A peptide targeting TREM-1 protein and a radioactive tracer derived therefrom

Abstract

The present invention relates to the field of biomedical technologies, and particularly relates to a polypeptide targeting TREM-1 protein and a radioisotope tracer derived therefrom. The present invention provides a polypeptide NOTA-C-IPB-YIT targeting TREM-1 positive tumors, which is labeled with <supgt;68< / supgt;Ga to construct a radioisotope tracer [<supgt;68< / supgt;Ga]Ga-NOTA-C-IPB-YIT targeting TREM-1 protein. Through in vitro radiochemical property studies and in vivo stability studies, it is found that the radioisotope tracer [<supgt;68< / supgt;Ga]Ga-NOTA-C-IPB-YIT has strong stability and is hydrophilic; through cell uptake studies and receptor binding studies, it is found that the radioisotope tracer [<supgt;68< / supgt;Ga]Ga-NOTA-C-IPB-YIT has good affinity for the TREM-1 protein expressed by U87MG cells; by investigating the tumor imaging effect of the radioisotope tracer [<supgt;68< / supgt;Ga]Ga-NOTA-C-IPB-YIT with a small animal PET / CT, it is found that the radioisotope tracer [<supgt;68< / supgt;Ga]Ga-NOTA-C-IPB-YIT can be used for in vivo imaging of TREM-1 positive tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a polypeptide targeting TREM-1 protein and a radioactive tracer derived therefrom. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The interactions between tumor cells, stromal cells, vascular endothelial cells, and infiltrating inflammatory cells shape a highly specialized tumor microenvironment (TME). Among them, inflammation is generally considered to be a core factor in promoting tumor development. Tumor cells use the power of the innate immune system to promote their own migration and the formation of new blood vessels, which are the basis for tumor spread.

[0004] Neutrophils are an important part of the tumor microenvironment and the most abundant type of white blood cells in the human body, accounting for 40% to 70% of human white blood cells. They are widely involved in important processes such as tumor immune circulation, immune editing, and immune escape. Tumor-associated macrophages (TAMs) are mainly derived from monocytes in the blood and participate in the regulation of a variety of complex immune responses in tumors. The number of TAMs in the tumor microenvironment is closely related to the patient's prognosis, and they play an important role in the occurrence, development and metastasis of tumors. TREM-1 (triggering receptor of myeloid cells-1) belongs to the immunoglobulin superfamily. It is expressed on neutrophils and macrophages / monocytes and can trigger and amplify inflammatory responses. TREM-1 is greatly upregulated in bacterial infection, fungal infection or sepsis, and also plays an important role in the pathological process of various non-infectious acute and chronic inflammatory diseases, including atherosclerosis, tissue damage caused by ischemia-reperfusion, colitis, fibrosis and cancer. TREM-1 is highly expressed in a variety of tumor tissues, so it is expected to become a new target for tumor molecular imaging.

[0005] In the field of oncology, PET / CT significantly improves diagnostic accuracy by overcoming the limitations of traditional biopsy in identifying the heterogeneity of primary tumors and their metastases. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a polypeptide targeting TREM-1 protein and a radioactive tracer derived therefrom.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a polypeptide targeting TREM-1 protein, the structural formula of which is shown in the following formula (I):

[0009]

[0010] Formula (I).

[0011] The second aspect of the present invention provides a method for preparing the polypeptide targeting TREM-1 protein according to the first aspect, comprising the following steps:

[0012] (1) synthesizing the polypeptide shown in SEQ ID NO.1 by solid phase synthesis method; the solid phase synthesis method comprises: using RinkAmide MBHA Resin as the starting resin and Fmoc-amino acid as the raw material, and synthesizing by deprotection and condensation in sequence;

[0013] (2) Cyclizing the polypeptide to obtain a cyclic polypeptide;

[0014] (3) condensing the cyclic polypeptide with compound 1, then condensing with polyethylene glycol-acetic acid, and then coupling with a chelating agent NOTA to obtain the polypeptide targeting TREM-1 protein;

[0015] Among them, the structural formula of the cyclic polypeptide is as follows:

[0016] ;

[0017] The structural formula of compound 1 is shown below:

[0018] .

