EphA2 targeting peptides and their applications

Abstract

The present invention discloses EphA2 targeting polypeptides and their applications. These high-affinity polypeptides can specifically bind to a variety of tumor cells, preferably liver cancer, breast cancer, lung cancer, colorectal cancer, glioma, pancreatic cancer, and prostate cancer. The high-affinity characteristics of the targeting peptides can be used for optical imaging and nuclear medicine imaging of malignant tumors. These high-affinity polypeptide monomers, polypeptide dimers or polypeptide polymers can be directly or indirectly coupled with fluorescent dyes to serve as tumor-specific targeting molecular probes, which are expected to achieve the effect of accurately locating the tumor boundary, and can bring real-time performance to preoperative and intraoperative image navigation, with the advantage of improving surgical accuracy. This series of polypeptide monomers, dimers or polymers can also be coupled with radionuclides to detect malignant tumors in real time in vivo, so as to achieve the purpose of disease diagnosis or treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and more particularly, to an EphA2 targeting polypeptide tumor targeting peptide and its application in tumor targeting screening, diagnosis, treatment or prognosis evaluation reagent. Background Art

[0002] Malignant tumors pose a serious threat to human health and are one of the main causes of death worldwide. Conventional tumor treatments include chemotherapy, radiotherapy, surgical resection, etc., but these methods usually cause damage to the body's normal tissues, have great toxic side effects, and bring great pain to tumor patients. Therefore, tumor targeted therapy with high specificity and significant efficacy is needed.

[0003] Tumor targeted therapy refers to a treatment method that inhibits tumor progression by interfering with specific molecules involved in the occurrence, development and spread of tumor cells. Tumor targeted therapy is different from traditional chemotherapy and has specific target molecules. People are also working hard to develop various tumor targeted therapies. It is currently believed that targeted peptides are an ideal means of tumor targeted therapy, with the following advantages: 1) fast plasma clearance, high affinity, and strong specificity; 2) good tissue penetration and can be taken up by tumor cells; 3) easy chemical synthesis and low immunogenicity, which can avoid the shortcomings of monoclonal antibody therapy. Therefore, specific targeted peptides are ideal and attractive targets for medical research, clinical applications, and molecular imaging.

[0004] Erythropoietin-producing hepatocyte receptors (Eph) are the most important class of receptor tyrosine kinases (RTKs). Currently, there are 14 Eph receptors and 8 related ligands (ephrins). Eph receptors are divided into Eph-A and Eph-B subfamilies based on their sequence homology and binding affinity to their cognate ephrin ligands. Eph receptor signaling contributes to a variety of biological events, mainly leading to cell-to-cell rejection or adhesion. Therefore, Eph receptors and corresponding ligands play an important role in embryonic tissue patterning, neuronal targeting, and vascular development. At the same time, high levels of Eph proteins are found in a variety of malignant tumors, and this overexpression significantly promotes carcinogenesis. Some Eph receptors, especially EphA2, are widely expressed in a variety of aggressive cancer types, including lung cancer, prostate cancer, breast cancer, ovarian cancer, gastric cancer, and colon cancer, while they are almost not expressed in normal tissues. Therefore, EphA2 has become a strong candidate for targeted diagnosis and treatment of malignant tumors.

[0005] At present, the most commonly used detection method is immunohistochemistry (IHC), but IHC examination has fundamental limitations: the characteristics of tumor heterogeneity in spatial expression make it insensitive to marker detection, and factors such as incomplete tumor sampling may lead to false positive results. In addition, tumor tissue biopsy is damaging and cannot repeatedly and dynamically monitor the expression level during tumor progression. The staining of biopsy tissue can only represent the expression status of a certain small area and cannot fully and accurately reflect the situation of the entire tumor tissue and lesion metastasis, which may cause false negative test results. In comparison, molecular imaging plays an increasingly important role in personalized tumor medicine. Among them, the preparation of molecular probes is the key to molecular imaging. After entering the body, highly sensitive and specific molecular probes can bind to specific targets in cells, and capture signals in vitro through special imaging equipment, such as positron emission tomography (PET-CT), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI) and fluorescence imaging (FL), so as to achieve specific diagnosis and highly specific diagnosis.

