PD-l1-targeting nuclide protein probe and use thereof

Abstract

A PD-L1-targeting nuclide protein probe and use thereof. Specifically, provided are a radionuclide-labeled PD-L1-targeting small protein probe and a preparation method therefor. The present invention is used for the diagnosis of malignant tumors, the precise localization of lesions, the monitoring of efficacy, and radioimmunotherapy.

Description

A radionuclide protein probe targeting PD-L1 and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, the present application relates to a radionuclide protein probe targeting PD-L1 and application thereof. BACKGROUND

[0002] Programmed death 1 (PD-1) / ligand (PD-L1) is expressed in various malignant tumors such as non-small cell lung cancer, triple-negative breast cancer, gastric cancer, esophageal cancer, glioma and malignant melanoma. Anti-PD-1 / PD-L1 pathway immunotherapy shows very good application prospect in the treatment of advanced solid tumors and becomes an important means of tumor treatment, but also shows certain limitations in the application process. It is generally believed that high PD-L1 expression level is related to the positive response rate of cancer treatment and clinical benefit of anti-PD-1 / anti-PD-L1 treatment. Therefore, it is of great significance to screen patients who have positive response to this treatment method according to the expression level of PD-L1 in the lesion site of patients before treatment. Accurate assessment of PD-L1 status of tumor patients is the key to improve the efficacy. In addition, there are problems of recurrence and drug resistance in targeted therapy, so individualization and precision of tumor treatment will become the most concerned problem in tumor targeted diagnosis and treatment.

[0003] Positron emission computed tomography (PET) and single photon emission computed tomography (SPECT) show advantages in the diagnosis and treatment of tumors. By tracing the changes of receptors in lesion tissues and the abnormalities of cell signal transduction, it provides basis for early diagnosis, clinical staging and efficacy evaluation of tumors, evaluates the prognosis, and can also be applied to targeted therapy of tumors.

[0004] Many studies have shown that PET / SPECT molecular imaging targeting PD-L1 can be used for lesion imaging of tumors and can be used for screening patients who respond to anti-PD-1 / anti-PD-L1 immunotherapy. PET imaging technology can non-invasively, quantitatively and in real time detect the PD-L1 expression in tumor patients by using molecular probes targeting PD-L1, thereby providing a possibility for screening patients suitable for anti-PD-1 / PD-L1 pathway tumor immunotherapy and efficacy monitoring.

[0005] Tumor radioimmunoimaging is to use specific antibodies or fragments thereof against tumor-related antigens with diagnostic radionuclides, such as 18 F、 45 Ti,52 Fe、 62 Cu、 64 Cu、 67 Ga、 68 Ga、 86 Y、 89 Zr、 94 Tc, 99m Tc, 111 ln、 124 I, 186 Re、 189 Re、 211 After being labeled with alpha (at) and injected into the human body, the radioactive nuclides travel through the bloodstream to the tumor tissue. There, they bind to tumor-related antigens, causing a localized concentration of radioactivity in the tumor tissue that exceeds that of normal tissue. Nuclear medicine imaging equipment is then used to qualitatively and locally visualize the primary lesion and metastases throughout the body, particularly enabling the detection of occult lesions that are difficult to identify using other diagnostic techniques. During decay, alpha or beta radionuclides can release alpha or beta rays, which have a strong ionizing radiation effect and a strong killing effect on tumor cells or abnormally proliferating tissues. Radionuclide therapy refers to the use of radioactive nuclides with therapeutic effects, such as... 32 P, 47 Sc、 67 Cu、 77 As、 90 Y、 89 Sr、 105 Rh、 111 Ag、 123 I, 125 I, 131 I, 142 Pr、 153 Sm、 166 Ho、 169 Er、 175 Lu、 177 Lu、 188 Re、 194 lr、 198 Au、 199 Au、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra、 225 Ac and other radionuclides or labeled drugs can be used to precisely kill diseased cells and tissues at close range, thereby achieving the purpose of treatment.

[0006] However, due to the large molecular weight (~150kDa) of traditional monoclonal antibodies, the antibodies labeled with radionuclides are cleared very slowly in vivo, resulting in a high background of radioactivity in normal tissues. This not only significantly reduces the tumor / non-tumor ratio (i.e., imaging contrast) but also increases the possibility of radiation damage to normal tissues.

[0007] Therefore, it is urgent to develop more PET and SPECT probes targeting PD-L1 with high specificity and high sensitivity, which can realize rapid, non-invasive, real-time and quantitative detection of PD-L1 expression in whole body lesions of tumor patients, obtain whole body quantitative information in vivo which cannot be obtained by conventional laboratory PD-L1 detection, and provide auxiliary detection means for clinical PD-1 / PD-L1 immunotherapy patient screening, efficacy monitoring and the like. SUMMARY

[0008] The purpose of the present application is to provide a radionuclide protein probe targeting PD-L1 and application thereof.

[0009] In a first aspect of the present application, a radionuclide conjugate is provided, which comprises:

[0010] (a) a small protein targeting PD-L1, the amino acid sequence of the small protein being shown as SEQ ID NO.: 3; and

[0011] (b) a conjugation moiety, the conjugation moiety being a radionuclide.

[0012] In another preferred embodiment, the radionuclide is a diagnostic or therapeutic radionuclide.

[0013] In another preferred embodiment, the radionuclide is selected from the group consisting of:

[0014] 68 Ga, 99m Tc and 177 Lu.

[0015] In another preferred embodiment, the radionuclide is 68 Ga.

[0016] In another preferred embodiment, the small protein and the conjugation moiety are connected by a linker.

[0017] In another preferred embodiment, the linker is selected from the group consisting of a polypeptide, a small molecule compound or a combination thereof.

[0018] In another preferred embodiment, the linker is a tricarbonyl compound.

[0019] In another preferred embodiment, the linker is a chelator of radionuclide.

