MUC1 protein targeted nuclide probe system and application

Abstract

The invention belongs to the field of radiopharmaceuticals and nuclear medicine, and relates to an MUC1 protein targeted nuclide probe system and application. The system comprises an MUC1 antibody and radionuclide, the radionuclide is labeled on the MUC1 antibody directly or through a bifunctional chelating agent, or the radionuclide realizes the labeling of the MUC1 antibody through a biological orthogonal system. The probe provided by the invention can be used for detecting or visualizing MUC1 protein, diagnosing individuals with MUC1 related diseases and screening people who may respond to MUC1 targeted therapy, and can also be used for differential diagnosis and staging of the MUC1 related diseases and malignant tumors, accurate positioning of focuses, curative effect monitoring and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of radiopharmaceuticals and nuclear medicine, and in particular relates to a MUC1 protein-targeted nuclide probe system and application. Background Art

[0002] Sugar is crucial in the body's energy metabolism. In 1922, German physiologist and Nobel Prize winner Prof. Otto Warburg discovered that, unlike most normal tissues, tumor cells tend to "ferment" glucose into lactic acid even when there is enough oxygen to support mitochondrial oxidative phosphorylation. This means that tumor cells prefer anaerobic glycolysis to consume glucose at a high rate. In 1954, Sols described the properties of the various substrates of hexokinase and proposed that 2-deoxyglucose (2-DG) has unique advantages in the study of glucose metabolism, laying the foundation for the development of the field of glucose metabolism. 18 F-FDG( 18 The metabolic activity of F-fluorodeoxyglucose (F-fluorodeoxyglucose) has been incorporated into the theoretical basis of tumor cell research. In 1976, Prof. Abass Alavi first 18 F-FDG was used on two normal human volunteers to initiate glucose metabolism imaging and was hailed as the "molecule of the century". 18 F-FDG rapidly circulates throughout the body's blood vessels and accumulates in large quantities in cancerous cells. Cancerous cells appear as "bright spots" when scanned using PET-CT (positron emission tomography). 18 F-FDG-PET / CT has brought tumor diagnosis and treatment into the "molecular era", and can detect primary and metastatic lesions of tumors throughout the body at an early stage, providing doctors with a reliable basis for accurately judging whether they are benign or malignant. 18 F-FDG-PET / CT is still the most widely performed functional imaging of tumor glucose metabolism in the world.

[0003] However, 18 The application of F-FDG-PET / CT in gastric cancer is relatively rare, not only because of objective reasons such as physical uptake of gastric mucosa and autonomous motility, but also because gastric cancer is highly heterogeneous, and the specific effects of tumor cavity tissue and specific types of gastric malignancies can affect the imaging of gastric cancer. 18 F-FDG accurate diagnosis. The symptoms of early gastric cancer are extremely hidden, and most patients (90%) are diagnosed as advanced at the time of initial diagnosis. And because of the high heterogeneity of gastric cancer, although new treatment methods such as targeted, immune, and CART are changing with each passing day, the methods that can clearly bring therapeutic benefits to gastric cancer are extremely limited, and the median survival time of advanced gastric cancer is only 11.1 to 14.4 months. Early diagnosis and personalized targeted treatment of gastric cancer remain important challenges.

[0004] Tumor biotherapy is a very important treatment method for malignant tumors at present. This emerging treatment method is often listed as the fourth major treatment method for tumors, alongside traditional surgery, radiotherapy, and chemotherapy. The basis of tumor biological therapy is the existence of tumor antigens. Glycosylation plays an important role in immune regulation and the tumor microenvironment. Tumor-specifically expressed glycoconjugate molecules are biological macromolecules with a common function of inhibiting immune surveillance, among which abnormally glycosylated tumor mucin molecules are the research focus.

[0005] Mucins are a class of macromolecular glycoproteins, mainly present on the surface of mucosal epithelial cells and some glandular cells. The positive expression rate of mucin MUC1 in gastric cancer tissues is 82%. Its expression varies in different types of gastric cancer tissues (P<0.05), with the highest expression rate in moderately and well-differentiated adenocarcinoma cases (91%, 30 / 33); its expression level is closely related to the patient's age, lymph node metastasis, and tumor size (P<0.05). In addition, MUC1 is highly expressed and highly O-glycosylated (long sugar chains) on normal cells, and these glycosylations mask many sites on the MUC1 protein; while in tumor cells, due to metabolic disorders, COMSC gene mutations, or transcriptional inhibition, etc., the sugar chains on MUC1 become shorter, forming some truncated sugar chains including Tn / S and Tn / T antigen structures (short sugar chains), thereby exposing the hidden protein backbone, and these structures can be used as targets for tumor immunotherapy.

[0006] Currently, multiple mucins have become popular targets for tumor immunotherapy, such as MUC1, MUC16 / 17, etc. ClinicalTrials data shows that a recent dual-antibody clinical project based on the MUC17 target (AMG199) is conducting a Phase I study (NCT04117958) in the United States for patients with metastatic gastric cancer and gastroesophageal junction cancer (G / GEJ), which is the first clinical trial to use MUC17 as a potential anti-tumor target. 18 Functional molecular imaging represented by F-FDG (deoxyglucose) positron emission tomography (PET) has advantages such as high sensitivity and quantitative analysis. It plays a crucial role in the early diagnosis of tumors and the evaluation of the efficacy of immunotherapy. However, it cannot meet the new requirements of tumor biotherapy and targeted therapy. Summary of the Invention

[0007] The object of the present invention is to provide a MUC1 protein-targeted radionuclide probe system and its application.

