HER2 targeting polypeptide and molecular probe

Abstract

The invention provides a polypeptide targeting HER2 (human epidermal growth factor receptor 2) and a molecular probe, and belongs to the technical field of nuclear medicine. The invention provides a polypeptide targeting HER2 (human epidermal growth factor receptor 2). The amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1 (NPNSGFSIWNWC). Research shows that HER2 is an important target for diagnosing lung cancer, meanwhile, the research shows that the polypeptide can be specifically combined with HER2, and the polypeptide has high receptor affinity with HER2, so that the polypeptide has great application prospects in preparation of HER2 imaging agents, products for evaluating the expression level of HER2 or products for imaging and diagnosing HER2 expression positive tumors such as lung cancer.

Description

Technical Field

[0001] This invention relates to a polypeptide and molecular probe targeting HER2, belonging to the field of nuclear medicine technology. Background Technology

[0002] Lung cancer is the leading cause of cancer-related death worldwide, with a five-year survival rate of less than 20% (IARC, 2022). Early and accurate diagnosis and dynamic monitoring of treatment response are crucial for improving prognosis. Studies have shown that overexpression of human epidermal growth factor receptor 2 (HER2) (IHC 3+ or FISH amplification) occurs in approximately 2-5% of NSCLC, and is significantly associated with enhanced tumor metastatic potential, targeted therapy resistance, and shortened overall survival. Therefore, in vivo non-invasive quantitative assessment of HER2 expression has significant clinical value for molecular subtyping, personalized treatment, and prognostic stratification of lung cancer.

[0003] Traditional HER2 testing relies on tissue biopsies, but this method has limitations such as spatial heterogeneity, lack of dynamic monitoring (treatment-induced HER2 expression downregulation occurs in 29.5%), and invasive risks. Breakthroughs in molecular imaging technology offer innovative solutions to these problems: HER2-targeted molecular imaging technology based on positron emission tomography (PET) can achieve real-time three-dimensional quantitative visualization of HER2 expression in lesions throughout the body using radionuclide-labeled specific probes (such as antibodies, nanobodies, and peptides). 68 Ga-labeled Affibody molecules ( 68 For example, Ga-ABY-025 exhibits excellent tumor uptake and rapid blood clearance characteristics in HER2+ breast cancer models. However, such probes face significant challenges in lung cancer applications: high physiological uptake by the liver and kidneys not only leads to interference with background signals but may also mask liver metastases, severely limiting their application value in lung cancer staging and efficacy assessment. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a HER2-targeting peptide and a molecular probe. The HER2-targeting peptide has a high affinity for HER2 and can specifically bind to it. The molecular probe prepared from this peptide can be used to prepare HER2 imaging agents, products for evaluating HER2 expression levels, or products for imaging diagnosis of HER2-positive tumors such as lung cancer.

[0005] Therefore, the present invention provides the following technical solution:

[0006] This invention provides a polypeptide targeting HER2, the amino acid sequence of which is shown in SEQ ID NO.1 (NPNSGFSIWNWC).

[0007] In one embodiment of the present application, the amino acid in the polypeptide is an L-form amino acid.

[0008] The present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned polypeptide.

[0009] As used herein, the terms "polynucleotide" and "nucleic acid molecule" can be used interchangeably and include DNA molecules or RNA molecules. The DNA molecules can be single-stranded or double-stranded.

[0010] Due to the degeneracy of the genetic code, a large number of polynucleotides that can be used to encode the polypeptide of the present application can be obtained, and thus, in the case where a specific amino acid sequence has been identified, any number of different nucleic acids can be made by a person skilled in the art by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. The more preferred polynucleotides can be selected by codon optimization according to the preference of the host cell used in the actual preparation process.

[0011] The nucleic acid molecule can be obtained by conventional methods such as PCR amplification or artificial synthesis method, etc. At present, the polynucleotide sequence can be obtained completely by chemical synthesis.

[0012] The present application also provides a recombinant vector expressing the above-mentioned polypeptide; or, the recombinant vector carrying the above-mentioned nucleic acid molecule.

[0013] As used herein, "vector" refers to a construct capable of delivering, preferably expressing in a host cell, one or more genes or sequences of interest.

[0014] The present application also provides a host cell expressing the above-mentioned polypeptide; the genome of the host cell is integrated with the above-mentioned nucleic acid molecule; or, the host cell carries the above-mentioned recombinant plasmid.

