Polypeptide and radioactive polypeptide targeting HER2 protein as well as preparation method and application of polypeptide and radioactive polypeptide

Abstract

The invention belongs to the technical field of polypeptides, and discloses a polypeptide targeting HER2 protein, a radioactive polypeptide and a preparation method and application of the radioactive polypeptide, and the structural formula of the radioactive polypeptide is as follows: # imgabs0 #. Through in-vitro radiochemical property research and in-vivo stability research, the 68Ga-DOTA-AON is found to be high in stability and hydrophilic; through cell uptake research and receptor binding force research, the 68Ga-DOTA-AON is found to have relatively high affinity to HER2 protein expressed by HT-29 cells; the tumor imaging effect of the radioactive polypeptide 68Ga-DOTA-AON is investigated through small animal PET / CT, and it is found that [68Ga] Ga-DOTA-AON can be used for in-vivo imaging of HER2 positive colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides, and in particular relates to a polypeptide targeting HER2 protein, a radioactive polypeptide, and a preparation method and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. In recent years, the incidence of CRC and its early-onset forms has shown an upward trend. Currently, the detection methods of CRC include colonoscopy and traditional anatomical imaging techniques. As the gold standard for CRC detection, colonoscopy has some limitations, such as the inability to accurately determine the nature of submucosal lesions, as well as patient tolerance and contraindications to examination. In addition, traditional anatomical imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI), have a certain lag and can only be identified when the tumor undergoes morphological changes. Compared with these traditional methods, molecular imaging technology can improve the detection rate of CRC and help to gain a deeper understanding of the extent and characteristics of the lesions. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a polypeptide targeting HER2 protein, a radioactive polypeptide, and a preparation method and application thereof.

[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions: In a first aspect, the present invention provides a polypeptide targeting HER2 protein, the structural formula of which is as follows: .

[0006] In a second aspect, the present invention provides a radioactive polypeptide targeting HER2 protein, the structural formula of which is shown below and is named 68 Ga-DOTA-AON: .

[0007] Human epidermal growth factor receptor 2 (HER2) is a transmembrane protein that belongs to the epidermal growth factor receptor (EGFR) family. HER2 lacks endogenous ligands. When the extracellular domain of HER2 binds to its ligand (such as epidermal growth factor or transforming growth factor), it activates tyrosine kinase activity by forming heterodimers with other HER family proteins, self-assembling into ligand-independent homodimers under HER2 overexpression conditions, or undergoing autophosphorylation, thereby triggering the cascade activation of downstream signaling pathways such as Ras / Raf / MEK / ERK and PI3K / Akt, promoting tumor cell cycle progression, promoting cell transition from G1 phase to S phase, and accelerating tumor cell division and proliferation; in addition, it also plays a role in inhibiting cell apoptosis, regulating tumor cell metabolism, promoting tumor cell invasion and metastasis, affecting tumor angiogenesis, and participating in tumor immune escape. Therefore, abnormal expression of HER2 is closely related to the occurrence and development of CRC and is an important target for the treatment of HER2-positive CRC patients.

[0008] PET imaging has high sensitivity and strong targeting specificity. It can non-invasively, quickly, whole-body, and visualize lesions, and is a commonly used clinical examination method. Peptide radiopharmaceuticals are important tools for PET imaging, with advantages such as high affinity and low immunogenicity, easy synthesis and modification, and high targeting. Depending on whether the two ends of the polypeptide chain are connected to form a closed ring structure, polypeptides can be divided into linear peptides and cyclic peptides. Compared with linear peptides, cyclic peptides improve their affinity with ligands by improving structural rigidity and optimizing their pharmacokinetic and pharmacodynamic properties.

[0009] The present invention synthesized the peptide DOTA-AON targeting HER2-positive colorectal cancer for the first time and carried out 68 Ga-labeled, radioactive peptides targeting HER2 protein were constructed [ 68 Ga]Ga-DOTA-AON.