[0019] In one or more embodiments, in step (2), the method of cyclizing the polypeptide to obtain a cyclic polypeptide comprises:

[0020] The peptide is mixed with dimethyl sulfoxide (DMSO) and reacted to obtain a cyclic peptide.

[0021] In one or more embodiments, in step (3), the method of condensing the cyclic polypeptide with compound 1, then condensing with diethylene glycol-acetic acid, and then coupling with the chelating agent NOTA comprises the following steps:

[0022] When the cyclic peptide is condensed with compound 1, the solid phase method of N,N-diisopropylethylamine / benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate / 1-hydroxybenzotriazole condensation system is used for synthesis;

[0023] When condensing with diethylene glycol-acetic acid, N,N-diisopropylethylamine is added as a condensing agent;

[0024] When coupling the chelating agent NOTA, N,N-diisopropylethylamine was added as a condensing agent.

[0025] According to a third aspect of the present invention, there is provided a radioactive tracer comprising the polypeptide targeting TREM-1 protein as described in the first aspect or the polypeptide targeting TREM-1 protein prepared by the preparation method as described in the second aspect and a radionuclide.

[0026] In one or more embodiments, the radionuclide is selected from 18 F. 94 Tc, 99m Tc, 90 In, 111 In, 67 Ga, 68 Ga, 86 Y. 90 Y. 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co. 57 Co. 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd or 166 One of Dy; preferably 68 Ga.

[0027] Preferably, the structure of the radioactive tracer is as shown in formula (II),

[0028]

[0029] Formula (II).

[0030] The fourth aspect of the present invention provides a method for preparing the radioactive tracer according to the third aspect, comprising the following steps:

[0031] After the radioactive nuclide is mixed and reacted with a polypeptide targeting the TREM-1 protein, the radioactive tracer is obtained.

[0032] In one or more embodiments, the reaction temperature is 70-90° C., and the reaction time is 15-20 min.

[0033] A fifth aspect of the present invention provides use of the polypeptide targeting TREM-1 protein as described in the first aspect or the radioactive tracer as described in the third aspect in the preparation of a preparation for detecting the expression level of TREM-1 protein in a tumour.

[0034] A sixth aspect of the present invention provides use of the polypeptide targeting TREM-1 protein as described in the first aspect or the radioactive tracer as described in the third aspect in the preparation of a preparation for diagnosing and / or treating tumours with high TREM-1 protein expression.

[0035] The beneficial effects of the present invention are:

[0036] The present invention provides a polypeptide NOTA-C-IPB-YIT targeting TREM-1 positive tumors, and carries out 68 Ga-labeled, radioactive tracers targeting TREM-1 protein were constructed [ 68 Ga]Ga-NOTA-C-IPB-YIT. Through in vitro radiochemical properties and in vivo stability studies, it was found that the radioactive tracer [ 68 Ga]Ga-NOTA-C-IPB-YIT is highly stable and hydrophilic; through cellular uptake studies and receptor binding studies, it was found that the radioactive tracer [ 68 Ga]Ga-NOTA-C-IPB-YIT has a good affinity for TREM-1 protein expressed in U87MG cells; the radiotracer [ 68 Ga]Ga-NOTA-C-IPB-YIT tumor imaging effect, found that the radiotracer [ 68 Ga]Ga-NOTA-C-IPB-YIT can be used for in vivo imaging of TREM-1 positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 is the mass spectrum of NOTA-C-IPB-YIT;

[0039] Figure 2 For radioactive tracers 68 Radiochemical purity test diagram of Ga]Ga-NOTA-C-IPB-YIT;

[0040] Figure 3For radioactive tracers 68 Results of the cellular uptake experiments of Ga]Ga-NOTA-C-IPB-YIT and TREM-1 protein;

[0041] Figure 4 For radioactive tracers 68 Saturation experimental curve of Ga]Ga-NOTA-C-IPB-YIT and TREM-1 protein;