[0015] The host cell of the present application can be a prokaryotic cell, a lower eukaryotic cell or a higher eukaryotic cell, etc.

[0016] The transformation of the vector into the host cell can be carried out by conventional methods well known to those skilled in the art. For example, CaCl2method, electroporation method, calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. The obtained transformants can be cultured by conventional methods well known to those skilled in the art, and the culture medium can be a conventional culture medium. The polypeptide produced by the transformants can be separated and purified by physical, chemical, etc. methods, and can be separated and purified by conventional methods well known to those skilled in the art such as salting out, centrifugation, cell disruption, chromatography, etc.

[0017] The application further provides a HER2-targeting molecular probe, which comprises the polypeptide, a bifunctional chelating group and a radionuclide labeling group; the bifunctional chelating group is connected to the N-terminal asparagine of the polypeptide; and the radionuclide labeling group is combined with the bifunctional chelating group.

[0018] In an embodiment of the application, the bifunctional chelating group is connected to the amino group of the N-terminal asparagine of the polypeptide.

[0019] In an embodiment of the application, the radionuclide labeling group comprises 68 Ga 3+ , [Al 18 F] 2+ , 64 Cu 2 + and / or 177 Lu 3+ .

[0020] In an embodiment of the application, the radionuclide labeling group comprises 68 Ga 3+ , [Al 18 F] 2+ and / or 64 Cu 2+ .

[0021] In an embodiment of the application, the radionuclide labeling group is [Al 18 F] 2+ , 68 Ga 3+ or 64 Cu 2 + .

[0022] In an embodiment of the application, the bifunctional chelating group comprises 1,4,7-triazacyclononane 4,7-diacetic acid 1 acetyl (NOTA) and / or 1,4,7,10 tetraazacyclododecane 4,7,10 triacetic acid 1 acetyl (DOTA);

[0023] The 1,4,7-triazacyclononane 4,7-diacetic acid 1 acetyl has the following structure:

[0024]

[0025] Alternatively, the 1,4,7,10 tetraazacyclododecane 4,7,10 triacetic acid 1 acetyl has the following structure:

[0026]

[0027] wherein * is the attachment site of the bifunctional chelating group to the polypeptide.

[0028] In one embodiment of the present application, the bifunctional chelating group is 1,4,7-triazacyclononane 4,7-diacetic acidyl 1 acetyl or 1,4,7,10-tetraazacyclododecane 4,7,10-triacetic acidyl 1 acetyl.

[0029] In one embodiment of the present application, the bifunctional chelating group with radionuclide labeling group has the following structure:

[0030]

[0031] Alternatively, the bifunctional chelating group with radionuclide labeling group has the following structure:

[0032]

[0033] Alternatively, the bifunctional chelating group with radionuclide labeling group has the following structure:

[0034]

[0035] Alternatively, the bifunctional chelating group with radionuclide labeling group has the following structure:

[0036]

[0037] Alternatively, the bifunctional chelating group with radionuclide labeling group has the following structure:

[0038]

[0039] Alternatively, the bifunctional chelating group with radionuclide labeling group has the following structure:

[0040]

[0041] In one embodiment of the present application, the HER2-targeting molecular probe has the following structure:

[0042]

[0043] The present application also provides a method for preparing the above-mentioned molecular probe, which comprises: mixing the above-mentioned polypeptide with a bifunctional chelating agent 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 a radionuclide to obtain a HER2-targeting molecular probe.

[0044] In one embodiment of the present invention, the method is as follows: the above-mentioned polypeptide, bifunctional chelating agent, and condensing agent are added to an organic solvent and a condensation reaction is carried out to obtain a reaction mixture; the reaction mixture is separated and purified to obtain a bifunctional chelating agent-polypeptide; the bifunctional chelating agent-polypeptide is radiolabeled to obtain a molecular probe targeting HER2.

[0045] In some embodiments, the condensing agent includes, but is not limited to, N,N-diisopropylethylamine (DIPEA).

[0046] In some embodiments, the organic solvent includes, but is not limited to, N,N-dimethylformamide (DMF).

