Application of endogenous polypeptide medicine JM2 and target thereof in tumor treatment

Abstract

The invention discloses an endogenous polypeptide medicine JM2 and application of a target spot of the endogenous polypeptide medicine JM2 in tumor treatment, and relates to the field of biological medicine. The polypeptide drug JM2 is small in molecular weight and easy to absorb, has no obvious toxicity in a cellular level experiment, and has good drug development value and clinical application prospect. The polypeptide medicine JM2 disclosed by the invention has good activity of inhibiting tumor cell proliferation, so that the polypeptide medicine JM2 is used as a tumor inhibitor to help to control growth and diffusion of tumors. The polypeptide medicine JM2 disclosed by the invention has an efficient macrophage phenotype repolarization function, and can generate a remarkable proinflammatory reaction and generate stronger anti-tumor activity, which is possibly crucial for improving the tumor recognition and removal capability of an immune system. The method can be further used for developing infectious diseases and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an endogenous polypeptide for anti-tumor use, the preparation of a modified drug containing the polypeptide, and the use of the endogenous polypeptide in the preparation of drugs or vaccines for preventing and / or treating tumors. Background Art

[0002] Affected by unhealthy lifestyles such as an aging population, environmental pollution, smoking, lack of exercise, and a high-calorie diet, the incidence of new cancers is increasing year by year, indicating a growing demand for anti-tumor drugs.

[0003] Due to the abnormal and rapid metabolism of tumor cells, the vast majority of solid tumors foster a unique reductive immunosuppressive microenvironment. This immunosuppressive microenvironment is extremely complex. In addition to tumor cells, it also contains numerous immune cells that are unable to recognize mutated tumor cells and secrete cytokines to fuel their insatiable proliferation. Tumor-associated macrophages are divided into M1 anti-tumor macrophages and M2 pro-tumor macrophages. In malignant solid tumors, the majority of macrophages are M2 macrophages, which play a crucial role in angiogenesis, extracellular matrix remodeling, tumor cell proliferation, and metastasis. Therefore, designing therapies that can guide macrophages toward a beneficial state is an effective anti-tumor strategy.

[0004] The tumor microenvironment (TME) is a dynamic ecosystem composed of tumor cells, immune cells, stromal cells, and complex signaling networks. Its immunosuppressive properties are a core obstacle limiting the efficacy of existing immunotherapies. In recent years, the abnormal activation of the coagulation system in the TME has attracted much attention, especially the significant increase in the production of thrombin, which is not only directly related to cancer-associated thrombosis (CAT), but also participates in tumor progression and immune regulation by activating protease-activated receptors (PARs). PAR4 belongs to the G protein-coupled receptor (GPCR) family and is widely expressed on the surface of immune cells such as macrophages and T cells. Its activation can trigger Gq, Gi / o, and other signaling pathways to regulate immune cell function.

[0005] Peptides, a hot topic in the post-genetic era, have been widely used in drug development. Their molecular size lies between small-molecule chemical drugs and protein drugs, creating a unique niche for them and cleverly filling the gap between them. Compared to conventional small-molecule drugs, peptide drugs offer higher activity and enhanced selectivity, particularly in the treatment of complex diseases. Compared to protein drugs, peptide drugs offer advantages such as improved stability, higher purity, and lower production costs. In summary, peptide drugs approach the quality control level of small-molecule drugs and similar activity to protein drugs, combining the advantages of both, making them more suitable for treating complex diseases that are difficult for small-molecule drugs to address. Peptide drugs have attracted attention in the development of anti-tumor drugs due to their targeted, safe, and specific properties. Different peptide drugs have diverse mechanisms of action. They can achieve direct anti-tumor effects by inhibiting tumor cell proliferation and promoting tumor cell apoptosis, or indirectly by enhancing and stimulating the body's immune response to tumor cells and inhibiting tumor angiogenesis. Furthermore, the diversity and specificity of their mechanisms of action also allow for peptide modification and fusion, achieving highly effective, targeted, and specific anti-tumor effects. Summary of the Invention

[0006] To overcome the problems of the prior art, one of the objectives of the present invention is to provide an anti-tumor endogenous polypeptide or a pharmaceutically acceptable salt thereof. The polypeptide, JM2, exhibits excellent stability and biological activity. Experimental results demonstrate that JM2 can activate PAR4 in tumor-associated macrophages, promote M1 macrophage polarization and TME remodeling, and exert effective anti-tumor effects.

