Disialic acid ganglioside GD2 affinity peptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a disialic acid ganglioside GD2 affinity peptide and application thereof. The series of polypeptides of the GD2 affinity peptide YQAG-n (n = 1-6) provided by the invention can be combined with tumor cells with high expression of GD2, and in-vivo optical imaging and results prove that the modified polypeptides prepared from the GD2 affinity peptide subjected to optical labeling have excellent imaging results on various tumors. Tumor detection reagents and surgical navigation imaging agents prepared on the basis of the YQAG-n (n = 1-6) series can be better applied to early tumor screening, early tumor diagnosis and intraoperative navigation of tumor operations.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and particularly relates to a class of ganglioside GD2 affinity peptides and their applications. Background Art

[0002] Early diagnosis of tumors is of great significance for improving the survival rate of patients. Currently, the conventional imaging techniques for tumor diagnosis are mainly X-CT, magnetic resonance imaging, ultrasonic diagnosis, etc. Among them, molecular probes, as powerful tools for analytical sensing and optical imaging, can directly visualize biological analytes at the molecular level and provide useful information for complex biological structures and processes. The basic imaging principle of molecular probes is to inject the prepared fluorescent probes into living tissues in a way such as injection, so that the target interacts with the molecular probe, and then the information emitted by the molecular probe is detected by a suitable imaging system. With the help of molecular probes targeting tumors, early screening and early diagnosis of tumors can be achieved.

[0003] Surgical treatment is one of the means of tumor treatment. Patients can obtain a good prognosis after surgical resection. However, the precise localization of the tumor boundary has always been a scientific research problem to be overcome. Providing the surgical boundary for surgeons to completely remove the tumor can reduce the possibility of postoperative recurrence in patients. However, the currently FDA-approved surgical navigation imaging agents for clinical use have low sensitivity and weak specificity. For example, indocyanine green, an imaging agent for liver cancer surgical navigation, is difficult to meet the clinical needs. Molecular probes targeting tumors have the advantages of strong specificity and high sensitivity, providing hope for the accurate localization of tumor boundaries.

[0004] Sphingolipids are important components of cell membranes. Sphingolipids are divided into two categories: sphingomyelin and glycosphingolipids. Glycosphingolipids are composed of two parts: ceramide and sugar chains. According to the nature of the sugar chains, they can be divided into neutral glycosphingolipids and acidic glycosphingolipids. Acidic glycosphingolipids generally refer to glycosphingolipids in which the sugar moiety contains sialic acid, also known as gangliosides.

[0005] Gangliosides are widely expressed in normal tissues, making most subtypes unsuitable as cancer treatment targets. The ganglioside GD2 subtype is expressed limitedly in normal tissues but overexpressed in a wide range of tumors. Ganglioside GD2 can be considered a tumor-associated antigen and is suitable as a cancer treatment target. Depending on the type of tumor, ganglioside GD2 is involved in tumor development and malignant phenotypes by enhancing cell proliferation, motility, migration, adhesion, and invasion. GD2 in the ganglioside family is a cell surface glycolipid composed of galactose, glucose, N-acetylgalactosamine, sialic acid, and ceramide, and is highly expressed on cancer cells of neuroectodermal origin, including neuroblastoma, retinoblastoma, melanoma, sarcoma, brain tumors, small cell lung cancer, and breast cancer.

[0006] For example, refer to the patent with the publication number CN116023432A, which is the prior research result of this research group. It discloses a disialoganglioside GD2 affinity peptide and its application, providing that the YQAF-X series of polypeptides can bind to tumor cells with high expression of GANGLIOSIDE. Through in vivo optical imaging and results verification, it has excellent imaging results for various tumors. The tumor detection reagent and surgical navigation imaging agent prepared based on the YQAF-X series can be better applied to early tumor screening, early tumor diagnosis, and intraoperative navigation of tumor surgery. Summary of the Invention

[0007] The purpose of the present invention is to provide a novel type of GD2 affinity peptide and its application published for the first time. The series of polypeptides provided by the present invention can bind to tumor cells with high expression of ganglioside, achieve targeting of tumor lesions, and can realize the imaging of tumor cells with high expression of ganglioside described in the background technology or the early diagnosis of cancer cells originating from the neuroectoderm.

