Targeting Trop2 polypeptide and molecular probe

Abstract

The invention relates to a Trop2 targeting polypeptide and a molecular probe, and belongs to the technical field of nuclear medicine. The invention provides a polypeptide targeting Trop2. The amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1. The polypeptide is intercepted from Trop2 targeting peptide T2-2 developed by a phage display technology and is structurally optimized. Research shows that Trop2 is an important target for diagnosing pancreatic cancer, meanwhile, the research shows that the polypeptide can be specifically combined with Trop2, and the polypeptide and Trop2 have high receptor affinity, so that the polypeptide has great application prospects in preparation of Trop2 imaging agents, products for evaluating the expression level of Trop2 or products for imaging and diagnosing Trop2 expression positive tumors such as pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to a polypeptide and a molecular probe targeting Trop2, belonging to the technical field of nuclear medicine. Background Art

[0002] Trophoblast cell-surface antigen 2 (Trop2) is a cell membrane glycoprotein composed of a 36 kDa nascent polypeptide modified by N-linked glycosylation. It is known to regulate tumor growth, invasion, and migration through multiple signaling pathways, and plays a role in stem cell biology and other diseases. Studies have shown that Trop2 can serve as a prognostic biomarker for various tumors. Compared with other tumor markers, Trop2 is highly expressed in more solid tumor types. Furthermore, a retrospective study analyzing Trop2 expression in 197 primary pancreatic cancers found that Trop2 was highly expressed in 55% of pancreatic cancers. Trop2 overexpression was significantly associated with lymph node metastasis, poor tumor differentiation, and worse prognosis. Furthermore, elevated Trop2 expression is associated with poor prognosis in malignancies such as gastric cancer, prostate cancer, and colorectal cancer, suggesting a role for this oncoprotein in promoting tumor progression. Given the critical role of Trop2 in cancer and the increasing clinical significance of Trop2-targeted therapy, it is crucial to develop tracking tools that can noninvasively identify, monitor, and quantify Trop2 expression in solid tumors.

[0003] Currently, there are 64 Cu / 90 Y-NOTA-AF650, 89 Zr-DFO-AF650, 64 Cu / 177 Lu-NOTA-hIMB1636 and [ 68 Trop2-targeted molecular probes such as Ga]Ga-NOTA-RTD01 / 98 / 161 have been used to image Trop2 expression in various tumor-bearing models. These molecular probes are all radiolabeled Trop2 antibodies. However, due to the large molecular weight, poor tissue penetration, and slow metabolism of monoclonal antibodies, these molecular probes require prolonged imaging and suffer from a long elimination half-life. Compared to monoclonal antibodies, small molecule peptides offer the advantages of small molecular weight, strong tissue penetration, and rapid metabolism. If small molecule peptides can be used as a basis while maintaining their relatively high affinity and selectivity for Trop2, it is expected that tracing tools with shorter elimination half-lives and the ability to rapidly and accurately detect Trop2 expression levels in solid tumors could be developed. Summary of the Invention

[0004] To solve the above problems, the present invention provides a polypeptide targeting Trop2, the amino acid sequence of the polypeptide is shown in SEQ ID NO.1 (GHSQECSEWL-NH2).

[0005] In one embodiment of the present invention, the amino acids in the polypeptide are L-amino acids.

[0006] The present invention also provides a nucleic acid molecule encoding the above polypeptide.

[0007] The present invention also provides a recombinant plasmid, which expresses the above polypeptide; or, the recombinant plasmid carries the above nucleic acid molecule.

[0008] The present invention also provides a host cell, wherein the host cell expresses the above polypeptide; the above nucleic acid molecule is integrated into the genome of the host cell; or the host cell carries the above recombinant plasmid.

[0009] The present invention also provides a molecular probe targeting Trop2, which comprises the above-mentioned polypeptide, a bifunctional chelating group and a radionuclide labeling group; the bifunctional chelating group is connected to the N-terminal glycine of the above-mentioned polypeptide; and the radionuclide labeling group is bound to the bifunctional chelating group.

