Application of polypeptide in preparation of DLL3 overexpressed tumor specific targeting molecular probe

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of polypeptide in preparation of a specific targeting molecular probe for DLL3 overexpressed tumors. The research finds that the molecular probe prepared from the polypeptide as shown in SEQ ID No.4 can effectively target DLL3 overexpressed tumor tissues, so that the application of the polypeptide or the dimer or polymer thereof in preparation of the specific targeting molecular probe for the DLL3 overexpressed tumor is provided, and the amino acid sequence of the polypeptide is as shown in SEQ ID No.4. The invention further provides the application of the polypeptide or the dimer or polymer thereof in preparation of the specific targeting molecular probe for the DLL3 overexpressed tumor. The molecular probe disclosed by the invention is high in imaging accuracy and excellent in affinity with DLL3, can realize early diagnosis and imaging of tumor tissues, can also be used for image navigation before and during an operation, and improves the operation accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more particularly, to the application of a polypeptide in the preparation of a specific targeting molecular probe for tumors with overexpression of DLL3. Background Art

[0002] Currently, cancer has become one of the major diseases threatening health. If early and accurate diagnosis of tumors can be achieved and timely intervention measures can be taken, it will greatly improve the survival probability of patients or their quality of life. However, most existing cancer diagnosis technologies have limitations. For example, commonly used clinical diagnostic methods include serum tests, metabolomics screening, and imaging examinations such as ultrasound contrast, CT, etc. Although imaging examinations can assist in diagnosis by comparing the differences between lesions and normal tissues, they still face problems such as high false positive rates and insufficient sensitivity. In addition, the current accuracy of whole-body examinations is not satisfactory. And some invasive techniques, such as puncture and biopsy, may cause additional trauma to patients. Therefore, it is of great significance to develop a diagnostic method that can reduce trauma and achieve high-precision and high-sensitivity real-time monitoring of tumors.

[0003] In the past few decades, significant progress has been made in cancer molecular imaging technology, and its research focus has gradually shifted from non-targeted imaging to targeted imaging. Diagnosis and treatment can be carried out through specific biomarkers highly expressed on tumor cells. Such targeted drugs mainly specifically bind to tumor tissues and have no targeting to normal tissues, thus achieving a diagnostic effect with high selectivity and low toxicity. In the field of nuclear medicine imaging, SPECT and PET, as the core means, have promoted the development of personalized drug therapy. Currently, a variety of molecular imaging tracers have been applied in clinical practice and research. These tracers include small molecules, peptides, antibodies, antibody fragments, and protein scaffolds, etc., which can achieve non-invasive target localization and molecular-level characterization of tumors.

[0004] Current research shows that targeted polypeptides are a highly promising tumor-targeted therapy strategy. Compared with antibodies and their fragments, polypeptide small molecules exhibit significant advantages: they have a lower molecular weight and a smaller volume, and can penetrate tumor tissues more effectively; polypeptides have a faster enrichment rate at the tumor site and can be rapidly cleared from the blood and non-target tissues, showing excellent pharmacokinetic properties; when used as molecular probes, polypeptides usually do not cause immune reactions or toxicity, thus avoiding immune-related problems that may occur in monoclonal antibody therapy; in addition, the design and synthesis process of polypeptides is relatively simple, can be rapidly and automatically produced by a polypeptide synthesizer, and can also be optimized for the binding affinity with specific receptors and in vivo pharmacokinetic behavior through extensive chemical or molecular modifications; and can be conveniently combined with various conventional or novel chelating agents for radionuclide labeling to further expand their application scope.

[0005] Delta-like Ligand 3 (DLL3) is a transmembrane protein attached to the cell membrane surface. It is not expressed or is expressed at low levels in healthy adult tissues, but is present on the surface of tumor cells such as small cell lung cancer. Given the abnormal upregulation of DLL3 expression, the development of targeted molecular probes can be used to guide the early diagnosis of cancer, accurately locate lesions, and assist in pathological staging and intraoperative navigation. Summary of the Invention

[0006] One of the objectives of the present invention is to provide a new polypeptide capable of preparing a specific targeted molecular probe for tumors with overexpression of DLL3 and its application.