[0042] Figure 5 For radioactive tracers 68 Blood clearance curve of Ga]Ga-NOTA-C-IPB-YIT;

[0043] Figure 6 For radioactive tracers 68 In vivo biodistribution of Ga]Ga-NOTA-C-IPB-YIT in U87MG tumor-bearing mice;

[0044] Figure 7 PET / CT images of U87MG tumor-bearing mice in the experimental group and the inhibition group;

[0045] Figure 8 These are immunohistochemical images of TREM-1 expression in tumors of U87MG tumor-bearing mice, where a is a 100-fold magnification and b is a 400-fold magnification. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0047] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0048] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0049] Example 1

[0050] Preparation of peptide NOTA-C-IPB-YIT targeting TREM-1 protein:

[0051] The polypeptide represented by SEQ ID NO.1 (CGFLSKSLVFC) was synthesized by solid phase method using Rink Amide MBHA Resin as starting resin and Fmoc-amino acid as raw material by adopting N,N-diisopropylethylamine (DIPEA) / benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (HOBT) condensation system.

[0052] Specifically, the amino protecting group on Rink Amide MBHA Resin is removed with a piperidine N,N-dimethylformamide (DMF) solution, the resin is washed, and unreacted sites are blocked with a dichloromethane blocking solution containing DIPEA / acetic anhydride; the solvent is removed, the resin is washed, and Fmoc-cysteine ​​(Cys) is connected, using the Cys bound to the resin as the starting point for synthesis; the Fmoc protecting group is removed, and the deprotection and condensation reactions are repeated in sequence according to the amino acid sequence Cys-Gly-D-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe-Cys from the carboxyl end to the amino end to synthesize the polypeptide shown in SEQ ID NO.1.

[0053] Wherein, the concentration of piperidine in the piperidine N,N-dimethylformamide solution is 20% (volume percentage);

[0054] The blocking solution contained 0.2 M DIPEA in dichloromethane and 0.2 M acetic anhydride in dichloromethane.

[0055] During the condensation reaction, the reactants are calculated based on the molar ratio of Fmoc-amino acid: DIPEA: HOBT: HBTU: RinkAmide MBHA Resin = 3.5:7:4:4:1.

[0056] (2) Remove the Fmoc protecting group, and the two cysteines form a disulfide bond to complete the cyclization and obtain a cyclic peptide. The cyclization conditions are: at room temperature, mix the resin coupled with the peptide with dimethyl sulfoxide (DMSO) and maintain the reaction for 24 hours.

[0057] (3) The cyclic peptide and compound 1 are condensed by coupling the carboxyl group of lysine in compound 1 with the amino group of cysteine ​​in the cyclic peptide. The cyclic peptide and compound 1 are condensed by a solid phase method using N,N-diisopropylethylamine (DIPEA) / benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (HOBT) condensation system, using N,N-dimethylformamide (DMF) as solvent.

[0058] When the product of the condensation of the cyclic polypeptide and compound 1 is condensed with polyethylene glycol-acetic acid, N,N-diisopropylethylamine (DIPEA) is added as a condensation agent to carry out a condensation reaction with the polyethylene glycol-acetic acid.

[0059] Coupling chelating agent NOTA: Add chelating agent N,N',N''-triacetic acid-1,4,7-triazacyclononane-N-hydroxysuccinimide ester (NOTA-NHS) and N,N-diisopropylethylamine (DIPEA) to carry out condensation reaction.

[0060] The peptide was cleaved from the resin and further purified by high performance liquid chromatography to obtain the peptide NOTA-C-IPB-YIT targeting the TREM-1 protein.

[0061] When the cyclic polypeptide is condensed with compound 1, the reactants are in a substance molar ratio of compound 1: DIPEA: HOBT: HBTU: resin grafted with the cyclic polypeptide = 1:7:4:4:1.

[0062] When coupling the chelating agent NOTA, the molar ratio of the resin grafted with the polypeptide: NOTA-NHS:DIPEA is 1:2:5.