[0006] Based on the above considerations, the applicant has designed a new type of EphA2-targeted peptide, which can specifically target EphA2. The peptide can be coupled with fluorescent dyes for optical imaging to assist doctors in accurately locating tumor boundaries during surgery when using molecular imaging surgical navigation equipment, so as to achieve the purpose of accurate tumor resection, thereby reducing trauma to patients and reducing the risk of postoperative recurrence. EphA2-targeted peptides can also be coupled with radionuclides for radionuclide imaging to achieve the purpose of early diagnosis and treatment of tumors. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides an EphA2 targeting polypeptide.

[0008] Another object of the present invention is to provide a modified polypeptide.

[0009] Another object of the present invention is to provide applications of EphA2 targeting polypeptides and modified polypeptides.

[0010] The purpose of the present invention can be achieved through the following technical solutions:

[0011] An EphA2 targeting polypeptide, wherein the EphA2 targeting polypeptide sequence comprises the following amino acid sequence:

[0012] YQ-EP-1:Pro-Glu-Ser-Thr-Pro-Tyr(SEQ ID NO.1)

[0013] YQ-EP-2:Pro-Asp-Ser-Thr-Pro-Tyr(SEQ ID NO.2)

[0014] YQ-EP-3:Pro-Glu-Ser-Val-Pro-Tyr(SEQ ID NO.3)

[0015] YQ-EP-4:Pro-Asp-Ser-Val-Pro-Tyr(SEQ ID NO.4)

[0016] YQ-EP-5:Tyr-Pro-Thr-Ser-Glu-Pro(SEQ ID NO.5)

[0017] YQ-EP-6:Tyr-Pro-Thr-Ser-Asp-Pro(SEQ ID NO.6)

[0018] YQ-EP-7:Tyr-Pro-Val-Ser-Glu-Pro(SEQ ID NO.7)

[0019] YQ-EP-8:Tyr-Pro-Val-Ser-Asp-Pro(SEQ ID NO.8)

[0020] YQ-EP-9:Arg-Ala-Trp-Leu-Tyr-Ala-Pro-Glu-Ser-Thr-Pro-Tyr (SEQ ID NO.9)

[0021] YQ-EP-10:Asn-Arg-Ala-Trp-Leu-Tyr-Ala-Pro-Glu-Ser-Thr-Pro-Tyr (SEQ IDNO.10)

[0022] YQ-EP-11:Cycle(Lys-Val-Met-Glu)-Glu-Ser-Thr-Pro-Tyr

[0023] YQ-EP-12:Cycle(Lys-Val-Met-Pro-Glu)-Ser-Thr-Pro-Tyr

[0024] YQ-EP-13: Cycle(Lys-Val-Met-Pro-Glu-Glu)-Thr-Pro-Tyr.

[0025] The present invention further provides a method for preparing the EphA2 targeting polypeptide, comprising: synthesizing the EphA2 targeting polypeptide by an Fmoc solid phase polypeptide synthesis method.

[0026] As a preferred specific implementation, the preparation method comprises:

[0027] The amino acids were coupled to the Rink Amide MBHA resin one by one according to the preset amino acid sequence. During the coupling process, the HCTU and Fmoc-protected amino acids were dissolved in DMF containing 0.4 mol / L DIPEA. Each coupling time was >1 h.

[0028] Then the deprotection process was carried out: the Fmoc group was removed using a DMF solution containing 20% ​​piperidine, each deprotection time was 5 min, and it was repeated twice.

[0029] Then, the side chain protecting groups are removed by reacting for 2-3 hours under the action of a strong acid, wherein the strong acid comprises TFA 90-95%, H 2 O 2-5%, TIS 2-5%, EDT 2%-5%.

[0030] The present invention further provides a multimer, wherein the multimer comprises at least two polypeptides; the polypeptides are selected from the EphA2 targeting polypeptides.

[0031] Furthermore, the multimer is formed by at least two EphA2 targeting polypeptides in a linear or D-form or after cyclization.

[0032] Furthermore, different polypeptides are connected by covalent linkage, non-covalent linkage or multimer mixing;

[0033] Preferably, the covalent connection is connected via a linker molecule, and the linker molecule includes one or more of 6-tert-butyloxycarbonylhydrazinonicotinic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or N-hydroxysuccinimide;

[0034] The polymer is any one of polyethylene glycol, polyvinyl alcohol, cyclodextrin, polyamide-amine dendrimer, polylactic acid or polylactic acid-ethanolamine, or a combination of at least two thereof.

[0035] A modified polypeptide characterized by having the following general formula:

[0036] ML-YQ-EP-X, or M-YQ-EP-X,

[0037] Wherein, M represents a fluorescent label or a radionuclide label;

[0038] L is a linking group;

[0039] YQ-EP-X is the EphA2-specific targeting polypeptide.