[0010] In one embodiment of the present invention, the bifunctional chelating group is connected to the amino group of the glycine at the N-terminus of the polypeptide.

[0011] In one embodiment of the present invention, the radionuclide labeling group comprises 68 Ga 3+ 、[Al 18 F] 2+ 、 64 Cu 2 + and / or 177 Lu 3+ .

[0012] In one embodiment of the present invention, the radionuclide labeling group comprises 68 Ga 3+ 、[Al 18 F] 2+ and / or 64 Cu 2+ .

[0013] In one embodiment of the present invention, the radionuclide labeling group is [Al 18 F] 2+ 、 68 Ga 3+ or 64 Cu2 + .

[0014] In one embodiment of the present invention, the bifunctional chelating group comprises 1,4,7-triazacyclononane-4,7-diacetoxy-1-acetyl (NOTA) and / or 1,4,7,10-tetraazacyclododecyl-4,7,10-triacetoxy-1-acetyl (DOTA);

[0015] The 1,4,7-triazacyclononane-4,7-diacetyl-1-acetyl group has the following structure:

[0016]

[0017] Alternatively, the 1,4,7,10-tetraazacyclododecyl-4,7,10-triacetoxy-1-acetyl group has the following structure:

[0018]

[0019] Wherein, * is the connection position between the bifunctional chelating group and the polypeptide.

[0020] In one embodiment of the present invention, the bifunctional chelating group is 1,4,7-triazacyclononane-4,7-diacetyl-1-acetyl or 1,4,7,10-tetraazacyclododecyl-4,7,10-triacetyl-1-acetyl.

[0021] In one embodiment of the present invention, the bifunctional chelating group coupled with a radionuclide labeling group has the following structure:

[0022]

[0023] Alternatively, the bifunctional chelating group coupled with a radionuclide labeling group has the following structure:

[0024]

[0025] Alternatively, the bifunctional chelating group coupled with a radionuclide labeling group has the following structure:

[0026]

[0027] Alternatively, the bifunctional chelating group coupled with a radionuclide labeling group has the following structure:

[0028]

[0029] Alternatively, the bifunctional chelating group coupled with a radionuclide labeling group has the following structure:

[0030]

[0031] In one embodiment of the present invention, the molecular probe targeting Trop2 has the following structure:

[0032]

[0033] The present invention also provides a method for preparing the above-mentioned molecular probe, which comprises: mixing the above-mentioned polypeptide with a bifunctional chelating agent and reacting the mixture to obtain a reaction mixture; separating and purifying the reaction mixture to obtain a bifunctional chelating agent-polypeptide; and labeling the bifunctional chelating agent-polypeptide with radioactive nuclides to obtain a molecular probe targeting Trop2.

[0034] In one embodiment of the present invention, the method is: adding the above-mentioned polypeptide, bifunctional chelating agent, and N,N-diisopropylethylamine (DIPEA) to anhydrous N,N-dimethylformamide (DMF), reacting at 20-30°C for 3-10 hours to obtain a reaction mixture; after the reaction mixture is separated and purified by chromatography, the product peak product is collected and freeze-dried to obtain a bifunctional chelating agent-polypeptide; after the bifunctional chelating agent-polypeptide, radionuclide and glacial acetic acid are mixed, reacting at 30-40°C for 10-30 minutes to obtain a molecular probe targeting Trop2.

[0035] The present invention also provides the use of the above polypeptide, nucleic acid molecule, recombinant plasmid, host cell or molecular probe in preparing a product for evaluating Trop2 expression level or preparing a product for imaging diagnosis of tumors, wherein the tumor is a Trop2 expression-positive tumor.

[0036] In one embodiment of the present invention, the imaging is PET imaging.

[0037] In one embodiment of the present invention, the product is a kit.

[0038] In one embodiment of the present invention, the tumor is a solid tumor positive for Trop2 expression.