[0008] Starting from clinical problems, the present invention selects the mucin (MUC) family, which is the key glycopeptide antigen determinant for the synthesis of abnormal glycoproteins in gastric cancer tumors, as the key research object. By analyzing and detecting specimens from a cohort of patients undergoing clinical immunotherapy, an immunotherapy-related murine tumor immune model (PDX model), an in vitro PDO immune model, and a (immune / tumor cell) dual-humanized tumor model, the characteristics of the MUC family in the development, metastasis, and heterogeneous microenvironment of gastric cancer are analyzed, key MUC family proteins are screened, the differential recognition mechanism of antibodies against COSMC-deficient cells is discovered, and a MUC1-targeted specific monoclonal antibody (currently, the A16 monoclonal antibody with dual targeting of glycan and glycopeptide has been obtained) is developed. Taking the MUC1-targeted monoclonal antibody 16A as an example, the present invention uses a solid target radionuclide 124 I / 64 Cu / 89 Zr or a therapeutic radionuclide 177 Lu to label the MUC1-targeted monoclonal antibody. The results of animal experiments show that it has significantly high uptake in various tumors with high MUC1 expression, indicating that the radionuclide-labeled MUC1-targeted monoclonal antibody, as a specific probe targeting MUC1, exhibits clinical translation potential in preclinical studies and is expected to become an imaging agent for the screening of patients undergoing tumor immunotherapy, the monitoring and evaluation of the treatment process, and radioimmunotherapy.

[0009] To achieve the above object, the present invention provides a MUC1 protein-targeted radionuclide probe system, which includes a MUC1 antibody and a radionuclide;

[0010] The radionuclide is directly labeled on the MUC1 antibody or through a bifunctional chelating agent (BCA), or the radionuclide labels the MUC1 antibody through a bioorthogonal system.

[0011] According to a preferred embodiment of the present invention, the MUC1 antibody is 16A, and its sequence is shown in SEQ ID NO: 1.

[0012] MEVKLHQSGGGLVQPGGFLKISCVVSGIDFSRYWMSWVRRAPGKGLEWIGEITPDSNTINYVPSLKDNFGISRDNAKNTLFLQMTKVRSEDTALYFCASYYEGFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTENLYFQSHHHHHHHHHH(SEQ ID NO: 1).

[0013] The radionuclide described in the present invention may be a diagnostic radionuclide, and the diagnostic radionuclide is preferably 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, <� 177 Lu, 186 Re, 188 Re, 189 Re, 194 lr, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 At least one of Ac; or, the radionuclide may be a therapeutic radionuclide, and the therapeutic radionuclide is preferably 32 P, 47 Sc, 57 Co, It should be noted that there is a misspelling in the original text. "<�00004>" should be " 177 ". The above translation has been corrected accordingly.89 Sr. 90 Y. 103 Pd, 106 Such as 124 I. 125 I. 131 I. 131 Cs, 137 Cs, 177 Lu, 192 Ir, 212 Bihe 225 At least one of Ac.

[0014] According to the present invention, when radionuclides are used to label MUC1 antibodies through a bioorthogonal system, the system comprises:

[0015] A targeting unit, wherein the targeting unit is a MUC1 antibody coupled with a clicker structure;

[0016] A targeting unit, wherein the targeting unit is a click-targeting probe labeled with a radionuclide;

[0017] The clicker targeting probe can undergo a catalytic-free in vivo click chemistry reaction with the clicker structure.

[0018] According to a preferred embodiment of the present invention, the click-targeting probe includes a click reaction portion and an optional bifunctional chelator portion, wherein the click reaction portion is used to react with the click-substructure, and the bifunctional chelator portion is used to covalently link with the click reaction portion and chelate with the radionuclide.

[0019] According to a preferred embodiment of the present invention, the clicker structure is a trans-cyclooctene structure (d-TCO), and accordingly, the targeting unit is a TCO-antibody, such as TCO-16A, which can be prepared by amidation reaction of d-TCO and 16A.

[0020] According to a preferred embodiment of the present invention, the click reaction portion is PEG7-Tz, having the structure shown in Formula I:

[0021]

[0022] According to a preferred embodiment of the present invention, the bifunctional chelating agent is selected from DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTP (di-2-ethylhexylterephthalate), DTPA (NR-diethylenetriaminepentacetic acid), CB-DO2A

[0023] (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DO3A (1,4,7,10-tetraazacyclododecane), NOTA (1,4,7-triazacyclononane-1,4,7-acetic acid), NODGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid), NO2A (1,4,7-triazacyclononane), ΝΕΤΑ ({4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), DFO / df (derferoxamine), TRITA (1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid), ATSM (diacetyl-bis(N4-methylthiosemicarbazone)), HETA (1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid), TACN-TM (N,N′,N″,tris(2-mercaptoethyl)1,4,7-triazacyclononane), HBED-CC (N,N'-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid), TPEN (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine), BAPTA-AM (O,O'-Bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid,tetraacetoxymethyl ester), TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]), CP256, PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid), PTSM (Methyl p-toluenesulfonate), Cyclen (1,4,7,10-tetraazacyclododecane), porphyrin, polyamine, crown ether, dithiocarbazone, polyoxime and at least one of their derivatives.,

[0024] According to a specific embodiment of the present invention, the probe is 68 Ga, 64 Cu, 89 Zr, 124 I, 125 I, 131 I or 177 Lu-labeled DFO-16A, NOTA-16A, DOTA-16A or 16A; preferably 89 Zr]Zr-DFO-16A, 64 Cu]Cu-NOTA-16A, 177 Lu]Lu-DOTA-16A, 124 I]I-16A, 125 I]I-16A or 131 I]I-16A.