[0040] Furthermore, the fluorescent label is one or more of fluorescent dyes such as IR Dye800, CY7, CY5, rhodamine, ICG, etc., and the radioactive isotope is 18 F, 68 Ga, 64 Cu, 99m Tc, 90 Y, 111 In, 125 I, 131 I or 177 Lu;

[0041] Furthermore, the fluorescent marker and the radioactive isotope are labeled by a chelating agent such as HYNIC, DOTA, NOTA or DTPA, and can also be labeled by click chemistry such as NHS, EDC, MAL, etc.

[0042] The EphA2-specific targeting polypeptide, the polymer, and the modified polypeptide of the present invention are used in the preparation of tumor targeted screening, diagnosis, tracing, prognosis assessment reagents or therapeutic drugs; preferably, they are used in the preparation of tumor diagnostic imaging agents; further preferably, they are used in the preparation of tumor boundary precise positioning and surgical navigation imaging reagents or in the preparation of radionuclide imaging reagents.

[0043] The tumor is an EphA2-positive tumor, that is, all tumors that overexpress EphA2, such as one or more of liver cancer, breast cancer, lung cancer, colorectal cancer, glioma, pancreatic cancer, and prostate cancer.

[0044] The present invention has the following beneficial effects:

[0045] (1) The present invention provides a polypeptide that can target EphA2-positive tumor cells. The polypeptide can specifically bind to the EphA2 receptor and can be used as a targeting polypeptide to couple with a fluorescent dye for fluorescent imaging for surgical navigation. In addition, the polypeptide can also be connected with a radionuclide or a preparation that can kill cancer cells for early diagnosis and treatment of EphA2-overexpressing tumors.

[0046] (2) The present invention further provides a modified polypeptide based on the discovered EphA2 targeting polypeptide, which can be used as a molecular probe, and has the advantages of good targeting, small molecular weight, fast metabolic rate, no immunogenicity, good permeability, etc. The modified polypeptide provided by the present invention can be used for clinical SPECT / PET detection, which is conducive to later clinical transformation, and can dynamically and non-invasively monitor the expression level of EphA2, and has good application prospects and clinical guidance significance.

[0047] (3) The preparation method of the modified polypeptide provided by the present invention is simple, easy, low-cost, highly practical, and has good biosafety and high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Structure of polypeptide YQ-EP-1.

[0049] Figure 2 The structures of MPA and YQ-EP-1-MPA in Example 3.

[0050] Figure 3 Flow cytometry of YQ-EP-X (X = 1 to 13) -MPA in PC3 cells.

[0051] Figure 4 Confocal images of peptide YQ-EP-1 labeled with FITC in MCF7 and PC3 cells.

[0052] Figure 5 Fluorescence imaging of the peptide MPA-YQ-EP-1 in mice bearing both A549 and PC3 tumors (the tumor on the left in each photo is A549 and the tumor on the right is PC3).

[0053] Figure 6 Structure of HYNIC-PEG4-YQ-EP-1.

[0054] Figure 7 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-1 in colorectal cancer cell HCT-116 tumor-bearing mice.

[0055] Figure 8 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-1 in mice bearing both lung cancer A549 cells and breast cancer MCF7 tumors (the three pictures on the left, middle and right are three sections of the same mouse. Two tumors can be seen in the middle section. The tumor on the left is A549 and the tumor on the right is MCF7).

[0056] Fig. 9 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-1 in mice bearing brain glioma cells U87.

[0057] Fig.10 99mSPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-1 in mice bearing both liver cancer cells HepG2 and pancreatic cancer cells BxPC3 (the three pictures on the left, middle and right are three sections of the same mouse. The middle section has two tumors. The tumor on the left is HepG2 and the tumor on the right is BxPC3).

[0058] Fig.11 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-1 in prostate cancer cell PC3 tumor-bearing mice.

[0059] Fig.12 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-2 in prostate cancer cell PC3 tumor-bearing mice.

[0060] Fig.13 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-3 in prostate cancer cell PC3 tumor-bearing mice.

[0061] Fig.14 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-4 in prostate cancer cell PC3 tumor-bearing mice.

[0062] Fig.15 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-5 in mice bearing prostate cancer cells PC3.

[0063] Fig.16 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-6 in mice bearing prostate cancer cells PC3.

[0064] Fig.17 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-7 in mice bearing prostate cancer cells PC3.

