions:

[0091] Solvent gradient: solvent A, deionized water; solvent B, acetonitrile (0.1% TFA); flow time: 20 min, acetonitrile from 10% to 90%; flow rate: 1.0 mL / min.

[0092]

[0093] The diagnostic radiopharmaceutical prepared in this example [ 64 The results of Radio-HPLC test of QM-2309 were as follows: 64 The radiochemical yield of Cu]QM-2309 was greater than 98% (Table 1) and the retention time was approximately 9.297 minutes ( Figure 2 The molar activity of the therapeutic radiopharmaceutical was greater than 37 GBq / μmol (Table 1).

[0094] Example 3 Diagnostic and therapeutic radiopharmaceuticals targeting tumor PD-L1 64 Synthesis of Cu]QM-2310

[0095] 20 μg of QM-2308 was dissolved in sodium acetate solution (0.1 mol / L, 100 mL, pH 4.5), and then a four-arm PEG molecule with a molecular weight of 5000 Da (the PEG molecular structure is shown in the figure below) was added. 64 CuCl2 (10 mCi) solution, incubated at 80°C for 6 hours. Diluted with physiological saline and sterilized by filtration using a 0.22 μm filter. Radio-HPLC analysis using a YMC-tria-C18 filter column 64 The radiochemical purity of QM-2310 was determined by the following analysis conditions:

[0096] Solvent gradient: solvent A, deionized water; solvent B, acetonitrile (0.1% TFA); flow time: 20 min, acetonitrile from 10% to 90%; flow rate: 1.0 mL / min.

[0097]

[0098] The diagnostic radiopharmaceuticals prepared in this example [ 64 The results of Radio-HPLC detection of QM-2310 were as follows: 64 The radiochemical yield of Cu]QM-2310 was greater than 78.3% (Table 1) and the retention time was approximately 9.307 minutes (Table Figure 2 The molar activity of the therapeutic radiopharmaceutical was greater than 37 GBq / μmol (Table 1).

[0099] Example 4 Diagnostic and therapeutic radiopharmaceuticals targeting tumor PD-L1 64 Synthesis of Cu]QM-2311

[0100] 20 μg of QM-2308 was dissolved in sodium acetate solution (0.1 mol / L, 100 mL, pH 4.5), and then an eight-arm PEG molecule with a molecular weight of 5000 Da (the molecular structure of PEG is shown in the figure below) was added. 64 CuCl2 (10 mCi) solution, incubate at 80°C for 6 hours. Dilute with physiological saline and filter sterilize using a 0.22 μm filter. Radio-HPLC equipped with a YMC-tria-C18 filter column is used to analyze the radiochemical purity of [64Cu]QM-2311; the analysis conditions are:

[0101] Solvent gradient: solvent A, deionized water; solvent B, acetonitrile (0.1% TFA); flow time: 20 min, acetonitrile from 10% to 90%; flow rate: 1.0 mL / min.

[0102]

[0103] The diagnostic radiopharmaceuticals prepared in this example [ 64 Cu]QM-2311 was tested by Radio-HPLC, and the results were: 64 The radiochemical yield of Cu]QM-2311 was greater than 78.2% (Table 1) and the retention time was approximately 9.760 minutes (Table Figure 2 The molar activity of the therapeutic radiopharmaceutical was greater than 37 GBq / μmol (Table 1).

