Radionuclide-labeled PD-l1-targeting small protein, preparation method therefor, and use thereof

Abstract

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

Description

Radionuclide-labeled targeting PD-L1 small protein and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, the present application relates to radionuclide-labeled targeting PD-L1 small protein and preparation method 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 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 with 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 responding 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 of traditional monoclonal antibody (~150 kDa), radionuclide-labeled antibody is slowly cleared in vivo, resulting in a high radioactivity background in normal tissues, which not only significantly reduces the tumor / non-tumor ratio (i.e. imaging contrast), but also increases the possibility of radioactivity 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 that cannot be obtained by conventional laboratory PD-L1 detection, and provide auxiliary detection means for clinical PD-1 / PD-L1 immunotherapy patient screening and efficacy monitoring. SUMMARY

[0008] The purpose of the present application is to provide a radionuclide-labeled small protein targeting PD-L1 and a preparation method 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] 18 F, 99m Tc and 177 Lu.

[0015] In another preferred embodiment, the radionuclide is 18 F.

[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 the 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] Nb1 is 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, a PD-L1 protein detection reagent is provided, which comprises 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, a pharmaceutical composition is provided, which comprises 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 injection 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).

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

[0085] 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.

[0086] In another preferred embodiment, the linker is selected from the group consisting of: a polypeptide, a small molecule compound or a combination thereof.

[0087] In another preferred embodiment, the linker is a chelator of radionuclide.

[0088] In another preferred embodiment, the linker is selected from the group consisting of: tricarbonyl compound, THP, RESCA, NOTA.

[0089] In a seventh aspect of the present application, a method for preparing the radionuclide conjugate according to the first aspect of the present application is provided, which comprises the steps of:

[0090] reacting the conjugate precursor according to the sixth aspect of the present application with a radionuclide under suitable conditions, thereby obtaining the radionuclide conjugate.

[0091] In another preferred embodiment, the suitable conditions include:

[0092] i) temperature conditions of 0-60 °C; and / or

[0093] ii) pH 4.0-6.0.

[0094] In another preferred embodiment, the method has a radionuclide labeling efficiency of ≥ 95%, preferably ≥ 99%.

[0095] In an eighth aspect of the present application, there is provided a method of treating a disease, comprising administering to a subject in need thereof the conjugate of the first aspect of the present application or the pharmaceutical composition of the third aspect of the present application.

[0096] In another preferred embodiment, the subject includes a mammal, such as a human.

[0097] 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 (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0098] The following drawings serve to illustrate specific embodiments of the present application and are not intended to limit the scope of the present application as defined by the claims.

[0099] Figure 1 shows a schematic diagram of the synthesis of the radionuclide-labeled targeting PD-L1 small protein

[0100] Figure 2 shows a Radio-TLC assay 18 F-RESCA-Nb1 labeling rate, radiochemical purity, and in vitro stability.

[0101] Figure 3 shows 18 F-RESCA-Nb1 metabolic imaging in normal mice.

[0102] Figure 4 shows 18 F-RESCA-Nb1 imaging in KM mice injected with humanized PDL1 and mouse PDL1, the location of humanized PDL1 is shown as a yellow circle, and the location of mouse PDL1 is shown as a white circle.

[0103] Figure 5 shows 18 F-RESCA-Nb1 imaging results in A549-PDL1 negative model mice, the location of the tumor is shown as a white circle. DETAILED DESCRIPTION

[0104] Through 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 utilizes the characteristics of the small protein, i.e., smaller molecular weight and better tumor penetration than traditional antibodies, to construct a tumor PD-L1 surface display tracer probe. Experiments show that the PD-L1 surface display tracer probe of the present application can accurately locate PD-L1 positive tumors in animals. On this basis, the present application is completed.

[0105] 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 the super-high affinity of the small protein to PD-L1 is retained, but also the small protein can be efficiently coupled with the maleimide group of Mal-RESCA, and the product 18 After purification, the radiochemical purity of F-RESCA-Nb1 reaches more than 99%.