[0008] In the first aspect, the present invention provides a class of GD2 affinity peptides, and the sequence of the GD2 affinity peptide is: Ac-Asn-Thr-Ile-X1-X2-X3-Trp-NH2; wherein, -X1-X2-X3- is selected from any one of -Gly-Gly-Ala-, -Gly-Gly-Arg-, -Gly-Gly-Met-, -Gly-Gly-Ser-, -Met-Gly-Arg- or -Gly-Gly-Tyr-. It should be noted that Ac at one end of the GD2 affinity peptide sequence represents an acetyl group.

[0009] That is, the GD2 affinity peptide is selected from any one of the following polypeptides:

[0010] YQAG-1 containing the amino acid sequence shown in SEQ ID NO: 1:

[0011] Ac-Asn-Thr-Ile-Gly-Gly-Ala-Trp-NH2;

[0012] YQAG-2 containing the amino acid sequence shown in SEQ ID NO: 2:

[0013] Ac-Asn-Thr-Ile-Gly-Gly-Arg-Trp-NH2;

[0014] YQAG-3 containing the amino acid sequence shown in SEQ ID NO: 3:

[0015] Ac-Asn-Thr-Ile-Gly-Gly-Met-Trp-NH2;

[0016] YQAG-4 comprising the amino acid sequence shown in SEQ ID NO: 4:

[0017] Ac-Asn-Thr-Ile-Gly-Gly-Ser-Trp-NH2;

[0018] YQAG-5 comprising the amino acid sequence shown in SEQ ID NO: 5:

[0019] Ac-Asn-Thr-Ile-Met-Gly-Arg-Trp-NH2;

[0020] YQAG-6 comprising the amino acid sequence shown in SEQ ID NO: 6:

[0021] Ac-Asn-Thr-Ile-Gly-Gly-Tyr-Trp-NH2.

[0022] Use of the GD2 affinity peptide or its dimer or multimer according to the present invention in the preparation of tumor diagnostic reagents or tumor therapeutic drugs.

[0023] Use of the GD2 affinity peptide or its dimer or multimer according to the present invention in the preparation of tumor diagnostic imaging agents; preferably in the preparation of precise positioning and surgical navigation imaging reagents for tumor boundaries or in the preparation of radionuclide imaging reagents.

[0024] Another object of the present invention is to provide a modified polypeptide, the modified polypeptide having a tumor-targeted imaging function and having the following general formula:

[0025] M-L-YQAG-n

[0026] Wherein, M represents a photo label or a radionuclide label; L is a linking group;

[0027] YQAG-n is any one of the polypeptides according to the present invention or its dimer or multimer.

[0028] The photo label M according to the present invention is selected from organic chromophores, organic fluorophores, light-reflecting compounds, light-scattering compounds, and bioluminescent molecules.

[0029] As an embodiment of the modified polypeptide according to the present application, the photo label M is an organic fluorophore, and the modified polypeptide is a fluorescence probe based on the GD2 affinity peptide. Further, when the organic fluorophore is a near-infrared fluorescent dye such as MPA, IRDye800, Cy7.5, Cy5.5, the modified polypeptide according to the present invention is a near-infrared fluorescence imaging probe, and the near-infrared fluorescent dye is further preferably MPA.

[0030] As an embodiment of the modified polypeptide described in the present application, when the photo-label M is a radionuclide label, the modified polypeptide is a radionuclide probe based on a GD2 affinity peptide, and the radionuclide in the radionuclide label M is preferably 99m Tc, 68 Ga, 64 Cu, 67 Ga, 90 Y, 111 In or 177 Lu, 125 I. The chelating agent used to chelate the radionuclide in the radionuclide label M is any one of HYNIC, DOTA, NOTA or DTPA.