[0020] In another preferred embodiment, the chelator of radionuclide is selected from the group consisting of:

[0021] DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid);

[0022] DOTP (di-2-ethylhexyl terephthalate);

[0023] DTPA (NR-diethylenetriaminepentacetic acid);

[0024] CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane);

[0025] DO3A (1,4,7,10-tetraazacyclododecane);

[0026] NOTA (1,4,7-triazacyclononane-1,4,7-acetic acid);

[0027] NODGA (1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid);

[0028] NO2A (1,4,7-triazacyclononane);

[0029] NETA ({4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid);

[0030] DFO / df (derferoxamine);

[0031] TRITA (1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid);

[0032] TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid);

[0033] ATSM (diacetyl-bis(N4-methylthiosemicarbazone));

[0034] HETA (1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid);

[0035] EDTA (ethylenediaminetetraacetic acid);

[0036] TACN-TM (N,N',N",tris(2-mercaptoethyl) 1,4,7-triazacyclononane);

[0037] HBED-CC (N,N'-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'- diacetic acid);

[0038] TPEN (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine);

[0039] BAPTA-AM (O,O'-Bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester);

[0040] TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]);

[0041] THP (tris(hydroxypyridinone));

[0042] RESCA ((±)H3 RESCA-Mal);

[0043] CP256;

[0044] PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid);

[0045] PTSM (Methyl p-toluenesulfonate);

[0046] Cyclen (1,4,7,10-tetraazacyclododecane);

[0047] porphyrin, polyamine, crown ether, dithiosemicarbazone, polyoxime and derivatives thereof.

[0048] In another preferred embodiment, the linker is selected from the group consisting of tricarbonyl compound, THP, RESCA, NOTA, DFO.

[0049] In another preferred embodiment, the conjugate contains a multivalent (e.g., divalent) small protein. The multivalent means that the small protein is repeated in the amino acid sequence of the conjugate.

[0050] In another preferred embodiment, the radionuclide conjugate has the structure of Formula I:

[0051] wherein,

[0052] Nb1is a small protein targeting PD-L1;

[0053] L is a linker;

[0054] R is a radionuclide.

[0055] In another preferred embodiment, the radionuclide conjugate has a structure selected from the group consisting of:

[0056] 99m Tc(CO)3(H2O)3-Nb1;

[0057] 68 Ga-THP-Nb1;

[0058] 68 Ga-NOTA-Nb1;

[0059] 18 F-RESCA-Nb1;

[0060] 177 Lu-DOTA-Nb1; and

[0061] wherein,

[0062] 99m Tc, 68 Ga, 18 F and 177 Lu is a radionuclide;

[0063] Nb1is the small protein targeting PD-L1;

[0064] (CO)3(H2O)3 is the tricarbonyl compound intermediate;

[0065] THP, RESCA, NOTA, DOTA are chelating agents as described above.

[0066] In another preferred embodiment, the conjugate is used for detecting PD-L1 expression in prognosis, diagnosis of cancer.

[0067] In another preferred embodiment, the detection is in vivo.

[0068] In another preferred embodiment, the conjugate is used for treating or preventing tumors expressing PD-L1 protein (i.e. PD-L1 positive).

[0069] In a second aspect of the present application, there is provided a PD-L1 protein detection reagent, comprising the conjugate as described in the first aspect of the present application and a detection-acceptable carrier.

[0070] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracer, contrast agent, flow detection reagent, cellular immunofluorescence detection reagent, nano-magnetic particle and imaging agent.

[0071] In another preferred embodiment, the detection reagent is a contrast agent, and the contrast agent further comprises other agents for contrast.

[0072] In another preferred embodiment, the contrast agent is a contrast agent for MRI (magnetic resonance imaging) or CT (computed tomography).

[0073] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0074] In another preferred embodiment, the dosage form of the detection reagent is a liquid dosage form.

[0075] In a third aspect of the present application, there is provided a pharmaceutical composition, comprising the conjugate as described in the first aspect of the present application and a pharmaceutically acceptable carrier.

[0076] In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating tumors, such as cytotoxic drugs.

[0077] In another preferred embodiment, the pharmaceutical composition is used for treating or preventing tumors expressing PD-L1 protein (i.e. PD-L1 positive).

[0078] In another preferred embodiment, the tumor comprises: gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, or adrenal gland tumor.

[0079] In another preferred embodiment, the pharmaceutical composition is in an injectable form.

[0080] In a fourth aspect of the present application, a kit for detecting PD-L1 molecule is provided, which comprises the conjugate according to the first aspect of the present application or the PD-L1 protein detection reagent according to the second aspect of the present application.

[0081] In another preferred embodiment, the kit further comprises an instruction, which records that the kit is used for non-invasively detecting PD-L1 expression of a subject.

[0082] In another preferred embodiment, the kit is used for detecting a tumor expressing PD-L1 protein (i.e. PD-L1 positive).

[0083] In a fifth aspect of the present application, a use of the conjugate according to the first aspect of the present application for preparing (a) a detection reagent, a kit or a detection plate for detecting PD-L1 molecule in vivo; (b) a pharmaceutical composition for treating or preventing a tumor expressing PD-L1 protein (i.e. PD-L1 positive) is provided.

[0084] In another preferred embodiment, the conjugate is used for preparing a contrast agent for detecting PD-L1 molecule in vivo.

[0085] In a fifth aspect of the present application, a use of the conjugate according to the first aspect of the present application for preparing (a) a detection reagent, a kit or a detection plate for detecting PD-L1 molecule in vivo; (b) a pharmaceutical composition for treating or preventing a tumor expressing PD-L1 protein (i.e. PD-L1 positive) is provided.

[0086] In another preferred embodiment, the conjugate is used for preparing a contrast agent for detecting PD-L1 molecule in vivo.

[0087] In a sixth aspect of the present application, a conjugate precursor is provided, which comprises a small protein targeting PD-L1 with an amino acid sequence as shown in SEQ ID NO: 3; and a linker moiety.