[0106] In particular, the small protein conjugate of the present application 18 F-RESCA-Nb1 can be labeled and prepared at room temperature to a temperature as high as 60 DEG C. Compared with other radionuclides or linkers (such as 89 Zr-DFO), the coupling efficiency is higher, and the finished product is more stable.

[0107] The terms

[0108] 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 have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application. Before the present application is described, it is to be understood that this application is not limited to the particular methodology and experimental conditions described, as such methodology 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.

[0109] 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.).

[0110] As used herein, the terms "comprising", "including", "containing", are interchangeable and are meant to be non-limiting. In other words, the terms include "consisting of", "consisting essentially of".

[0111] High-affinity small protein targeting PD-L1

[0112] The radionuclide conjugate of the present application comprises a high-affinity small protein targeting PD-L1.

[0113] The main advantages of high-affinity small proteins over monoclonal antibodies are:

[0114] (1) High affinity and high specificity, the affinity is 10-100 times higher than that of polypeptides, close to that of monoclonal antibodies;

[0115] (2) Very low immunogenicity and toxicity, and not prone to sticking like scFv;

[0116] (3) Good tissue penetration, can fully bind to target organs or tumor tissues;

[0117] (4) Small relative molecular mass, unbound parts can be quickly cleared by the kidneys, reducing background interference in the blood;

[0118] (5) The high-affinity small protein can be used for room temperature labeling, and also for labeling at temperatures up to 60°C and above.

[0119] (6) Using modern genetic engineering techniques, high-yield protein probes can be obtained, and the structure can be modified or modified, which is more conducive to molecular level image detection.

[0120] Radionuclide

[0121] 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.

[0122] 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.

[0123] 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 and the compound are easy to coordinate, 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.

[0124] As an ideal PET radionuclide, compared with 68 Ga (T1 / 2 = 68 min), 18F has a longer half-life (T1 / 2 = 109 min), and 18 F is prepared by accelerator, which is more suitable for batch preparation of multiple patients, and is also more suitable for developing automated synthesis preparation method.

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

[0126] In one embodiment, the conjugate of the present application comprises a radionuclide 18 F.

[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, all referring 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: 18 F, 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 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 chelators as described above.

[0146] In another preferred embodiment, the structure of the radionuclide conjugate is as shown in 18 F-RESCA-Nb1.

[0147] Method for preparing the conjugate

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

[0149] (1) 99m Method for labeling PD-L1 targeting Nb1 with Tc:

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

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

[0152] ① 68 Ga-labeled THP-Nb1

[0153] The concentration of 2-3 mg / mL precursor THP-Nb1 (200 μg) is quickly dissolved, 300 μL of 2.0 mol / L sodium acetate solution is added, and 1 mL of 68 Ga eluent (259-740 MBq) is added, the reaction system pH = 5.0-6.5, mixed and placed in 0-60°C for 15 min. Plus 0.22 μm microfiltration membrane, to the system added 2.0 mL of normal saline, the product is filtered to a sterile vacuum bottle, the product is obtained 68 Ga-THP-Nb1; preferably the product obtained is 68 Ga-THP-Nb1 radiochemical purity is greater than 95%;

[0154] ② 68 Ga-labeled NOTA-Nb1

[0155] Eluted with 4 mL of 0.05M HCl 68 Ge / 68 Ga generator, the eluate is received in 1.5 mL EP tubes, and 1 mL of the intermediate eluate is transferred to another new 1.5 mL EP tube. 65 μL (1.0M) of sodium acetate solution and 200 μg of NOTA-Nb1 are added to the above EP tube, mixed, the reaction system pH = 4.0-5.0; placed in 0-60°C for 15 min. The reaction solution is purified by PD-10 column, plus 0.22 μm microfiltration membrane, to the system added 2.0 mL of normal saline, the product is filtered to a sterile vacuum bottle, the target product is obtained 68 Ga-NOTA-Nb1, preferably the product obtained is 68Ga-NOTA-Nb1 radiochemical purity is greater than 95%;