[0031] In the probe of the present invention, L is selected from azidovaleric acid, 6-aminohexanoic acid, propargylic acid, polyethylene glycol, 1,4,7-triazacyclononane-1,4,7-triacetic acid, 7-[(4-hydroxypropyl)methylene]-1,4,7-triazacyclononane-1,4-diacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, mercaptoacetyltriglycine, MAG2, N3S, N2S2 ligands, diethylenetriaminepentaacetic acid, 1,4-succinic acid, 5-aminopentanoic acid, polyethyleneimine, 6-hydrazinopyridine-3-carboxylic acid, benzyl bromoformate, N-(2-aminohexanoic acid) maleimide or a combination thereof. L is further preferably any one or more of 6-aminohexanoic acid, PEG4, and PEG6. It should be noted that both PEG4 and PEG6 belong to polyethylene glycol, PEG4 is tetraethylene glycol, and PEG6 is hexaethylene glycol.

[0032] The ganglioside GD2 affinity peptide of the present invention has a good tumor targeting effect. After entering the body, it can efficiently bind to GD2 on the cell membrane of tumor cells, has good aggregation and retention at the tumor site, has a high target-to-non-target ratio, and is suitable for being prepared into an imaging agent and used as an optical imaging agent for accurate localization of the tumor boundary and an imaging agent for intraoperative navigation during tumor surgery. Therefore, another object of the present invention is to provide the application of the modified polypeptide in the preparation of a tumor diagnostic imaging agent or an intraoperative navigation imaging agent.

[0033] The beneficial effects of the present invention are:

[0034] The present invention has developed a series of polypeptides with high affinity for ganglioside GD2, which can be used to target ganglioside GD2. GD2 is highly expressed on the cell membranes of various tumors, such as neuroblastoma, retinoblastoma, melanoma, sarcoma, brain tumor, small cell lung cancer, and breast cancer. Based on the principle of the binding of YQAG-n (n = 1-6) polypeptides to GD2, early diagnosis and intraoperative navigation of various tumors, including neuroblastoma, retinoblastoma, melanoma, sarcoma, brain tumor, small cell lung cancer, and breast cancer, can be achieved.

[0035] The polypeptides of the YQAG-n series are all low-molecular-weight polypeptides. The short peptides of this series are composed of natural amino acids, with easily available raw materials, low synthesis costs, and are all reported for the first time. The synthesis method is simple and the acquisition channels are convenient.

[0036] The imaging probes prepared by optically labeling and modifying the YQAG-n series polypeptides have excellent imaging effects on various tumors, including neuroblastoma, retinoblastoma, melanoma, sarcoma, brain tumor, small cell lung cancer, and breast cancer. The circulation time of the polypeptides in the body is appropriate, which can enable the imaging probes to aggregate and retain at the tumor site, thereby obtaining better tumor imaging effects and facilitating the promotion of clinical applications. For example, the present invention utilizes the advantages of the near-infrared fluorescent dye MPA prepared from the YQAG-n series, such as deeper penetration depth and weaker autofluorescence of background tissues, and has good application prospects in fluorescence imaging and fluorescence-guided surgery. The YQAG-n series polypeptides can be prepared into radioactive drugs for tumor screening and early diagnosis, and can also non-invasively monitor early malignant tumors and treatment in real time in situ. Description of the Drawings

[0037] Figure 1 It is the test result of the affinity of the near-infrared fluorescent probe for neuroblastoma cells SK-N-SH;

[0038] Figure 2 It is the optical imaging diagram of the near-infrared fluorescent probe MPA-YQAG-1 in neuroblastoma SK-N-SH tumor-bearing nude mice;

[0039] Figure 3 It is the optical imaging diagram of the near-infrared fluorescent probe MPA-YQAG-2 in breast cancer MCF-7 tumor-bearing nude mice;

[0040] Figure 4 It is the optical imaging diagram of the near-infrared fluorescent probe MPA-YQAG-3 in non-small cell lung cancer A549 tumor-bearing nude mice;

[0041] Figure 5 It is the optical imaging diagram of the near-infrared fluorescent probe MPA-YQAG-5 in breast cancer 4T1 tumor-bearing BalB / C white mice;

[0042] Figure 6 is a radionuclide probe 99m Tc-HYNIC-(PEG 4)2 Optical imaging of E-(YQAG-1)2 in nude mice bearing 4T1 breast cancer tumors. Detailed implementation manners

[0043] The present invention will be further illustrated by specific examples and application examples below: All chemical substances used in the synthesis steps are existing substances or commercially available products. All polypeptides involved in the examples were synthesized by Hangzhou Genscript Biotech Co., Ltd.