[0063] When the peptide is cleaved, the mass ratio of the resin grafted with the peptide: the cleavage solution is 1:10; in the cleavage solution, the volume ratio is trifluoroacetic acid (TFA): triisopropylsilane (TIS): phenol (PhOH): methyl phenyl sulfide (Thioanisole): H 2 O=30:2:2:1:1.

[0064] The amino acid sequence of the polypeptide NOTA-C-IPB-YIT targeting the TREM-1 protein is: Cys-Gly-D-Phe -Leu-Ser-Lys-Ser-Leu-Val-Phe-Cys-OH (Cys&Cys Bridge), and the ring structure is composed of 11 amino acids CGFLSKSLVFC. A disulfide bond is formed by two cysteines in the head and tail sequences to form a cyclic polypeptide. At the cysteine ​​near glycine in the cyclic polypeptide, compound 1 (Lys-Glu-C4-p-iodophenylbutyric acid structure) is introduced by forming an amide bond to improve the affinity of the polypeptide. The linker is composed of polyethylene glycol-acetic acid, and the carboxyl group of the acetic acid undergoes a condensation reaction with the amino group of the lysine introduced into the structure. The amino group in the polyethylene glycol undergoes a condensation reaction with the coupling agent NOTA-NHS through a coupling technology to obtain the polypeptide NOTA-C-IPB-YIT targeting the TREM-1 protein.

[0065] The mass spectrum of the polypeptide NOTA-C-IPB-YIT targeting the TREM-1 protein obtained in this example is as follows Figure 1As shown, the experimental results show that the molecular weight of NOTA-C-IPB-YIT is consistent with the theoretical value and its purity is higher than 98%.

[0066] Example 2

[0067] Radioactive tracers 68 Preparation of [Ga]Ga-NOTA-C-IPB-YIT:

[0068] Dissolve NOTA-C-IPB-YIT in NaOAc buffer and add [ 68 Ga]GaCl 3 The solution was reacted at 80 °C for 15 minutes to obtain a radioactive tracer targeting TREM-1 protein. 68 Ga]Ga-NOTA-C-IPB-YIT.

[0069] The pH of NaOAc buffer was 4.0-4.6; after NOTA-C-IPB-YIT was dissolved in NaOAc buffer, the concentration was 10 μM; [ 68 Ga]GaCl 3 The solution is eluted with 0.01M HCl, which is 18 ± 1 mCi; 68 When Ga is labeled, NOTA-C-IPB-YIT solution and [ 68 Ga]GaCl 3 The volume ratio of the solution is 1:1.

[0070] The radioactive tracer prepared in this example [ 68 The radiochemical purity of Ga]Ga-NOTA-C-IPB-YIT was tested by analytical HPLC with a radioactivity detector. Figure 2 shown.

[0071] Among them, HPLC mobile phase (A = 0.1% TFA / water, B = 0.1% TFA / acetonitrile), Zorbax C18 100 Å (250 × 4.6 mm, 5 µm), see Table 1 for details.

[0072] Table 1 HPLC elution conditions

[0073]

[0074] Depend on Figure 2 It is known that radioactive tracers [ 68 The radiochemical purity of Ga]Ga-NOTA-C-IPB-YIT was greater than 95%.

[0075] Example 3

[0076] Radioactive tracers 68 Study on the radiochemical properties of Ga]Ga-NOTA-C-IPB-YIT in vitro:

[0077] In vitro stability refers to the ability of a radiotracer to maintain its performance characteristics over a certain period of time under specified storage conditions.

[0078] The radioactive tracer prepared in Example 2 [ 68 Ga]Ga-NOTA-C-IPB-YIT was mixed with PBS buffer (pH = 7.4) and human serum (volume ratio of 1:3) and incubated at 37 °C for 1 h and 2 h, respectively. After incubation, the PBS sample was directly taken out and injected into Radio-HPLC for detection; the serum sample was added with anhydrous ethanol, centrifuged, filtered, and then detected by Radio-HPLC; the experimental results were analyzed by radiotracer [ 68 The results are shown in Table 2.

[0079] Table 2 In vitro stability

[0080]

[0081] The experimental results show that the radioactive tracer [ 68 Ga]Ga-NOTA-C-IPB-YIT has good in vitro stability in PBS buffer and human serum.