[0007] A second object of the present invention is to provide the use of the above-mentioned anti-tumor endogenous polypeptide or its pharmaceutically acceptable salt.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] An anti-tumor endogenous polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide has the amino acid sequence shown in I or II below:

[0010] I:GGTQTPSVYDESGSTGGGGDDSTPSILPAPR;

[0011] II: GGTQTPSVYDESGSTGGGDDSTPSILPAPR an amino acid sequence that has been substituted, deleted or added with one or more amino acids and has the same function.

[0012] Furthermore, the II is a shortened sequence SGST of GGTQTPSVYDESGSTGGGDDSTPSILPAPR.

[0013] Furthermore, the II is a sequence obtained by adding 1 to 5 amino acid residues to the N-terminus and / or C-terminus of GGTQTPSVYDESGSTGGGDDSTPSILPAPR.

[0014] Furthermore, the salt is acetate, hydrochloride, hydrobromide, sulfate, phosphate, nitrate, oxalate or tartrate.

[0015] A polyethylene glycol-modified polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide is the above-mentioned endogenous anti-tumor polypeptide. The polypeptide can be modified with mPEG to increase its half-life in vivo.

[0016] Furthermore, the molecular weight of the mPEG is in the range of 2000-5000.

[0017] A nucleic acid encoding the above-mentioned anti-tumor endogenous polypeptide.

[0018] A biological material comprising the above nucleic acid, which is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0019] Use of the above-mentioned anti-tumor endogenous polypeptide or its pharmaceutically acceptable salt, polyethylene glycol-modified polypeptide or its pharmaceutically acceptable salt, nucleic acid or biological material in the preparation of an immunomodulator.

[0020] Use of the above-mentioned anti-tumor endogenous polypeptide or its pharmaceutically acceptable salt, polyethylene glycol-modified polypeptide or its pharmaceutically acceptable salt, nucleic acid or biological material in the preparation of drugs for preventing and / or treating diseases caused by immunosuppression.

[0021] Use of the above-mentioned anti-tumor endogenous polypeptide or its pharmaceutically acceptable salt, polyethylene glycol-modified polypeptide or its pharmaceutically acceptable salt, nucleic acid or biological material in the preparation of antibacterial preparations and / or anti-infective preparations.

[0022] Use of the above-mentioned anti-tumor endogenous polypeptide or its pharmaceutically acceptable salt, polyethylene glycol-modified polypeptide or its pharmaceutically acceptable salt, nucleic acid or biological material in the preparation of anti-tumor drugs.

[0023] Furthermore, the tumor is any one or more of a solid tumor and a metastatic tumor, including but not limited to: melanoma, non-small cell lung cancer, and breast cancer.

[0024] Furthermore, the anti-tumor drug is an anti-melanoma drug.

[0025] Furthermore, the anti-tumor drug is a drug for inhibiting melanoma lung metastasis.

[0026] The present invention has the following advantages and effects compared to the prior art:

[0027] (1) The polypeptide drug JM2 of the present invention has a small molecular weight and is easily absorbed. It has no obvious toxicity in cell-level experiments and has good drug development value and clinical application prospects.

[0028] (2) The polypeptide drug JM2 of the present invention has good activity in inhibiting tumor cell proliferation. Therefore, the polypeptide drug JM2 is used as a tumor suppressor to help control the growth and spread of tumors.

[0029] (3) The polypeptide drug JM2 of the present invention has the function of highly effectively repolarizing macrophage phenotypes, which can produce a significant pro-inflammatory response and generate stronger anti-tumor activity. This may be crucial for improving the immune system's ability to recognize and eliminate tumors. It can be further developed for infectious diseases and has broad application prospects.

[0030] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without making any creative efforts.

[0032] Figure 1 This is the H NMR spectrum of mPEG-JM2.

[0033] Figure 2 This is a statistical analysis chart of the effects of different JM2s on tumor cell survival rate.

[0034] Figure 3 This is a statistical analysis of the proportion of macrophages regulated by JM2 from M2 phenotype to M1 phenotype by flow cytometry.

[0035] Figure 4 This is a statistical analysis of the proportion of macrophages repolarized from M2 phenotype to M1 phenotype regulated by the shortened form of JM2, SGST, by flow cytometry.