[0044] MPA is ICG-Der-02 prepared in the patent CN101440282 previously disclosed by this research group; human neuroblastoma cell line SK-N-SH was purchased from Nanjing Kebai Biotechnology; human breast cancer cell line MCF-7 was purchased from Nanjing Kebai Biotechnology; non-small cell lung cancer A549 was purchased from Nanjing Kebai Biotechnology; murine breast cancer cell line 4T1 was purchased from Nanjing Kebai Biotechnology; nude mice bearing tumors were purchased from Nanjing Qinglongshan Animal Farm; the linker connection form of (PEG4)2E-HYNIC is YONG1-PEG4-E-PEG4-YONG1, where E is glutamic acid, HYNIC is connected to the glutamic acid amino group, and two carboxyl groups are connected to PEG4. The preparation process of (PEG4)2E-HYNIC refers to the published literature: Wang L, Shi J, Kim Y S, et al. Improving tumor-targeting capability and pharmacokinetics of 99mTc-labeled cyclic RGD dimers with PEG4 linkers[J]. Molecular pharmaceutics, 2009, 6(1): 231-245.

[0045] Example 1 Preparation of polypeptide YQAG-1

[0046] The preparation of polypeptide YQAG-1 includes the following steps:

[0047] Step S101, resin swelling: Add a certain amount of RinkAmide MBHA resin to the reaction column, then add an appropriate amount of dichloromethane (DCM), and gently blow nitrogen for 10 - 30 minutes to fully swell the resin. Drain the dichloromethane solution, and then wash it 3 times with dimethylformamide (DMF) and drain it;

[0048] Step S102, removal of Fmoc: Add a 20% solution of piperidine in DMF to the reaction column, deprotect once every 5 minutes and once every 8 minutes. After the reaction, wash the resin 3 times with DMF, DCM, and DMF in sequence;

[0049] Step S103, Coupling: Weigh accurately Fmoc-Asn-OH and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU) which are 3 times the molar amount of the charged resin. Completely dissolve them in DMF, add N,N-diisopropylethylamine (DIPEA) to activate the carboxyl group, then add the solution into the reaction column for reaction. After reacting for 30 minutes, wash it 3 times successively with DMF, DCM, and DMF respectively, and then drain the solvent. Take a small amount of resin and add one drop each of 6% ninhydrin / ethanol solution and 80% phenol / ethanol solution for detection. If the condensation is complete and there is no free amino group, the solution will be colorless or light yellow; otherwise, the resin or the solution will turn blue or reddish-brown, indicating that the reaction is incomplete. After the reaction is completed, wash it 3 times successively with DCM, DCM, and DMF. Repeat the above operation to couple other amino acids in turn until the last amino acid Fmoc-Trp-OH is coupled. Wash the obtained peptidylresin with methanol and dry it thoroughly in a vacuum drying oven;

[0050] Step S104, Cleavage A: Take 120 mL of cleavage solution (87.5% trifluoroacetic acid + 5% benzenemethanethiol + 2.5% ethanedithiol + 2.5% phenol + 2.5% water) and add it to the resin. Shake it at low temperature for 2 h, then separate the cleavage solution from the resin with a fritted funnel and retain the filtrate. Slowly drip the filtrate into ice-cold anhydrous ether. After dripping, let it settle naturally for 30 min. Then centrifuge to obtain the solid, wash the solid three times with ether, and dry the obtained precipitate to get the dry powder crude product.

[0051] Step S105, Purification: Purify it by high performance liquid chromatography. The chromatographic packing material for purification is a 10-μm C18 preparative column, and the mobile phase system is 0.1% TFA / aqueous solution - 0.1% TFA / acetonitrile solution. Use gradient elution and perform cyclic injection purification. Load the crude product solution onto the chromatographic column, start the mobile phase elution, collect the main peak, evaporate the acetonitrile, obtain the target polypeptide concentrate, then lyophilize it to obtain the target polypeptide, and finally measure the mass-to-charge ratio to determine the molecular weight [M-H] - = 718.