[0025] According to a specific implementation method of the present invention, the target seeking unit is 68 Ga, 64 Cu, 89 Zr, 124 I, 125 I, 131 I or 177 Lu-labeled DOTA-PEG7-Tz, NOTA-PEG7-Tz or PEG7-Tz. Preferably 68 Ga]Ga-DOTA-PEG7-Tz, 64 Cu]Cu-NOTA-PEG7-Tz, 177 Lu]Lu-DOTA-PEG7-Tz, 124 I]I-PEG7-Tz, 125 I]I-PEG7-Tz or 131 I]I-PEG7-Tz.

[0026] According to the present invention, the probe in which a radionuclide is directly or indirectly labeled on the MUC1 antibody through a bifunctional chelating agent can be prepared by a conventional radionuclide labeling method. For example, a labeling precursor 16A / BCA-16A, a radionuclide solution, and a buffer are mixed for radionuclide labeling to obtain 16A / BCA-16A with radionuclide labeling, followed by separation and purification after condition optimization.

[0027] For the probe in which a radionuclide labels the MUC1 antibody through a bioorthogonal system, it can be prepared by a method including the following steps:

[0028] React the MUC1 antibody with a click moiety to obtain a targeting unit;

[0029] Mix a labeling precursor click moiety targeting probe, a radionuclide solution, and a buffer for radionuclide labeling to obtain a click moiety targeting probe with radionuclide labeling. When the labeling rate is insufficient, separation and purification can be optionally performed, such as separation and purification using a PD-10 column. The radiochemical purity of the purified probe is greater than 95%, preferably greater than 98%. According to a specific embodiment, a labeling precursor BCA-PEG7-Tz / PEG7-Tz, a radionuclide eluate, and a buffer are mixed for radionuclide labeling to obtain PEG7-Tz / BCA-PEG7-Tz with radionuclide labeling, followed by separation and purification after condition optimization; wherein, the labeling precursor BCA-PEG7-Tz is prepared by amidation reaction of BCA and PEG7-Tz.

[0030] The radionuclide solution can be prepared by a conventional method in the art. For example, 89 The Zr solution can be produced by an accelerator and purified and separated by corresponding resins and elution reagents, and the present invention has no particular limitation thereto.

[0031] According to the present invention, preferably, the buffer is a HEPES-Na2CO3 buffer, a NaAc-HCl buffer, or a PB buffer, and corresponding optimized purification methods are adopted.

[0032] The bifunctional chelating agent can bind to the MUC1 targeting monoclonal antibody (taking 16A as an example) or the targeting probe (taking PEG7-Tz as an example) through a specific buffer (such as HEPES-Na2CO3 buffer, NaAc-HCl buffer, PB buffer, etc.) and optimized reaction conditions (such as pH value, temperature, reaction time, etc.) to achieve an efficient labeling process.

[0033] According to the position and type of the labeled radionuclide, the labeling of the radioactive probe includes the following steps:

[0034] (1) 89Method for labeling MUC1-targeted monoclonal antibody with Zr (taking 16A as an example):

[0035] Add 0.1 - 1.0 ml of 0.1 M HEPES-Na2CO3 buffer with pH 6.5 - 7.0 and 25 - 100 MBq of freshly prepared 89 Zr solution to 15 μg - 2 mg of labeling precursor DFO-16A, adjust the pH value to 7.0, and incubate at 37 °C for 30 - 60 min. The reaction product is optionally separated and purified by a PD-10 column to obtain 89 89 Zr]Zr-DFO-16A; preferably, the radiochemical purity of the obtained

[0036] (2) 64 Method for labeling MUC1-targeted monoclonal antibody with Cu (taking 16A as an example):

[0037] Add 0.1 - 1.0 ml of 0.05 M NaAc-HCl buffer with pH 4.5 - 5.5 and 25 - 100 MBq of freshly prepared 64 Cu solution to 15 μg - 2 mg of labeling precursor NOTA-16A, adjust the pH value to 5.0, and incubate at 37 °C for 30 - 60 min. The reaction product is optionally separated and purified by a PD-10 column to obtain 64 Cu]Cu-NOTA-16A; preferably, the radiochemical purity of the obtained 64 Cu]Cu-NOTA-16A is greater than 95%;

[0038] (3) 177 Method for labeling MUC1-targeted monoclonal antibody with Lu (taking 16A as an example):

[0039] Add 0.1 - 1.0 ml of 0.05 M NaAc-HCl buffer with pH 4.5 - 5.5 and 25 - 100 MBq of freshly prepared 177 Lu solution to 15 μg - 2 mg of labeling precursor DOTA-16A, adjust the pH value to 5.0, and incubate at 37 °C for 30 - 60 min. The reaction product is optionally separated and purified by a PD-10 column to obtain 177 Lu]Lu-DOTA-16A; preferably, the radiochemical purity of the obtained 177 Lu]Lu-DOTA-16A is greater than 95%;

[0040] (4) 124 I / 125 I / 131 Method for labeling MUC1-targeted monoclonal antibody with I (taking 16A as an example):

[0041] ​Add 0.5 - 1.5 mL of 0.1 M PB buffer with pH 7.2, 0.5 - 1.0 mL of 124 I / 125 I / 131 I]NaI solution and 6 - 120 μg of N - bromosuccinimide (NBS) to 0.5 - 5.0 mg of labeled precursor 16A, adjust the pH value to 7.0 - 7.5, and react at 37 °C for 60 s. The reaction product is optionally separated and purified by a PD - 10 column to obtain 124 I / 125 I / 131 I]I - 16A; preferably, the obtained 124 I / 125 I / 131 I]I - 16A has a radiochemical purity greater than 95%.