[0007] DLL3 is a transmembrane protein belonging to the Notch ligand family, mainly expressed intracellularly, but abnormally expressed on the cell surface in certain tumors. The DLL3 gene is located in the 19q13 region of human chromosome 19. The DLL3 protein contains a transmembrane domain that can anchor to the cell surface. DLL3 is an inhibitory ligand of the Notch signaling pathway and mainly regulates neurogenesis during embryonic development. DLL3 is expressed at low levels in normal tissues, but shows abnormal expression in a variety of tumors, mainly including the following tumor types: 1. Small cell lung cancer (SCLC): DLL3 is highly expressed in approximately 80% - 96% of patients with small cell lung cancer. This high expression is mainly concentrated in the SCLC subtype with significant neuroendocrine characteristics. DLL3 is hardly expressed in normal lung tissue, so it is considered an ideal target for SCLC treatment.

[0008] 2. Neuroendocrine neoplasms (NENs): DLL3 is abnormally expressed in a variety of neuroendocrine neoplasms, including gastric neuroendocrine carcinoma, large cell neuroendocrine lung cancer (LCNEC), neuroendocrine prostate cancer, etc. The expression of DLL3 in these tumors is closely related to the enhanced proliferation, migration, and invasion abilities of tumor cells.

[0009] 3. Melanoma: DLL3 also shows abnormal expression in melanoma and promotes the proliferation and migration of tumor cells by activating the MAPK signaling pathway.

[0010] 4. Glioblastoma multiforme (GBM): DLL3 is highly expressed in certain types of glioblastoma and is associated with the inhibition of the Notch signaling pathway.

[0011] 5. Other tumors: There are also reports of abnormal expression of DLL3 in tumors such as endometrial cancer and ovarian cancer.

[0012] In summary, DLL3 protein shows abnormal expression in various tumors, especially significantly high expression in small cell lung cancer and other neuroendocrine tumors, making it a potential target for tumor treatment and diagnosis.

[0013] The present invention provides an application of a polypeptide or its dimer or multimer in preparing a specific targeting molecular probe for tumors with overexpression of DLL3, and the amino acid sequence of the polypeptide is as shown in SEQ ID No. 4.

[0014] Research has found that molecular probes with good targeting performance for DLL3 are expected to become an effective means for the diagnosis / tracer of related tumors. However, in addition to good targeting properties, tumor molecular probes also need to take into account aspects such as specificity, stability, biocompatibility, safety, and good pharmacokinetic properties. Through repeated experiments, the present invention provides a new polypeptide, which, when used as a molecular probe for tumor tissues with overexpression of DLL3, has a high target / non-target ratio, good uptake and retention effects at the tumor site, and can be rapidly cleared from non-target tissues, with good pharmacokinetic properties. Combined with optical labeling or radionuclide labeling, it can be used for intraoperative imaging navigation of tumors and precise localization of tumor boundaries.

[0015] The present invention also provides a specific targeting molecular probe for tumors with overexpression of DLL3, and the specific targeting molecular probe is a polypeptide or its dimer or multimer linked with an optical label or a radionuclide label; the polypeptide is as described above.

[0016] In the specific targeting molecular probe of the present invention, the radionuclide label is a diagnostic radionuclide or a therapeutic radionuclide; the optical label is selected from organic chromophores, organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, photochromic compounds, phosphorescent compounds or bioluminescent molecules.

[0017] The polypeptide of the present invention can be simply modified with various conventional and novel chelating agents to label radionuclides or link optical labels.

[0018] Those skilled in the art can select the specific label types included in the molecular probe of the present invention according to the application target.

[0019] Preferably, the optical label is a near-infrared fluorescent dye; the radionuclides are selected from 99m Tc, 68 Ga, 64 Cu, 67 Ga, 90 Y, 111 In, 177 Lu, 225 Ac or 125 I.