[0010] The radioactive polypeptide targeting HER2 protein 68The amino acid sequence of Ga]Ga-DOTA-AON is: His-Tyr -Gln-cyclo(Lys-Ser-Pro-Asn-Pro-Arg-Phe-Gly-Ser-Lys), and the ring structure is composed of 10 amino acids KSPNPRFGSK (SEQ ID NO: 1), and two lysines in its amino acid sequence form an amide bond to form a cyclic polypeptide; the linker is composed of 3 amino acids HYQ, and its amino acid sequence is sequentially condensed, and the carboxyl group of histidine is condensed with the amino group of lysine in the ring structure. The amino group at the N-terminus of the cyclic polypeptide AON is condensed with the coupling agent DOTA-NHS through the bioconjugation technology to obtain the polypeptide conjugate DOTA-AON. The chelating agent DOTA in DOTA-AON reacts with the radionuclide 68 Ga chelation, construction of radioactive peptides [ 68 Ga]Ga-DOTA-AON.

[0011] In a third aspect, the present invention provides a method for preparing the polypeptide targeting HER2 protein, comprising the following steps: The Lys-Ser-Pro-Asn-Pro-Arg-Phe-Gly-Ser-Lys polypeptide sequence was synthesized by solid phase synthesis; The polypeptide sequence is cyclized, and the obtained ring structure is subjected to dehydration condensation with His-Tyr-Gln, and then connected with a coupling agent DOTA to obtain the product.

[0012] In a fourth aspect, the present invention provides a method for preparing the radioactive polypeptide targeting HER2 protein, comprising the following steps: The Lys-Ser-Pro-Asn-Pro-Arg-Phe-Gly-Ser-Lys polypeptide sequence was synthesized by solid phase synthesis; The polypeptide sequence is cyclized, and the obtained cyclic structure is dehydrated and condensed with His-Tyr-Gln, and then connected with a coupling agent DOTA to obtain a polypeptide conjugate DOTA-AON; The peptide conjugate DOTA-AON was dissolved in a buffer solution to obtain a DOTA-AON solution, to which [ 68 Ga]GaCl 3 The solution is reacted at 70-90°C for a set time to obtain a radioactive polypeptide.

[0013] In some embodiments, the buffer is NaOAc buffer with a pH value of 4.0-4.6.

[0014] In some embodiments, the concentration of the DOTA-AON solution is 20-25 μM.

[0015] Preferably, the [68 Ga]GaCl 3 The solution is the solution eluted with 0.01N HCl.

[0016] Further preferably, the DOTA-AON solution and [ 68 Ga]GaCl 3 The volume ratio of the solution is 0.6-1.5:1.

[0017] In some embodiments, the reaction is carried out at 70-90° C. for 15-20 min.

[0018] In a fifth aspect, the present invention provides the use of the radioactive polypeptide targeting HER2 protein in HER2-positive colorectal cancer tumor imaging.

[0019] The beneficial effects achieved by one or more embodiments of the present invention are as follows: The present invention synthesized the peptide DOTA-AON targeting HER2-positive colorectal cancer for the first time and carried out 68 Ga-labeled, radioactive peptides targeting HER2 protein were constructed [ 68 Ga]Ga-DOTA-AON. Through in vitro radiochemical property studies and in vivo stability studies, it was found 68 Ga-DOTA-AON is highly stable and hydrophilic; through cell uptake studies and receptor binding studies, it was found that 68 Ga-DOTA-AON has a high affinity for HER2 protein expressed in HT-29 cells; radioactive peptides were investigated by small animal PET / CT 68 The tumor imaging effect of Ga-DOTA-AON was found to be 68 Ga]Ga-DOTA-AON can be used for in vivo imaging of HER2-positive colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 It is the ESI-MS mass spectrum of DOTA-AON in Example 1 of the present invention.

[0022] Figure 2 In Example 1 of the present invention, 68 Radiochemical purity detection diagram of Ga]Ga-DOTA-AON.

[0023] Figure 3 In Example 3 of the present invention, 68Bar graph of the cellular uptake experiment of Ga]Ga-DOTA-AON and HER2 protein.

[0024] Figure 4 In Example 3 of the present invention, 68 Saturation experimental curve of Ga]Ga-DOTA-AON and HER2 protein.

[0025] Figure 5 In Example 5 of the present invention, 68 Blood clearance curve of Ga]Ga-DOTA-AON.

[0026] Figure 6 In Example 6 of the present invention, 68 In vivo biodistribution of Ga]Ga-DOTA-AON in HT-29 tumor-bearing mice.