[0042] (5) 68 Method for labeling click - based targeting probe with

[0043] Add 0.1 - 1.0 ml of 0.1 M NaAc - HCl buffer with pH 4.0 - 5.0 and 25 - 100 MBq of freshly prepared 68 Ga solution to 15 μg - 2 mg of labeled precursor DOTA - PEG7 - Tz, adjust the pH value to 4.5, and incubate at 95 °C for 10 - 20 min. The reaction product is optionally separated and purified by a C - 18 column to obtain 68 Ga]Ga - DOTA - PEG7 - Tz; preferably, the obtained 68 Ga]Ga - DOTA - PEG7 - Tz has a radiochemical purity greater than 95%;

[0044] (6) 64 Method for labeling click - based targeting probe with

[0045] [[ID=4D]]Add 0.1 - 1.0 ml of 0.05 M NaAc - HCl buffer with pH 4.5 - 5.5 and 25 - 100 MBq of freshly prepared 64 Cu solution to 15 μg - 2 mg of labeled precursor NOTA - PEG7 - Tz, adjust the pH value to 5.0, and incubate at 95 °C for 10 - 20 min. The reaction product is optionally separated and purified by a C - 18 column to obtain 64 Cu]Cu - NOTA - PEG7 - Tz; preferably, the obtained 64 Cu]Cu - NOTA - PEG\(_7\) - Tz has a radiochemical purity greater than 95%;

[0046] (7) 177Method for labeling click sub-targeting probe with Lu (taking PEG7-Tz as an example):

[0047] Add 0.1 - 1.0 ml of 0.05 M NaAc-HCl buffer solution with pH 4.5 - 5.5 and 25 - 100 MBq of freshly prepared 177 Lu solution to 15 μg - 2 mg of pre-labeling precursor DOTA-PEG7-Tz, adjust the pH value to 5.0, and incubate at 95 °C for 10 - 20 min. The reaction product is optionally separated and purified by a C-18 column to obtain 177 177 Lu]Lu-DOTA-PEG7-Tz; preferably, the radiochemical purity of the obtained

[0048] (8) 124 I / 125 I / 131 Method for labeling click sub-targeting probe with

[0049] Add 0.5 - 1.5 mL of 0.1 M PB buffer solution with pH 7.2, 0.5 - 1.0 mL of 25 - 90 kBq / μL 124 I / 125 I / 131 I]NaI solution and 6 - 120 μg of N-bromosuccinimide (NBS) to 0.5 - 5.0 mg of pre-labeling precursor PEG7-Tz, adjust the pH value to 7.0 - 7.5, and react at 37 °C for 60 s. The reaction product is optionally separated and purified by a C-18 column to obtain 124 I / 125 I / 131 I]I-PEG7-Tz; preferably, the radiochemical purity of the obtained 124 I / 125 I / 131 I]I-PEG7-Tz is greater than 95%.

[0050] According to a specific embodiment of the present invention, the method for conjugating d-TCO with MUC1-targeting monoclonal antibody (taking 16A as an example) is as follows:

[0051] ​Take a PD-10 column equilibrated with 25 mL of Na2CO3 / NaHCO3 (0.1 M 10% Na2CO3 - 90% NaHCO3 buffer, pH = 9.0), load the monoclonal antibody standard solution of 16A, exchange the solvent, collect several tubes with the highest concentration and add them to an EP tube. Calculate the molar amount of the monoclonal antibody in the tube, and then add approximately 20-fold molar amount of d-TCO-DMSO solution (10 mg / mL). React at 37 °C for 1 h, and gently shake the reaction tube every 10 - 15 min. Take another PD-10 column activated with PBS for purification, and collect 1 - 2 tubes of the product with the highest concentration (detected by nano-drop) for standby.

[0052] The probe prepared by the above method can be tested by the following quality control methods: Measure the radioactive purity of the probe by radio-TLC and radio-HPLC. Radio-TLC detection: Take 2 μL of the free radionuclide and the purified radioactive probe sample containing 37 - 74 kBq (1 - 2 μCi) of radioactivity and drop them at a position 1 cm from the bottom end of the Xinhua No. 1 filter paper, place them in the developing system (saturated EDTA: 0.9% normal saline = 1:1), after complete development, take out the filter paper and dry it, and perform radio-TLC detection; Radio-HPLC detection: Take 2 μL of the free radionuclide and the purified radioactive probe sample containing 37 - 74 kBq (1 - 2 μCi) of radioactivity and dilute them to 50 μL in 0.01 M pH 7.4 PBS for radio-HPLC analysis. Analysis conditions: Agilent Bio SEC-3 gel filtration / size exclusion chromatography column, flow rate 1.0 mL / min; mobile phase 0.01 M pH 7.4 PBS.

[0053] The second aspect of the present invention provides any of the following applications of the above bioorthogonal system:

[0054] (1) Preparation of a targeting MUC1 imaging agent;

[0055] (2) Preparation of a screening reagent for people in need of MUC1-targeted therapy;

[0056] (3) Preparation of a tumor diagnostic reagent; the tumor diagnosis includes tumor staging, lesion localization, and efficacy monitoring;

[0057] (4) Preparation of a tumor therapeutic drug.

[0058] By the above technical solutions, the present invention has at least the following advantages and beneficial effects: The MUC1-targeted radioactive probe provided by the present invention has stable properties, good imaging effects, high affinity and functional activity for MUC1. In particular, it can identify the expression of MUC1 in COSMC-deficient tumor cells, and is expected to be used for real-time and non-invasive monitoring of the MUC1 expression in systemic lesions, monitoring the MUC1 expression heterogeneity in the same lesion and different lesions, observing the changes in MUC1 expression during the treatment process, providing early warnings for the treatment process of MUC1-highly expressed tumors, patient screening, efficacy monitoring, drug resistance and / or recurrence and metastasis, and realizing individualized and precision treatment of targeted tumor drugs.