[0020] More preferably, the near-infrared fluorescent dye is MPA, IRDye800, Cy7.5 or Cy5.5.

[0021] The polypeptide modified by radioactive labeling can be used for tumor screening and early diagnosis, and can also monitor early malignant tumors and treatments in real time and non-invasively in situ.

[0022] The present invention also provides the use of the polypeptide shown in SEQ ID No.4 or its dimer or multimer or the above-mentioned specific targeting molecular probe in any of the following aspects: (1) Preparing a tumor diagnostic reagent; DLL3 is overexpressed in the tumor; (2) Preparing a tumor-targeted drug; DLL3 is overexpressed in the tumor; (3) Application in preparing a reagent or kit for diagnosing, treating and / or preventing a disease; the disease is characterized by overexpression of DLL3.

[0023] The polypeptide of the present invention has a low molecular weight, a simple preparation method and low cost. The tumor molecular probe prepared therefrom is relatively stable in vivo, can promote the accumulation and retention of the imaging probe at the tumor site, and thus obtain a better tumor imaging effect, which is conducive to clinical promotion and application.

[0024] The polypeptide targeting DLL3 of the present invention can also be used to construct a diagnostic and therapeutic agent, which can be combined with a substance having drug activity to guide the precise positioning of the drug, improve the targeting of the drug carrier, thereby reducing the toxic and side effects caused by systemic free drug and enhancing the therapeutic effect.

[0025] The diagnostic reagent or kit of the present invention can effectively trace tumors with overexpression of DLL3, and thus can be used to assist in the diagnosis of tumors or intraoperative navigation.

[0026] The present invention also provides a targeted drug, which comprises the polypeptide shown in SEQ ID No.4 or its dimer or multimer or the above-mentioned specific targeting molecular probe and a pharmaceutically acceptable excipient.

[0027] In the targeted drug of the present invention, the polypeptide shown in SEQ ID No.4 or its dimer or multimer or the above-mentioned specific targeting molecular probe is also conjugated with an active substance; the active substance can prevent, inhibit and / or treat a disease characterized by overexpression of DLL3.

[0028] The present invention also provides a diagnostic reagent or kit, which comprises the above-mentioned specific targeting molecular probe.

[0029] The present invention also provides a method for preparing the above-mentioned specific targeting molecular probe, which includes the step of connecting a photo-label or a radionuclide label to the polypeptide shown in SEQ ID No. 4 or its dimer or multimer.

[0030] Those skilled in the art can synthesize the polypeptide of the present invention according to common general knowledge in the art, and the amino acids used for its synthesis can be L-type or D-type.

[0031] The beneficial effects of the present invention are at least as follows: The present invention provides a new polypeptide that can specifically target tumors with overexpressed DLL3. After binding a photo-label or a radionuclide label, it can be used as a specific targeting molecular probe for tumors with overexpressed DLL3, having a high target / non-target ratio, high imaging accuracy, and good uptake and retention effects at the tumor site, with good pharmacokinetic properties, enabling in vivo diagnosis of tumors, and providing a new option for early diagnosis of tumors, precise localization of tumor boundaries, or intraoperative image navigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the structural formula of the polypeptide YQF-3 of the present invention.

[0033] Figure 2 It is the binding energy analysis diagram of the kinetic simulation of the polypeptide YQF-3 of the present invention.

[0034] Figure 3 It is the affinity analysis diagram of the tumor affinity probe (fluorescent targeting probe) prepared with the polypeptide YQF-3 of the present invention and DLL3.

[0035] Figure 4 It is the representative optical imaging diagram of the fluorescent targeting probe MPA-YQF-3 in nude mice bearing SHP-77 small cell lung cancer tumors.

[0036] Figure 5 It is the radionuclide probe 99M The SPECT / CT imaging diagram of 99mTc-YQF-3 in nude mice bearing SHP-77 small cell lung cancer tumors, and the tumor is circled by the red dotted line in the figure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The following will specifically describe the preferred embodiments of the present invention in conjunction with the examples. It should be understood that the following examples are only for illustrative purposes and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained commercially or prepared by conventional methods in the art.