[0027] Figure 7 These are PET / CT images of HT-29 tumor-bearing mice in the experimental group and the inhibition group in Example 7 of the present invention, and PET / CT images of MDA-MB-468 tumor-bearing mice in the negative control group.

[0028] Figure 8 This is the immunohistochemistry diagram of HER2 expression in the tumor of HT-29 tumor-bearing mice in Example 8 of the present invention, wherein a is magnified 400 times; b is magnified 200 times.

[0029] Fig. 9 This is the immunohistochemistry diagram of HER2 expression in the tumor of MDA-MB-468 tumor-bearing mice in Example 8 of the present invention, wherein a is magnified 400 times; b is magnified 200 times. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are illustrative and 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.

[0031] DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid.

[0032] DOTA-NHS: hydroxysuccinimide-tetraazacyclododecanetetraacetic acid.

[0033] Rink amide-MBHA resin: Rink amide MBHA resin; DIPEA: N,N-diisopropylethylamine; HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBT: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid; TIS: triisopropylsilane; PhOH: phenol; Thioanisole: methyl phenyl thioether; DIPEA: N,N-diisopropylethylamine; DCM: dichloromethane.

[0034] The present invention will be further described below in conjunction with the embodiments.

[0035] Example 1 (1) Rink amide-MBHA resin was used as the starting resin and Fmoc-amino acid as the raw material. The DIPEA / HBTU / HOBT condensation system was used for solid phase synthesis. Then, TFA / TIS / PhOH / Thioanisole / H 2 O cleavage buffer from the resin.

[0036] The amino protecting group on the Rink amide-MBHA resin is removed with a piperidine DMF solution, the resin is washed, and the unreacted sites are blocked with a DCM blocking solution containing DIPEA / acetic anhydride; the solvent is removed, the resin is washed, and Lys with a protecting group is connected, and the Lys bound to the resin is used 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 Lys-Ser-Pro-Asn-Pro-Arg-Phe-Gly-Ser-Lys from the carboxyl end to the amino end until all the sequences required for the ring structure are coupled to the resin; Wherein, the concentration of piperidine DMF solution is 20% (volume percentage); In the blocking solution, the volume ratio of DIPEA: acetic anhydride = 1:2; During the condensation reaction, the reactants were calculated by the molar ratio of Fmoc-amino acid: DIPEA: HOBT: HBTU: Rinkamide-MBHA resin = 3.5:7:4:4:1; (2) Remove the Fmoc protecting group, condense two lysines to form a peptide bond, complete the cyclization, and then continue to dehydrate and condense with His-Tyr-Gln. Then connect the coupling agent DOTA, cleave the peptide from the resin, and obtain the DOTA-His-Tyr-Gln-cyclo(Lys-Ser-Pro-Asn-Pro-Arg-Phe-Gly-Ser-Lys) peptide sequence, which is further purified by high performance liquid chromatography to obtain DOTA-AON. Among them, in the lysis buffer, by volume ratio, TFA:TIS:PhOH:Thioanisole:H 2 O=30:2:2:1:1; In the case of DOTA coupling, the reactants were calculated by the molar ratio of resin: DOTA-NHS: DIPEA = 2:8:1; When the peptide was cleaved, the mass ratio of DOTA-His-Tyr -Gln-cyclo(Lys-Ser-Pro-Asn-Pro-Arg-Phe-Gly-Ser-Lys) peptide resin: cleavage solution was 1:10; The cyclization conditions are as follows: at room temperature, the peptide is mixed with dimethyl sulfoxide (DMSO) and reacted for 24 hours to promote the cyclization reaction of the peptide.

[0037] (3) Dissolve DOTA-AON in NaOAc buffer and add [ 68 Ga]GaCl 3 The solution was reacted at 80°C for 15 minutes to obtain a radioactive peptide targeting HER2 protein. 68 Ga]Ga-DOTA-AON.

[0038] The concentration of NaOAc buffer was 0.1 M and the pH was 4.0-4.6; after DOTA-AON was dissolved in NaOAc buffer, the concentration was 20 μM; [ 68 Ga]GaCl 3 The solution is the solution eluted with 0.01N HCl; 68 When Ga is labeled, DOTA-AON solution and [ 68 Ga]GaCl 3 The volume ratio of the solution is 1:1.