[0065] Fig.18 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-8 in mice bearing prostate cancer cells PC3.

[0066] Fig.19 99mSPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-9 in mice bearing prostate cancer cells PC3.

[0067] Fig. 20 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-10 in mice bearing prostate cancer cells PC3.

[0068] Fig.21 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-11 in mice bearing prostate cancer cells PC3.

[0069] Fig. 22 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-12 in mice bearing prostate cancer cells PC3.

[0070] Fig.23 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-13 in mice bearing prostate cancer cells PC3.

[0071] Fig.24 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-1-dimer in mice bearing prostate cancer cells PC3. DETAILED DESCRIPTION

[0072] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0073] N,N-diisopropylethylamine (DIPEA), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), N,N-dimethylformamide (DMF), methanol, phenol, ninhydrin, anhydrous ether, resin, triisopropylsilane (TIS), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), dimethyl sulfoxide (DMSO), various Fmoc-protected amino acids, peptide synthesis tubes, shakers, vacuum water pumps and other instruments involved in the following examples were obtained from commercial sources.

[0074] Example 1

[0075] This example provides a method for preparing polypeptide YQ-EP-X (X=1-10). The polypeptide is synthesized by solid phase synthesis. The specific synthesis method is as follows:

[0076] 1) Resin swelling

[0077] Weigh n equivalents of Rink Amide MBHA resin into a peptide synthesis tube, add dichloromethane (DCM) to swell for 30 minutes, remove the DCM solution, wash with DMF, and dry.

[0078] 2) Removal of Fmoc

[0079] Add 20% piperidine in DMF solution to the synthesis tube, deprotect for 5 min, repeat twice, and wash with DMF after the reaction.

[0080] 3) Coupling

[0081] Add 2n equivalents of amino acid, 2n equivalents of DIPEA, 2n equivalents of HCTU and DMF to the synthesis tube, shake and react for 1 hour, remove the reaction solution and wash with DMF, then add the method of step 2) to remove Fmoc, wash, and detect with ninhydrin.

[0082] 4) Add different amino acids in the sequence in sequence to perform various modifications in the same manner as in step 3. The amino acid residues involved can be L-type, D-type, or a mixture of L and D-types, proline (Pro) can be replaced by hydroxyproline (Hyp), arginine (Arg) can be replaced by homoarginine (homo-Arg), and alanine can be replaced by β-alanine.

[0083] 5) Lysis

[0084] The resin was blown dry with nitrogen, and a cutting solution (87.5% TFA + 5% thioanisole + 2.5% ethanedithiol + 2.5% phenol + 2.5% water) was added to the polypeptide synthesis tube. The ratio of the cutting solution volume to the resin was approximately 10 ml / g. After reacting for 2-3 hours, the filtrate was filtered out, a large amount of ether was added, and then centrifuged. The solid was washed three times with ether to obtain a crude polypeptide.

[0085] 6) Separation and purification

[0086] Reverse phase high performance liquid chromatography was used for purification, the chromatographic filler for purification was 10 μm reverse phase C18, the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution, the gradient system was used for elution, and the ultraviolet absorption of the polypeptide was measured by ultraviolet spectrophotometry for quantification. The results showed that the polypeptide was successfully synthesized and the purity was above 95%. The collected eluate was placed in a freeze dryer for concentration and freeze-dried into a white powder.

[0087] YQ-EP-X (X = 1 to 9) was confirmed by mass spectrometry. The structure of YQ-EP-1 is as follows Figure 1 As shown, its [MH]- :691.30; YQ-EP-2 [MH] - :677.29; YQ-EP-3 [MH] - :689.30; YQ-EP-4 [MH] - :675.30; YQ-EP-5 [MH] - :691.30; YQ-EP-6 [MH] - :677.29; YQ-EP-7 [MH] - :689.30; YQ-EP-8 [MH] - :675.30; YQ-EP-9 [MH] - :1451.7; YQ-EP-10 [MH] - :1565.75.

[0088] Example 2

[0089] This example provides a method for preparing polypeptide YQ-EP-X (X=11-13). The polypeptide is synthesized by solid phase synthesis. The specific synthesis method is as follows:

[0090] 1) Resin swelling

[0091] Weigh n equivalents of Rink Amide MBHA resin into a peptide synthesis tube, add dichloromethane (DCM) to swell for 30 minutes, remove the DCM solution, wash with DMF, and dry.