[0059] The solid target PET radionuclides 124 I / 64 Cu / 89 Zr and therapeutic radionuclides 177 Lu are used in the present invention. Such radionuclides can achieve the labeling of monoclonal antibodies with less influence on the activity of monoclonal antibodies; in addition, the non-specific uptake in the liver is relatively low, which is beneficial to the detection of liver lesions; and, the whole body background is relatively low, with a good tumor-to-non-tumor ratio, which is beneficial to the observation of tumor lesions; finally, it is convenient to replace the imaging radionuclide with a therapeutic radionuclide to achieve the integration of diagnosis and treatment.

[0060] The present invention first applies the in vivo click chemistry (bioorthogonal) reaction to the radionuclide labeling and in vivo imaging of MUC1-targeted antibodies. By coupling the clickable structure (taking trans-cyclooctene d-TCO as an example) that can undergo catalyst-free in vivo click to the MUC1 antibody (taking TCO-16A as an example), and using the radiolabeled clickable targeting probe (taking PEG7-Tz as an example) or the clickable targeting probe conjugated with a bifunctional chelating agent (taking BCA-PEG7-Tz as an example) for in vivo click reaction, high-sensitivity and high-specificity imaging of MUC1-highly expressed tumors is achieved, the imaging background is reduced, the imaging time is optimized, and radiation damage is greatly avoided. The probe of the present invention can not only be used for the high-sensitivity and high-specificity diagnosis of MUC1-related diseases, but also for the precise positioning, staging and efficacy monitoring of tumor lesions, and has significant clinical application prospects.

[0061] Other features and advantages of the present invention will be described in detail in the subsequent specific implementation part. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] By describing the exemplary embodiments of the present invention in more detail in conjunction with the drawings, the above and other objects, features and advantages of the present invention will become more obvious.

[0063] Figure 1 They are the mass spectrometry quality control charts of 16A and DFO-16A.

[0064] Figure 2 shows 89 Results of the radiolabeling rate and radiochemical purity of the [Zr]Zr-DFO-16A probe: A) Radiolabeling rate after labeling; B) Radiochemical purity after purification; C) Radiochemical purity within 120 h in 0.01 M PBS at pH 7.2; D) Radiochemical purity within 120 h in 5% HSA.

[0065] Figure 3 shows 89 Uptake experiments of [Zr]Zr-DFO-16A and its blocking control group in 293T-MUC1 / 293T-COSMCKO-MUC1 / A549 / B16-OVA-MUC1 / B16-OVA-COSMCKO-MUC1 cells: A) 89 Uptake of [Zr]Zr-DFO-16A in 293T-MUC1 / 293T-COSMCKO-MUC1 cells; B) 89 Uptake of [Zr]Zr-DFO-16A in A549 / B16-OVA-MUC1 / B16-OVA-COSMCKO-MUC1 cells.

[0066] Figure 4 shows 89 Internalization rate test of [Zr]Zr-DFO-16A in B16-OVA-COSMCKO-MUC1 / B16-OVA-MUC1 cells.

[0067] Figure 5 shows 89 Enzyme-linked immunosorbent assay between [Zr]Zr-DFO-16A and MUC1 recombinant protein.

[0068] Figure 6 shows 89 Biodistribution experiment of [Zr]Zr-DFO-16A in normal Kunming mice.

[0069] Figure 7 shows in the present invention 89 The [Zr]Zr-DFO-16A labeled compound and the control probe 89 2-120 h Micro-PET imaging comparison of the [Zr]Zr-IgG labeled compound in a xenograft model with MUC1 transfection / COSMC gene knockout.

[0070] Figure 8 shows in the present invention 89 The [Zr]Zr-DFO-16A labeled compound and the control probe 892 - 120 h Micro - PET imaging comparison of [[Zr]]Zr - IgG labeled compounds in a xenograft model of allogeneic MUC1 - positive / negative gastric cancer cells.

[0071] Figure 9 Shows in the present invention 89 2 - 120 h Micro - PET imaging comparison of [[Zr]]Zr - DFO - 16A labeled compounds and the blocking group with additional unlabeled DFO - 16A in a xenograft model of MUC1 - positive gastric cancer cells.

[0072] Figure 10 Shows in the present invention 89 2 - 120 h Micro - PET imaging results of [[Zr]]Zr - DFO - 16A labeled compounds in other xenograft models of MUC1 - positive / negative tumor cells.

[0073] Figure 11 Mass spectrometry quality control charts for 16A and TCO - 16A.

[0074] Figure 12 For click reaction of TCO - 16A and 68 radio - TLC detection spectrum of in vitro click reaction of [[Ga]]Ga - DOTA - PEG7 - Tz.

[0075] Figure 13 Shows in the present invention 68 30 min micro - PET imaging of [[Ga]]Ga - DOTA - PEG7 - Tz labeled compounds and clicker TCO - 16A (injected 30 min / 24 h in advance) in a xenograft model of MUC1 - transfected / COSMC gene - knockout. Detailed implementation mode

[0076] The preferred implementation modes of the present invention will be described in more detail below. Although the preferred implementation modes of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the implementation modes set forth herein.

[0077] Precursor 16A in the present invention is provided by the School of Pharmacy, Tongji University.

[0078] For those not specified in the examples, all are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified for the manufacturer, all are conventional products that can be obtained through commercial purchase.