[0039] The DOTA-AON prepared in step (2) of this example was characterized by ESI-MS mass spectrometry. Figure 1 As shown, the experimental results show that the molecular weight of DOTA-AON is consistent with the theoretical value and its purity is higher than 98%.

[0040] The radioactive polypeptide targeting HER2 protein prepared in step (3) of this example [ 68 The radiochemical purity of Ga]Ga-DOTA-AON was tested by analytical HPLC with a radioactivity detector. Figure 2 shown.

[0041] Among them, HPLC mobile phase (A = 0.1% TFA / water, B = 0.1% TFA / acetonitrile), Zorbax 5 μ C18 100 Å (250 × 4.6 mm, 5 μm), see Table 1 for details.

[0042] Table 1

[0043] Depend on Figure 2 It can be seen that 68 The radiochemical purity of Ga]Ga-DOTA-AON is greater than 99%.

[0044] Example 2 Radiopeptide targeting HER2 protein 68 Study on the Radiochemical Properties of Ga]Ga-DOTA-AON in Vitro In vitro stability: refers to the ability of a radioactive peptide to maintain its performance characteristics for a certain period of time under specific storage conditions.

[0045] The [ 68 Ga]Ga-DOTA-AON was mixed with PBS buffer (pH = 7.4) and human serum (volume ratio of 1:3), and incubated at 37 °C for 30 minutes and 60 minutes, 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 are shown in [ 68 The percentage of radionuclides in the chemical form of Ga]Ga-DOTA-AON to the total radionuclides is shown in Table 2.

[0046] Table 2

[0047] The experimental results showed that in vitro 68 The radiochemical purity of Ga]Ga-DOTA-AON did not change significantly, all of which were greater than 90%, indicating that [ 68 Ga]Ga-DOTA-AON has good in vitro stability in PBS buffer and human serum.

[0048] Hydrophilicity and lipophilicity test: 10 μL (about 0.1 MBq) of the [ 68 Ga]Ga-DOTA-AON was diluted to 500 µL with HEPES buffer (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.

[0049] The lipid-water partition coefficient is calculated by the formula Log D = [Log 10 (radioactivity count of organic phase / radioactivity count of aqueous phase)].

[0050] The experimental results showed that radioactive peptides[ 68 The lipid-water partition coefficient of Ga]Ga-DOTA-AON is Log D It is -3.28 ± 0.14, indicating good hydrophilicity.

[0051] Example 3 Cellular uptake and receptor binding studies of DOTA-AON targeting HER2 protein Cell culture: In 5% CO 2 The human colorectal adenocarcinoma cell line HT-29 was cultured in McCoy's 5A medium containing 10% fetal bovine serum in a 37°C cell culture incubator.

[0052] In 5% CO 2 The human triple-negative breast cancer cell line MDA-MB-468 was cultured in L-15 medium containing 10% fetal bovine serum in a 37°C cell culture incubator.

[0053] Cellular uptake assay: HT-29 cells were cultured in 24-well plates to a volume of 1 × 10 5 After 10 cells / well, wash with PBS.

[0054] The experimental group was heated at 37°C with the [ 68 Ga]Ga-DOTA-AON (20 nM) was incubated for 5 min, 15 min, 30 min, 60 min, 90 min, and 120 min.

[0055] The binding of HER2 protein in the inhibitory group was blocked by adding 1000-fold concentration of DOTA-AON (20 μmol) at the same time, and then [ 68 The cells were further incubated with Ga]Ga-DOTA-AON (20 nM) for 15 min, 30 min, 60 min, and 120 min.

[0056] After the incubation, the cells were washed three times with cold PBS, the cells were collected and the intracellular radioactivity counts were detected by a gamma counter to calculate the cell uptake histogram, e.g. Figure 3 shown.

[0057] like Figure 3 As shown, the experimental results show that within 60 minutes, the cells 68The radioactive uptake of Ga]Ga-DOTA-AON gradually increased and stabilized after 60 minutes; the radioactive uptake of the inhibition group was significantly lower than that of the experimental group, indicating that [ 68 The uptake of Ga]Ga-DOTA-AON in HT-29 cells was significantly inhibited by unlabeled DOTA-AON, indicating its binding specificity.