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

[0157] 100ul of normal saline was added to the labeling precursor DOTA-Nb1 / NOTA-Nb1, and 370MBq of 18 F elution (about 1110MBq) was added to 4ul of 2mmol / L AlCl3 solution, reacted for 5min, 11ul of KHP (pH=4.0) solution was added, about 100ul of RESCA-Nb1 solution was added, the reaction system pH=4.6~4.8, the reaction liquid was evenly divided into three parts, and comparative experiments were carried out at 37℃, 60℃ and 80℃ reaction temperature respectively; the reaction time was appropriately shortened, but it was not less than 12min. Purified by molecular exclusion column PD-10, 1~2ml of normal saline was added to the purified product, mixed, and filtered to a sterile vacuum bottle with a 0.22um sterile needle filter, 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] 100ul of NaAc / HAc (pH=5.5) solution was added to the labeling precursor DOTA-Nb1 / NOTA-Nb1, and 370MBq of 177 LuCl3 solution, 37℃ reaction for 30min. Purified by molecular exclusion column PD-10, 1~2ml of normal saline was added after purification, mixed, and filtered to a sterile vacuum bottle with a 0.22um sterile needle filter, 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 visual tool for detecting and monitoring the efficacy of PD-L1 expression of tumors. 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, has a 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 a small protein conjugate 18 F-RESCA-Nb1 can be labeled and prepared at room temperature to a temperature of up to 60°C, compared with other radionuclides or linkers (such as 89 Zr-DFO), the coupling efficiency is higher, and the finished product is more stable.

[0165] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, and the methods are generally carried out according to 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 according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

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

[0167] (1) Plasmid synthesis

[0168] The E. coli expression vector used in this study is pET29b. In a conventional method, the following nucleotide sequence (SEQ ID NO: 2) is inserted into the NdeI and XhoI enzyme cutting sites to construct a recombinant plasmid. MSG is added at the N-terminal of the sequence, wherein M is the start codon; GSCGSENLYFQSLEHHHHHH (SEQ ID NO: 5) sequence is added at the C-terminal 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 high-affinity small protein targeting PD-L1 (SEQ ID NO: 1)

[0170] Nucleotide sequence of high-affinity small protein targeting PD-L1 (SEQ ID NO: 2)

[0171] The amino acid sequence (SEQ ID NO: 3) is as follows:

[0172] The nucleotide sequence (SEQ ID NO: 4) is as follows:

[0173] (2) Plasmid transformation

[0174] After the construction of the small protein recombinant plasmid, the full-length gene is sequenced to verify that there is no error, and then the plasmid is transformed into E. coli expression strain lemo21 (purchased from Beijing Zhuangmeng International Biological Gene Technology Co., Ltd.). Take 100 μL of competent cells lemo21 and thaw on ice. Lyophilized expression plasmid is dissolved 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.

[0175] (3) Induced expression and purification

[0176] After successful transformation, single colonies are picked for protein induction and expression, as follows:

[0177] 1. Small shaking: use a sterilized 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.

[0178] 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.

[0179] 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.

[0180] 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, discard the supernatant, and obtain the bacterial cell precipitate.

[0181] 5. Ultrasonic lysis: add 50 mL lysis buffer to resuspend the bacteria, then add protease inhibitor (100 μg / mL PMSF), mix on a shaker, then pour into a small beaker. Fix the beaker in an ice water mixture at 0°C, then use an ultrasonic disrupter to break the bacteria. The ultrasonic conditions are: power 150 w, ultrasonic for 6 s, stop for 6 s, working time 1 h. After ultrasonic treatment, collect the bacteria solution in a centrifuge tube, centrifuge at 9000 rpm for 20 min, collect the supernatant for use.