[0039] The amino acids used in the present invention were purchased from GL Biochem (Shanghai) Ltd., the Rink Amide MBHA resin was purchased from Jiangsu Jitai Peptide Co., Ltd. (JT-08-2235, 10 mg), the 1640 medium used was purchased from Jiangsu Kaygene Biotechnology Co., Ltd., and the SHP-77 cells were purchased from Wuhan Shang'en Biotechnology Co., Ltd.; DMSO, EDCI, and NHS were all purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0040] Example 1 Preparation of Polypeptides In this example, multiple peptides (YQF-0, YQF-1, YQF-2, YQF-3, YQF-4) were synthesized: The amino acid sequence of YQF-0 is (SEQ ID No.1): SYYWS; The amino acid sequence of YQF-1 is (SEQ ID No.2): YVYYSGTTNYNPSLKS; The amino acid sequence of YQF-2 is (SEQ ID No.3): IAVTGFYFDY; The amino acid sequence of YQF-3 is (SEQ ID No.4): RASQRVNNNYLA; The amino acid sequence of YQF-4 is (SEQ ID No.5): QQYDRSPLT.

[0041] Each peptide was synthesized by the solid-phase synthesis method, and the specific example is as follows: (1) Resin swelling Weigh 1 mmol equivalent of Rink Amide MBHA resin and put it into a polypeptide synthesis tube. Add an appropriate amount of dichloromethane (DCM) to cover the resin, and swell for 30 min. Drain the DCM solution, wash with DMF, and drain dry.

[0042] (2) Removal of Fmoc Add a DMF solution containing 20% piperidine by volume to the synthesis tube to cover the resin. The deprotection time is 5 min, and repeat twice. After the reaction, wash with DMF.

[0043] (3) Coupling Add 2 mmol equivalent of amino acid, 4 mmol equivalent of DIPEA, 2 mmol equivalent of HCTU, and DMF to the synthesis tube, shake and react for 1 h. Drain the reaction solution and wash with DMF, then remove Fmoc in the same way as in step (2), wash clean, and detect with ninhydrin.

[0044] (4) Couple different amino acids in the sequence successively in the manner of step (3). The amino acid residues involved can be of the L-type or the D-type. Proline (Pro) can also be replaced by hydroxyproline (Hyp), arginine (Arg) can be replaced by homo-arginine (homo-Arg), and alanine can be replaced by β-alanine.

[0045] (5) Cleavage Dry the resin with nitrogen. Add the cleavage solution (87.5% TFA + 5% benzyl methyl sulfide + 2.5% ethanedithiol + 2.5% phenol + 2.5% water) to the polypeptide synthesis tube. The ratio of the volume of the cleavage solution to the resin is approximately 10 ml / g. After reacting for 2 - 3 h, filter by suction to obtain the filtrate. Add a large amount of methyl tert-butyl ether, then centrifuge. Wash the solid three times with methyl tert-butyl ether to obtain the crude polypeptide.

[0046] (6) Separation and purification Purify by reverse-phase high-performance liquid chromatography. The chromatographic packing material for purification is 10-μm reverse-phase C18. The mobile phase system is 0.1% TFA / aqueous solution and acetonitrile solution. Elute using a gradient system (see Example 3). Determine the ultraviolet absorption of the polypeptide by ultraviolet spectrophotometry for quantification. The results show that each polypeptide is successfully synthesized and the purity is above 95%. Put the collected eluate into a freeze dryer for concentration and freeze-dry it into a white powder.

[0047] The structural formula of YQF-3 is shown in Figure 1 , and the molecular weight is 1404.72.

[0048] Example 2 The present invention also uses the AUTODOCK software to perform virtual docking between each peptide prepared in Example 1 and the receptor (DLL3), and uses the gmx grompp, gmx trjconv, gmx mdrun, and gmx energy commands to extract the docking energy data. The energy data is shown in Figure 2 . Among them, the polypeptide with the highest free energy of binding shows stronger affinity.