[0036] Figure 5 The figures are the results of detecting the ability of macrophages treated with polypeptide JM2 to phagocytose tumor cells by flow cytometry (left) and fluorescence imaging (right).

[0037] Figure 6These are photographs of in vitro tumors, tumor volume change curves, and growth status graphs of mice in the PBS control group, intratumoral macrophage depletion group, JM2-treated group after intratumoral macrophage depletion, and JM2 group.

[0038] Figure 7 These are H&E sections of isolated tumors and lungs of mice in the PBS control group and the JM2-treated group.

[0039] Figure 8 Figure 2 is the flow cytometric analysis result of M1 macrophages (CD80+) and M2 macrophages (CD206+) in the tumor site after JM2 treatment.

[0040] Figure 9 This is a diagram showing the infiltration of M1 macrophages (CD68) and cytotoxic T cells (CD8) in the tumor site after JM2 treatment.

[0041] Figure 10 The figure shows the in vivo imaging of the lung metastasis model of melanoma cells (luciferase marker, B16-LUC) treated with JM2 on days 15, 18, 20, and 22, and the corresponding lung fluorescence quantitative curves.

[0042] Figure 11 These are actual photos and H&E sections of lung tissue after JM2 was used to treat a melanoma cell lung metastasis model.

[0043] Figure 12 This is the flow cytometric analysis result of memory T cells in mice with melanoma cell lung metastasis model after JM2 treatment. DETAILED DESCRIPTION

[0044] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0045] Example 1: Preparation of mPEG-modified JM2 polypeptide

[0046] 1. Activate carboxyl-terminated polyethylene glycol (mPEG-COOH) (molecular weight 2000) with EDC / NHS. Dissolve 1 μM mPEG-COOH, 1 μM EDC, and 1 μM NHS in 1 ml of PBS buffer and stir at room temperature for 15 minutes to obtain solution 1.

[0047] 2. The company was commissioned to synthesize JM2 (amino acid sequence: GGTQTPSVYDESGSTGGGDDSTPSILPAPR), and JM2 was dissolved in 500 μl PBS buffer at a concentration of 1 μM to obtain solution 2.

[0048] 3. Adjust the pH of solution 1 to 8.5, then add it dropwise to solution 2 while stirring continuously. Place the mixture in a shaker at 4°C for overnight reaction.

[0049] 4. The obtained product was purified by ultrafiltration.

[0050] 5. The purified polypeptide was preserved using freeze-drying technology to obtain mPEG-modified JM2 polypeptide, named mPEG-JM2.

[0051] The chemical structure of the peptide mPEG-JM2 was verified by nuclear magnetic resonance hydrogen spectrum (1H-NMR) ( Figure 1 Spectral analysis focused on characteristic peaks of mPEG (e.g., ethylene glycol unit signals at 3.5-3.7 ppm) and characteristic signals of amino acid residues of the JM2 polypeptide backbone (e.g., aromatic amino acid signals at 6.5-8.0 ppm). Comparison with standard spectra confirmed the structural integrity of mPEG-JM2 and the success of the modification.

[0052] Example 2: Study on the effect of JM2 on tumor cell survival rate

[0053] Non-small cell lung cancer cell line A549 cells were cultured at 8×10 3 Cells were plated in 96-well plates and incubated at 37°C overnight. JM2 (150 mM) was added to the cells and incubated for 24 hours. The PAR4 agonist PAR4-AP (150 mM) was used as a control group. Cell viability was assessed using a CCK-8 assay, with absorbance measured at 450 nm. Triplicate wells were used in the experiment, and data are expressed as relative viability (%). Statistical analysis was performed using a t-test or one-way analysis of variance (ANOVA).

[0054] Figure 2 This is a statistical analysis of the effects of different concentrations of JM2 on the survival rate of A549 tumor cells. Compared with the existing PAR4 receptor agonist PAR4-AP, JM2 significantly inhibited the growth of tumor cells, and the inhibitory efficiency increased with increasing concentration.

[0055] Example 3: Verification of JM2's ability to regulate macrophage phenotype in vitro

[0056] Materials and methods are as follows: 1×10 4 Macrophages Raw264.7 or BMDM were seeded into six-well plates the night before. Then, mPEG-JM2 and the shortened form of JM2, SGST, were added and co-cultured with macrophages for 24 hours until the peptide concentrations reached 0, 62.5, 125, 250, 500, and 1000 μg / mL, respectively. Cells were then collected. + CD80 + , F4 / 80 + CD206 + Flow cytometry was used to evaluate the regulation of macrophage phenotype by JM2 using fluorescent antibody labeling.