[0182] 6. Protein column: take 1 mL nickel beads and add to a 10 mL empty affinity chromatography column, use gravity to naturally flow out the ethanol preservative, then add 10 mL PBS to wash the nickel column to ensure that the ethanol is completely removed. Add 1 mL PBS to resuspend the nickel beads, then add to the ultrasonic lysis supernatant, rotate the column in a 4°C chromatography cabinet shaker for 2 h.

[0183] 7. Nickel column purification: add the mixture of lysis supernatant and nickel beads to an empty affinity chromatography column, connect a rubber tube to the liquid outlet of the affinity chromatography column, use a peristaltic pump to pump the flow-through liquid into a centrifuge tube at a constant flow rate. After 3 times of flow-through, discard the supernatant. Elute with 20 mL eluent (50 mmol / L Tris HCl), 20 mL eluent containing 20 mmol / L imidazole, 10 mL eluent containing 50 mmol / L imidazole, 1 mL eluent containing 100 mmol / L imidazole, then add 4 mL eluent containing 500 mmol / L imidazole to the nickel beads, mix well and stand for 5 min, then collect the liquid by gravity. Repeat this process once, and collect a total of 8 mL eluent. Add the collected eluent to a 3K dialysis bag, place the dialysis bag in a beaker containing PBS solution, add a magnetic rotor, and dialyze overnight on a magnetic stirrer in a 4°C chromatography cabinet.

[0184] 8. Molecular exclusion chromatography purification: add the dialyzed protein solution to a 3K molecular cut-off ultrafiltration tube, centrifuge at 4500 rpm and 4°C to concentrate, centrifuge to a final concentration of 10 mg / mL, then use Superdex 75 Increase molecular exclusion chromatography for purification. Use an Agilent 1260 high-performance liquid chromatography system, with PBS solution as the mobile phase and a flow rate of 0.8 mL / min. After purification, the protein solution is stored at -80°C for use.

[0185] The amino acid protein sequence obtained in this example is shown in SEQ ID NO: 3, i.e. the Nb1 sequence of the application. Remove the MSG added at the N-terminus of the sequence and the GSCGSENLYFQSLEHHHHHH sequence added at the C-terminus of the sequence, as shown in SEQ ID NO: 1.

[0186] The molecular structure was characterized by mass spectrometry and HPLC; HPLC analysis conditions: Superdex75 Increase 10 / 300 gel filtration / volume exclusion chromatographic column, flow rate 0.8 mL / min; mobile phase A is 0.1 mol / L PBS solution; mobile phase gradient setting: 0.0-30.0 min 100% A, Nb1 structure has good stability, pharmacokinetic properties and target specificity.

[0187] Example 2 68 Ga elution

[0188] The germanium-gallium generator was eluted with a 4 mL syringe of 0.05 mol / L HCl at a speed of 1-2 mL / min, the first mL of HCl was discarded, and the remaining 3 mL of eluent was collected and the radioactivity was recorded.

[0189] Example 3 Preparation of tricarbonyl compound intermediate 99m Tc(CO)3(H2O)3] +

[0190] 5 mg of Na2CO3, 10 mg of NaBH4, and 15 mg of potassium sodium tartrate were added to a 10 mL penicillin vial, 1 mL of normal saline was added, shaken well, and vacuumed with a 10 mL disposable needle tube and then carbon monoxide gas was introduced, the air in the bottle was exhausted, and then 2 mL of Na 99 mTcO4 eluent (about 13 mCi) was injected, heated at 80°C for 30 min in an oil bath (carbon monoxide gas was introduced during the reaction process), cooled, and the intermediate 99m Tc(CO)3(H2O)3] + was obtained and was ready for use.