[0047] In some embodiments, the condensation reaction is carried out at 20–30°C for 3–10 hours. The condensation temperature can be any one of 20°C, 23°C, 25°C, 28°C, or 30°C, or a range between any two of these values. The condensation reaction time can be…

[0048] In some embodiments, the ratio of the polypeptide, bifunctional chelating agent, and condensing agent is 0.1–2 mg: 0.1–3 mg: 20–40 μL. Further, the ratio of the polypeptide, bifunctional chelating agent, and condensing agent can be any one of the following values ​​or a range between any two values: 0.1 mg: 3 mg: 20 μL, 2 mg: 0.1 mg: 40 μL, 0.5 mg: 2.5 mg: 38 μL, 0.8 mg: 2 mg: 33 μL, 1 mg: 1.5 mg: 30 μL, 1.5 mg: 1 mg: 25 μL, or 2 mg: 0.5 mg: 20 μL.

[0049] In some embodiments, during the labeling step, the bifunctional chelating agent-peptide, radionuclide, and acidic solution are mixed and reacted at 70–100°C for 10–30 min. In some embodiments, the radionuclide solution is taken, an acidic solution is added, the pH is adjusted, and then the bifunctional chelating agent-peptide is added, reacting at 70–100°C for 10–30 min. In some embodiments, the pH is adjusted to 4–5.

[0050] In some embodiments, the radioactivity intensity in the mixed solution of the radionuclide solution and the acidic solution is 10 to 30 mCi, which can be any one of 10, 15, 20, 25, and 30 mCi or a range between any two values.

[0051] In some embodiments, the concentration range of the aqueous solution of the bifunctional chelating agent-peptide is 0.5 to 1 mg / mL, which can be any one of 0.5, 0.6, 0.8, 1 mg / mL or a range between any two values.

[0052] In some embodiments, the volume ratio of the mixed solution of the radionuclide solution and the acidic solution to the aqueous solution of the bifunctional chelating agent-peptide is (1-5):(1-5), which can be any one of the values ​​of 1:1, 1:5, 5:1, 2:3, 3:2 or a range between any two values.

[0053] In one embodiment of the present invention, the method is as follows: the above-mentioned polypeptide, bifunctional chelating agent, and N,N-diisopropylethylamine (DIPEA) are added to anhydrous N,N-dimethylformamide (DMF), and reacted at 20-30°C for 3-10 h to obtain a reaction mixture; the reaction mixture is separated and purified by chromatography, the product peak is collected, and lyophilized to obtain the bifunctional chelating agent-polypeptide; the bifunctional chelating agent-polypeptide, radionuclide, and glacial acetic acid are mixed and reacted at 70-100°C for 10-30 min to obtain a molecular probe targeting HER2.

[0054] The present invention also provides the application of the above-mentioned polypeptide, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned host cell or the above-mentioned molecular probe in the preparation of products for evaluating HER2 expression levels or in the preparation of products for imaging diagnosis of tumors, wherein the tumor is a HER2-positive tumor.

[0055] In one embodiment of the present invention, the imaging is PET imaging.

[0056] In one embodiment of the present invention, the product is a reagent kit.

[0057] In one embodiment of the present invention, the tumor is a HER2-positive solid tumor.

[0058] In one embodiment of the present invention, the HER2-positive solid tumor includes lung cancer, breast cancer, and / or gastric cancer.

[0059] The present invention also provides a product for evaluating HER2 expression levels, the product comprising 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.

[0060] The present invention also provides a product for imaging diagnosis of tumors, wherein the tumor is a HER2-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.

[0061] In one embodiment of the present invention, the imaging is PET imaging.

[0062] In one embodiment of the present invention, the product is a reagent kit.

[0063] In one embodiment of the present invention, the tumor is a HER2-positive solid tumor.

[0064] In one embodiment of the present invention, the HER2-positive solid tumor includes lung cancer, breast cancer, and / or gastric cancer.

[0065] The technical solution of this invention has the following advantages:

[0066] 1. This invention provides a HER2-targeting polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1 (NPNSGFSIWNWC). This invention demonstrates that HER2 is an important target for diagnosing lung cancer. Furthermore, this invention shows that this polypeptide can specifically bind to HER2 and exhibits a high receptor affinity for HER2. Therefore, this polypeptide shows great promise for applications in the preparation of HER2 imaging agents, products for evaluating HER2 expression levels, or products for the imaging diagnosis of HER2-positive tumors such as lung cancer.