[0088] In another preferred embodiment, the linker is selected from the group consisting of: a polypeptide, a small molecule compound or a combination thereof.

[0089] In another preferred embodiment, the linker is a chelator of a radionuclide.

[0090] In another preferred embodiment, the linker is selected from the group consisting of triscarbonyl compound, THP, RESCA, NOTA.

[0091] In a seventh aspect of the present application, there is provided a method for preparing the radionuclide conjugate as described in the first aspect of the present application, the method comprising the steps of:

[0092] reacting the conjugate precursor as described in the sixth aspect of the present application and a radionuclide under suitable conditions, thereby obtaining the radionuclide conjugate.

[0093] In another preferred embodiment, the suitable conditions comprise:

[0094] i) temperature conditions of 0-60 °C; and / or

[0095] ii) pH 4.0-6.0.

[0096] In another preferred embodiment, the method has a radionuclide labeling efficiency of > 95%, preferably > 99%.

[0097] In an eighth aspect of the present application, there is provided a method for treating a disease, the method comprising administering to a subject in need thereof the conjugate as described in the first aspect of the present application or the pharmaceutical composition as described in the third aspect of the present application.

[0098] In another preferred embodiment, the subject comprises a mammal, such as a human.

[0099] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0100] The following drawings are used to illustrate specific embodiments of the present application and are not used to limit the scope of the present application as defined by the claims.

[0101] Figure 1 shows a schematic diagram of the synthesis of the radionuclide-labeled targeting PD-L1 small protein

[0102] Figure 2 shows the determination by Radio-TLC 68 Ga-THP-Nb1 labeling rate and radiochemical purity.

[0103] Figure 3 shows 68 Ga-THP-Nb1 in A549 PDL1 tumor-bearing mice.

[0104] Figure 4 shows 68 Ga-THP-Nb1 in A549 and A549 PDL1The tumor location of the tumor-bearing mouse model is shown by arrows. DETAILED DESCRIPTION

[0105] After extensive and in-depth research, the present inventors provide a radionuclide-labeled small protein targeting PD-L1 and a preparation method and application thereof. The present application couples a super-high affinity small protein targeting PD-L1 with a radionuclide, and constructs a tumor PD-L1 surface imaging tracer probe by taking advantage of the characteristics of the small protein, i.e., smaller molecular weight and better tumor penetration than traditional antibodies. Experiments show that the PD-L1 surface imaging tracer probe of the present application can accurately locate PD-L1 positive tumors in animals. On this basis, the present application is completed.

[0106] The present application performs sequence modification on the existing super-high affinity small protein targeting PD-L1, and adds a specially designed sequence at the C-terminus of the small protein. Not only does it retain the super-high affinity of the small protein to PD-L1, but also enables it to be coupled with the maleimide group of (±)THP-Mal with high efficiency, thereby obtaining the product 68 The radiochemical purity of Ga-THP-Nb1 after purification is more than 99%.

[0107] In particular, the small protein conjugate of the present application 68 Ga-THP-Nb1 can be labeled and prepared at room temperature to a temperature as high as 60°C, and compared with other radionuclides or linkers (such as 89 Zr-DFO), the coupling efficiency is higher, and the finished product is more stable.

[0108] Terminology

[0109] In order that the application can be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the application pertains. Before the present application is described, it is to be understood that this application is not limited to the particular methodologies and experimental conditions described, as such methodologies and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the application will be limited only by the appended claims.

[0110] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0111] As used herein, the terms "comprise", "comprising", "containing" and "containing" are used interchangeably and include both closed and open-ended definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0112] High-affinity small protein targeting PD-L1

[0113] The radionuclide conjugate of the present application comprises a high-affinity small protein targeting PD-L1.

[0114] The main advantages of high-affinity small proteins relative to monoclonal antibodies are:

[0115] (1) High affinity and high specificity, the affinity is 10-100 times higher than that of polypeptides, close to that of monoclonal antibodies;

[0116] (2) Very low immunogenicity and toxicity, and not easy to stick like scFv;

[0117] (3) Good tissue penetration, can fully bind to target organs or tumor tissues;

[0118] (4) Small relative molecular mass, unbound parts can be quickly cleared by the kidney, reducing the background interference in the blood;

[0119] (5) The high-affinity small protein can be used for room temperature labeling, and also can be used for labeling at temperatures up to 60°C and above.

[0120] (6) Using modern genetic engineering technology, high-yield protein probes can be obtained, and the structure can be modified or modified, which is more conducive to molecular level image detection.

[0121] Radionuclide

[0122] DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTP (di-2- ethylhexyl terephthalate), DTPA (NR-diethylenetriaminepentacetic acid), CB-DO2A (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), NETA ({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), THP (tris(hydroxypyridinone)), RESCA ((±)H3 RESCA-Mal), porphyrin, polyamine, crown ether, dithiosemicarbazone, polyoxime and its derivatives, etc. after modification, can be diagnostic radionuclide, 99m Tc, 68 Ga, 18 F or therapeutic radionuclide 177 Lu, etc. labeled.

[0123] 99m Tc is the most widely used single-photon radionuclide, which is prepared by 99 Mo- 99m Tc generator, and the cost of SPECT examination is cheap and easy to promote.

[0124] 68 Ga is a widely used positron emitting radionuclide, and the radionuclide 68 Ga is prepared by 68 Ge- 68 Ga generator, and the source is convenient, 68 Ga is easy to coordinate with compounds, and the labeling operation method is relatively simple; compared with the cyclotron, the radioactivity of the elution labeled 68 Ga is relatively small, which is convenient for quality control at any time; and 68 Ga has a short half-life, which can diagnose diseases non-invasively in real time and obtain whole-body metabolic images.