[0058] Saturation experiment: HT-29 cells were cultured in 24-well plates to a volume of 1 × 10 5 cells / well, different concentrations of [ 68 Ga]Ga-DOTA-AON,[ 68 The final concentrations of Ga]Ga-DOTA-AON were 0.3125nM, 0.625nM, 1.25nM, 2.5nM, 5nM, 10nM, 20nM, and 40nM. The cells were incubated at 37°C for 60 minutes, and the radioactivity counts on the cell surface and inside the cells were measured. 68 Binding capacity of Ga-DOTA-AON to HER2 protein (specific binding + nonspecific binding).

[0059] Nonspecific binding was detected by co-incubation [ 68 Ga]Ga-DOTA-AON and 1000 times the concentration of unlabeled DOTA-AON (20 μmol), and then the saturation binding curve was fitted by Graphpad Prism software to obtain [ 68 The maximum specific binding capacity of Ga]Ga-DOTA-AON and receptor ( B max ) and binding force ( K d ), the results are shown in Table 3 and Figure 4 (Saturation experimental curve) shown.

[0060] Table 3

[0061] The saturation test results show that [ 68 Receptor binding ability of Ga]Ga-DOTA-AON with HER2 protein in HT-29 cells K d The value reached 25.12±3.32nM, [ 68 Maximum binding capacity of Ga]Ga-DOTA-AON to the receptor of HT-29 cells B max Approximately 1398 ± 103.5 fmol / mg protein.

[0062] Example 4 Radiopeptide targeting HER2 protein 68 Study on the in vivo stability of Ga]Ga-DOTA-AON Three healthy female Balb / c mice were used to evaluate the [ 68 In vivo stability of Ga]Ga-DOTA-AON.

[0063] Each mouse was injected via the tail vein with about 37 MBq of [ 68 Ga]Ga-DOTA-AON, 60 minutes after injection, kidney, liver, urine and blood samples were collected from three mice, processed and injected into Radio-HPLC to observe [ 68 The radiochemical purity of different samples of Ga]Ga-DOTA-AON is shown in Table 4.

[0064] Table 4

[0065] The experimental results showed that radioactive peptides [ 68 The stability of Ga]Ga-DOTA-AON in kidney, liver, urine and blood after 60 min was higher than 80%, and its in vivo stability was good.

[0066] Example 5 Radiopeptide targeting HER2 protein 68 Study on the pharmacokinetics of Ga]Ga-DOTA-AON Three healthy female Balb / c mice were used to evaluate the [ 68 Pharmacokinetic properties of Ga]Ga-DOTA-AON. Each mouse was injected via the tail vein with about 3.7 MBq of [ 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-DOTA-AON. The percentage injected dose rate per gram of tissue (%ID / g) was corrected for radioactive decay. The pharmacokinetic curves were fitted using DAS2.0 software. The results are shown in Figure 5 .

[0067] The experimental results showed that radioactive peptides [ 68 The pharmacokinetic curve of Ga]Ga-DOTA-AON in healthy mice conforms to the two-compartment model. 1 / 2β =32.18 ± 2.63 minutes, indicating that [ 68 The metabolic rate of Ga]Ga-DOTA-AON in vivo is moderate.

[0068] Example 6 Radiopeptide targeting HER2 protein 68 Study on the biodistribution of Ga]Ga-DOTA-AON in vivo Establishment of NSG subcutaneous tumor model: HT-29 cells were cultured in vitro, washed with serum-free culture medium 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 colorectal adenocarcinoma cells was injected into the left axilla of the mouse forelimb, and the general condition of the mouse and the size of the tumor were monitored. When the tumor grew to 5-8 mm in diameter, subsequent experiments could be performed.

[0069] The same method was used to construct the MDA-MB-468 tumor-bearing mouse model.

[0070] Six HT-29 tumor-bearing mice were randomly divided into two groups. Both groups were injected with about 3.7 MBq of radioactive peptide [ 68 Ga]Ga-DOTA-AON. Three mice were killed 15 minutes after injection in one group, and three mice were killed 30 minutes after injection in the other group. Blood, tumors and other major organs and tissues were collected, weighed and radioactive counts were measured. After correction for radioactive decay, the percentage injected dose rate per gram of tissue (%ID / g) was calculated. The results are shown in Table 5 and Figure 6 shown.