[0049] Example 3 Preparation of the fluorescent targeting probe MPA-YQF-3 The preparation method of the fluorescent targeting probe MPA-YQF-3 is as follows: (1) Take 0.02 mmol of MPA and dissolve it in 200 μL of 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 for carboxyl activation reaction.

[0050] (2) Take 0.02 mmol of the polypeptide YQF-3 synthesized by solid-phase synthesis in Example 1, 0.1 mmol of triethylamine and 200 μL of ultradry DMSO and add them to a 5 mL reaction flask, and react for 10 min under nitrogen protection; add the reaction solution in the above step (1) to the reaction solution in step (2), and stir and react at room temperature for 12 h; (3) 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.

[0051] The preparative liquid chromatography conditions are as follows: Use an Agilent 1220 Infinity II series HPLC system equipped with an Agilent ZORBAX SB-C18 semi-preparative column (9.4×250 mm, 5 μm), gradient elution for 60 minutes, flow rate 2 mL / min, where mobile phase A is ultrapure water (volume fraction 0.01% TFA / aqueous solution), and B is acetonitrile.

[0052] 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; at 60 minutes, 10% A and 90% B.

[0053] The finally obtained green product is confirmed to be the expected product MPA-YQF-3 by analytical HPLC and ESI-MS mass spectrometry analysis.

[0054] Example 4 Radionuclide Probe 99M Preparation of Tc-YQF-3 Radionuclide Probe 99M The preparation method of Tc-YQF-3 is specifically as follows: (1) Take 0.03 mmol of HYNIC and dissolve it in 200 μL of DMSO, add 6.87 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4.72 mg of N-hydroxysuccinimide (EDCI / NHS) (molar ratio HYNIC:EDCI:NHS = 1:1.2:1.2), react in the dark for 4 h to carry out carboxyl activation reaction.

[0055] (2) Take 0.02 mmol of the polypeptide YQF-3 synthesized by solid-phase synthesis in Example 1, 0.1 mmol of triethylamine and 200 μL of ultradry DMSO and add them to a 5 mL reaction flask, and react for 10 min under nitrogen protection; add the reaction solution in the above step (1) to the reaction solution in step (2), and stir and react at room temperature for 12 h; (3) 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.

[0056] The conditions of preparative liquid chromatography are as follows: An Agilent 1220Infinity II series HPLC system equipped with an Agilent ZORBAX SB-C18 semi-preparative column (9.4×250mm, 5um) is used for gradient elution for 60 minutes at a flow rate of 2 mL / min. Among them, mobile phase A is ultrapure water (0.01% TFA / aqueous solution by volume), and B is acetonitrile.

[0057] 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; at 60 minutes, 10% A and 90% B.

[0058] The finally obtained green product is confirmed to be the process product HYNIC-YQF-3 by analytical HPLC and ESI-MS mass spectrometry.

[0059] 20 mg of triphenylphosphine trisodium sulfonate (TPPtS) is dissolved in 200 μL of normal saline, 26 mg of N-(tris(hydroxymethyl)methyl)glycine (Tricine) is dissolved in 200 μL of normal saline, 10 μL of each is taken, 10 μg of the process product HYNIC-YQF-3 is taken, and they are put into a glass bottle reactor together with sodium pertechnetate with an activity value of 400 microcuries and heated at 100 °C for 30 minutes.

[0060] The finally obtained green product is confirmed by analytical HPLC to be 99M Tc-YQF-3.

[0061] Example 5 In vitro Affinity Experiment of Fluorescent Targeting Probe MPA-YQF-3 and Other Peptide Fluorescent Probes for SHP-77 Cells According to the method described in Example 3, each peptide YQF-0, YQF-1, YQF-2, YQF-3, YQF-4 described in Example 1 is prepared into a fluorescent targeting probe, which are respectively named MPA-YQF-0, MPA-YQF-1, MPA-YQF-2, MPA-YQF-3, MPA-YQF-4. And their in vitro affinities for SHP-77 cells are tested. The specific method is as follows: (1) First, prepare to lay a 12-well plate. After resuspending SHP-77 cells with good growth status and no contamination with fresh 1640 medium, count them by the counting plate counting method. Add the same amount of cells into each well of the 12-well plate, and then place it in a cell culture incubator at 37 °C with 5% CO2 for 24 h.