[0057] Figure 3 This is a statistical analysis of the proportion of macrophages that repolarize from the M2 phenotype to the M1 phenotype regulated by JM2, as measured by flow cytometry. As can be seen, JM2 can repolarize M2 macrophages, and its efficiency increases with increasing concentration.

[0058] Figure 4 This is a statistical analysis of the proportion of macrophages that repolarize from the M2 phenotype to the M1 phenotype by flow cytometry using the shortened form of JM2, SGST. As can be seen, SGST can repolarize M2 macrophages.

[0059] Example 4: Verification of JM2's ability to regulate macrophage phagocytosis in vitro

[0060] Raw 264.7 cells were plated at 1×10 4 100 cells / well were plated in 24-well plates and incubated with JM2 for 24 hours. Meanwhile, mouse melanoma cell line B16 cells stably expressing EGFP green fluorescence were plated at 8×10 3 100 cells / well of a 96-well plate was plated and cultured overnight. Then, JM2-treated and untreated macrophages were added to the 96-well plate at a ratio of 1:2 (macrophage:tumor cell), and the phagocytosis of tumor cells by macrophages was observed under a fluorescence microscope.

[0061] Figure 5 The results of flow cytometry (left) and fluorescence imaging (right) are used to detect the ability of macrophages treated with peptide JM2 to phagocytose tumor cells. It can be seen that JM2-treated macrophages have a significantly increased ability to recognize and phagocytose tumor cells.

[0062] Example 5: Verification of the therapeutic effect of JM2 in a mouse model of malignant tumor melanoma

[0063] Materials and methods: First, a mouse model of malignant melanoma was established: C57BL / 6 mice (6-8 weeks old) were selected and subcutaneously inoculated with mouse skin melanoma cell line B16-F10 cells (2×10 5 The experiment was divided into four groups: a PBS control group, an intratumoral macrophage depletion group (macrophages were depleted using clodronate disodium), a macrophage depletion + JM2 treatment group, and a JM2 treatment group (JM2 at a dose of 10 mg / kg, injected intratumorally once every three days), with seven mice in each group. To evaluate the therapeutic effect, tumor growth and body weight of the mice were monitored, and tumor volume was measured every three days using the formula: volume = (length × width²) / 2. On day 21 of the experiment, the mice were sacrificed, and the tumors were dissected, photographed, and weighed. Tumor volume curves were plotted using GraphPad Prism, and inter-group comparisons were performed using two-way analysis of variance.

[0064] Figure 6 This is a graph showing the growth of tumors treated with JM2. It can be seen that JM2 treatment can effectively inhibit the growth of malignant melanoma tumors.

[0065] Figure 7 These are H&E sections of tumor and lung tissue treated with JM2. The subcutaneous tumor sections show that JM2 treatment effectively induces tumor cell necrosis or apoptosis. The lung sections also show that JM2 treatment significantly reduces lung metastasis.

[0066] Example 6: Verification of the ability of JM2 to improve the tumor immune microenvironment

[0067] Materials and methods: A mouse model of malignant melanoma was established as described in Example 5. After treatment, tumor tissue was harvested and processed into single-cell suspensions. Intratumoral immune cell activation was assessed by antibody labeling and flow cytometry. Tumor tissue sections were incubated with anti-CD68 and anti-CD8 antibodies, counterstained with DAPI, and images were captured using a fluorescence microscope.

[0068] Figure 8 This figure shows the proportion of M1 macrophages in the tumor site after JM2 treatment. It can be seen that JM2 can repolarize tumor-associated macrophages in the tumor into pro-inflammatory M1 macrophages.

[0069] Figure 9 This figure shows the infiltration of M1 macrophages (CD68) and cytotoxic T cells (CD8) in the tumor site after JM2 treatment. As can be seen, JM2 treatment increased the number of anti-tumor immune cells in the tumor site, indicating that JM2 can effectively activate the anti-tumor immune response.