[0071] The tumor / muscle ratio (T / M) was defined as the ratio of radioactivity counts in the tumor to those in the muscle.

[0072] Table 5

[0073] The experimental results showed that in the HT-29 tumor-bearing mouse model, injection of [ 68 After 15 and 30 minutes, the radioactivity uptake of Ga]Ga-DOTA-AON was highest in the kidney, followed by the tumor, indicating that [ 68 Ga]Ga-DOTA-AON is mainly metabolized through the renal pathway and has tumor targeting in HER2-positive tumors. The T / M ratios were 3.70 ± 0.35 and 6.43 ± 0.17 at 15 and 30 minutes after injection, respectively, and the tracer had a good tumor-to-background ratio at 30 minutes.

[0074] Three MDA-MB-468 tumor-bearing mice were injected with about 3.7 MBq of radioactive peptide via the tail vein. 68Ga]Ga-DOTA-AON. Three mice were killed 30 minutes after the peptide injection, and blood, tumors and other major organs and tissues were collected, weighed and radioactive counts were measured, and %ID / g was calculated. The results are shown in Table 6.

[0075] Table 6

[0076] The experimental results showed that in the MDA-MB-468 tumor-bearing mouse model, injection of [ 68 Ga]Ga-DOTA-AON, the radioactivity uptake in the kidney was the highest after 30 minutes, indicating that [ 68 Ga]Ga-DOTA-AON is mainly metabolized by the kidney; radioactivity uptake is low in tumors, suggesting that [ 68 Ga]Ga-DOTA-AON has no obvious targeting activity in HER2-negative tumors.

[0077] Example 7 [ 68 PET / CT imaging of Ga]Ga-DOTA-AON in tumor-bearing mice Six HT-29 tumor-bearing mice described in Example 6 were randomly divided into two groups: an experimental group and an inhibition group, with 3 mice in each group.

[0078] The experimental group was injected directly via the tail vein with approximately 7.4 MBq [ 68 Ga]Ga-DOTA-AON, and 60 minutes after the injection of unlabeled DOTA-AON in the inhibition group, about 7.4 MBq of [ 68 Ga]Ga-DOTA-AON, anesthetized mice were induced and maintained with oxygen containing 2% isoflurane, and PET / CT imaging was performed, and images were dynamically collected in three-dimensional mode for 15, 30, and 60 minutes.

[0079] 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.

[0080] Observe the imaging of the experimental group and the inhibition group. The results are as follows Figure 7 shown.

[0081] Three MDA-MB-468 tumor-bearing mice described in Example 6 were taken as the HER2 negative control group and injected directly via the tail vein with about 7.4 MBq [ 68 Ga]Ga-DOTA-AON. injection[ 68After the infusion of Ga]Ga-DOTA-AON, the mice were anesthetized with oxygen containing 2% isoflurane, and PET / CT imaging was performed. Images were collected dynamically in three-dimensional mode for 15, 30, and 60 minutes. Nucline NanoScan3.00 was used to reconstruct and obtain attenuation-corrected PET / CT fusion images, and InterView FUSION 3.0 was used for image analysis. The tumor imaging was observed, and the results were as follows: Figure 7 shown.

[0082] The experimental results showed that the tumor sites in the experimental group had obvious [ 68 Ga]Ga-DOTA-AON radioactive uptake, and the radioactive accumulation of tumors in the inhibition group and negative control group was significantly reduced; indicating that [ 68 Ga]Ga-DOTA-AON specifically targets HER2 protein and can be used for imaging and efficacy monitoring of HER2 protein-expressing colorectal adenocarcinoma.