[0092] 2) Removal of Fmoc

[0093] Add 20% piperidine in DMF solution to the synthesis tube, deprotect for 5 min, repeat twice, and wash with DMF after the reaction.

[0094] 3) Coupling

[0095] Add 2n equivalents of amino acid, 2n equivalents of DIPEA, 2n equivalents of HCTU and DMF to the synthesis tube, shake and react for 1 hour, remove the reaction solution and wash with DMF, then add the method of step 2) to remove Fmoc, wash, and detect with ninhydrin.

[0096] 4) Add different amino acids in the sequence in sequence according to step 3 to perform various modifications.

[0097] 5) Removal of OALL (allyl ester protecting group)

[0098] Add 0.2n tetrakistriphenylphosphine palladium, phenylsilane and an appropriate amount of dichloromethane to the synthesis tube, shake and react at room temperature for 2 hours, repeat twice, draw off the reaction liquid and wash with DMF.

[0099] 6) Side chain coupling

[0100] Add 2n equivalents of PYBOP, 2n equivalents of DIPEA and DMF to the synthesis tube, shake and react for 6 hours, remove the reaction solution and wash with DMF, then add Fmoc removal according to step 2), wash, and detect with ninhydrin.

[0101] 7) Lysis

[0102] The resin was blown dry with nitrogen, and a cutting solution (87.5% TFA + 5% thioanisole + 2.5% ethanedithiol + 2.5% phenol + 2.5% water) was added to the polypeptide synthesis tube. The ratio of the cutting solution volume to the resin was approximately 10 ml / g. After reacting for 2-3 hours, the filtrate was filtered out, a large amount of ether was added, and then centrifuged. The solid was washed three times with ether to obtain a crude polypeptide.

[0103] 8) Separation and purification

[0104] Reverse phase high performance liquid chromatography was used for purification, the chromatographic filler for purification was 10 μm reverse phase C18, the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution, the gradient system was used for elution, and the ultraviolet absorption of the polypeptide was measured by ultraviolet spectrophotometry for quantification. The results showed that the polypeptide was successfully synthesized and the purity was above 95%. The collected eluate was placed in a freeze dryer for concentration and freeze-dried into a white powder.

[0105] YQ-EP-X (X = 10 ~ 13) was confirmed by mass spectrometry, YQ-EP-11 [MH] - :1064.5; YQ-EP-12 [MH] - :1032.5; YQ-EP-13 [MH] - :1074.5.

[0106] Example 3

[0107] This example provides a method for preparing a dimer of the YQ-EP-1 polypeptide. The specific synthesis method is as follows:

[0108] 2.0 mg of the YQ-EP-1 polypeptide prepared in Example 1, 2.0 mg of fluorenylmethoxycarbonyl-6-aminocaproic acid, and 3.0 mg of HATU were dissolved in DMSO, and 1 μL of N, N-diisopropylethylamine (DIPEA) was added to shake at room temperature for 1 hour, and a 20% piperidine dichloromethane solution was added to shake for 10 minutes after the reaction was completed, and finally the intermediate was purified. 1 mg of the intermediate, 1.0 mg of Fmoc-Glu, and 2.0 mg of HATU were dissolved in DMSO, and 0.5 μL of N, N-diisopropylethylamine (DIPEA) was added to shake at room temperature for 1 hour, and a 20% piperidine dichloromethane solution was added to shake for 10 minutes after the reaction was completed, and finally the YQ-EP-1-dimer was purified. Confirmed by mass spectrometry, its [M-2H] 2- / 2:859.5.

[0109] Example 4

[0110] This example provides a method for preparing a polypeptide probe of MPA-YQ-EP-X (X=1-13). MPA is a near-infrared fluorescent dye from an invention patent (CN101440282) applied by our research group. The specific synthesis steps are as follows:

[0111] 2.0 mg of the YQ-EP-X (X=1-13) polypeptide prepared in Example 1 and Example 2, 2.0 mg of HATU and 3.0 mg of the fluorescent dye MPA were dissolved in 300 μL of dimethyl sulfoxide (DMSO), and then 1.0 μL of N,N-diisopropylethylamine (DIPEA) was added, and the mixture was shaken and reacted at room temperature for 1 hour. After the reaction, the mixture was separated and purified by preparative liquid phase, and the preparative liquid phase conditions were as follows: an Agilent 1220 Infinity II series HPLC system equipped with an Agilent ZORBAX SB-C18 semi-preparative column (9.4×250 mm, 5 um) was used, with a gradient elution of 60 minutes and a flow rate of 2 mL / min, wherein mobile phase A was ultrapure water (0.01% TFA) and mobile phase B was acetonitrile (0.01% TFA). The elution gradient was set to: 95% A and 5% B at 0-5 minutes, 80% A and 20% B at 15 minutes, 50% A and 50% B at 45 minutes, 5% A and 95% B at 60 minutes, and finally the product was collected and purified by MPA-YQ-EP-X (X=1-13). Taking MPA-YQ-EP-1 as an example, its structure is as follows Figure 2 shown.