[0067] 2. This invention provides a molecular probe targeting HER2, comprising a HER2-targeting polypeptide with the amino acid sequence shown in SEQ ID NO. 1, a bifunctional chelating group, and a radiolabeled group; wherein the bifunctional chelating group is attached to the N-terminal asparagine of the polypeptide, and the radiolabeled group is bound to the bifunctional chelating group. Currently, HER2-targeting PET imaging molecular probes are mainly radiolabeled HER2 antibodies, which suffer from large molecular weight, weak tissue penetration, and slow biological metabolism. This molecular probe, as a small molecule polypeptide probe, has advantages such as small molecular weight, strong tissue penetration, and rapid blood clearance (mainly metabolized by the kidneys). Furthermore, research in this invention shows that this molecular probe, as a tumor receptor-targeting probe, exhibits significant uptake in HER2-positive lung cancer-bearing mice, high contrast in tumor lesion imaging, and strong tumor resolution, compared to existing... 18 Tumor clinical diagnostic imaging probes such as F-FDG have higher tumor targeting specificity. Therefore, these molecular probes have great application potential in the preparation of products for evaluating HER2 expression levels or for imaging diagnosis of HER2-positive tumors such as lung cancer.

[0068] Furthermore, the labeling method of this molecular probe is simple and universal, the reaction system is stable and the conditions are mild, and it has high radiochemical purity, specific activity and radiochemical yield, making it easy to produce and apply in clinical practice. Attached Figure Description

[0069] Figure 1 HPLC chromatogram of precursor DOTA-NC12.

[0070] Figure 2 High-resolution mass spectrum of precursor DOTA-NC12.

[0071] Figure 3 Molecular probes 68 Radioactive HPLC chromatogram of Ga]Ga-DOTA-NC12.

[0072] Figure 4 Molecular probes 68 Radiometric HPLC chromatograms of Ga]Ga-DOTA-NC12 in PBS and FBS over 2 hours in vitro.

[0073] Figure 5 Molecular probes 68 Dynamic microPET images of Ga-DOTA-NC12 over a 60-minute period in NCI-H2170 tumor-bearing mice and NCI-H2170 tumor-bearing mice with DOTA-NC12 blockade.

[0074] Figure 6 Molecular probes 68 Dynamic microPET images of Ga-DOTA-NC12 in NCI-H520 tumor-bearing mice over a period of 60 minutes.

[0075] Figure 7 Molecular probes 68 Tumor uptake values ​​(%ID / g) of Ga-DOTA-NC12 at various time points within 60 min in NCI-H2170 tumor-bearing mice, NCI-H2170 tumor-bearing mice with DOTA-NC12 blockade, and NCI-H520 tumor-bearing mice.

[0076] Figure 8 Molecular probes 68 Biodistribution map of Ga-DOTA-NC12 in NCI-H2170 tumor-bearing mice and NCI-H520 tumor-bearing mice at 60 min time points.

[0077] Figure 9 Molecular probes 68 Pharmacokinetic analysis results of Ga]Ga-DOTA-NC12. Detailed Implementation

[0078] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0079] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0080] Example 1: A polypeptide targeting HER2

[0081] This embodiment provides a polypeptide that targets HER2, the amino acid sequence of which is shown in SEQ ID NO.1 (NPNSGFSIWNWC).

[0082] Example 2: A molecular probe targeting HER2 [ 68 Ga]Ga-DOTA-NC12

[0083] This embodiment provides a molecular probe targeting HER2. 68 Ga]Ga-DOTA-NC12, the molecular probe [ 68 Ga]Ga-DOTA-NC12 has the following structure:

[0084]

[0085] Example 3: A method for preparing molecular probes [ 68 Ga]Ga-DOTA-NC12 method

[0086] This embodiment provides the preparation of the molecular probe described in Example 2. 68 The method for Ga-DOTA-NC12 is as follows:

[0087] Using L-type amino acids as raw materials, NC12, with the amino acid sequence shown in SEQ ID NO.1, was synthesized via solid-phase peptide synthesis. 1 mg of NC12 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 h to obtain a reaction solution. After the reaction was complete, 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 then subjected to semi-preparative high-performance liquid chromatography (HPLC) (chromatographic conditions: semi-preparative reversed-phase C18 column (Waters XBridge)). (C18, 5μm, 10×250mm) Mobile phase composition: Phase A is an aqueous solution containing 0.1% (v / v) trifluoroacetic acid (TFA), and Phase B is an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid (TFA). Gradient elution conditions: 0–10 min, Phase A 90% (v / v), Phase B 10% (v / v), flow rate 3 mL / min; 10–30 min, Phase A decreases to 10% (v / v), Phase B increases to 90% (v / v), flow rate 3 mL / min; 30–40 min, Phase A increases to 90% (v / v), Phase B decreases to 10% (v / v), flow rate 3 mL / min. Detection wavelength: 220 nm. Column temperature: 25℃. Separation and purification were performed, and the eluent (i.e., the product peak) was collected and lyophilized to obtain the precursor DOTA-NC12 (i.e., a bifunctional chelating agent-peptide). The precursor DOTA-NC12 was determined using HPLC and high-resolution mass spectrometry. Figure 1 The high-performance liquid chromatography (HPLC) chromatogram of the precursor DOTA-NC12; Figure 2 This is the high-resolution mass spectrum of the precursor DOTA-NC12. The retention time (Rt) of the HPLC chromatogram is 4.727 min, the molecular weight (m / z) of the MS mass spectrometry is 1811.4, and the purity determined by HPLC is greater than 95%.

[0088] Use 0.05M hydrochloric acid solution (5 mL) as the fractionation eluent from 68 Ge / 68 Eluting radionuclides in a Ga generator 68 Ga, obtained 68 Ga 3+ Elution buffer; 68 Ga 3+ The eluent (1.4 mL) was transferred to a new Eppendorf tube, and after testing the isotope dose, it was eluted with 0.25 M sodium acetate solution (320 μL). 68 Ga 3+The pH of the eluent was adjusted to 4.0 to obtain a mixture; 50 μg of the precursor DOTA-NC12 was added to the mixture, and the mixture was incubated at 80 °C for 15 min, with shaking every 5 min during incubation, to obtain a reaction solution; 10 mL of water for injection was added to the reaction solution and mixed well to obtain a diluent; the diluent was transferred to a Sep-pak C18 column, and the Sep-pak C18 column was first rinsed three times with 10 mL of water for injection and dried, and then eluted with 10 mM hydrochloric acid ethanol (300 μL) to obtain a molecular probe containing HER2 targeting [ 68 The eluent for Ga-DOTA-NC12 was prepared; the eluent 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 required product. 68 Ga-DOTA-NC12 injection solution. The determination was performed by radio-HPLC (same as Example 4). 68 Ga]Ga-DOTA-NC12 injection. Figure 3 Molecular probes targeting HER2 [ 68 The radiometric HPLC chromatogram of Ga]Ga-DOTA-NC12 showed a target imaging agent retention time of 11.3 min and a product radiochemical purity greater than 95%.

[0089] Example 4: Molecular probe [ 68 Stability experiment of Ga-DOTA-NC12

[0090] This embodiment provides the molecular probe described in Embodiment 2. 68 The stability experiment of Ga-DOTA-NC12 is as follows:

[0091] Take 2 mL of PBS buffer (pH 7.4, 0.1 M) or 2 mL of fetal bovine serum (FPS) and mix with 50 μCi of the solution prepared in Example 3. 68 After thoroughly mixing the Ga-DOTA-NC12 injection solution, it was incubated at 37℃ for 0, 0.5, 1.5, and 2 hours, respectively, to obtain the incubation solution. The incubation solution was then analyzed using high-performance liquid chromatography (HPLC) to evaluate its properties. 68 The in vitro stability of Ga]Ga-DOTA-NC12 was tested by repeating the experiment more than 3 times. Figure 4 Molecular probes 68 Radiometric HPLC chromatograms of Ga]Ga-DOTA-NC12 in PBS and FBS over 2 hours in vitro.

[0092] HPLC analysis conditions: The analytical column was a ZORBAX Eclipse XDBC18 column; the mobile phases were: Phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid (TFA), and 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 decreased to 10%, Phase B increased to 90%, flow rate 1 mL / min; 16–20 min, Phase A increased to 90%, Phase B decreased to 10%, flow rate 1 mL / min. The UV detection wavelength was 220 nm, and the radioactivity detector was from LabLogic Systems Ltd.

[0093] Figure 4 The results show that molecular probes [ 68 Ga]Ga-DOTA-NC12 is stable in both PBS and FBS, exhibiting good in vitro stability.