[0052] Example 2 Preparation of polypeptide YQAG-n (n = 2 - 6)

[0053] Refer to the method of Example 1 to prepare the ganglioside GD2 affinity peptide YQAG-2. The difference between the preparation of YQAG-2 and Example 1 is only that: in the coupling step of Step S103, the amino acids are coupled in turn according to the amino acid sequence of YQAG-2 shown in SEQ ID NO: 2. Confirm by mass spectrometry [M-H] - = 803.

[0054] Prepare the ganglioside GD2 affinity peptide YQAG-3 according to the method of Example 1. The difference in the preparation of YQAG-3 from Example 1 is only that: in the coupling step of step S103, the amino acids are sequentially coupled according to the amino acid sequence of YQAG-3 shown in SEQ ID NO: 3. Mass spectrometry confirmation [M-H] - = 778.

[0055] Prepare the ganglioside GD2 affinity peptide YQAG-4 according to the method of Example 1. The difference in the preparation of YQAG-4 from Example 1 is only that: in the coupling step of step S103, the amino acids are sequentially coupled according to the amino acid sequence of YQAG-4 shown in SEQ ID NO: 4. Mass spectrometry confirmation [M-H] - = 734.

[0056] Prepare the ganglioside GD2 affinity peptide YQAG-5 according to the method of Example 1. The difference in the preparation of YQAG-5 from Example 1 is only that: in the coupling step of step S103, the amino acids are sequentially coupled according to the amino acid sequence of YQAG-5 shown in SEQ ID NO: 5. Mass spectrometry confirmation [M-H] - = 877.

[0057] Prepare the ganglioside GD2 affinity peptide YQAG-6 according to the method of Example 1. The difference in the preparation of YQAG-6 from Example 1 is only that: in the coupling step of step S103, the amino acids are sequentially coupled according to the amino acid sequence of YQAG-6 shown in SEQ ID NO: 6. Mass spectrometry confirmation [M-H] - = 810.

[0058] Example 3 Prepare fluorescent targeting compounds

[0059] Taking MPA-YQAG-1 as an example, the specific preparation process is as follows:

[0060] Step S301, take 0.02 mmol of MPA and dissolve it in 200 μL of ultradry DMSO. Add 3.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.2 mg of N-hydroxysuccinimide (EDCI / NHS) (molar ratio MPA:EDCI:NHS = 1:1.5:1.5), and react in the dark for 4 h to carry out the carboxyl activation reaction;

[0061] Step S302, take 0.02 mmol of the solid-phase synthesized polypeptide YQAG-1 (n = 1-6), 0.1 mmol of triethylamine and 200 μL of ultradry DMSO and add them into a 5 mL reaction flask, and react under nitrogen protection for 10 min; add the solution in the above reaction (1) to the reaction solution in (2), and stir and react at room temperature for 12 h;

[0062] Step S303: After the reaction is completed, the reaction solution is concentrated by freeze-drying, then diluted with distilled water, and separated and purified by preparative liquid chromatography. The preparative liquid chromatography conditions are as follows: An Agilent 1220InfinityⅡ series HPLC system equipped with an Agilent ZORBAX SB-C18 semi-preparative column (9.4×250mm, 5μm) is used, gradient elution is carried out for 60 minutes, and the flow rate is 2 mL / min. Among them, mobile phase A is ultrapure water (0.01% TFA), and B is acetonitrile (0.01% TFA). The elution gradient is set as follows: at 0 - 5 minutes, 95% A and 5% B; at 15 minutes, 85% A and 15% B; at 30 minutes, 70% A and 30% B; at 45 minutes, 50% A and 50% B. The finally obtained green product is confirmed to be the expected product MPA-YQAG-1 by analytical HPLC and ESI-MS mass spectrometry analysis.