[0112] Example 5

[0113] In this example, the affinity of MPA-YQ-EP-X (X=1-13) in the EphA2 high-expressing cell line PC3 was detected by flow cytometry. The specific process is as follows:

[0114] PC3 cells were plated at 1×10 6 Cells were seeded at 100 μL density in six-well plates. After 24 h of culture, the culture medium was replaced with fresh culture medium containing 5.0 μM concentration of MPA-YQ-EP-1 or MPA and incubated at 37 °C for 1 h. The cells were washed three times with PBS (pH 7.4) to remove unbound drugs. The cells were then trypsinized, centrifuged at 2000 rpm for 3 min, resuspended in 400 μL PBS, and stored in a flow tube in the dark. The samples were loaded and analyzed by a flow cytometer equipped with a near-infrared FL 4 channel. Finally, the raw data were analyzed using FlowJo 7.0 software and GraphPad Prism 8.0 software to express the mean fluorescence intensity (MFI).

[0115] The results are as follows Figure 3 As shown, it indicates that the polypeptide MPA-YQ-EP-X (X=1-13) has a strong affinity for EphA2 high-expressing cell lines.

[0116] Example 6

[0117] Taking YQ-EP-1 as an example, this example detects the interaction between YQ-EP-1 and EphA2 high-expressing cell lines PC3 and MCF7 cells, respectively. The specific process is as follows:

[0118] The density of 3×10 5 A549 or PC3 cells were cultured at 5% CO 2 Grow at 37°C for 24 hours. After the cells adhere, add 10μM probe or dye to the cell culture medium, and set the incubation time gradient to 10, 30 and 60min. And add 100μL Hoechst 33342 reagent and incubate for 10min. After incubation, wash the cells three times with PBS (pH 7.4). For receptor blocking experiments, add 200μM polypeptide and incubate with the cells for 60min, fix, and wash. Place under a Leica DM 4000B inverted fluorescence microscope to collect fluorescence images at different time points, and then use Leica Application Suite X software to process the images. The results are shown in the figure. Figure 4 As shown, it indicates that polypeptide YQ-EP-1 can specifically target EphA2.

[0119] Example 7

[0120] This example uses the probe MPA-YQ-EP-1 prepared in Example 2 to perform in vivo fluorescence imaging in tumor-bearing mice. The specific process is as follows:

[0121] A549 and PC3 cells (A549 on the left and PC3 on the right) were subcutaneously inoculated into female mice aged 6-8 weeks. When the tumor volume grew to 100 mm 3 One group of mice was injected with 100 μL of the probe MPA-YQ-EP-1 through the tail vein, and the other group of mice was injected with 100 μL of the pure dye MPA through the tail vein. Fluorescence detection was performed on the small animal in vivo imaging system at 1h, 2h, 4h, 6h, 9h, 12h and 24h after administration. The results are shown in Figure 5 As shown, the probe MPA-YQ-EP-1 has a strong fluorescence signal in the tumor after 1 hour, and the fluorescence intensity of the mouse tumor injected with the probe is stronger than that of the pure dye, which proves that the probe of the present invention has EphA2 targeting and can achieve high-sensitivity in vivo imaging of tumors. The distribution of major organs also shows that the probe has good biocompatibility and safety.

[0122] Example 8

[0123] This example provides the preparation of HYNIC-PEG4-YQ-EP-X (X = 1 to 13)

[0124] (1) Synthesis of bifunctional chelating agent HYNIC-NHS

[0125] 6-Chloronicotinic acid and 80% hydrazine hydrate are added to ethanol, heated to reflux for reaction, and after the reaction is completed, the solvent is evaporated under reduced pressure, and the obtained viscous material is added to distilled water, and the pH is adjusted to about 5.5, solid is precipitated, and suction filtration is dried to obtain a yellow solid, and the product is confirmed to be 6-hydrazinic acid by ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum. The obtained 6-hydrazinic acid and p-aminobenzaldehyde are added to dimethyl sulfoxide (DMSO), heated to react for 5-6 hours, and after the reaction is completed, they are added to water to precipitate, and suction filtration is obtained to obtain a solid, and after drying, the solid is added to DMSO together with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) to react at room temperature, and after the reaction is completed, the solid is added to water to precipitate a solid, and the solid is purified by silica gel column and confirmed to be the target product by ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum.