[0104] Example 5

[0105] The diagnostic and therapeutic radiopharmaceutical of the present invention [ 64Cu]QM-2308, 64 Cu]QM-2309, 64 Cu]QM-2310 and [ 64 In vivo PET imaging studies of Cu]QM-2311 in MC38 tumor-bearing mice, specifically:

[0106] PET scanning was performed using Inveon Micro-PET / CT (Siemens Medical Solutions, Knoxville, Munich, Germany). Each tumor-bearing mouse was injected with 3.7 MBq (100 μL) of [ 64 Cu]QM-2308, dynamic PET scanning was started immediately after injection and continued for 60 minutes (n=3) to analyze the uptake of diagnostic radioactive drugs in tumors and their metabolism in vivo. Each tumor-bearing mouse was injected with 3.7MBq (100μL) [ 64 Cu]QM-2309, static PET scans were performed at 1 hour, 3 hours, 16 hours, 24 hours and 40 hours after injection (n = 3). Each tumor-bearing mouse was injected with 3.7 MBq (100 μL) [ 64 Cu]QM-2310 or [ 64 Cu]QM-2311, static PET scans were performed at 1 hour, 5.5 hours, 18 hours, 28 hours, 43 hours, 52 hours, and 65 hours after injection (n=3). PET series images showed that the PEG polymerized radioactive therapeutic drug [ 64 Cu]QM-2310 and [ 64 Cu]QM-2311 enrichment in tumor sites was significantly increased; 64 Cu]QM-2311 was most abundant in the tumor site and was still significantly retained in the tumor site 65 hours after injection ( Figure 3 , Figure 4 ).

[0107] Example 6

[0108] The diagnostic and therapeutic radiopharmaceutical of the present invention [ 64 Cu]QM-2308, 64 Cu]QM-2309, 64 Cu]QM-2310 and [ 64 The therapeutic effect of Cu]QM-2311 on MC38 tumor-bearing mice was studied as follows:

[0109] Each tumor-bearing mouse was injected with 111MBq (100μL) via the tail vein. 64 Cu]QM-2308, 64Cu]QM-2309, 64 Cu]QM-2310 or [ 64 Cu]QM-2311 (n=3), and recorded the changes in mouse tumor volume, mouse survival rate and mouse weight on days 6, 9, 12, 15, 19, 21, 23 and 25 after injection, and analyzed the therapeutic and toxic effects of diagnostic radioactive drugs on tumor-bearing mice. The experimental results showed that [ 64 Cu]QM-2311 has the most significant inhibitory effect on mouse tumor growth, and [ 64 The survival rate of mice in the Cu]QM-2311 treatment group was the highest ( Figure 5 ).

[0110] Example 7

[0111] The diagnostic and therapeutic radiopharmaceutical of the present invention [ 64 Cu]QM-2311 and [ 64 Comparative study of the toxic effects of Cu]WL-12, specifically:

[0112] Each tumor-bearing mouse was injected with 111MBq (100μL) via the tail vein. 64 Cu]QM-2311 or [ 64 Cu]WL-12 (n=3), the control group tumor-bearing mice were injected with 100 μL PBS solution; the weight of the mice was recorded on the day before injection, and the weight changes of the mice were recorded on the day of injection and 3 days, 7 days, 17 days, 30 days and 50 days after injection, and the blood of the mice was collected for blood cell composition analysis ( Figure 6 ).

Claims

1. A labeled precursor of a radioactive diagnostic and therapeutic drug targeting tumor PD-L1, It is characterized in that The labeling precursor consists of a polypeptide compound, a connecting arm and a chelating agent, wherein the amino side of the connecting arm is connected to the chelating agent, and the carboxyl side of the connecting arm is connected to the N-terminal serine amino group of the polypeptide compound, and the amino acid sequence of the polypeptide compound is SGQYASYHCWCWRDPGRSGGSK.

2. The labeled precursor of the radioactive diagnostic and therapeutic drug targeting tumor PD-L1 according to claim 1, It is characterized in that The connecting arm is selected from one of the following compounds: Wherein, n is an integer in the range of 1-20.

3. The labeled precursor of the radioactive diagnostic and therapeutic drug targeting tumor PD-L1 according to claim 1, It is characterized in that The chelating agent is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane, 1,4,7,10-tetraazacyclododecane-1-pentanedioic acid-4,7,10-triacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, N,N-bis(2-mercaptoethyl)-N',N'-diethylethylenediamine, 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine, diethylenetriamine pentaacetate or deferoxamine.