[0039] In one embodiment of the present invention, the Trop2-positive solid tumors include pancreatic cancer, lung cancer, breast cancer and / or gastric cancer.

[0040] The present invention also provides a product for evaluating the expression level of Trop2, wherein the product contains the above polypeptide, the above nucleic acid molecule, the above recombinant plasmid, the above host cell and / or the above molecular probe.

[0041] The present invention also provides a product for imaging and diagnosing tumors, wherein the tumor is a Trop2-positive tumor, and the product contains the above-mentioned polypeptide, the above-mentioned nucleic acid molecule, the above-mentioned recombinant plasmid, the above-mentioned host cell and / or the above-mentioned molecular probe.

[0042] In one embodiment of the present invention, the imaging is PET imaging.

[0043] In one embodiment of the present invention, the product is a kit.

[0044] In one embodiment of the present invention, the tumor is a solid tumor positive for Trop2 expression.

[0045] In one embodiment of the present invention, the Trop2-positive solid tumors include pancreatic cancer, lung cancer, breast cancer and / or gastric cancer.

[0046] The technical solution of the present invention has the following advantages:

[0047] 1. The present invention provides a polypeptide targeting Trop2, the amino acid sequence of which is shown in SEQ ID NO. 1 (GHSQECSEWL-NH2). This polypeptide is extracted from the Trop2-targeting peptide T2-2 (CCPPCGRSVGEECSEWLLLWCYC) developed using phage display technology and has undergone structural optimization. Studies have shown that Trop2 is an important target for diagnosing pancreatic cancer. Furthermore, studies have shown that this polypeptide can specifically bind to Trop2 and has a high receptor affinity for Trop2. Therefore, this polypeptide has great application prospects in the preparation of Trop2 imaging agents, products for evaluating Trop2 expression levels, or products for imaging and diagnosing Trop2-positive tumors such as pancreatic cancer.

[0048] 2. The present invention provides a molecular probe targeting Trop2, which includes a polypeptide targeting Trop2 with an amino acid sequence as shown in SEQID NO.1, a bifunctional chelating group and a radionuclide labeling group; wherein the bifunctional chelating group is connected to the N-terminal glycine of the above-mentioned polypeptide, and the radionuclide labeling group is bound to the bifunctional chelating group. At present, the PET imaging molecular probes for Trop2 are mainly radioactively labeled Trop2 antibodies, which have the problems of large molecular weight, weak tissue penetration and slow biological metabolism. However, this molecular probe, as a small molecule polypeptide probe, has the advantages of small molecular weight, strong tissue penetration and fast blood clearance rate in the body (mainly metabolized by the kidneys). In addition, studies have shown that this molecular probe, as a tumor receptor targeting probe, has the characteristics of obvious uptake in pancreatic cancer-bearing mice with positive Trop2 expression, high imaging contrast of tumor lesions and strong tumor resolution. Compared with the existing 18Clinical diagnostic imaging probes for tumors such as F-FDG have higher tumor targeting specificity. Therefore, this molecular probe has great application prospects in the preparation of products for evaluating Trop2 expression levels or for imaging and diagnosing Trop2-positive tumors such as pancreatic cancer.

[0049] Furthermore, the labeling method of this molecular probe is simple and universal, the reaction system is stable, the conditions are mild, and it has high radiochemical purity, specific activity and radiochemical yield, and is easy to produce and apply clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : GL10 obtained by computer-aided drug design optimization, and the molecular docking diagram of GL10 and Trop2.

[0051] Figure 2 : Results of affinity determination between GL10 and Trop2.

[0052] Figure 3 : HPLC profile of precursor NOTA-GL10.

[0053] Figure 4 : High-resolution mass spectrum of the precursor NOTA-GL10.

[0054] Figure 5 :Molecular Probes[ 68 Radioactive HPLC spectrum of Ga]Ga-NOTA-GL10.

[0055] Figure 6 :Molecular Probes[ 68 Radioactive HPLC profile of [Ga]Ga-NOTA-GL10 in PBS in vitro over 2 h.