[0079] Example 1 This example is used to illustrate the preparation of precursor DFO - 16A

[0080] Take 1.5 mg of 16A monoclonal antibody (30 mg / mL, 50 μL) and mix it with 8 - 12 times the molar amount of DFO - DMSO solution (10 mmol / L). Add 1 mL of 0.1 M 10% Na2CO3 - 90% NaHCO3 buffer (pH = 9.0), and react at 37 °C for 1 h, gently shaking the reaction tube every 10 - 15 min. Take a PD - 10 column, first equilibrate it with 25 mL (5 mL × 5) of 0.01 M PBS, load the reacted DFO - 16A solution, make up the volume to 2.5 mL with 0.01 M PBS, let it drain completely, then add another 2.5 mL of 0.01 M PBS, and collect 5 tubes of the eluate, about 0.5 mL in each tube. Detect the antibody concentration in each tube successively by Nano - Drop ultraviolet spectrophotometer, and combine the products in several collected tubes with the highest concentration for MALDI - TOF mass spectrometry monitoring( Figure 1 )

[0081] Example 2 This example is used to illustrate 89 The labeling of Zr]Zr - DFO - 16A

[0082] Take 1.0 mL of HEPES - Na2CO3 buffer solution (a mixture of 0.25 M HEPES solution and 2.0 M Na2CO3 solution in a ratio of 10:1), and add oxalic 89 Zr]zirconium solution into a 10 mL sterile vial, and gently shake to mix. Then add 1.0 ml of DFO - 16A solution, gently shake to mix and place it at 37 °C for 1 h, gently shaking the reaction flask every 10 min. Add the reacted solution to the PD - 10 column equilibrated according to the method of Example 1, make up the volume to about 2.5 ml, wait for all the samples to enter the column, discard the first 2.5 mL of the eluate, add 0.01 M PBS buffer solution (pH = 7.2) for elution, and collect the next eluted solution as the sample to be tested and the product before sterilization.

[0083] Radio - TLC detection: Take 2 μL of free oxalic acid 89 Zr]zirconium and the purified 89 Zr]Zr - DFO - 16A samples containing 37 - 74 kBq (1 - 2 μCi) of radioactivity respectively, and drop them at a position 1 cm from the bottom of the No. 1 Whatman filter paper. Place it in the developing system (saturated EDTA: 0.9% normal saline = 1:1). After complete development, take out the filter paper and let it dry, and carry out radio - TLC detection. Radio - HPLC detection: Take 2 μL of free oxalic acid 89 Zr]zirconium and the purified 89Zr]Zr-DFO-16A was diluted to 50 μL in 0.01 M PBS at pH 7.4 for radio-HPLC analysis. Analysis conditions: Agilent Bio SEC-3 gel filtration / size exclusion chromatography column, flow rate 1.0 mL / min; mobile phase 0.01 M PBS at pH 7.4.

[0084] Finally, the target product was obtained 89 The labeling rate of Zr]Zr-DFO-16A was 97.3%, and the radiochemical purity was greater than 99%. After the labeled product was placed in 0.01 M PBS at pH 7.2 / 5% HSA for 120 h, the radiochemical purity did not change significantly and remained above 99% ( Figure 2 ).

[0085] Example 3 This example is used to illustrate 89 The cellular uptake study of Zr]Zr-DFO-16A

[0086] The cell line B16-OVA-MUC1 with high MUC1 expression and its COSMC gene knockout group B16-OVA-COSMCKO-MUC1 (heterogeneous glycoprotein expression, facilitating the targeting of 16A) were obtained by transfecting MUC1 into 293T / B16-OVA cells and performing stable screening. 293T-MUC1 and its COSMC gene knockout group cells were used as negative control cells. Cells were evenly seeded in a 24-well plate and cultured overnight to allow them to adhere well. The next day, 1 mCi 89 Zr]Zr-DFO-16A was added or and cultured for an additional 60 min, 120 min, 180 min, and 240 min, respectively. In the Blocking group, 30 μg / well of cold precursor 16A was added simultaneously to block the uptake signal. After the culture ended, the cells were washed three times with PBS, and the cell lysates were collected for detection and analysis of the uptake signal using a γ-Counter.

[0087] The experimental results showed ( Figure 3 ), at 60 min, the cells transfected with MUC1 showed a relatively high uptake rate of 89 Zr]Zr-DFO-16A, and it increased with the prolongation of time. When the cold precursor was added for blocking, the uptake rate decreased significantly, indicating that the uptake signal was MUC1-specific. Among them, the uptake of 89 Zr]Zr-DFO-16A by the COSMC gene knockout group cells was significantly higher than that of the wild group.

[0088] Example 4 This example is used to illustrate 89 The cellular internalization experiment of Zr]Zr-DFO-16A in B16-OVA-COSMCKO-MUC1 / B16-OVA-MUC1 cells

[0089] Add 89 Zr]Zr-DFO-16A to B16-OVA-MUC1 and its COSMC knockout group cells (24-well plate, 2×10 5 cells per well, 0.5 mL of serum-free 1640 medium, 1 μCi of radioactive probe, 40 μL), and perform cell internalization experiments at 0 °C and 37 °C (sampling at 60 min, 120 min, 180 min, and 240 min).

[0090] The experimental results show that ( Figure 4 ), the internalization rate generally decreases with time, and the internalization rate of the COSMC KO group is higher than that of the WT group, but there is no significant difference between different temperatures.

[0091] Example 5 This example is used to illustrate 89 the enzyme-linked immunosorbent assay of

[0092] Add 89 Zr]Zr-DFO-16A to a 96-well plate coated with MUC1 protein (0.1 μg / well), and add the antibody at 12 gradient concentrations (antibody concentrations are 0.4875, 0.975, 1.95, 3.9, 7.8, 15.625, 31.25, 62.5, 125, 250, 500, 1000 nM, n = 4), incubate at room temperature for 2 h, and measure the absorbance (OD 450nm value) of each well to calculate the 50% maximum biological effect concentration (EC 50 ).

[0093] As Figure 5 shown, the EC 50 is approximately 64.31 nM.