[0062] (2) After the cells had grown for 24 h, the culture medium in the 12-well plate was discarded, and 500 μL of fresh RPMI 1640 medium without serum was added. Different groups were set up: blank group Control (CON), single dye group MPA, and peptide-added group. In the peptide-added group, 5 μL of MPA and 5 μL of MPA-YQF-0, MPA-YQF-1, MPA-YQF-2, MPA-YQF-3, and MPA-YQF-4 fluorescent targeting probes were added respectively. The initial concentration of the probes was 500 μM, and the final concentration in the well plate was 5 μM. Then, the cells were continued to be cultured and incubated in the incubator for 2 h.

[0063] (3) Sample preparation before flow cytometry: Transfer the cell suspension into a 1.5 mL EP tube, centrifuge at 1200 rpm for 5 min, wash 3 times with 500 μL of PBS buffer (pH 7.2), and finally resuspend with 500 μL of PBS buffer (pH 7.2) for standby.

[0064] (4) Measuring the fluorescence intensity of cells by flow cytometry: Set the parameters of the flow cytometer, with the flow rate at medium flow rate and 40,000 cells for cell injection. Load the samples and measure the fluorescence intensity of each group, as well as the fluorescence intensity relative to the control group and the single dye group.

[0065] (5) Data processing: Use FlowJo 7.0 software to generate a peak graph of the original data, and calculate the mean fluorescence intensity (MFI). Use GraphPad Prism software to quantitatively plot the mean fluorescence intensity and perform data difference analysis.

[0066] When the affinity between the probe and the receptor on the cells is strong, the average fluorescence intensity value of the cells detected by the flow cytometer is high. See Figure 3 . The results of the in vitro affinity experiment showed that after the fluorescent targeting probes with the same concentration were incubated with SHP-77 cells with high DLL3 expression respectively, the affinity intensity between MPA-YQF-3 of the present invention and SHP-77 was the largest (47.06×10 3 ).

[0067] Example 6 Optical imaging of the fluorescent targeting probe MPA-YQF-3 in SHP-77 tumor-bearing mice with small cell lung cancer The fluorescent targeting probe MPA-YQF-3 prepared in Example 3 was dissolved in a physiological saline solution to prepare a solution with a concentration of 1 mg / 1 mL. 15 μL of the probe MPA-YQF-3 solution was injected into 3 nude mice bearing SHP-77 small cell lung cancer tumors (weighing about 20 grams) via the tail vein, and optical signal acquisition was performed at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h after injection. Observe the distribution of the MPA-YQF-3 fluorescent targeting probe in the mouse body and its enrichment in the tumor region.

[0068] The imaging results of the fluorescent targeting probe MPA-YQF-3 in 3 tumor-bearing nude mice were basically the same. The representative imaging results are as Figure 4 shown. It can be seen from the imaging map at 1 h that the probe has significantly accumulated in the tumor, and the outline of the tumor margin is relatively clear. The probe still remains in the tumor until 48 h. Among them, the probe accumulates the most in the tumor at 1 h, while the uptake and clearance in other background organs are relatively fast. It can be inferred from the signal of the bladder that this probe is mainly metabolized through the kidneys.

[0069] The above-mentioned pharmacodynamic experiments show that: the polypeptide of the present invention has the ability to specifically bind to small cell lung cancer cells. The high-affinity characteristics of the targeting peptide can be used for optical imaging of malignant tumors. These high-affinity polypeptide monomers, polypeptide dimers, or polypeptide polymers directly or indirectly conjugated with fluorescent dyes can be used as tumor-specific targeting molecular probes, and are expected to achieve the effect of accurately positioning the tumor boundary, which can bring real-time imaging for preoperative and intraoperative image navigation and has the advantage of improving the surgical accuracy.