[0056] Figure 7 :Molecular Probes[ 68 Radioactive HPLC profile of Ga]Ga-NOTA-GL10 in serum at 2 h in vivo.

[0057] Figure 8 :Molecular Probes[ 68 MicroPET imaging of Ga]Ga-NOTA-GL10 at 60 min in BxPC-3 tumor-bearing mice.

[0058] Figure 9 :Molecular Probes[ 68 Uptake value (%ID / g) of Ga]Ga-NOTA-GL10 in organs of BxPC-3 tumor-bearing mice at 60 min.

[0059] Figure 10 :Molecular Probes[ 68MicroPET imaging and competitive inhibition imaging of Ga]Ga-NOTA-GL10 at 60 min in BxPC-3 tumor-bearing mice.

[0060] Figure 11 :Molecular Probes[ 68 Biodistribution of Ga]Ga-NOTA-GL10 in BxPC-3 tumor-bearing mice at 60 min.

[0061] Figure 12 :Molecular Probes[ 68 Pharmacokinetic test results of Ga]Ga-NOTA-GL10. DETAILED DESCRIPTION

[0062] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0063] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0064] Example 1: A polypeptide targeting Trop2

[0065] This example provides a polypeptide targeting Trop2, the amino acid sequence of which is shown in SEQ ID NO. 1 (GHSQECSEWL-NH2).

[0066] Experimental Example 1: Truncation and Optimization of Peptides Targeting Trop2

[0067] This experimental example provides an experiment to extract and optimize a peptide targeting Trop2. The specific process is as follows:

[0068] Referring to the literature “Jinjing Li, Hongtan Liu, Shuling Xiao, Shihui Fan, Xueting Cheng, and Chuanliu Wu. De Novo Discovery of Cysteine ​​Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery. Journal of the American Chemical Society 2023 145(51), 28264-28275”, a peptide T2-2 specifically targeting Trop2 with an amino acid sequence as shown in SEQ ID NO. 2 (CCPPCGRSVGEECSEWLLLWCYC) was developed using phage display technology and the K affinity between the peptide and the Trop2 protein was determined. D The affinity between peptide T2-2 and Trop2 was evaluated by measuring the K D The value was 97 nM; the polypeptide T2-2 was truncated into 10 amino acid fragments to establish a polypeptide library. Based on molecular docking and computer simulation screening, alanine scanning and point saturation mutagenesis techniques were used to optimize the polypeptide sequence and screened by molecular dynamics simulation to obtain a polypeptide specifically targeting Trop2 with an optimized amino acid sequence as shown in SEQ ID NO.1 (GHSQECSEWL-NH2), which was named GL10. Figure 1 GL10 optimized for computer-aided drug design, and the molecular docking diagram of GL10 and Trop2. Figure 1 It can be seen that the structural optimization improves the affinity between the peptide and Trop2. D The affinity between it and Trop2 was further evaluated. Figure 2 It can be seen that the K of GL10 D The value is K D =12.9nM. K D The results of the determination of the values ​​further showed that the affinity between the structure-optimized GL10 and Trop2 was significantly improved compared with that of the peptide T2-2.

[0069] Example 2: A molecular probe targeting Trop2 68 Ga]Ga-NOTA-GL10

[0070] This embodiment provides a molecular probe targeting Trop2 [ 68 Ga]Ga-NOTA-GL10, the molecular probe [ 68Ga]Ga-NOTA-GL10 has the following structure:

[0071]

[0072] Example 3: A method for preparing a molecular probe [ 68 Ga]Ga-NOTA-GL10 method

[0073] This example provides a method for preparing the molecular probe described in Example 2 [ 68 The method of Ga]Ga-NOTA-GL10, the specific steps are as follows:

[0074] GL10 (having an amino acid sequence as shown in SEQ ID NO. 1) was synthesized using L-amino acids as raw materials via solid-phase peptide synthesis. 1 mg of GL10 and 1.5 mg of NOTA were added to 200 μL of anhydrous DMF, followed by the addition of 30 μL of DIPEA. The mixture was then reacted at room temperature (25°C) for 8 hours to obtain a reaction solution. After the reaction, 300 μL of ddH2O containing 0.1% (v / v) trifluoroacetic acid (TFA) was added to the reaction solution to obtain a mixed solution. The mixed solution was separated and purified by semi-preparative high-performance liquid chromatography, and the eluate (i.e., the product peak) was collected and lyophilized to obtain the precursor NOTA-GL10 (i.e., a bifunctional chelator-peptide). The precursor NOTA-GL10 was characterized by HPLC and high-resolution mass spectrometry. Figure 3 is the high performance liquid chromatography (HPLC) spectrum of the precursor NOTA-GL10; Figure 4 This is a high-resolution mass spectrum of the precursor NOTA-GL10. The HPLC retention time Rt is 4.23 min, the mass spectrum MS (m / z) molecular weight is 1459.6, and the HPLC test purity is greater than 98.9%.

[0075] 0.05 M hydrochloric acid solution (5 mL) was used as the fractionation eluent. 68 Ge / 68 Elution of radionuclides from Ga generators 68 Ga, obtain 68 Ga3+ eluent; 68 The Ga3+ eluate (1.4 mL) was transferred to a new Eppendorf tube and after testing the isotope dose, 0.25 M sodium acetate solution (320 μL) was used to 68The pH of the Ga3+ eluate was adjusted to 4.0 to obtain a mixed solution; 50 μg of the precursor NOTA-GL10 was added to the mixed solution, and the mixture was incubated at 37°C for 15 minutes. During the incubation process, the mixture was shaken once every 5 minutes to obtain a reaction solution; 10 mL of injection water was added to the reaction solution and mixed to obtain a dilution solution; the dilution solution was transferred to a Sep-pak C18 column, and the Sep-pak C18 column was first rinsed 3 times with 10 mL of injection water and blown dry, and then eluted with 10 mM hydrochloric acid ethanol (300 μL) to obtain a molecular probe containing targeting Trop2 [ 68 The eluate of Ga]Ga-NOTA-GL10 was diluted with water for injection to a product solution containing 5% (v / v) ethanol; the product solution was filtered through a sterile filter membrane to obtain the product that meets the requirements. 68 Ga]Ga-NOTA-GL10 injection. Determination by radio-HPLC [ 68 Ga]Ga-NOTA-GL10 injection. Figure 5 Molecular probe targeting Trop2[ 68 The radioactive HPLC spectrum of Ga]Ga-NOTA-GL10 showed that the retention time of the target imaging agent was 9.6 min, and the radiochemical purity of the product was greater than 95%.

[0076] Experimental Example 2: Molecular probes[ 68 Stability experiment of Ga]Ga-NOTA-GL10

[0077] This experimental example provides the molecular probe described in Example 2 [ 68 The stability experiment of Ga]Ga-NOTA-GL10 is as follows:

[0078] Experiment 1: 2 mL of PBS buffer (pH 7.4, 0.1 M) was mixed with 50 μCi of [ 68 After the Ga]Ga-NOTA-GL10 injection solution was fully mixed, it was incubated at 37°C for 2 h to obtain an incubation solution; the incubation solution was measured using high performance liquid chromatography (HPLC) to evaluate [ 68 The in vitro stability of Ga]Ga-NOTA-GL10 was tested by repeating the experiment more than three times. Figure 6 For molecular probes 68 Radioactive HPLC profile of [Ga]Ga-NOTA-GL10 in PBS in vitro over 2 h.