[0082] Example 4

[0083] Hydrophilicity and lipophilicity test:

[0084] 10 µL (about 1 MBq) of the radioactive tracer prepared in Example 2 [ 68 Ga]Ga-NOTA-C-IPB-YIT was diluted to 500 µL with HEPES buffer at pH 7.4, and then 500 µL of n-octanol was added and the mixture was shaken vigorously. After centrifugation, 200 µL of liquid was taken from each of the aqueous and organic phases to measure their radioactivity counts.

[0085] The lipid-water partition coefficient is given by the formula Log D = [Log 10 (radioactivity count of organic phase / radioactivity count of aqueous phase)].

[0086] The experimental results showed that the radioactive tracer [ 68 The lipid-water partition coefficient Log D of Ga]Ga-NOTA-C-IPB-YIT is -0.85 ± 0.09, indicating that it is hydrophilic.

[0087] Example 5

[0088] Cellular uptake and receptor binding studies of NOTA-C-IPB-YIT targeting TREM-1 protein:

[0089] Cell culture:

[0090] In 5% CO 2 The human glioblastoma cell line U87MG was cultured in MEM (containing NEAA) basal medium containing 10% fetal bovine serum in a 37°C cell culture incubator.

[0091] (1) Cell uptake experiment:

[0092] U87MG cells were cultured in 12-well plates to a volume of 5 × 10 5 After 10 cells / well, wash with PBS buffer.

[0093] Experimental group: at 37 ℃ with radioactive tracer [ 68 The cells were incubated with Ga]Ga-NOTA-C-IPB-YIT (20 nM) for 15 min, 30 min, 60 min, 90 min, and 120 min.

[0094] Inhibition group: TREM-1 protein binding was blocked by the simultaneous addition of 1000-fold concentration of NOTA-C-IPB-YIT (20 μM), followed by the addition of radioactive tracers at 37 °C. 68 The cells were further incubated with Ga]Ga-NOTA-C-IPB-YIT (20 nM) for 15 min, 30 min, 60 min, 90 min, and 120 min.

[0095] After the incubation, the cells were washed three times with cold PBS buffer, 400 μL of NaOH was added to each well to lyse the cells, the cells were collected and the intracellular radioactivity counts were detected by a gamma counter to calculate the cell uptake histogram, as shown in Figure 3 shown.

[0096] like Figure 3 As shown, the experimental results show that within 2 hours, cells respond to the radioactive tracer [ 68 The radioactive uptake of the inhibitory group was significantly lower than that of the experimental group, indicating that the radioactive tracer [ 68 The uptake of [Ga]Ga-NOTA-C-IPB-YIT in U87MG cells was significantly inhibited by unlabeled NOTA-C-IPB-YIT, indicating its binding specificity.

[0097] (2) Saturation experiment:

[0098] U87MG cells were cultured in 12-well plates to a volume of 5 × 10 5 cells / well, and different concentrations of radiotracer were added to U87MG cells. 68 Ga]Ga-NOTA-C-IPB-YIT, radiotracer [ 68 The final concentrations of Ga]Ga-NOTA-C-IPB-YIT were 0.15625 nM, 0.3125 nM, 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM and 40 nM. The cells were incubated at 37 °C for 1.5 h, and the radioactivity counts on the cell surface and inside the cells were measured to obtain the radioactive tracer [ 68 Binding capacity of Ga]Ga-NOTA-C-IPB-YIT to TREM-1 protein (specific binding + non-specific binding).

[0099] Nonspecific binding was detected by co-incubation with a radioactive tracer [ 68 Ga]Ga-NOTA-C-IPB-YIT and 1000-fold concentration of unlabeled NOTA-C-IPB-YIT (20 μM), and then the saturation binding curve was fitted by Graphpad Prism software to obtain the radioactive tracer [ 68 The maximum specific binding capacity of Ga]Ga-NOTA-C-IPB-YIT to the receptor ( B max ) and binding force ( K d ), the results are shown in Table 3 and Figure 4 (Saturation experimental curve) shown.