[0070] Example 7 Radionuclide Probe 99M SPECT / CT Imaging of The radionuclide probe prepared in Example 4 99M Tc-YQF-3 was dissolved in a physiological saline solution to prepare a solution with a concentration of 10 μg / 1 mL. 100 μL of the radionuclide probe 99M Tc-YQF-3 solution was injected into SHP-77 tumor-bearing nude mice (weighing about 20 grams) via the tail vein, and radioactive signals were collected by SPECT at 30 min, 1 h, and 2 h after injection. Observe 99M the distribution of the

[0071] The imaging results are as Figure 5 shown. It can be seen from the imaging map at 0.5 h that the radionuclide probe 99MTc-YQF-3 has shown obvious accumulation in tumors, with relatively clear tumor margin contours. The probe accumulates the most in the tumor site at 1 h, while the uptake and clearance in other background organs are relatively fast. It can be inferred from the signal in the bladder that this probe is mainly metabolized through the kidneys.

[0072] The above-mentioned pharmacodynamic experiments show that the polypeptide of the present invention has the ability to specifically bind to small cell lung cancer cells with high specificity. The high affinity of the targeting peptide can be used for SPECT imaging of malignant tumors. These high affinity polypeptide monomers, polypeptide dimers or polypeptide polymers directly or indirectly chelating radionuclides can be used as tumor-specific targeting molecular probes, and it is expected to achieve the effect of accurately locating the tumor boundary. Replacing different radionuclides has different functions such as diagnosis and treatment.

[0073] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Use of a polypeptide or a dimer or multimer thereof in preparing a specific targeting molecular probe for tumors with overexpression of DLL3, wherein the amino acid sequence of the polypeptide is shown in SEQ ID No.

4.

2. A specific targeting molecular probe for tumors with overexpression of DLL3, characterized in that: The specific targeting molecular probe is a polypeptide or a dimer or polymer thereof connected with a light label or a radionuclide label; the polypeptide is as described in claim 1.

3. The specific targeting molecular probe according to claim 2, characterized in that: The radionuclide label is a diagnostic radionuclide or a therapeutic radionuclide; the optical label is selected from an organic chromophore, an organic fluorophore, a light absorbing compound, a light reflecting compound, a light scattering compound, a photochromic compound, a phosphorescent compound or a bioluminescent molecule.

4. The specific targeting molecular probe according to claim 3, characterized in that: The radionuclide label is selected from 99m Tc, 68 Ga, 64 Cu, 67 Ga, 90 Y. 111 In, 177 Lu, 225 Ac or 125 I.

5. Use of the polypeptide shown in SEQ ID No. 4 or its dimer or polymer or the specific targeting molecular probe according to any one of claims 2 to 4 in the preparation of tumor diagnostic reagents or tumor targeted drugs; DLL3 is overexpressed in the tumor.

6. Use of the polypeptide represented by SEQ ID No. 4 or its dimer or multimer or the specific targeting molecular probe according to any one of claims 2 to 4 in the preparation of a reagent or kit for diagnosing, treating and / or preventing a disease characterized by overexpression of DLL3.

7. A targeted drug, characterized in that: It comprises the polypeptide shown in SEQ ID No. 4 or its dimer or polymer or the specific targeting molecular probe according to any one of claims 2 to 4 and pharmaceutically acceptable excipients.

8. The targeted drug according to claim 7, characterized in that: The polypeptide shown in SEQ ID No. 4 or its dimer or multimer or the specific targeting molecular probe according to any one of claims 2 to 4 is also coupled with an active substance; the active substance can prevent, inhibit and / or treat a disease characterized by overexpression of DLL3.

9. A diagnostic reagent or kit, characterized in that: The method comprises the specific targeting molecular probe according to any one of claims 2 to 4.

10. A method for preparing the specific targeting molecular probe according to any one of claims 2 to 4, characterized in that: The method comprises the step of connecting a light label or a radionuclide label to the polypeptide shown in SEQ ID No. 4 or its dimer or multimer.

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