[0191] Example 4 Preparation of precursor THP-Nb1

[0192] According to the molar ratio of purified Nb1 (SEQ ID No: 3) to Mal-THP of 1:10, Mal-THP solid powder was weighed into a 1.5 mL EP tube. The concentrated Nb1 solution was added, shaken uniformly, and then placed in an incubator for reaction at 37°C for 2 h. After the reaction was completed, PD-10 column was used for purification with 25 mL PBS. 1-1.5 mL of the reacted solution was added, supplemented with PBS to 2.5 mL, and then flowed dry. Another 2.5 mL of PBS was added, and 5 tubes of effluent were collected, about 0.5 mL per tube. The tubes with higher concentration were combined to obtain THP-Nb1, which was divided into 200 μg / tube and stored in a -80°C refrigerator.

[0193] Example 5 Preparation of precursor NOTA-Nb1 ​

[0194] Nb1 (SEQ ID No: 3) solution 0.5-1.0 mL was added to an EP tube, and about 10-20 times molar amount of NOTA-DMSO solution (10 mmol / L) was added, and the reaction was carried out at 37°C for 1 h, and the reaction tube was gently shaken every 10-15 min. PD-10 column was used for purification with 25 mL PBS. After adding 1-1.5 mL of the reaction solution, PBS was added to 2.5 mL, and the flow was dried. Then 2.5 mL of PBS was added, and 5 tubes of effluent were collected, about 0.5 mL per tube. The tubes with higher concentration were combined and divided into 200 μg / tube, and stored in a -80°C refrigerator.

[0195] Example 6 Preparation of precursor RESCA-Nb1

[0196] The coupling of Nb1 (SEQ ID No: 3) and Mal-RESCA was achieved by specific reaction of the maleimide group of (±)-H3 RESCA-Mal with the thiol group in the cysteine of the small protein Nb1 to form a thioether bond. Mal-RESCA (structure shown in Figure 1) solid powder was weighed according to the molar ratio of Mal-RESCA to Nb1 (SEQ ID No: 3) 1:10 into a 1.5 mL EP tube, and Nb1 solution was added to the reaction tube with a pipette, shaken and mixed, NaHCO3 / Na2CO3 buffer solution was added to adjust the pH to 6.5-7.5, and placed in an incubator at 37°C for 1 h. After the reaction was completed, the reaction solution was filtered through a 0.22 μm water phase needle filter, and the precursor was obtained by HPLC system purification. The purification chromatographic column was Superdex75 Increase 10 / 300, the detection wavelength was 210 nm and 280 nm, the injection amount was 500 μL, and the flow rate was 0.8 mL / min. The molecular weight of RESCA-Nb1 was detected by MALDI-TOF. The precursor sample was divided according to 200 μg, and stored in a -80°C refrigerator.

[0197] Example 7 Preparation of DFO-Nb1

[0198] Take 0.5 mg of small protein Nb1 (SEQ ID No: 3), and draw the liquid into a 1.5 mL EP tube according to the molar ratio of DFO to Nb1 of 1:12, and mix well. 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 completed, purify the product using a PD-10 column equilibrated with Metal-free PBS 5 times, add 1-1.5 mL of the reaction solution, supplement with PBS to 2.5 mL, and let it flow dry. 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 it into 200 μg / tube, and store it in a -80°C refrigerator.

[0199] Example 8 68 Preparation of Ga-THP-Nb1

[0200] Take the divided precursor THP-Nb1 (200 μg) from the -80°C refrigerator, and dissolve it quickly to a concentration of 2-3 mg / mL. Add 300 μL of 2.0 mol / L sodium acetate solution, and then add 1 mL of 68 Ga(259-740 MBq) freshly eluted in step (1) 68 Ga, and mix well. Divide the reaction system into 2 1.5 mL EP tubes, and react at 37°C and 60°C respectively for 15 min. Add a 0.22 μm microporous filter, add 2.0 mL of normal saline to the system, filter the product into a sterile vacuum bottle, and obtain the product 68 Ga-THP-Nb1.