4. A method for preparing a labeling precursor according to any one of claims 1 to 3, It is characterized in that The following steps are involved: synthesizing the polypeptide compound; Carrying out coupling reaction between the polypeptide compound, the connecting arm and the chelating agent to obtain a crude product of the labeling precursor; Purifying the crude product of the labeling precursor to obtain the labeling precursor; a) The molar ratio of the polypeptide compound, the linker arm and the chelating agent is 1:(1-5):(1-5); b) The purification reagent is a mixture of diethyl ether and n-hexane, and the volume ratio of the diethyl ether to the n-hexane is 1:

1.

5. A radioactive diagnostic and therapeutic drug targeting tumor PD-L1, It is characterized in that The radioactive diagnostic and therapeutic drug is obtained by labeling the labeling precursor according to claim 1 with a radionuclide, and the radionuclide is selected from 18 F. 64 Cu, 67 Cu, 68 Ga, 89 Zr, 99m Tc, 111 In, 177 Lu, 186 Re, 188 Re, 203 Pb, 212 Pb, 213 Bi, 225 Ac, 227 Th.

6. A method for preparing a radioactive diagnostic and therapeutic drug targeting tumor PD-L1 as claimed in claim 5, It is characterized in that Mixing and incubating the labeled precursor with a radionuclide solution to prepare the radioactive diagnostic and therapeutic drug targeting tumor PD-L1; a) The incubation conditions are 20-110°C for 5-60 minutes; b) Before the labeling precursor is mixed with the nuclide solution, the labeling precursor is first dissolved in a buffer solution, wherein the buffer solution is a mixture of one or more of sodium acetate, water, ethanol, phosphate buffer solution or dimethyl sulfoxide, and the pH value is 4.0-10.

0.

7. PEGylated radioactive therapeutic drugs targeting tumor PD-L1, It is characterized in that Connecting the radionuclide-labeled compound of claim 5 with polyethylene glycol to form a pegylated radioactive diagnostic and therapeutic drug targeting tumor PD-L1; The polyethylene glycol molecule is selected from one of the following compounds: Wherein, n is an integer in the range of 1-1000; R is a common chemical group such as H, hydroxyl, amino, azido, alkynyl, NHS, etc.

8. The method for preparing the PEGylated tumor PD-L1 targeted radioactive diagnostic and therapeutic drug according to claim 7, It is characterized in that The labeled precursor, the polyethylene glycol and the radionuclide solution are mixed and incubated to prepare a PEGylated radioactive diagnostic and therapeutic drug targeting tumor PD-L1; a) The preparation method is characterized in that, before the labeling precursor is mixed with the nuclide solution, the labeling precursor is first dissolved in a buffer solution, wherein the buffer solution is a mixture of one or more of sodium acetate, water, ethanol, phosphoric acid, saline buffer solution or dimethyl sulfoxide, and the pH value is 4.0-10.0; b) The incubation condition is 20-110°C for 5-60 minutes.

9. Use of the labeled precursor according to claim 1, the radioactive therapeutic drug targeting tumor PD-L1 according to claim 5, or the pegylated radioactive therapeutic drug targeting tumor PD-L1 according to claim 7 in the preparation of an agent, drug, and / or drug combination for diagnosing and / or treating one or more tumors or cells expressing PD-L1.

10. The use according to claim 9, in, The one or more tumors or cells expressing PD-L1 are selected from the group consisting of non-small cell lung cancer and cells, ovarian cancer and cells, osteosarcoma and cells, nasopharyngeal cancer and cells, liver cancer and cells, kidney cancer and cells, bladder cancer and cells, colon cancer and cells, breast cancer and cells, esophageal cancer and cells, liver cancer and cells; The one or more tumors or cells expressing PD-L1 are in vitro, in vivo or ex vivo.

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