[0063] In the above preparation process, by replacing the YQAG-1 polypeptide used in the step with the solid-phase synthesized YQAG-n (n = 1 - 6) polypeptide, other various polypeptide compounds with tumor-targeted optical imaging functions, namely MPA-YQAG-1, MPA-YQAG-2, MPA-YQAG-3, MPA-YQAG-4, MPA-YQAG-5, and MPA-YQAG-6, can be obtained.

[0064] Example 4 Preparation of Radionuclide Probe

[0065] Taking 99m Tc-HYNIC-(PEG 4)2 E-(YQAG-1)2 as an example, the specific preparation process is as follows:

[0066] Dissolve 5 mg of the synthesized and purified intermediate (PEG4)2E-HYNIC in 0.3 mL of DMSO, then add 2.1 mg of EDCI and 1.25 mg of NHS, react at room temperature for 5 hours, detect the reaction progress by analytical high-performance liquid chromatography. After the reaction is completed, add 7.8 mg of the mimic peptide YQAG-1, then add 5.6 mg of DIPEA, react at room temperature for 3 hours. After the reaction is completed, separate and purify by preparative liquid chromatography, and finally obtain 6.5 mg of yellow solid, which is confirmed to be the target product by mass spectrometry.

[0067] Prepare TPPTS (sodium triphenylphosphine trisulfonate) solution with a concentration of 100 mg / mL, Tricine (trimethylglycine) with a concentration of 130.0 mg / mL, and succinic acid-sodium succinate buffer with a concentration of 102.4 mg / mL (where succinic acid is 77.0 mg and sodium succinate is 25.4 mg) respectively. Take 10.0 μL of TPPTS solution, 10.0 μL of Tricine solution, and 10.0 μL of succinic acid-sodium succinate buffer and mix them with 10.0 μL (1.0 g / mL) of the (YQAG-1)2-(PEG4)2E-HYNIC in a vial respectively. Then add 10 mCi Na 99m TcO4 and heat it in a metal bath at 100 °C for 20 minutes. After the reaction is completed, cool it to room temperature to obtain the radionuclide probe (YQAG-1)2-(PEG4)2E-HYNIC- 99m Tc. The product is analyzed and identified by an Agilent ZORBAX SB-Aq analytical column. The HPLC method used is an Agilent 1220Infinity II series HPLC system equipped with a radioactive on-line detector (Flow-RAM) and an Agilent ZORBAX SB-Aq analytical column (4.6×250 mm, 5 μm). Gradient elution is carried out for 45 minutes at a flow rate of 1 mL / min. Among them, mobile phase A is ultrapure water (0.01% TFA), and B is acetonitrile (0.01% TFA). The elution gradient is set as follows: at 0 - 5 minutes, 95% A and 5% B; at 15 minutes, 70% A and 30% B; at 20 minutes, 65% A and 35% B; at 25 minutes, 45% A and 55% B; at 45 minutes, 5% A and 95% B.

[0068] Example 5 Affinity test of MPA-YQAG-n (n = 1 - 6) for neuroblastoma cell SK-N-SH

[0069] After eluting the cultured human neuroblastoma cell SK-N-SH from the 12-well plate, resuspend it in PBS solution, and co-incubate it with MPA-YQAG-n (n = 1 - 6) with a concentration of 10 μmol / L prepared in Example 1 and Example 2 for 2 hours respectively. Then detect its mean fluorescence intensity by flow cytometry. The stronger the fluorescence intensity, the stronger the affinity for the cells. When the affinity between the probe and the receptor on the cells is strong, the mean fluorescence intensity value of the cells detected by the flow cytometer is high. See Figure 1 . The results of the in vitro affinity experiment show that after the probes of MPA-YQAG-n (n = 1 - 6) with the same concentration are co-incubated with the neuroblastoma cell SK-N-SH with high expression of GD2 respectively, the affinity of YQAG-1 of the present invention for SK-N-SH cells is the strongest and the affinity intensity is the largest.