[0125] 90 Y and 177 Lu is a beta radionuclide, 90Y has higher energy (2280 keV) and stronger penetration ability (12 mm), and has stronger killing effect on tumor cells or abnormal proliferative tissues, etc. 90 Y has higher energy (2280 keV) and stronger penetration ability (12 mm), and has stronger killing effect on tumor cells or abnormal proliferative tissues, etc. 177 Lu can emit small ion range β rays (0.5 MeV), which can not only ensure that the ray energy is transmitted to the tumor area, but also reduce the damage to the surrounding normal tissues.

[0126] In one embodiment, the conjugate of the present application comprises a radionuclide 68 Ga.

[0127] The conjugate of the present application

[0128] As used herein, the terms "radionuclide conjugate", "radionuclide conjugate of the present application", "conjugate of the present application", "PD-L1 imaging tracer probe" are used interchangeably, and all refer to the radionuclide conjugate of the first aspect of the present application.

[0129] The radionuclide conjugate of the present application comprises:

[0130] (a) a small protein targeting PD-L1, the amino acid sequence of which is shown as SEQ ID NO.: 3; and

[0131] (b) a conjugation moiety, which is a radionuclide.

[0132] In another preferred embodiment, the radionuclide is a diagnostic or therapeutic radionuclide.

[0133] In another preferred embodiment, the radionuclide is selected from the group consisting of: 68 Ga, 99m Tc and 177 Lu.

[0134] In another preferred embodiment, the small protein and the conjugation moiety are connected by a linker; preferably, the linker is selected from the group consisting of THP, RESCA, NOTA shown above.

[0135] In another preferred embodiment, the radionuclide conjugate has a structure selected from the group consisting of:

[0136] 99m Tc(CO)3(H2O)3-Nb1;

[0137] 68 Ga-THP-Nb1;

[0138] 68 Ga-NOTA-Nb1;

[0139] 18 F-RESCA-Nb1;

[0140] 177 Lu-DOTA-Nb1

[0141] wherein,

[0142] 99m Tc、 68 Ga、 18 F、 177 Lu is a radionuclide;

[0143] Nb1 is the small protein targeting PD-L1;

[0144] (CO)3(H2O)3 is a tricarbonyl compound as described above;

[0145] THP, RESCA, NOTA are chelating agents as described above.

[0146] In another preferred embodiment, the structure of the radionuclide conjugate is as shown in 68 Ga-THP-Nb1.

[0147] Preparation method of the conjugate

[0148] According to the type of the labeled radionuclide, the labeling of the radionuclide can include the following steps:

[0149] (1) 99m Tc labeling method of PD-L1 targeting Nb1

[0150] Into a 10 mL penicillin vial, 5 mg Na2CO3, 10 mg NaBH4, 15 mg potassium sodium tartrate are added, 1 mL of normal saline is added, shaken well, vacuumed with a 10 mL disposable needle tube, and carbon monoxide gas is introduced, the air in the bottle is exhausted, and then 2 mL of Na 99 mTcO4 eluent (about 13 mCi) is injected, heated at 80°C for 30 min (carbon monoxide gas is introduced during the reaction process), cooled, and the intermediate 99m Tc(CO)3(H2O)3) is obtained. + The small protein Nb1 is prepared at a concentration of 1 mg / mL, 200 μL is added to 1 mL (about 2-3 mCi) of prepared 99m Tc(CO)3(H2O)3, the pH is adjusted to 8 with hydrochloric acid, and the reaction is carried out in an oil bath at 80°C for 30 min, then 1 mL of 0.8M NaOH solution is added and reacted for 30 min, after the reaction is completed, the pH is adjusted to acid with 1M HCl, distilled under reduced pressure, dissolved in an appropriate amount of methanol; add a 0.22 μm microporous filter membrane, add 2.0 mL of normal saline to the system, filter the product into a sterile vacuum bottle, and the product radioactivity is obtained.99m Tc] 99m Tc(CO)3(H2O)3-Nb1; the preferred [ 99m Tc] 99m Tc(CO)3(H2O)3-Nb1 has a radiochemical purity greater than 95%;

[0151] (2) 68 Ga-labeled PD-L1 targeting Nb1 method:

[0152] ① 68 Ga-labeled THP-Nb1

[0153] The precursor THP-Nb1 (200 μg) with a concentration of 2–3 mg / mL was rapidly dissolved and then added to 300 μL of 2.0 mol / L sodium acetate solution, followed by 1 mL of [other solution]. 68 Ga eluent (259-740 MBq), reaction system pH = 5.0-6.5, mixed well and reacted at 0-60℃ for 15 min. A 0.22 μm microporous membrane was added, and 2.0 mL of physiological saline was added to the system. The product was filtered into a sterile vacuum bottle to obtain the radioactive product. 68 Ga-THP-Nb1; Optimally obtained 68 Ga-THP-Nb1 has a radiochemical purity greater than 95%;

[0154] ② 68 Ga-marked NOTA-Nb1

[0155] Wash with 4 mL of 0.05 M HCl 68 Ge / 68 The Ga generator was used to collect the eluent in 1.5 mL EP tubes, with the middle 1 mL of eluent transferred to a new 1.5 mL EP tube. 65 μL (1.0 M) sodium acetate solution and 200 μg NOTA-Nb1 were added to the EP tubes, mixed thoroughly, and the pH of the reaction system was adjusted to 4.0-5.0. The reaction was incubated at 0-60 °C for 15 min. After purification using a PD-10 column, a 0.22 μm microporous membrane was added, and 2.0 mL of physiological saline was added to the system. The product was then filtered into a sterile vacuum bottle to obtain the target product. 68 Ga-NOTA-Nb1, the preferred choice 68 The radiochemical purity of Ga-NOTA-Nb1 is greater than 95%;

[0156] (3) 18 F-labeled PD-L1 targeting Nb1 method:

[0157] 100 μL of physiological saline 18F elution (about 1110 MBq), 4 μL of 2 mmol / L AlCl3 solution was added, and the reaction was carried out for 5 min, 11 μL of KHP (pH = 4.0) solution was added, about 100 μL of RESCA-Nb1 solution was added, the pH of the reaction system was 4.6-4.8, and the reaction liquid was evenly divided into three parts, and comparative experiments were carried out at 37℃, 60℃ and 80℃, respectively; the reaction was carried out for 15 min, and the reaction time was appropriately shortened, but it was not less than 12 min. The purified product was added with 1-2 mL of normal saline, mixed, and filtered through a 0.22 μm sterile needle filter into a sterile vacuum bottle, to obtain 18 F-RESCA-Nb1, preferably obtained 18 The radiochemical purity of F-RESCA-Nb1 is greater than 95%.