[0100] Table 3 Saturation test results

[0101]

[0102] The results of saturation experiments showed that the radioactive tracer [ 68 Receptor binding ability of Ga]Ga-NOTA-C-IPB-YIT with TREM-1 protein in U87MG cells K d The value reached 10.26 ± 1.57 nM, and the radiotracer [ 68 Maximum binding capacity of Ga]Ga-NOTA-C-IPB-YIT to receptors of U87MG cells B max Approximately 21872 ± 293.5 fmol / mg protein.

[0103] Example 6

[0104] Radioactive tracers 68 In vivo stability study of Ga]Ga-NOTA-C-IPB-YIT:

[0105] Three healthy female Balb / c mice were used to evaluate the radiotracer [ 68 In vivo stability of Ga]Ga-NOTA-C-IPB-YIT.

[0106] Each mouse was injected via the tail vein with approximately 37 MBq of radioactive tracer [ 68 Ga]Ga-NOTA-C-IPB-YIT, 2 hours after injection, kidney, liver, urine and blood samples were collected from 3 mice, processed and injected into Radio-HPLC to observe the radiotracer [ 68 The radiochemical purity of Ga]Ga-NOTA-C-IPB-YIT in different samples is shown in Table 4.

[0107] Table 4 In vivo stability

[0108]

[0109] The experimental results showed that the radiotracer [ 68 The stability of Ga]Ga-NOTA-C-IPB-YIT in kidney, liver, urine and blood after 2 hours was higher than 85%, and its in vivo stability was good.

[0110] Example 7

[0111] Radioactive tracers 68 Pharmacokinetic study of Ga]Ga-NOTA-C-IPB-YIT:

[0112] Three healthy female Balb / c mice were used to evaluate the radiotracer [ 68 Pharmacokinetic properties of Ga]Ga-NOTA-C-IPB-YIT. Each mouse was injected via the tail vein with approximately 3.7 MBq of radiotracer [ 68 Blood samples were collected, weighed and radioactive counts were determined after 1, 3, 5, 10, 15, 30, 60, 90 and 120 minutes of injection of Ga]Ga-NOTA-C-IPB-YIT. The percentage injected dose rate per gram of tissue (%ID / g) was calculated after correction for radioactive decay. The pharmacokinetic curves were fitted using DAS 2.0 software. The results are shown in Figure 5The drug concentration changes obtained by DAS software fitting reflect the summary of the distribution phase (α phase) and the elimination phase (β phase). In the distribution phase (α phase), the blood drug concentration drops rapidly after administration, which is the fast elimination stage. The drug concentration in the elimination phase (β phase) decreases proportionally according to the law of dynamic equilibrium, which is the slow elimination stage.

[0113] The experimental results showed that the radioactive tracer [ 68 The pharmacokinetic curve of Ga]Ga-NOTA-C-IPB-YIT in healthy mice conformed to the two-compartment model, and the half-life of the distribution phase (T 1 / 2α ) was 15.707 ± 2.45 minutes, and the elimination phase half-life (T 1 / 2β ) was 69.315 ± 3.41 minutes, indicating that the radioactive tracer [ 68 Ga]Ga-NOTA-C-IPB-YIT has a longer retention time in the body.

[0114] Example 8

[0115] Radioactive tracers 68 In vivo biodistribution study of Ga]Ga-NOTA-C-IPB-YIT:

[0116] Establishment of NSG subcutaneous tumor model:

[0117] U87MG cells were cultured in vitro, washed with PBS buffer and centrifuged. After counting the viable cells, NSG immunodeficient mice were anesthetized by oxygen induction with 2% isoflurane. 100 μL of 2×10 6 The serum-free suspension of U87MG cells was injected into the axilla of the back of mice, and the general condition of the mice and the size of the tumor were monitored. When the tumor grew to 5-8 mm in diameter, subsequent experiments were carried out.