[0201] Example 9 68 Preparation of Ga-NOTA-Nb1

[0202] Take the divided precursor NOTA-Nb1 (200 μg) from the -80°C refrigerator, and transfer 1 mL of the eluate with the highest activity in step (2) to another new 1.5 mL EP tube. Add 65 μL (1.0 M) of sodium acetate solution and 200 μg of NOTA-Nb1 to the EP tube, mix well, and adjust the pH of the reaction system to 4.0-5.0. Divide the reaction solution into 2 1.5 mL EP tubes, and react at 37°C and 60°C respectively for 15 min. After the reaction solution is purified by a PD-10 column, add a 0.22 μm microporous filter, add 2.0 mL of normal saline to the system, filter the product into a sterile vacuum bottle, and obtain the target product 68 Ga-NOTA-Nb1.

[0203] Example 10 Labeling rate, radiochemical purity, and quality control

[0204] 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 was spotted onto an iTLC-SG strip from 1-2 tubes with high activity, developed with physiological saline, and the radiochemical purity was analyzed by scanning on Radio-TLC. 100 μL of PBS was added to one 1.5 mL EP tube, and 100 μL of 5% HSA was added to another tube; 100 μL of the reaction solution was then added to each of these tubes, and in vitro stability was measured at 0.5 h, 1 h, 2 h, 4 h, and 6 h. The resulting product had a labeling rate >95%, radiochemical purity >99%, and in vitro stability maintained above 95%.

[0205] Example 11 [ 99m Tc] 99m Preparation of Tc(CO)3(H2O)3-Nb1

[0206] Prepare a 1 mg / mL solution of the small protein Nb1, and add 200 μL of this solution to 1 mL (approximately 2-3 mCi). 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.

[0207] Example 12 177 Preparation of Lu-DOTA-Nb1

[0208] 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.

[0209] Example 13 18 Preparation of F-RESCA-Nb1

[0210] Take 100 μL of normal saline 18 F elution (about 1110 MBq), add 4 μL of 2 mmol / L AlCl3 solution, react for 5 min, add 110 μL of NaAc / Hac (pH = 4.1) solution, 100 μL of RESCA-Nb1 solution, the system pH = 4.6-4.8, react for 15 min at 37°C. Purify with a PBS solution pretreated molecular exclusion column PD-10, add 1-2 mL of normal saline to the purified product, mix, and filter with a 0.22 μm sterile needle filter into a sterile vacuum bottle to obtain the product.

[0211] Example 14 18 F-RESCA-Nb1 quality control

[0212] iTLC: take 2 μL of the reaction solution, spot on iTLC-SG paper, develop with normal saline, and perform label rate scanning analysis on Radio-TLC. After purification with a PD-10 column, take 2 μL of the sample with the highest radioactivity and spot on iTLC-SG paper to show radiochemical purity.

[0213] F is a radionuclide with a relatively low label rate for proteins. The results show that the present application 18 The F-RESCA-Nb1 label rate is 14.19%. But the radiochemical purity detected after purification is 99.53%.

[0214] Incubate in PBS and 5% HSA for 4 hours, and detect the radioactivity probe in vitro stability by Radio-TLC. The probe has good stability at 0.5 h, 1 h, 2 h, and 4 h. It shows that the probe has high stability in vitro. The results are shown in Figure 2.

[0215] Example 15 18 High-temperature preparation of F-RESCA-Nb1

[0216] Take 400 μL of normal saline 18F elution (about 1110 MBq), 6 μL of 2 mmol / L AlCl3 solution was added, and the reaction was allowed to proceed for 5 min, 11 μL of 0.5 mol / L KHP (pH = 4.0) solution was added, 500 μL of RESCA-Nb1 solution was added, the pH of the system was 4.6-4.8, the reaction solution was evenly divided into three parts, and comparative experiments were carried out at 37 °C, 60 °C and 80 °C, respectively. The reaction time was appropriately shortened, but it was not less than 12 min; the molecular exclusion column PD-10 pretreated with PBS solution was used for purification, 1-2 mL of normal saline was added to the purified product, and the mixture was filtered through a 0.22 μm sterile needle filter into a sterile vacuum bottle, to obtain the product. The labeling rate and in vitro stability of the product labeled under high temperature conditions were basically consistent with those at 37 °C.