[0094] Example 6 This example is used to illustrate 89 the biodistribution of

[0095] Add 89 Zr]Zr-DFO-16A to normal Kunming mice via the tail vein. At 2 h, 24 h, 48 h, 72 h, 96 h, and 120 h after injection, separate the heart, liver, spleen, lung, kidney, stomach, small intestine, large intestine, muscle, bone, brain, blood and other organ tissues of the mice. After grinding, use a γ-Counter to detect and analyze the uptake signal to obtain 89 the ID% / g of the

[0096] The experimental results show that ( Figure 6) In normal mice, the uptake rate of blood is the highest at all time points. Except for blood, the uptake in the liver, lungs, and spleen is relatively higher than that in other organs and tissues at 2 h. The uptake in all organs decreases with the prolongation of time. Compared with blood, the uptake in other organs tends to be at a lower level.

[0097] Example 7 This example is used to illustrate 89 PET imaging study of

[0098] Cells with high expression of MUC1 (melanoma mouse models B16-OVA-MUC1 / B16-OVA-COSMCKO-MUC1 transfected with humanized MUC1, lung cancer model A549, bladder cancer model SW780, pancreatic cancer model BxPC3, gastric cancer model SGC7901) and low expression (gastric cancer model MKN45) were respectively selected for inoculation to construct xenograft tumor models. All tumor tissues were subcutaneously inoculated into the right hind limb of nude mice. A total of 7 model mice were injected with 3.7 - 7.4 MBq 89 Zr]Zr-DFO-16A, and 4 positive (high uptake) models were injected with 3.7 - 7.4 MBq 89 Zr-IgG as a control. At 2 h, 24 h, 48 h, 72 h, 96 h, and 120 h after injection, the mice were anesthetized with isoflurane (2% isoflurane - 30% oxygen / air), and imaging studies were performed on a small animal Micro-PET / CT.

[0099] Micro-PET / CT imaging is as Figures 7 - 10 shown, 89 The distribution of 89 Zr]Zr-DFO-16A in MUC1-positive tumor-bearing mice changes with time. At 2 h imaging, there is a relatively high uptake in the blood pool, comparable to that in the tumor area. Starting from 24 h, the background signals in the heart and lungs decrease significantly, and there is a significant increase in uptake in the tumor area. At 120 h, the signal in the blood pool almost disappears, and only a relatively high uptake signal remains in the tumor area; 89 Zr]Zr-DFO-IgG as a control group shows a relatively high uptake signal in the heart and lungs at 2 h in MUC1-positive tumor-bearing mice, and significant liver retention is observed compared with 89 Zr]Zr-DFO-16A. Only a small amount of uptake signal is present in the tumor area, and the uptake level is significantly lower than that of 89 Zr]Zr-DFO-16A; the tumor uptake in the control group with unlabeled 16A added decreases significantly, and the liver uptake increases slightly, which indirectly confirms the

[0100] Example 8 This example is used to illustrate the preparation of clicker TCO-16A and the click reaction process

[0101] Take 1.5 mg of 16A monoclonal antibody (30 mg / mL, 50 μL) and mix it with 20-fold molar amount of solution TCO-DMSO solution (10 mmol / L). Add 1 mL of 0.1 M 10% Na2CO3 - 90% NaHCO3 buffer (pH = 9.0), and react at 37 °C for 1 h. Gently shake the reaction tube every 10 - 15 min. Take a PD-10 column, first equilibrate it with 25 mL (5 mL × 5) of 0.01 M PBS, load the reacted DFO-16A solution, make up the volume to 2.5 mL with 0.01 M PBS, let it drain, then add another 2.5 mL of 0.01 M PBS, and collect 5 tubes of the eluate, about 0.5 mL per tube. Sequentially detect the antibody concentration in each tube through a Nano-Drop ultraviolet spectrophotometer, and combine the products in several collected tubes with the highest concentration for MALDI-TOF mass spectrometry monitoring ( Figure 11 )

[0102]

[0103] Example 9 This example is used to illustrate 68 Preparation of [68Ga]Ga-DOTA-PEG7-Tz

[0104] Dissolve 40 μg of the precursor DOTA-PEG7-Tz before labeling in DMSO (2 mg / mL), add 0.3 mL of 0.1 M NaAc solution and 100 - 500 MBq of freshly prepared 68 [68Ga]Ga hydrochloric acid solution (3 mL), adjust the pH value to 4.5, and incubate at 95 °C for 10 - 20 min. The product obtained from the reaction is separated and purified by a C-18 column to obtain 68 [68Ga]Ga-DOTA-PEG7-Tz, as shown in Formula II; the radiochemical purity is greater than 95%.

[0105]

[0106] Example 10 This example is used to illustrate the click reaction of TCO-16A and 68 [68Ga]Ga-DOTA-PEG7-Tz in vitro

[0107] Mix freshly prepared 68 [68Ga]Ga-DOTA-PEG7-Tz and TCO-16A in a molar ratio of 1 - 2:1 in 0.01 M PBS, and the click product can be obtained within 1 min at room temperature. Detect the radiochemical purity by radio-TLC ( Figure 12 ) Figure 12 The left peak in 68Ga]Ga-DOTA-PEG7-Tz, the right peak indicates 68 Ga]Ga-DOTA-PEG7-Tz-TCO-16A. It can be seen that the reaction occurred successfully and the reaction product had a high radiochemical purity.

[0108] Example 11 This example is used to illustrate the PET imaging study of the click reaction between TCO-16A and 68 Ga]Ga-DOTA-PEG7-Tz in animals

[0109] Inject TCO-16A into mice with MUC1-overexpressing B16-OVA-MUC1 / B16-OVA-COSMCKO-MUC1 tumor models. After waiting for 30 min / 24 h, inject 68 Ga]Ga-DOTA-PEG7-Tz and perform imaging within 30 min ( Figure 13 ). The results show that at 30 min, obvious in vivo click reaction can be observed in the tumor, and the radioactive metabolism basically follows the metabolism law of small molecules, improving the imaging specificity and reducing the radiation risk at the same time.