[0118] Nine U87MG tumor-bearing mice were randomly divided into three groups, with 3 mice in each group. About 7.4 MBq of radioactive tracer was injected into the tail vein. 68 Ga]Ga-NOTA-C-IPB-YIT, one group was killed 1 hour after injection, blood, tumors and other major organs and tissues were taken, weighed and radioactive counts were measured. One group was killed 2 hours after injection, and the operation was the same as above. The 2-hour inhibition group was injected with an overdose of non-labeled NOTA-C-IPB-YIT in advance, and was killed 2 hours later. Other operations were the same as above. It is expressed as the percentage of radioactive counts per gram of tissue to the total radioactive counts (% ID / g).

[0119] The tumor / muscle ratio (T / M) was defined as the ratio of radioactivity counts in the tumor to those in the muscle.

[0120] Table 5 Radioactive tracers [ 68Biodistribution of Ga]Ga-NOTA-C-IPB-YIT in vivo

[0121]

[0122] The experimental results showed that in the U87MG tumor-bearing mouse model, injection of radioactive tracer [ 68 Ga]Ga-NOTA-C-IPB-YIT. After 1 and 2 hours, significant radioactive uptake was observed in the kidney and liver, indicating that the radiotracer [ 68 Ga]Ga-NOTA-C-IPB-YIT is mainly metabolized through the kidney and liver. And over time, the radioactive uptake of the tumor increased continuously and reached the highest at 2 hours, which was 7.63 ± 2.00% ID / g. In addition, after co-injection of unlabeled prodrug, the radioactive uptake of the tumor was significantly reduced, indicating that the radiotracer [ 68 Ga]Ga-NOTA-C-IPB-YIT has specific tumor targeting to TREM-1 positive tumors. The T / M ratios were 2.00 ± 0.13 and 10.39 ± 2.75 at 1 and 2 hours after injection, respectively, and the tracer had a good tumor-to-background ratio at 2 hours.

[0123] Example 9

[0124] Radioactive tracers 68 PET / CT imaging of Ga]Ga-NOTA-C-IPB-YIT in tumor-bearing mice:

[0125] Six U87MG tumor-bearing mice in Example 8 were randomly divided into two groups, one as an experimental group and the other as an inhibition group, with 3 mice in each group.

[0126] The experimental group was injected directly into the tail vein with approximately 7.4 MBq of radioactive tracer [ 68 The inhibition group was injected with about 7.4 MBq of [ 68 The mice were anesthetized with 2% isoflurane in oxygen and anesthetized with overdose of unlabeled NOTA-C-IPB-YIT. PET / CT was performed, and images were dynamically acquired at 0.5, 1, 1.5, and 2 h in three-dimensional mode.

[0127] Nucline NanoScan 3.00 was used to reconstruct and obtain attenuation-corrected PET / CT fusion images, and InterView FUSION 3.0 was used for image analysis.

[0128] Observe the imaging of the experimental group and the inhibition group. The results are as follows Figure 7 shown.

[0129] The experimental results showed that there was obvious radioactive tracer in the tumor site of the experimental group. 68 Ga]Ga-NOTA-C-IPB-YIT radioactive uptake, and the radioactive accumulation of tumors in the inhibition group was significantly reduced; this indicates that the radiotracer [ 68 Ga]Ga-NOTA-C-IPB-YIT specifically targets TREM-1 protein and can be used for imaging of TREM-1 protein-expressing tumors and monitoring of therapeutic efficacy.

[0130] Example 10

[0131] The ex vivo tumor tissue of the U87MG tumor-bearing mice in Example 8 was fixed in 4% paraformaldehyde for 24 hours, then embedded and paraffin sections were prepared. The prepared paraffin sections were placed in an oven at 60 ° C for 30 minutes, and then the sections were immersed in xylene I and xylene II in turn for 10 minutes each. Next, the sections were immersed in ethanol solutions of different concentrations in turn, and each concentration of ethanol solution was immersed for 2 minutes (the ethanol concentration gradient was 100%, 95%, 90%, 80%, and 70%). After that, the sections were placed in citric acid antigen repair solution, heated to boiling in a microwave oven, and then continued to heat for 15 minutes, then the microwave oven was turned off and naturally cooled to room temperature.