[0094] Example 5: Molecular probe [ 68 Micro-PET Imaging Experiment with Ga-DOTA-NC12

[0095] This embodiment provides the molecular probe described in Embodiment 3. 68 Micro-PET imaging experiments using Ga-DOTA-NC12 were conducted. Micro-PET / CT imaging studies utilized Siemens Inveon MicroPET / CT, with an Inveon Acquirision workplace (IAW) 2.2 for acquisition and an Inveon Research workplace (IRW) for data analysis. The specific procedures are as follows:

[0096] HER2-high expressing human lung cancer cells NCI-H2170 and HER2-low expressing human lung cancer cells NCI-H520 (both purchased from the Cell Bank of the Chinese Academy of Sciences) were used at 5×10⁻⁶ cells per cell line. 6 The tumors were subcutaneously injected into the right axilla of immunodeficient mice (BALB / c nude mice) (purchased from Changzhou Cavens Laboratory Animal Co., Ltd., 4-5 weeks old, weighing 18-20g) at a density of 1 / mouse, until the tumor volume reached 200mm. 3 Micro-PET imaging experiments were performed on NCI-H2170 tumor-bearing mice and NCI-H520 tumor-bearing mice.

[0097] HER2-overexpressing tumor imaging: NCI-H2170 tumor-bearing mice were anesthetized with isoflurane and fixed on the scanning table. 100 μCi of the tumor sample prepared in Example 3 was collected. 68Ga-NOTA-GL10 injection solution was administered to tumor-bearing mice via the tail vein. Immediately after injection, a 60-minute dynamic PET scan was performed. Imaging suppression: [Ga-NOTA-GL10 injection solution was administered to tumor-bearing mice via the tail vein.] 68 30 minutes before administration of Ga-DOTA-NC12 (100 μCi), tumor-bearing mice were injected via the tail vein with the precursor DOTA-NC12 (50 μg). Other procedures were the same as for “HER2-overexpressing tumor imaging”. Figure 5 Molecular probes 68 Dynamic microPET images of Ga-DOTA-NC12 over a 60-minute period in NCI-H2170 tumor-bearing mice and NCI-H2170 tumor-bearing mice with DOTA-NC12 blockade.

[0098] HER2-low expression tumor imaging: NCI-H520 tumor-bearing mice were anesthetized with isoflurane and fixed on the scanning table. 100 μCi of the tumor sample prepared in Example 3 was collected. 68 Ga]Ga-DOTA-NC12 injection solution was injected into tumor-bearing mice via the tail vein. Immediately after the injection, a dynamic PET scan was performed for 60 minutes. Figure 6 Molecular probes 68 Dynamic microPET images of Ga-DOTA-NC12 in NCI-H520 tumor-bearing mice over a period of 60 minutes.

[0099] in, Figures 5-6 The upper part shows a cross-sectional PET image of the tumor site. Figures 5-6 The lower part shows a coronal PET image of the tumor site. The white circle indicates the location of the tumor. Figure 7 Molecular probes 68 Tumor uptake values ​​(%ID / g) of Ga-DOTA-NC12 at various time points within 60 min in NCI-H2170 tumor-bearing mice, NCI-H2170 tumor-bearing mice with DOTA-NC12 blockade, and NCI-H520 tumor-bearing mice.

[0100] Micro-PET imaging results indicate that the developer [ 68 Ga-DOTA-NC12 exhibited high uptake in NCI-H2170 tumor-bearing mice, with a maximum uptake value reaching 5.01±0.03% ID / g. This was 2.72 times and 2.60 times the maximum uptake values ​​in NCI-H520 tumors (1.84±0.16% ID / g) and the DOTA-NC12 blockade group tumors (1.93±0.14% ID / g), respectively, indicating that [Ga-DOTA-NC12 showed high uptake in NCI-H2170 tumors (1.84±0.16% ID / g) and DOTA-NC12 blockade group tumors (1.93±0.14% ID / g), respectively. 68Ga-DOTA-NC12 exhibits high binding specificity for NCI-H2170 tumors. Tumor uptake was significantly reduced in the blocking group, further validating the probe's binding targeting. Dynamic imaging showed rapid blood clearance of the imaging agent, primarily metabolized by the kidneys and liver. Imaging results indicate that the imaging agent can be specifically taken up at the tumor site, demonstrating promising application prospects.