[0070] Example 7: Verification of the therapeutic effect of JM2 in a mouse model of lung metastasis

[0071] Materials and methods are as follows: Melanoma cell lung metastasis model was established: 1×10 5 Mice were challenged with 150 μM cells / mL B16-Luc cells. Mice were then fed a normal diet for 14 days. On days 15, 17, and 19, mPEG-JM2 (150 μM, 100 μL) was intravenously administered. Tumor cell growth was assessed by measuring the luciferase expression in B16 cells using an in vivo imaging system.

[0072] Luciferin-based in vivo tumor imaging: Mice were anesthetized with isoflurane and injected intraperitoneally (ip) with 150 mg / kg of D-luciferin. Following injection, the mice were placed on the imaging platform of a PerkinElmer IVIS Spectrum imaging station and continuously inhaled isoflurane. Starting 8 minutes after D-luciferin injection, 40 seconds of white light and luciferase activity were recorded. Luciferase activity was quantified using Living Image software.

[0073] Figure 10 This is an in vivo imaging image of a melanoma cell lung metastasis model treated with the peptide drug JM2. It can be seen that JM2 treatment can effectively inhibit the lung metastasis of melanoma cells.

[0074] Lung Macroscopic Analysis and Histopathology: Lung lobes were harvested on day 25 after tumor inoculation. Visible B16 tumor nodules on the surface of all lung lobes were counted using a dissecting microscope. In selected experiments, lung vascular perfusion was performed to remove red blood cells and prepare for further immunohistochemical analysis. Lung lobes were subsequently cryofixed or paraffin-embedded. Paraffin-embedded lung sections stained with hematoxylin and eosin were scanned using the Olympus VS200ASW software and the VS200 research slide scanner for further analysis.

[0075] Figure 11 These are photos and H&E sections of lung tissue after JM2 treatment in a melanoma cell lung metastasis model. As can be seen, JM2 treatment can effectively reduce lung metastasis.

[0076] To explore the regulatory effect of JM2 on immune memory, on the 22nd day of the experiment, immune cells from mouse spleens were isolated and labeled with anti-CD3, anti-CD8, anti-CD44 and anti-CD62L fluorescent antibodies, and the proportions of central memory T cells (CD44+CD62L+) and effector memory T cells (CD44+CD62L-) were detected.

[0077] Figure 12 This figure shows flow cytometry analysis of the proportion of memory T cells in a mouse model of lung metastasis. The results demonstrate that JM2 can effectively activate memory T cells in mice, achieving long-term anti-tumor effects.

[0078] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An anti-tumor endogenous polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide has the amino acid sequence shown in I or II below: I:GGTQTPSVYDESGSTGGGGDDSTPSILPAPR; II: GGTQTPSVYDESGSTGGGDDSTPSILPAPR an amino acid sequence that has been substituted, deleted or added with one or more amino acids and has the same function.

2. The anti-tumor endogenous polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The II is a shortened sequence SGST of GGTQTPSVYDESGSTGGGDDSTPSILPAPR, or a sequence obtained by adding 1 to 5 amino acid residues to the N-terminus and / or C-terminus of GGTQTPSVYDESGSTGGGDDSTPSILPAPR.

3. The anti-tumor endogenous polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The salt is acetate, hydrochloride, hydrobromide, sulfate, phosphate, nitrate, oxalate or tartrate.

4. A polyethylene glycol-modified polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide is the anti-tumor endogenous polypeptide described in any one of claims 1-3.

5. The polyethylene glycol-modified polypeptide or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The molecular weight of the mPEG is in the range of 2000-5000.

6. A biomaterial, characterized in that: The biological material is a nucleic acid encoding the anti-tumor endogenous polypeptide according to any one of claims 1 to 3, or a recombinant DNA, an expression cassette, a transposon, a vector or a host cell comprising the nucleic acid.

7. Use of the anti-tumor endogenous polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, the polyethylene glycol-modified polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 4 to 5, or the biomaterial according to claim 6, wherein the use is any one of the following: (1) Application in the preparation of immunomodulators; (2) Application in the preparation of drugs for preventing and / or treating diseases caused by immunosuppression (3) Application in the preparation of antibacterial preparations and / or anti-infective preparations; (4) Application in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumor is any one or more of a solid tumor and a metastatic tumor, including melanoma, non-small cell lung cancer, and breast cancer.

9. The use according to claim 7, characterized in that: The anti-tumor drug is an anti-melanoma drug.

10. The use according to claim 7, characterized in that: The anti-tumor drug is a drug for inhibiting melanoma lung metastasis.

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