[0126] (2) Synthesis of YQ-EP-1-PEG4

[0127] 2 mg YQ-EP-X (X = 1 to 13), 2 mg PEG-BOC and 3 mg HATU were dissolved in DMSO, and then 1 μL triethylamine was added, and the reaction was heated at 40°C for 1 hour. After the reaction was completed, YQ-EP-1-PEG4-BOC was separated and purified by reverse phase preparative liquid phase and freeze-dried. 100 μL TFA was added to the product, and after reaction at room temperature for 30 minutes, YQ-EP-1-PEG4 was separated and purified by reverse phase preparative liquid phase.

[0128] (3) Synthesis of HYNIC-PEG4-YQ-EP-X (X = 1 to 13)

[0129] 1 mg of the purified intermediate YQ-EP-X (X = 1 to 13) -PEG4 and 1 mg of HYNIC-NHS were dissolved in 0.3 mL of DMSO, and 1 μL of DIPEA was added to react at 40°C for 2 h. The reaction progress was detected by analytical high performance liquid chromatography. After the reaction was completed, separation and purification were performed by preparative liquid chromatography to obtain the product. Taking HYNIC-PEG4-YQ-EP-1 as an example, its structure is as follows: Figure 6 In the above preparation process, YQ-EP-1-dimer is used to replace the YQ-EP-X polypeptide used in the step to obtain HYNIC-PEG4-YQ-EP-1-dimer.

[0130] Example 9

[0131] This embodiment provides 99m Preparation of Tc-HYNIC-PEG4-YQ-EP-X (X = 1 to 13)

[0132] Prepare 100.0 mg / mL TPPTS (triphenylphosphine trisulfonate) solution, 130.0 mg / mL Tricine (trimethylglycine), and 102.4 mg / mL succinic acid-sodium succinate buffer (77.0 mg succinic acid and 25.4 mg sodium succinate) respectively, take 10.0 uL TPPTS solution, 10.0 uL Tricine solution, 10.0 uL succinic acid-sodium succinate buffer and 10.0 uL (1.0 mg / mL) HYNIC-PEG4-YQ-EP-X (X=1-13) described in Example 5 and mix them in a vial, then add 10 mCi Na 99m TcO4 was heated in a 100°C metal bath for 20 minutes and cooled to room temperature after the reaction was completed to obtain a peptide radiopharmaceutical. 99mTc-HYNIC-PEG4-YQ-EP-X (X = 1 ~ 13), the product was analyzed and identified by Agilent ZORBAX SB-Aq analytical column. The HPLC method used was an Agilent 1220 Infinity II series HPLC system equipped with a radioactive online detector (Flow-RAM) and an Agilent ZORBAX SB-Aq analytical column (4.6 × 250mm, 5um). Gradient elution was 45 minutes, the flow rate was 1mL / min, and the mobile phase A was ultrapure water (0.01% TFA), and B was acetonitrile (0.01% TFA). The elution gradient was set as: 95% A and 5% B at 0-5 minutes, 70% A and 30% B at 15 minutes, 65% A and 35% B at 20 minutes, 45% A and 55% B at 25 minutes, and 5% A and 95% B at 45 minutes. 99m The purity of Tc-HYNIC-PEG4-YQ-EP-X (X = 1 to 13) is > 95%. In the above preparation process, YQ-EP-1-dimer is used to replace the YQ-EP-X polypeptide used in the step to obtain 99m Tc-HYNIC-PEG4-YQ-EP-1-dimer.