[0101] Example 6: Molecular probe [ 68 In vivo biodistribution experiment of Ga]Ga-DOTA-NC12

[0102] This implementation provides the molecular probe described in Example 2. 68 The in vivo biodistribution experiment of Ga-DOTA-NC12 was conducted as follows:

[0103] NCI-H2170 and NCI-H520 tumor-bearing mice from Example 5 were injected intravenously with 100 μCi of the PET imaging agent prepared in Example 3. 68 Ga]Ga-DOTA-NC12. Mice were euthanized by cervical dislocation 60 minutes after injection. Blood, brain, heart, lungs, liver, spleen, and kidneys, as well as other major organs and tissues, were collected, weighed, and their radioactivity was measured in a gamma counter to study the biodistribution of the imaging agent in the mice. Figure 8 Molecular probes 68 Biodistribution of Ga]Ga-DOTA-NC12 in NCI-H2170 tumor-bearing mice and NCI-H520 tumor-bearing mice at 60 min time points.

[0104] Figure 8 The results showed that the molecular probe [ 68 Ga]Ga-DOTA-NC12 is primarily metabolized by the kidneys and is rapidly cleared from the bloodstream.

[0105] Example 7: Molecular probe [ 68 Pharmacokinetics of Ga-DOTA-NC12

[0106] This implementation provides the molecular probe described in Example 3. 68 The pharmacokinetic experiment of Ga-DOTA-NC12 was conducted as follows:

[0107] Four Kunming rats (purchased from Changzhou Cavens Laboratory Animal Co., Ltd., 5-6 weeks old, weighing 20-25g) were injected intravenously with 100 μCi of the [prepared in Example 3] into the tail vein of the four Kunming rats. 68Ga-DOTA-NC12 injection solution. Blood samples were collected via 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. Results are as follows. Figure 9 As shown, the concentration-time curves indicate a good fit with the two-compartment pharmacokinetic model. Due to its significant hydrophilicity, [ 68 Ga-DOTA-NC12 exhibits rapid distribution dynamics, with a distribution half-life (t... 1 / 2α The elimination half-life (t) was 2.22 min, followed by efficient systemic clearance, with an elimination half-life of 2.22 min. 1 / 2β The duration of action was 31.08 min. These pharmacokinetic characteristics indicate that the molecular probe [ 68 Ga-DOTA-NC12 possesses dual advantages, including rapid exudation and effective penetration into tumor tissue, while simultaneously being effectively cleared from non-target tissues. Therefore, [ 68 The Ga-DOTA-NC12 rapidly achieves a high tumor-to-background ratio, which is crucial for tumor diagnostic imaging applications.

[0108] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A polypeptide targeting HER2, 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 includes a nucleotide sequence encoding the polypeptide of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector expresses the polypeptide of claim 1; or, the recombinant vector carries the nucleic acid molecule of claim 2.

4. A host cell, characterized in that, The host cell expresses the polypeptide of claim 1; or, the host cell's genome integrates the nucleic acid molecule of claim 2; or, the host cell carries the recombinant plasmid of claim 3.

5. A molecular probe targeting HER2, characterized in that, The molecular probe comprises the polypeptide of claim 1, a bifunctional chelating group, and a radionuclide labeling group; the bifunctional chelating group is attached to the N-terminal asparagine of the polypeptide of claim 1; and the radionuclide labeling group is attached to the bifunctional chelating group.

6. The molecular probe as described in claim 5, characterized in that, The bifunctional chelating group is attached to the amino group of the N-terminal asparagine of the polypeptide of claim 1.

7. A method for preparing the molecular probe according to claim 5 or 6, characterized in that, The method is as follows: the polypeptide of claim 1 is mixed with a bifunctional chelating agent and reacted to obtain a reaction mixture; the reaction mixture is separated and purified to obtain a bifunctional chelating agent-polypeptide; the bifunctional chelating agent-polypeptide is radiolabeled to obtain a molecular probe targeting HER2.

8. The use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, the host cell of claim 4, or the molecular probe of claim 5 or 6 in the preparation of products for evaluating HER2 expression levels or for the preparation of products for imaging diagnosis of tumors, characterized in that, The tumor was a HER2-positive tumor.

9. A product for evaluating HER2 expression levels, characterized in that, The product contains the polypeptide of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, the host cell of claim 4, and / or the molecular probe of claim 5 or 6.

10. A product for imaging diagnosis of tumors, characterized in that, The tumor is a HER2-positive tumor, and the product contains the polypeptide of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, the host cell of claim 4, and / or the molecular probe of claim 5 or 6.

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