[0132] Endogenous peroxidase blocker was added to the tissue sections and incubated at room temperature for 15 minutes, followed by washing with double distilled water. Next, normal goat serum for blocking was added and incubated at room temperature for 20 minutes. Subsequently, TREM-1 antibody (rabbit anti-mouse, dilution ratio 1:200) was added to the sections and incubated overnight at 4°C. After removal, the sections were rewarmed at room temperature for 30 minutes, and then washed with PBS buffer for 5 minutes, and repeated three times to complete the primary antibody incubation. Then, secondary antibody (goat anti-rabbit, dilution ratio 1:200) was added, incubated at 37°C for 30 minutes, and then washed with PBS buffer for 5 minutes, and repeated three times to complete the secondary antibody incubation. Next, DAB color development solution was added, incubated for 10 seconds, and then washed with water. Then, Mayor's hematoxylin was added and incubated at room temperature for 2 minutes. Then, hydrochloric acid alcohol differentiation was performed, alkaline solution blueing treatment was performed, and then washed with water. Finally, the sections were immersed in ethanol solutions of different concentrations for a few seconds (the ethanol concentration gradient was 80%, 90%, and 95%), then immersed in anhydrous ethanol for 10 minutes, and then immersed in xylene for 10 minutes, and finally 1 drop of neutral resin was added for sealing.

[0133] The results were observed under a microscope and photographed. The immunohistochemical results of TREM-1 tumors in U87MG-bearing mice were as follows: Figure 8As shown in a and b, according to the results of immunohistochemistry, TREM-1 receptor was positively expressed in U87MG tumor tissue.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide targeting TREM-1 protein, characterized in that Its structural formula is shown in the following formula (I): Formula (I).

2. The method for preparing a polypeptide targeting TREM-1 protein according to claim 1, characterized in that: The steps include: (1) synthesizing the polypeptide shown in SEQ ID NO.1 by solid phase synthesis method; the solid phase synthesis method comprises: using RinkAmide MBHA Resin as the starting resin and Fmoc-amino acid as the raw material, and synthesizing by deprotection and condensation in sequence; (2) Cyclizing the polypeptide to obtain a cyclic polypeptide; (3) condensing the cyclic polypeptide with compound 1, then condensing with polyethylene glycol-acetic acid, and then coupling with a chelating agent NOTA to obtain the polypeptide targeting TREM-1 protein; Among them, the structural formula of the cyclic polypeptide is as follows: ； The structural formula of compound 1 is shown below: 。 3. The preparation method according to claim 2, characterized in that: In step (2), the method of cyclizing the polypeptide to obtain a cyclic polypeptide includes: The polypeptide is mixed with dimethyl sulfoxide and reacted to obtain a cyclic polypeptide.

4. A radioactive tracer, characterized in that Comprising the polypeptide targeting TREM-1 protein according to claim 1 or the polypeptide targeting TREM-1 protein prepared by the preparation method according to claim 2 and a radionuclide; The radionuclide is selected from 111 In, 67 Ga, 68 Ga, 86 Y. 90 Y. 177 Lu, 64 Cu, 67 Cu, 55 Co. 57 Co. 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 153 Sm, 166 Ho, 153 Gd and 157 One of Gd.

5. The radioactive tracer according to claim 4, characterized in that The structure of the radioactive tracer is shown in formula (II), Formula (II).

6. The method for preparing a radioactive tracer according to claim 4 or 5, characterized in that: The steps include: After the radioactive nuclide is mixed and reacted with a polypeptide targeting the TREM-1 protein, the radioactive tracer is obtained.

7. The preparation method according to claim 6, characterized in that: The reaction temperature is 70~90 ℃, and the reaction time is 15~20 min.

8. Use of a polypeptide targeting TREM-1 protein according to claim 1 or a radiotracer according to any one of claims 4 to 6 in the preparation of a preparation for detecting the expression level of TREM-1 protein in a tumour.

9. Use of a polypeptide targeting TREM-1 protein according to claim 1 or a radioactive tracer according to any one of claims 4 to 6 in the preparation of a preparation for diagnosing tumours with high TREM-1 protein expression.

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