[0070] Optical Imaging Experiment of Compound MPA-YQAG-1 in Nude Mice Bearing Neuroblastoma SK-N-SH Tumors

[0071] The compound MPA-YQAG-1 prepared in Example 3 was formulated into a physiological saline solution with a concentration of 1 mg / mL. 15 μL of the drug, namely MPA-YQAG-1 solution, was injected into 3 nude mice bearing neuroblastoma SK-N-SH tumors, each weighing approximately 20 g, via the tail vein. Optical signal acquisition was performed at 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h after administration. The imaging results are as follows Figure 2 shown. The distribution of the probe in the mice and its enrichment in the tumor region were observed. The imaging results of the compound MPA-YQAG-1 in the 3 nude mice bearing tumors were basically the same. It can be seen from the imaging map at 1 h that the probe had significantly aggregated in the tumor, and the contour of the tumor edge was relatively clear. The probe still remained in the tumor until 24 h. Among them, the probe had the most enrichment in the tumor at 2 h, while the uptake and clearance in other background organs were relatively fast. It can be inferred from the information of the bladder that this probe was mainly metabolized through the kidneys.

[0072] Optical Imaging Experiment of MPA-YQAG-2 in Nude Mice Bearing Breast Cancer MCF-7 Tumors

[0073] The compound MPA-YQAG-2 prepared in Example 3 was formulated into a physiological saline solution with a concentration of 1 mg / mL. 15 μL of the drug, namely MPA-YQAG-2 solution, was injected into 3 nude mice bearing breast cancer MCF-7 tumors, each weighing approximately 20 g, via the tail vein. Optical signal acquisition was performed at 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h after administration. The distribution of the probe in the mice and its enrichment in the tumor region were observed. The imaging results are as follows Figure 3 shown. The imaging results of the compound MPA-YQAG-2 in the 3 nude mice bearing tumors were basically the same. It can be seen from the imaging map at 1 h that the probe had significantly aggregated in the tumor, and the contour of the tumor edge was relatively clear. The probe still remained in the tumor until 24 h. Among them, the probe had the most enrichment in the tumor at 2 h, while the uptake and clearance in other background organs were relatively fast. It can be inferred from the information of the bladder that this probe was mainly metabolized through the kidneys.

[0074] Optical Imaging Experiment of MPA-YQAG-3 in Nude Mice Bearing Non-Small Cell Lung Cancer A549 Tumors

[0075] The compound MPA-YQAG-3 prepared in Example 3 was formulated into a physiological saline solution (1 mg / mL). Three nude mice bearing non-small cell lung cancer A549 tumors, each weighing approximately 20 g, were injected with 15 μL of the drug, i.e., the MPA-YQAG-3 solution, via the tail vein, and optical signal collection was performed at 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h after administration. The distribution of the probe in the mice and its enrichment in the tumor region were observed. The imaging results were as follows Figure 4 shown. The imaging results of the compound MPA-YQAG-3 in the three tumor-bearing nude mice were basically consistent. It can be seen from the imaging map at 1 h that the probe had significantly accumulated in the tumor, and the contour of the tumor edge was relatively clear. The probe still remained in the tumor until 12 h. Among them, the probe had the highest enrichment in the tumor at 2 h, while the uptake and clearance in other background organs were relatively fast. It can be inferred from the information of the bladder that this probe was mainly metabolized through the kidneys.

[0076] Example 9 Optical Imaging Experiment of MPA-YQAG-5 in Mice Bearing 4T1 Breast Cancer Tumors

[0077] The compound MPA-YQAG-5 prepared in Example 3 was formulated into a physiological saline solution with a concentration of 1 mg / mL. Three BalB / C mice bearing 4T1 breast cancer tumors, each weighing approximately 20 g, were injected with 15 μL of the drug, i.e., the MPA-YQAG-5 solution, via the tail vein, and optical signal collection was performed at 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h after administration. The distribution of the probe in the mice and its enrichment in the tumor region were observed. The imaging results were as follows Figure 5 shown. The imaging results of the compound MPA-YQAG-5 in the three tumor-bearing BalB / C mice were basically consistent. It can be seen from the imaging map at 1 h that the probe had significantly accumulated in the tumor, and the contour of the tumor edge was relatively clear. The probe still remained in the tumor until 12 h. Among them, the probe had the highest enrichment in the tumor at 2 h, while the uptake and clearance in other background organs were relatively fast. It can be inferred from the information of the bladder that this probe was mainly metabolized through the kidneys.