[0158] (4) 177 Lu-labeled PD-L1 targeting Nb1 method:

[0159] 100 μL of NaAc / HAc (pH = 5.5) solution was added to the labeling precursor DOTA-Nb1 / NOTA-Nb1, 370 MBq of 177 LuCl3 solution was reacted at 37℃ for 30 min. The purified product was added with 1-2 mL of normal saline, mixed, and filtered through a 0.22 μm sterile needle filter into a sterile vacuum bottle, to obtain 177 Lu-DOTA-Nb1, preferably obtained 177 The radiochemical purity of Lu-DOTA-Nb1 is greater than 95%.

[0160] The main advantages of the present application include:

[0161] (1) The radionuclide protein probe preparation process of the present application is simple, convenient to operate, time-saving, high labeling rate, stable label, and convenient for further application in clinical, scientific research and drug development.

[0162] (2) The present application provides a visualization tool for tumor PD-L1 expression detection and efficacy monitoring. It can be used for screening, treatment prediction and efficacy monitoring of PD-L1 positive tumor patients.

[0163] (3) The radiolabeled probe of the present application belongs to a small protein labeled compound, which has small molecular weight, low immunogenicity, good tissue penetration ability and high affinity for tumor tissue.

[0164] (4) The present application is screened to find that the small protein conjugate 68 Ga-THP-Nb1 can be labeled and prepared at room temperature to a temperature as high as 60℃, and other radionuclides or linkers (such as 89Compared with Zr-DFO, the coupling efficiency is higher and the finished product is more stable.

[0165] The application will be further described below in connection with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0166] Example 1 Expression and purification of the small protein Nb1 targeting PD-L1

[0167] (1) Plasmid synthesis

[0168] The E. coli expression vector used in this study is pET29b. The following nucleotide sequence (SEQ ID NO: 2) is inserted into the recombinant plasmid by selecting NdeI and XhoI enzyme cutting sites in a conventional method. MSG is added at the N-terminus of the sequence, wherein M is the start codon; GSCGSENLYFQSLEHHHHHH (SEQ ID NO: 5) sequence is added at the C-terminus of the sequence, wherein ENLYFQS sequence (SEQ ID NO: 6) is used as a cutting site, and HHHHHH (SEQ ID NO: 7) tag can be used for nickel column protein purification.

[0169] Amino acid sequence of the high-affinity small protein targeting PD-L1 (SEQ ID NO: 1)

[0170] DRERARELARILLKVIKLSDSPEARRQLLRNLEELAEKYKDPEVRRILEE AERYIK

[0171] Nucleotide sequence of the high-affinity small protein targeting PD-L1 (SEQ ID NO: 2)

[0172] The amino acid sequence (SEQ ID NO: 3) is as follows:

[0173] The nucleotide sequence (SEQ ID NO: 4) is as follows:

[0174] (2) Plasmid transformation

[0175] After the construction of the small protein recombinant plasmid, the full-length gene was sequenced to verify that there was no error, and then the plasmid was transformed into E. coli expression strain lemo21 (purchased from Beijing Zhuangmeng International Biogene Co., Ltd.). Take 100 μL of competent cells lemo21 and thaw on ice. Dissolve the freeze-dried expression plasmid in ddH2O to make its concentration 25 ng / μL. Take 3 μL of plasmid and add it to the competent cells, and place it on ice for 30 min. Place the competent cells in a 42°C water bath for 60-90 s, then quickly place them on ice for 2 min. Then add 500 μL of LB liquid medium (without resistance) to the mixture, shake at 37°C, 220 rpm for 45 min. After the time, take 300 μL of medium and add it to a 10 cm ampicillin-resistant LB culture plate, add plate coating beads and shake in the cross direction for 10 times, and pour out the plate coating beads. Place the plate upside down in a 37°C incubator and incubate overnight.

[0176] (3) Induction expression and purification

[0177] After successful transformation, single colonies were picked for protein induction expression, as follows:

[0178] 1. Small shaking: use a sterile gun head to pick a single colony and punch into a shaking tube containing 5 mL of LB medium containing kanamycin (100 μg / mL), and incubate at 37°C, 220 rpm overnight.

[0179] 2. Large shaking: add 5 mL of medium to 1 L of LB medium containing kanamycin (100 μg / mL). Incubate at 37°C, 220 rpm for 1-2 h. After 1 h, measure OD600 every 30 min with a Nanodrop.

[0180] 3. Induction: OD 600 When the OD is about 0.6-0.9, add IPTG inducer to the large shaking liquid to a final concentration of 1 mmol / L. Incubate at 16°C, 180 rpm for 14-16 h.

[0181] 4. Collect the bacterial cells: after the induction is completed, pour the bacterial liquid into a 250 mL centrifuge bottle, centrifuge at 9000 rpm for 20 min, and discard the supernatant. The bacterial cell precipitate can be obtained.

[0182] 5. Ultrasonic lysis: add 50 mL of lysis buffer to resuspend the bacterial cells, and then add protease inhibitor (100 μg / mL PMSF). After mixing well on a shaker, pour into a small beaker. Fix the beaker in a 0°C ice-water mixture, and use an ultrasonic disrupter to break the bacterial cells. The ultrasonic conditions are: power 150 w, ultrasonic 6 s, stop 6 s, working time 1 h. After ultrasonic treatment, collect the bacterial liquid in a centrifuge tube and centrifuge at 9000 rpm for 20 min. Collect the supernatant for use.