[0079] Experiment 2: 2 mL of fetal bovine serum (FPS) and 50 μCi of [ 68 After the Ga]Ga-NOTA-GL10 injection solution was fully mixed, it was incubated at 37°C for 2 h to obtain an incubation solution; the incubation solution was measured using high performance liquid chromatography (HPLC) to evaluate [68 The in vitro stability of Ga]Ga-NOTA-GL10 was tested by repeating the experiment more than three times. Figure 7 For molecular probes 68 Radioactive HPLC profile of [Ga]Ga-NOTA-GL10 in FPS in vitro within 2 h.

[0080] In Experiments 1 and 2, HPLC analysis conditions were as follows: the analytical column was a ZORBAX Eclipse XDB-C18 column; the mobile phases were: phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid (TFA); phase B was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid (TFA). The elution gradient was as follows: 0-2 min, phase A 90%, phase B 10%, flow rate 1 mL / min; 2-16 min, phase A was reduced to 10%, phase B was increased to 90%, flow rate 1 mL / min; 16-20 min, phase A was increased to 90%, phase B was reduced to 10%, flow rate 1 mL / min. UV detection was at 220 nm, and the radioactivity detector was provided by LabLogic Systems Ltd.

[0081] Figure 6 and Figure 7 The results showed that the molecular probe [ 68 Ga]Ga-NOTA-GL10 is stable in both PBS and FBS and has good in vitro stability.

[0082] Experimental Example 3: Molecular probes[ 68 Experimental study on the lipid-water partition coefficient of Ga]Ga-NOTA-GL10

[0083] This experimental example provides the molecular probe described in Example 2 [ 68 The lipid-water partition coefficient experiment of Ga]Ga-NOTA-GL10 is as follows:

[0084] Take 10 μL of the [ 68 After the Ga]Ga-NOTA-GL10 injection solution was added to a 2.5 mL centrifuge tube containing 1 mL of n-octanol and 990 μL of water, it was sealed and placed in a dry box incubator for 10 minutes at room temperature (25°C), and then allowed to stand for 10 minutes to allow the two phases to separate. 500 μL of each phase was then taken from each phase using a pipette and placed in a γ counter tube, and the radioactivity was counted using a γ counter. Two batches of experiments were performed in parallel, with each batch repeated 3 times. According to the formula The molecular probe [ 68 [Ga]Ga-NOTA-GL10 Log P value; where counts in water represents the radioactivity counts in the aqueous phase; counts in 1-octanol represents the radioactivity counts in the 1-octanol phase; Log is the logarithm to base 10.

[0085] Molecular probes[ 68 The lipid-water partition coefficient (log P) of Ga]Ga-NOTA-GL10 was -2.14±0.23, indicating that it is a water-soluble substance with good hydrophilicity. It is predicted that after in vivo uptake as an imaging agent, it will be mainly metabolized by the kidneys, and the uptake in other soft tissues may be low, resulting in a relatively low imaging background uptake.

[0086] Experimental Example 4: Molecular probes[ 68 Micro-PET Imaging Experiment of Ga]Ga-NOTA-GL10

[0087] This experimental example provides the molecular probe described in Example 2 [ 68 Micro-PET imaging experiments of Ga]Ga-NOTA-GL10 were conducted using Siemens Inveon Micro-PET / CT. The acquisition workstation was the Inveon Acquirision Workplace (IAW) 2.2, and the data analysis workstation was the Inveon Research Workplace (IRW). The specific process is as follows:

[0088] Human pancreatic adenocarcinoma cells BxPC-3 with high expression of Trop2 and human pancreatic cancer cells PANC-1 with low expression of Trop2 (both purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured at 5×10 6 The cells were inoculated subcutaneously into the right armpit of nude mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd., 4-5 weeks old, weighing 18-20 g) at a density of 100 / mouse. The cells were then inoculated into the right armpit of nude mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd., 4-5 weeks old, weighing 18-20 g). The tumors were grown to 200 mm in size. 3 Micro-PET imaging experiments were performed on BxPC-3 and PANC-1 tumor-bearing mice.