[0083] Example 8 Immunohistochemical staining of HER2 expression in tumors of tumor-bearing mice The ex vivo tumor tissues of HT-29 and MDA-MB-468 tumor-bearing mice described in Example 6 were fixed in 4% paraformaldehyde for 24 h, and then embedded to prepare paraffin sections to complete the preparation of sections; the paraffin sections were placed in a 60°C oven for 30 minutes, and then the sections were immersed in xylene I and xylene II for 10 minutes each; the sections were placed in ethanol from high to low for 2 minutes each (ethanol gradient: 100%, 95%, 90%, 80%, 70%); then the sections were placed in citric acid antigen repair solution, heated in a microwave oven at high heat until boiling, and then turned off the heat for 15 minutes, and naturally cooled to room temperature; Endogenous peroxidase blocker was dripped on the tissue, incubated at room temperature for 15 minutes, and washed with double distilled water; normal goat serum for blocking was dripped and incubated at room temperature for 20 minutes; HER2 antibody (rabbit anti-mouse, 1:200) was dripped on the slices, and kept at 4°C overnight. After being taken out, it was warmed up at room temperature for 30 minutes, and washed with PBS for 5 minutes × 3 times to complete the primary antibody incubation; secondary antibody (goat anti-rabbit, 1:200) was dripped, incubated at 37°C for 30 minutes, and washed with PBS for 5 minutes × 3 times to complete the secondary antibody incubation; DAB color developing solution was dripped, incubated for 10 seconds and then washed with water; Mayor's hematoxylin was dripped and incubated at room temperature for 2 minutes; hydrochloric acid alcohol differentiation, alkaline solution turned blue, and washed with water; the slices were placed in ethanol from high to low for a few seconds (ethanol gradient: 80%, 90%, 95%), anhydrous ethanol for 10 minutes, and then immersed in xylene for 10 minutes, and 1 drop of neutral resin was added to seal the slices, and the results were observed under a microscope and photographed.

[0084] The results of HER2 immunohistochemistry in HT-29 and MDA-MB-468 tumor-bearing mice were as follows: Figure 8 a and b, Fig. 9 As shown in a and b. According to the results of immunohistochemistry, HER2 receptor was positively expressed in HT-29 tumor tissue and negatively expressed in MDA-MB-468 tumor tissue.

[0085] 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 HER2 protein, characterized in that: Its structural formula is as follows: 。 2. A radioactive polypeptide targeting HER2 protein, characterized in that: Its structural formula is as follows: 。 3. The method for preparing the polypeptide targeting HER2 protein according to claim 1, characterized in that: The steps include: The Lys-Ser-Pro-Asn-Pro-Arg-Phe-Gly-Ser-Lys polypeptide sequence was synthesized by solid phase synthesis; The polypeptide sequence is cyclized, and the obtained ring structure is subjected to dehydration condensation with His-Tyr-Gln, and then connected with a coupling agent DOTA to obtain the product.

4. The method for preparing the radioactive polypeptide targeting HER2 protein according to claim 2, characterized in that: The steps include: The Lys-Ser-Pro-Asn-Pro-Arg-Phe-Gly-Ser-Lys polypeptide sequence was synthesized by solid phase synthesis; The polypeptide sequence is cyclized, and the obtained cyclic structure is dehydrated and condensed with His-Tyr-Gln, and then connected with a coupling agent DOTA to obtain a polypeptide conjugate DOTA-AON; The peptide conjugate DOTA-AON was dissolved in a buffer solution to obtain a DOTA-AON solution, to which [ 68 Ga]GaCl3 solution, react at 70-90℃ for a set time to obtain a radioactive polypeptide.

5. The method for preparing a radioactive polypeptide targeting HER2 protein according to claim 4, characterized in that: The buffer solution is NaOAc buffer solution, and its pH value is 4.0-4.

6.

6. The method for preparing a radioactive polypeptide targeting HER2 protein according to claim 4, characterized in that: The concentration of the DOTA-AON solution is 20-25 μM.

7. The method for preparing a radioactive polypeptide targeting HER2 protein according to claim 5, characterized in that: Said 68 The Ga]GaCl3 solution is the solution eluted with 0.01N HCl.

8. The method for preparing a radioactive polypeptide targeting HER2 protein according to claim 7, characterized in that: The DOTA-AON solution and [ 68 The volume ratio of Ga]GaCl3 solution is 0.6-1.5:

1.

9. The method for preparing a radioactive polypeptide targeting HER2 protein according to claim 4, characterized in that: React at 70-90°C for 15-20 minutes.

10. Use of the radioactive polypeptide targeting HER2 protein according to claim 2 in HER2-positive colorectal cancer tumor imaging.

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