[0133] Example 10

[0134] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-1 in tumor-bearing mice

[0135] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-1 was prepared into physiological saline solution and injected into the tail vein of 7 tumor-bearing nude mice (HCT-116, A549, MCF-7, U87, HepG2, BxPC3, PC3) at 500 μCi. Three mice of each tumor-bearing type were injected. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Figure 2. Figure 7 ,8,9,10,11, the probe 99m Tc-HYNIC-PEG4-YQ-EP-1 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0136] Embodiment 11

[0137] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-2 in tumor-bearing mice

[0138] The probe prepared in Example 9 99mTc-HYNIC-PEG4-YQ-EP-2 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.12 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-2 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0139] Example 12

[0140] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-3 in tumor-bearing mice

[0141] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-2 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.13 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-3 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0142] Embodiment 13

[0143] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-4 in tumor-bearing mice

[0144] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-4 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.14 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-4 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0145] Embodiment 14

[0146] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-5 in tumor-bearing mice

[0147] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-5 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.15 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-5 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0148] Embodiment 15

[0149] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-6 in tumor-bearing mice

[0150] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-6 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.16 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-6 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0151] Example 16

[0152] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-7 in tumor-bearing mice

[0153] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-7 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.17 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-7 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0154] Embodiment 17

[0155] Radioactive probe99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-8 in tumor-bearing mice

[0156] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-8 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.18 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-8 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0157] Embodiment 18

[0158] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-9 in tumor-bearing mice

[0159] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-9 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.19 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-9 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0160] Embodiment 19

[0161] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-10 in tumor-bearing mice

[0162] The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-10 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig. 20 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-10 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0163] Embodiment 20

[0164] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-11 in tumor-bearing mice The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-11 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.21 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-11 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0165] Embodiment 21

[0166] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-12 in tumor-bearing mice The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-12 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig. 22 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-12 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0167] Embodiment 22

[0168] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-13 in tumor-bearing mice The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-13 was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.23 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-13 was obviously taken up in the tumor site, indicating that the probe had good targeting and could be cleared out of the body quickly.

[0169] Embodiment 23

[0170] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-PEG4-YQ-EP-1-dimer in tumor-bearing mice The probe prepared in Example 9 99m Tc-HYNIC-PEG4-YQ-EP-1-dimer was prepared into physiological saline solution and 500 μCi was injected into three PC3 tumor-bearing nude mice through the tail vein. SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The imaging results are shown in Fig.24 As shown, the probe 99m Tc-HYNIC-PEG4-YQ-EP-1-dimer was significantly taken up at the tumor site, indicating that the probe had good targeting and could be cleared from the body quickly.

Claims

1. EphA2-specific targeting polypeptide YQ-EP-6, characterized in that The amino acid sequence is shown in SEQ ID NO.

6.

2. Use of the EphA2-specific targeting polypeptide according to claim 1 in the preparation of prostate cancer targeted screening, diagnosis and tracing reagents.

3. The use according to claim 2, characterized in that Use of the EphA2-specific targeting polypeptide described in claim 1 in the preparation of a prostate cancer diagnostic imaging agent.

4. The use according to claim 3, characterized in that Use of the EphA2-specific targeting polypeptide described in claim 1 in the preparation of imaging agents for accurate positioning of tumor boundaries and surgical navigation or in the preparation of radionuclide imaging agents.

5. A modified polypeptide, characterized in that With the following general formula: ML-YQ-EP-6, or M-YQ-EP-6, Wherein, M represents photolabeling or radionuclide labeling; L is a linking group; YQ-EP-6 is the EphA2-specific targeting polypeptide according to claim 1.

6. The modified polypeptide according to claim 5, characterized in that The optical label is selected from organic chromophores, organic fluorophores, light absorbing compounds, light reflecting compounds, light scattering compounds or bioluminescent molecules.

7. The modified polypeptide according to claim 6, characterized in that The optical marker is selected from near-infrared fluorescent dyes MPA, IRDye800, Cy7.5, and Cy5.

5.

8. The modified polypeptide according to claim 5, characterized in that The radionuclide is selected from 99m Tc, 68 Ga, 64 Cu, 67 Ga, 90 Y, 111 In or 177 Lu, 125 I.

9. The modified polypeptide according to claim 8, characterized in that The radionuclide labeling is composed of a radionuclide ligand, a bifunctional chelating agent for radionuclide labeling and a radionuclide; the radionuclide ligand is N-tris(hydroxymethyl)methylglycine or triphenylphosphine tris-sulfonic acid sodium salt, and the bifunctional chelating agent for radionuclide labeling is HYNIC, DOTA, NOTA or DTPA.

10. The modified polypeptide according to claim 5, characterized in that The L is selected from any one or more of 6-aminocaproic acid, PEG4, PEG6, HYNIC-PEG4 or HYNIC.

11. Use of the modified polypeptide according to any one of claims 5 to 10 in the preparation of a reagent for diagnosing or tracing prostate cancer.

12. The use according to claim 11, characterized in that The polypeptide is used in preparing fluorescent imaging or radioactive imaging reagents for prostate cancer.

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