[0078] Example 10 Radionuclide Probe 99m Tc-HYNIC-(PEG 4)2 E-(YQAG-1)2 SPECT-CT Imaging Experiment in Mice Bearing 4T1 Breast Cancer Tumors

[0079] The 99m Tc-HYNIC-(PEG 4)2 E-(YQAG-1)2 prepared in Example 4 was formulated into a physiological saline solution of 1 mg / mL. Three nude mice bearing 4T1 breast cancer tumors, each weighing approximately 20 g, were injected with 15 μL of the drug, i.e., 99m Tc-HYNIC-(PEG4)2 E-(YQAG-1)2, and SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h after administration. The distribution of the probe in mice and its enrichment in the tumor region were observed. The imaging effect was as Figure 6 shown, and the distribution of the radionuclide probe in mice and its enrichment in the tumor region were observed. 99m Tc-HYNIC-(PEG 4)2 E-(YQAG-1)2 had obvious uptake in the tumor part and was mainly metabolized through the kidneys.

Claims

1. A disialylganglioside GD2 affinity peptide, referred to as GD2 affinity peptide, characterized in that: The GD2 affinity peptide sequence is: Ac-Asn-Thr-Ile-X1-X2-X3-Trp-NH2; wherein -X1-X2-X3- is selected from any one of -Gly-Gly-Ala-, -Gly-Gly-Arg-, -Gly-Gly-Met-, -Gly-Gly-Ser-, -Met-Gly-Arg- or -Gly-Gly-Tyr-.

2. Use of the GD2 affinity peptide or its dimer or polymer according to claim 1 in the preparation of tumor diagnostic reagents or tumor therapeutic drugs.

3. Use of the GD2 affinity peptide or its dimer or polymer according to claim 1 in the preparation of a tumor diagnostic imaging agent.

4. Use of the GD2 affinity peptide or its dimer or polymer according to claim 1 in preparing imaging agents for accurate positioning of tumor boundaries and surgical navigation or in preparing radionuclide imaging agents.

5. A modified polypeptide, characterized in that The modified polypeptide has the following general formula: ML-YQAG-n; wherein M represents a light label or a radionuclide label; L is a linking group; YQAG-n is any one of the GD2 affinity peptides described in claim 1 or its dimer or polymer; the light label M is selected from an organic chromophore, an organic fluorophore, a light reflecting compound, a light scattering compound and a bioluminescent molecule.

6. A modified polypeptide according to claim 5, characterized in that The optical label M is an organic fluorophore, and the organic fluorophore is selected from any one of MPA, IRDye800, Cy7.5, and Cy5.

5.

7. A modified polypeptide according to claim 5, characterized in that The optical label M is a radionuclide label, and the radionuclide in the radionuclide label M is 99m Tc, 68 Ga, 64 Cu, 67 Ga, 90 Y, 111 In or 177 Lu, 125 I; the chelating agent used to chelate the radionuclide in the radionuclide label M is any one of HYNIC, DOTA, NOTA or DTPA.

8. A modified polypeptide according to claim 5, characterized in that L is selected from azidopentanoic acid, 6-aminohexanoic acid, propiolic acid, polyethylene glycol, 1,4,7-triazacyclopentane-1,4,7-triacetic acid, 7-[(4-hydroxypropyl)methylene]-1,4,7-triazanonane-1,4-diacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, mercaptoacetyl triglycine, MAG2, N3S, N2S2 type ligands, diethyltriaminepentaacetic acid, 1,4-succinic acid, 5-aminopentanoic acid, polyethyleneimine, 6-hydrazinopyridine-3-carboxylic acid, benzyl bromoformate, N-(2-aminohexanoic acid)maleimide or a combination thereof.

9. A modified polypeptide according to claim 8, characterized in that The polyethylene glycol is PEG4 or PEG 6.

10. Use of the modified polypeptide according to any one of claims 5 to 9 in the preparation of a tumor diagnostic imaging agent or an intraoperative navigation imaging agent.

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