[0217] Example 16 18 F-RESCA-Nb1 mouse normal model imaging

[0218] According to the radioactivity 18 F-labeled PD-L1 targeting imaging agent 18 F-RESCA-Nb1 was prepared by the method. KM mice were injected with 0.2 mL (7.4 MBq) 18 F-RESCA-Nb1, MicroPET scanning imaging was performed after 1 h. The region of interest (ROI) of the whole body decay correction coronal image obtained by Micro-PET scanning was reconstructed. The results are shown in Figure 3, and there is no obvious nuclide signal except in the kidneys and bladder, which further indicates that 18 F-RESCA-Nb1 has targeting specificity, and there is no non-specific binding.

[0219] Example 17 18 F-RESCA-Nb1 mouse positive model imaging

[0220] According to the radioactivity 18 F-labeled PD-L1 targeting imaging agent 18 F-RESCA-Nb1 was prepared by the method. KM mice were injected with 0.2 mL (7.4 MBq) 18 F-RESCA-Nb1, MicroPET scanning imaging was performed after 1 h. The region of interest (ROI) of the whole body decay correction coronal image obtained by Micro-PET scanning was reconstructed. The results are shown in Figure 3, and there is no obvious nuclide signal except in the kidneys and bladder, which further indicates that 18F-RESCA-Nb1 has specific imaging capabilities for both human and mouse PDL1 in vivo.

[0221] Example 18 18 Imaging of F-RESCA-Nb1 mouse negative model

[0222] Nude mice were subcutaneously inoculated with A549 cells under the right axilla. After 6–8 days, the tumor grew to approximately 300 mm. 3 0.2 mL (5 GBq) was injected into the tail vein of mice. 18 F-RESCA-Nb1 was used for micro-PET imaging at 0.5h and 1h. The whole-body decay-corrected coronal region of interest (ROI) obtained from the micro-PET scans was reconstructed. The results are shown in Figure 5, indicating low uptake in mouse negative tumor tissue. This probe is rapidly metabolized and is a promising tumor imaging agent.

[0223] Comparative Example 1 89 Zr-DFO-Nb1 preparation

[0224] Take 70 μL (780 μCi) of oxalic acid 89 Zr was added to 10 times HEPES-Na2CO3 buffer solution and mixed well. After adding DFO-Nb1, oxalic acid was added to adjust the pH to 7.0. The mixture was placed in a constant temperature incubator and reacted at 37°C for 60 min. Intermediate quality control was performed after 30 min, and the labeling status was detected by iTLC. After the reaction, the product was purified using a equilibrated PD-10 column with a 0.22 μm microporous membrane. 2.0 mL of physiological saline was added to the system, and the product was filtered into a sterile vacuum bottle to obtain the target product. 89 Zr-DFO-Nb1. 2 μL of sample was spotted onto an iTLC-SG paper strip, showing radiochemical purity. The resulting product had a labeling rate of <50% and radiochemical purity of <50%, indicating poor labeling performance.

[0225] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A radionuclide conjugate, characterized in that, The radionuclide conjugate comprises: (a) a small protein targeting PD-L1, wherein 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: 18 F, 99m Tc and 177 Lu.

2. The radionuclide conjugate of claim 1, wherein, The radionuclide is 18 F.

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 RESCA, tricarbonyl compound, THP, 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 a 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 shown in the following formula: 18 F-RESCA-Nb1 wherein, 18 F is a radionuclide 18 F; Nb1 is the small protein targeting PD-L1; RESCA is a bifunctional chelator RESCA ((±)H3 RESCA-Mal).

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.

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