[0089] Imaging of Trop2-overexpressing tumors: BxPC-3 tumor-bearing mice were anesthetized with isoflurane and fixed on the scanning bed. 100 μCi of [ 68 The tumor-bearing mice were injected with Ga]Ga-NOTA-GL10 solution via the tail vein. After the injection, dynamic PET scanning lasting 60 minutes was performed immediately. Figure 8 For molecular probes 68 microPET images of Ga]Ga-NOTA-GL10 at 10 min, 30 min, and 60 min in BxPC-3 tumor-bearing mice.

[0090] Imaging of Trop2 low-expressing tumors: PANC-1 tumor-bearing mice were anesthetized with isoflurane and fixed on the scanning bed. 100 μCi of [ 68The tumor-bearing mice were injected with Ga]Ga-NOTA-GL10 solution via the tail vein. After the injection, dynamic PET scanning lasting 60 minutes was performed immediately. Figure 9 For molecular probes 68 microPET images of Ga]Ga-NOTA-GL10 at 10 min, 30 min, and 60 min in PANC-1 tumor-bearing mice.

[0091] Inhibition imaging: After injection [ 68 Precursor NOTA-GL10 (50 μg) was injected into tumor-bearing mice via the tail vein 30 min before administration of Ga]Ga-NOTA-GL10 (100 μCi). Other procedures were the same as those in “Imaging of Trop2-overexpressing tumors”. Figure 10 For molecular probes 68 microPET images of Ga]Ga-NOTA-GL10 at 10 min, 30 min, and 60 min in BxPC-3 tumor-bearing mice.

[0092] in, Figures 8 to 10 The upper side is a cross-sectional PET image of the tumor site. Figures 8 to 10 The lower side is a coronal PET image of the tumor site. The white arrow points to the location of the tumor.

[0093] Figure 8 The imaging results of Trop2 high expression tumors showed that [ 68 Ga]Ga-NOTA-GL10 has a high uptake in the tumors of BxPC-3 tumor-bearing mice, with an uptake value of 5.03±0.49%ID / mL at 60 min. Furthermore, the uptake at the tumor site is significantly higher than that in organs or tissues such as muscle, lung, and intestine. Furthermore, it is mainly metabolized through the kidneys. Time-dependent dynamic diagrams of various organs revealed that the tumor / muscle ratio is highest at around 60 min, with a ratio of 4.44±0.30. This characteristic of rapidly achieving a high signal-to-background ratio can provide an efficient diagnostic tool for pancreatic cancer patients. Figures 9 and 10 The results of Trop2 low expression tumor imaging and inhibition imaging showed that [ 68 The tumor uptake (1.41±0.13%ID / mL, 1.86±0.43%ID / mL) and tumor / muscle ratio (1.23±0.28, 1.49±0.46) of [Ga]Ga-NOTA-GL10 were significantly decreased, indicating that [ 68 Ga]Ga-NOTA-GL10 is specifically taken up at the tumor site. 68 Ga]Ga-NOTA-GL10 can specifically target Trop2-overexpressing tumors and has good application prospects.

[0094] Experimental Example 5: Molecular probes[ 68In vivo biodistribution experiment of Ga]Ga-NOTA-GL10

[0095] This experimental example provides the molecular probe described in Example 2 [ 68 The in vivo biodistribution experiment of Ga]Ga-NOTA-GL10 was conducted as follows:

[0096] Four Kunming mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd., 5-6 weeks old, weighing 20-25 g) were injected into the tail vein of the four Kunming mice with 100 μCi of the [ 68 Ga]Ga-NOTA-GL10 injection, 1 hour after normal intake, the mice were killed, and the blood, brain, heart, lung, liver, spleen and kidney and other major organs and tissues were weighed and γ counted to study the molecular probe [ 68 Biodistribution of Ga]Ga-NOTA-GL10 in Kunming mice. Figure 11 For molecular probes 68 Biodistribution of Ga]Ga-NOTA-GL10 in Kunming mice at 60 min.