[0183] 6. Protein column: 1 mL of nickel beads was added to 10 mL of an empty affinity column, and the ethanol preservative was naturally flowed out by gravity, and then 10 mL of PBS was added to flush the nickel column to ensure that the ethanol was completely removed. After adding 1 mL of PBS to resuspend the nickel beads, they were added to the supernatant of the ultrasonic lysis, and the column was rotated in a 4°C shaking incubator for 2 h.

[0184] 7. Nickel column purification: The mixture of the lysis supernatant and the nickel beads was added to the empty affinity column, a rubber tube was connected to the liquid outlet of the affinity column, and a peristaltic pump was used to pump the flow-through liquid into a centrifuge tube at a constant flow rate. After three such flow-throughs, the supernatant was discarded. After elution with 20 mL of eluent (50 mmol / L Tris HCl), 20 mL of eluent containing 20 mmol / L imidazole, 10 mL of eluent containing 50 mmol / L imidazole, and 1 mL of eluent containing 100 mmol / L imidazole, 4 mL of eluent containing 500 mmol / L imidazole was added to the nickel beads, mixed well, and allowed to stand for 5 min. The liquid was collected by gravity. This process was repeated once, and a total of 8 mL of eluent was collected. The collected eluent was added to a 3K dialysis bag, which was placed in a beaker containing PBS solution, a magnetic rotor was added, and the dialysis bag was dialyzed overnight on a magnetic stirrer in a 4°C incubator.

[0185] 8. Molecular exclusion chromatography purification: The protein solution after dialysis was added to a 3K ultrafiltration tube, and ultrafiltration concentration was performed at 4500 rpm and 4°C. After centrifugation to a final concentration of 10 mg / mL, Superdex75 Increase molecular exclusion chromatography was used for purification. An Agilent 1260 high-performance liquid chromatography system was used, with PBS solution as the mobile phase and a flow rate of 0.8 mL / min. The purified protein solution was stored in a -80°C refrigerator after the concentration was determined by Nanodrop.

[0186] The amino acid protein sequence obtained in this example is shown in SEQ ID NO: 3, i.e., the Nb1 sequence of the application. The MSG added at the N-terminus of the sequence and the GSCGSENLYFQSLEHHHHHH sequence added at the C-terminus of the sequence are removed, as shown in SEQ ID NO: 1.

[0187] The molecular structure was characterized by mass spectrometry and HPLC; the HPLC analysis conditions were as follows: Superdex75 Increase 10 / 300 gel filtration / volume exclusion chromatography column, flow rate 0.8 mL / min; mobile phase A was 0.1 mol / L PBS solution; mobile phase gradient setting: 0.0-30.0 min 100% A; the Nb1 structure had good stability, pharmacokinetic properties, and target specificity.

[0188] Example 268 Ga elution

[0189] Take 4mL 0.05mol / L HCl to elute the germanium-gallium generator with a syringe, the elution speed is 1-2mL / min, the first 1mL HCl is discarded, collect the remaining 3mL eluent, and record the radioactivity.

[0190] Example 3 Tricarbonyl compound intermediate 99m Tc(CO)3(H2O)3] + Preparation

[0191] Add 5mg Na2CO3, 10mg NaBH4, 15mg potassium sodium tartrate to a 10mL penicillin vial, add 1mL normal saline to it, shake well, vacuum with a 10mL disposable needle tube, and introduce carbon monoxide gas, exhaust the air in the bottle, then inject 2mL Na 99 mTcO4 eluent (about 13mCi), continue to heat at 80℃ oil bath for 30min (carbon monoxide gas is introduced during the reaction process), cool, and obtain the intermediate 99m Tc(CO)3(H2O)3] + , for later use.

[0192] Example 4 Preparation of precursor THP-Nb1

[0193] According to the molar ratio of purified Nb1 (SEQ ID No: 3) to Mal-THP of 1:10, weigh Mal-THP solid powder into a 1.5mL EP tube. Add the concentrated Nb1 solution, mix and shake uniformly, and then place it in an incubator for reaction at 37℃ for 2h. After the reaction is completed, use PD-10 column to purify with 25mL PBS. Add 1-1.5mL of the reacted solution, supplement with PBS to 2.5mL, and flow dry. Add another 2.5mL PBS, collect 5 tubes of effluent, about 0.5mL per tube. Combine the tubes with higher concentration to obtain THP-Nb1, and store it in the -80℃ refrigerator at 200μg / tube.

[0194] Example 5 Preparation of precursor NOTA-Nb1

[0195] Take 0.5-1.0 mL of Nb1 (SEQ ID No: 3) solution into an EP tube, then add about 10-20 times the molar amount of NOTA-DMSO solution (10 mmol / L), and react at 37°C for 1 h, with gentle shaking of the reaction tube every 10-15 min. Purify using a PD-10 column equilibrated with 25 mL of PBS. Add 1-1.5 mL of the reacted solution, make up with PBS to 2.5 mL, and let it flow through. Add another 2.5 mL of PBS, and collect 5 tubes of effluent, about 0.5 mL per tube. Combine the tubes with higher concentrations, and divide into 200 μg / tube, and store in a -80°C refrigerator.

[0196] Example 6 Preparation of precursor RESCA-Nb1

[0197] According to the molar ratio of Mal-RESCA to Nb1 (SEQ ID No: 3) of 1:10, weigh Mal-RESCA solid powder into a 1.5 mL EP tube, pipette Nb1 solution into the reaction tube, shake to mix, add NaHCO3 / Na2CO3 buffer solution to adjust the pH to 6.5-7.5, and place in an incubator at 37°C for 1 h. After the reaction is complete, filter the reaction solution through a 0.22 μm water-phase needle filter, and purify by HPLC system to obtain the precursor. The purification chromatographic column is Superdex75 Increase 10 / 300, the detection wavelength is 210 nm and 280 nm, the sample injection amount is 500 μL, and the flow rate is 0.8 mL / min. The molecular weight of RESCA-Nb1 is detected by MALDI-TOF. The precursor sample is divided into 200 μg, and stored in a -80°C refrigerator.