[0110] The above results indicate that the TCO-16A probe is specifically taken up by MUC1-positive tumors and concentrated in the tumor site. After injecting 68 Ga]Ga-DOTA-PEG7-Tz with a targeting effect, 68 Ga]Ga-DOTA-PEG7-Tz reacts with TCO-16A by click reaction, making the tumor site labeled and imaged. This method optimizes the imaging time, and obvious radionuclide imaging can be observed within 30 min, greatly avoiding radiation damage.

[0111] The present invention realizes high-specific targeting of MUC1 protein by the binding of specific radionuclides (such as 89 Zr, 64 Cu, 177 Lu, etc.) and the 16A antibody. Through the optimization of specific bifunctional chelating agents (such as DFO, DOTA, NOTA, etc.) and labeling conditions, the labeling efficiency and stability of the probe are significantly improved. In addition, the present invention applies in vivo click chemistry (bioorthogonal) reaction to the radionuclide labeling and in vivo imaging of MUC1-targeted antibodies for the first time. By coupling trans-cyclooctene (d-TCO) to the 16A antibody and using 68 Ga, 68The in vivo click reaction of the DOTA / NOTA-tetrazine-polypeptide structure (DOTA / NOTA-PEG7-Tz) labeled with nuclides such as Cu achieved highly sensitive and specific imaging of tumors with high expression of MUC1, reduced the imaging background, optimized the imaging time, and significantly avoided radiation damage. The probe of the present invention can not only be used for highly sensitive and specific diagnosis of MUC1-related diseases, but also for precise localization, staging, and efficacy monitoring of tumor lesions, and has significant clinical application prospects.

[0112] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is also not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A MUC1 protein-targeting radionuclide probe system, characterized in that, The system includes a MUC1 antibody and a radionuclide; The radionuclide is directly labeled on the MUC1 antibody or through a bifunctional chelating agent, or the radionuclide labels the MUC1 antibody through a bioorthogonal system.

2. The MUC1 protein-targeted radionuclide probe system according to claim 1, wherein The MUC1 antibody is 16A, and its sequence is shown in SEQ ID NO:

1.

3. The MUC1 protein-targeted radionuclide probe system according to claim 1, wherein The radionuclide is a diagnostic radionuclide, and the diagnostic radionuclide is preferably 18 F. 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As、 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln、 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Second, 175 Lu, 177 Lu, 186 Re、 188 Re、 189 Re、 194 lr、 198 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 At least one of Ac; Alternatively, the radionuclide is a therapeutic radionuclide, and the therapeutic radionuclide is preferably 32 P, 47 Sc, 57 Co, 89 Sr, 90 Y, 103 Pd, 106 Ru, 124 I, 125 I, 131 I, 131 Cs, 137 Cs, 177 Lu, 192 Ir, 212 Bi, and 225 at least one of Ac.

4. The MUC1 protein-targeted radionuclide probe system according to claim 1, wherein, When the radionuclide labels the MUC1 antibody through a bioorthogonal system, the system includes: A targeting unit, which is a MUC1 antibody conjugated with a click moiety; A target-seeking unit, which is a click moiety target-seeking probe labeled with a radionuclide; The click moiety target-seeking probe can undergo a catalyst-free in vivo click chemical reaction with the click moiety.

5. The MUC1 protein-targeted radionuclide probe system according to claim 4, wherein, The click moiety target-seeking probe includes a click reaction part and optionally a bifunctional chelating agent part. The click reaction part is used to react with the click moiety, and the bifunctional chelating agent part is used to covalently connect with the click reaction part and chelate with the radionuclide.

6. The MUC1 protein-targeted radionuclide probe system according to claim 4, wherein, The click moiety is a trans-cyclooctene structure.

7. The MUC1 protein-targeted radionuclide probe system according to claim 6, wherein, The click reaction part is PEG7-Tz.

8. The MUC1 protein-targeting radionuclide probe system according to claim 1 or 5, wherein The bifunctional chelating agent is selected from at least one of DOTA, DOTP, DTPA, CB-DO2A, DO3A, NOTA, NODGA, NO2A, ΝΕΤΑ, DFO / df, TRITA, TETA, ATSM, HETA, EDTA, TACN-TM, HBED-CC, TPEN, BAPTA-AM, TRAP, CP256, PCTA, PTSM, Cyclen, porphyrin, polyamine, crown ether, dithiocarbazone, and polyoxime and their derivatives.

9. The MUC1 protein-targeted radionuclide probe system according to claim 7, wherein, The target-seeking unit is 68 Ga, 64 Cu, 89 Zr, 124 I, 125 I, 131 I or 177 Lu-labeled DOTA-PEG7-Tz, NOTA-PEG7-Tz or PEG7-Tz; preferably, the target-seeking unit is 68 Ga]Ga-DOTA-PEG7-Tz, 64 Cu]Cu-NOTA-PEG7-Tz, 177 Lu]Lu-DOTA-PEG7-Tz, 124 I]I-PEG7-Tz, 125 I]I-PEG7-Tz or 131 I]I-PEG7-Tz.

10. Any of the following applications of the MUC1 protein-targeting radionuclide probe system according to any one of claims 1-9: (1) Preparation of a MUC1-targeted imaging agent; (2) Preparation of a screening reagent for people in need of MUC1-targeted treatment; (3) Preparation of a tumor diagnostic reagent; the tumor diagnosis includes tumor staging, lesion localization, and efficacy monitoring; (4) Preparation of a tumor treatment drug.

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