[0097] Figure 11 The results showed that molecular probes [ 68 Ga]Ga-NOTA-GL10 is mainly metabolized by the kidneys, cleared quickly from the blood, has low radioactivity in bones, and does not defluorinate in the body.

[0098] Experimental Example 6: Molecular probes[ 68 Pharmacokinetics of Ga]Ga-NOTA-GL10

[0099] This experimental example provides the molecular probe described in Example 2 [ 68 The pharmacokinetic experiment of Ga]Ga-NOTA-GL10 is as follows:

[0100] Four Kunming mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd., 5-6 weeks old, weighing 20-25 g) were injected into the tail vein of the four Kunming mice with 100 μCi of the [ 68 Ga]Ga-NOTA-GL10 injection. Blood samples were collected through the tail vein at predetermined time points (1, 2, 3, 5, 10, 15, 20, 30, 40, 60, 90, and 120 minutes after injection). Each blood sample was weighed and radioactivity was measured using a gamma counter. The results are shown in Figure 2. Figure 12 As shown in Figure 2, the concentration-time curves show that it fits well with the two-compartment pharmacokinetic model. Due to its significant hydrophilicity, [ 68 Ga]Ga-NOTA-GL10 exhibits rapid distribution kinetics, with a distribution half-life (t 1 / 2α) is 0.624 min, followed by efficient systemic clearance with an elimination half-life (t 1 / 2β ) was 25.422min. These pharmacokinetic characteristics indicate that the molecular probe [ 68 Ga]Ga-NOTA-GL10 has dual advantages, including rapid extravasation and effective penetration into tumor tissues, while being effectively cleared from non-target tissues. 68 Ga]Ga-NOTA-GL10 rapidly achieves high tumor-to-background ratios, which is crucial for tumor diagnostic imaging applications.

[0101] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A polypeptide targeting Trop2, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide of claim 1.

3. A recombinant plasmid, characterized in that: The recombinant plasmid expresses the polypeptide according to claim 1; or, the recombinant plasmid carries the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that The host cell expresses the polypeptide according to claim 1; or, the nucleic acid molecule according to claim 2 is integrated into the genome of the host cell; or, the host cell carries the recombinant plasmid according to claim 3.

5. A molecular probe targeting Trop2, characterized in that: The molecular probe comprises the polypeptide according to claim 1, a bifunctional chelating group and a radionuclide labeling group; the bifunctional chelating group is connected to the N-terminal glycine of the polypeptide according to claim 1; and the radionuclide labeling group is bound to the bifunctional chelating group.

6. The molecular probe according to claim 5, wherein The bifunctional chelating group is connected to the amino group of the N-terminal glycine of the polypeptide according to claim 1.

7. A method for preparing the molecular probe according to claim 5 or 6, characterized in that: The method comprises: mixing the polypeptide according to claim 1 with a bifunctional chelating agent and reacting the mixture to obtain a reaction mixture; separating and purifying the reaction mixture to obtain a bifunctional chelating agent-polypeptide; and labeling the bifunctional chelating agent-polypeptide with radioactive nuclides to obtain a molecular probe targeting Trop2.

8. Use of the polypeptide according to claim 1, the nucleic acid molecule according to claim 2, the recombinant plasmid according to claim 3, the host cell according to claim 4, or the molecular probe according to claim 5 or 6 in preparing a product for evaluating the expression level of Trop2 or preparing a product for imaging diagnosis of tumors, characterized in that: The tumor is a Trop2-positive tumor.

9. A product for evaluating the expression level of Trop2, characterized in that The product contains the polypeptide according to claim 1, the nucleic acid molecule according to claim 2, the recombinant plasmid according to claim 3, the host cell according to claim 4 and / or the molecular probe according to claim 5 or 6.

10. A product for imaging and diagnosing tumors, characterized in that: The tumor is a Trop2-positive tumor, and the product contains the polypeptide according to claim 1, the nucleic acid molecule according to claim 2, the recombinant plasmid according to claim 3, the host cell according to claim 4 and / or the molecular probe according to claim 5 or 6.

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