[0198] Prepare the precursor DOTA-Nb1 according to the method of Example 4.

[0199] Example 7 Preparation of DFO-Nb1

[0200] Put in 0.5 mg of small protein Nb1 (SEQ ID No: 3), and according to the molar ratio of DFO to Nb1 of 1:12, pipette liquid into a 1.5 mL EP tube, and mix. Add NaHCO3 / Na2CO3 buffer solution to adjust the pH to 9.0. React in a 37°C oven for 60 min, shake once every 10 min, and after the reaction is complete, purify using a PD-10 column equilibrated with Metal-free PBS 5 times, add 1-1.5 mL of the reacted solution, make up with PBS to 2.5 mL, and let it flow through. Add another 2.5 mL of PBS, and collect 5 tubes of effluent, about 0.5 mL per tube. Combine the tubes with higher concentrations to obtain DFO-Nb1, and divide into 200 μg / tube, and store in a -80°C refrigerator.

[0201] Example 8 68Preparation of Ga-THP-Nb1

[0202] The aliquoted precursor THP-Nb1 (200 μg) was taken out from -80 °C freezer, and was quickly dissolved in 300 μL of 2.0 mol / L sodium acetate solution, and then 1 mL of 0.9% NaCl was added. The solution was mixed well and was divided into two 1.5 mL EP tubes, which were placed in 37 °C and 60 °C respectively for 15 min. The reaction solution was purified by PD-10 column, and then 0.22 μm microporous filter was added. 2.0 mL of physiological saline was added to the system, and the product was filtered into a sterile vacuum bottle. The target product Ga-THP-Nb1 was obtained. 68 Ga (259-740 MBq) freshly eluted in step (1) 68 The reaction system pH was 5.0-6.5, and was mixed well and was divided into two 1.5 mL EP tubes, which were placed in 37 °C and 60 °C respectively for 15 min. The reaction solution was purified by PD-10 column, and then 0.22 μm microporous filter was added. 2.0 mL of physiological saline was added to the system, and the product was filtered into a sterile vacuum bottle. The target product Ga-THP-Nb1 was obtained. 68 Ga-THP-Nb1, the process is shown in Figure 1.

[0203] Example 9 68 Preparation of Ga-NOTA-Nb1

[0204] The aliquoted precursor NOTA-Nb1 (200 μg) was taken out from -80 °C freezer, and was quickly dissolved in 300 μL of 2.0 mol / L sodium acetate solution, and then 1 mL of 0.9% NaCl was added. The solution was mixed well and was divided into two 1.5 mL EP tubes, which were placed in 37 °C and 60 °C respectively for 15 min. The reaction solution was purified by PD-10 column, and then 0.22 μm microporous filter was added. 2.0 mL of physiological saline was added to the system, and the product was filtered into a sterile vacuum bottle. The target product Ga-THP-Nb1 was obtained. 68 Ga-NOTA-Nb1.

[0205] Example 10 68 Labeling rate, radiochemical purity and quality control of Ga-THP-Nb1

[0206] iTLC: After the reaction, 2 μL of the reaction solution was spotted onto an iTLC-SG strip, developed with physiological saline, and the labeling rate was analyzed by scanning on Radio-TLC. After purification, 2 μL of the reaction solution from 1-2 tubes with high activity was spotted onto an iTLC-SG strip, developed with physiological saline, and the radiochemical purity was analyzed by scanning on Radio-TLC. 100 μL of PBS was added to a 1.5 mL EP tube, and 100 μL of the reaction solution was added to the same tube. In vitro stability was measured at 0.5 h, 1 h, 2 h, and 4 h. 100 μL of 5% HSA was added to another tube, and 100 μL of the reaction solution was added to the same tube. In vitro stability was measured at 0 h and 4 h. The resulting product had a labeling rate >95%, radiochemical purity >99%, and in vitro stability maintained above 95%, as shown in Figure 2.

[0207] Example 11 [ 99m Tc] 99m Preparation of Tc(CO)3(H2O)3-Nb1

[0208] Prepare a 1 mg / mL concentration of the nanobody Nb1, and add 200 μL of the solution to 1 mL (approximately 2-3 mCi) of the prepared solution. 99m Tc(CO)3(H2O)3 was reacted with hydrochloric acid to adjust the pH to 8, and then in an oil bath at 80°C for 30 min. 1 mL of 0.8 M NaOH solution was then added, and the reaction continued for another 30 min. After the reaction, the pH was adjusted to acidic with 1 M HCl, and the mixture was distilled under reduced pressure and dissolved in an appropriate amount of methanol. A 0.22 μm microporous membrane was added, and 2.0 mL of physiological saline was added to the system. The product was then filtered into a sterile vacuum bottle to obtain the final product. 99m Tc] 99m Tc(CO)3(H2O)3-Nb1.

[0209] Example 12 177 Preparation of Lu-DOTA-Nb1

[0210] 100 μL of NaAc / Hac (pH = 5.5) solution was sequentially added to the labeled precursor DOTA-Nb1 / NOTA-Nb1 (200 μg), followed by 370 MBq of [a solution / concentration]. 177 LuCl3 solution, reacted at 37℃ for 30 min, yielded 177 Lu-DOTA-Nb1 has a radiochemical purity greater than 80%. It was purified using a PD-10 size exclusion column pretreated with PBS solution. The purified product was then added to 1–2 mL of physiological saline, mixed thoroughly, and filtered through a 0.22 μm sterile needle filter into a sterile vacuum bottle to obtain the final product.

[0211] Example 13 18 Preparation of F-RESCA-Nb1

[0212] Take 400 μL of physiological saline 18 Add F eluent (approximately 1110 MBq) to 6 μL of 2 mmol / L AlCl3 solution and react for 5 min. Then add 11 μL of 0.5 mol / L KHP (pH = 4.0) solution and 500 μL of RESCA-Nb1 solution. Adjust the system pH to 4.6–4.8 and react at 37°C for 15 min. Purify the product using a size exclusion column (PD-10) pretreated with PBS. Add 1–2 mL of physiological saline to the purified product, mix well, and filter through a 0.22 μm sterile syringe filter into a sterile vacuum bottle to obtain the final product.

[0213] Example 14 68 Biodistribution of Ga-THP-Nb1 in mouse tumor models

[0214] Nude mice were subcutaneously inoculated with A549 cells on the left side of the forelimb axilla and on the right side. PD-L1 Cells, the tumor grew to about 100 mm after 6-8 days. 3 (n=4). 0.2 mL (7.4 MBq) was injected into the tail vein of mice. 68 Mice were sacrificed after 1 hour of Ga-THP-Nb1 administration. Blood, brain, heart, liver, spleen, lung, kidney, stomach, intestine, pancreas, flesh, and tumor tissues were collected. After weighing, radioactivity was measured using a gamma counter, and the uptake (%ID / g) of each organ and tissue was calculated. Experimental results are expressed as mean ± SD. Injection 68 Figure 3 shows the radioactive uptake in various organs after Ga-THP-Nb1 treatment. 68 Ga-THP-Nb1 is mainly metabolized by the kidneys, and its metabolism is normal with a rapid clearance rate, demonstrating the probe's targeting ability for PD-L1.

[0215] Example 15 68 Imaging of Ga-THP-Nb1 mouse tumor model

[0216] Nude mice were subcutaneously inoculated with A549 cells on the right side of their hind legs, and A549 cells were inoculated on the right side of their legs. PD-L1 Cells, the tumor grew to about 100 mm after 6-8 days. 3 (n=4). 0.2 mL (7.4 MBq) was injected into the tail vein of mice. 68 Ga-THP-Nb1 was subjected to Micro-PET imaging 1 hour later. The whole-body decay-corrected coronal region of interest (ROI) obtained from the Micro-PET scan was reconstructed. The results are shown in Figure 4; positive tumor tissue in mice showed high uptake, while negative tumor tissue showed low uptake. This probe is metabolized rapidly and has a high target-to-probe ratio, indicating that this marker has good specificity and affinity, making it a promising tumor imaging agent.

[0217] Comparative Example 1 89 Preparation of Zr-DFO-Nb1

[0218] Take 70 μL (780 μCi) oxalic acid 89 Zr, add 10 times HEPES-Na2CO3buffer solution, mix well; after adding DFO-Nb1, adjust pH to 7.0 with oxalic acid. Place in constant temperature incubator, react at 37°C for 60 min. Perform intermediate control at 30 min, detect labeling condition with iTLC; after reaction, purify using equilibrated PD-10 column, add 0.22 μm microporous filter membrane, add 2.0 mL normal saline to the system, filter the product into a sterile vacuum bottle to obtain the target product 89 Zr-DFO-Nb1. Take 2 μL sample and spot on iTLC-SG paper strip to show radiochemical purity. The obtained product has a labeling rate < 50%, radiochemical purity < 50%, and poor labeling effect.

[0219] All the documents mentioned in the present application are cited as references in the present application as if each document is cited as a reference individually. In addition, it should be understood that various modifications or changes can be made to the present application by those skilled in the art after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A radionuclide conjugate, characterized in that, The radionuclide conjugate comprises: (a) a small protein targeting PD-L1, the amino acid sequence of the small protein is shown as SEQ ID NO.: 3; and (b) a coupling moiety, said coupling moiety being a radionuclide, said radionuclide being selected from the group consisting of: 68 G, 99m Tc and 177 Lu.

2. The radionuclide conjugate of claim 1, wherein, The radionuclide is 68 G.

3. The radionuclide conjugate of claim 1, wherein, The small protein and the conjugated part are connected by a linker, and the linker is selected from the group consisting of a polypeptide, a small molecule compound, or a combination thereof.

4. The radionuclide conjugate of claim 3, wherein, The linker is selected from the group consisting of THP, RESCA, tricarbonyl compound, NOTA, DFO.

5. The radionuclide conjugate of claim 1, wherein, The radionuclide conjugate has the structure shown in formula I: R-L-Nb1 (I) wherein, Nb1 is the small protein targeting PD-L1; L is a linker; R is a radionuclide.

6. The radionuclide conjugate of claim 1, wherein, The radionuclide conjugate structure is as shown in the following formula: 68 Ga-THP-Nb1 wherein, 68 Ga is a radionuclide; Nb1 is the small protein targeting PD-L1; THP is a chelating agent.

7. A PD-L1 protein detection reagent, characterized by, The detection reagent comprises the conjugate of claim 1 and a detectably acceptable carrier.

8. A kit for detecting a PD-L1 molecule, characterized by, The kit comprises the conjugate of claim 1 or the PD-L1 protein detection reagent of claim 7.

9. Use of a conjugate according to claim 1, characterized in that, A method for preparing (a) a detection reagent for detecting PD-L1 molecules in vivo, a detection reagent kit or a detection plate; (b) a pharmaceutical composition for treating tumors expressing PD-L1 protein (i.e. PD-L1 positive).

10. A method of preparing the conjugate of claim 1, wherein, The method comprises the steps of: reacting a conjugate precursor and a radionuclide under suitable conditions to obtain the radionuclide conjugate; The conjugate precursor comprises a small protein targeting PD-L1 with an amino acid sequence shown as SEQ ID NO: 3; and a linker part.

Images