A polypeptide with bidirectional regulation function of tumor immune microenvironment and its application

Abstract

The present invention discloses a polypeptide with the function of bidirectionally regulating the tumor immune microenvironment and its application, which belongs to the field of tumor immunology. The drug prepared based on the polypeptide is an anti-tumor drug, which can reduce PD-L1 on the surface of tumor cells and promote the expression of MHC-Ⅰ on the surface of tumor cells, thereby achieving bidirectional regulation of the tumor immune microenvironment. The excellent effects of this polypeptide drug are as follows: fewer amino acid sequences and lower molecular weight, which facilitate the entry of drugs into tumor tissues, and the preparation method is also simpler and less expensive; it can reduce PD-L1 and promote MHC-Ⅰ expression, while reducing the immune escape of tumor cells, increasing antigen presentation, and then promoting the capture of tumor cells by immune cells. This innovative move can significantly enhance the effect of tumor immunotherapy; it produces bidirectional regulation of the tumor immune microenvironment, which can increase patient compliance and is beneficial to obtaining ideal clinical treatment effects.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunity technology, and specifically relates to a polypeptide having the function of bidirectionally regulating the tumor immune microenvironment and its application. Background Art

[0002] The tumor immune microenvironment comprises tumor cells, immune cells, and cytokines. These components can be divided into tumor suppressors and tumor promoters based on their specific roles. The distribution of these components within the immune microenvironment determines the direction of tumor immunity. Although the human immune system can theoretically eliminate tumors, tumors evade immune surveillance and killing by forming an immunosuppressive microenvironment. This is manifested by elevated PD-L1 expression on the tumor cell surface to evade immune surveillance and decreased expression of MHC-I molecules to disrupt antigen presentation, thereby evading immune surveillance.

[0003] Monoclonal antibodies targeting PD-L1 have been approved for marketing in China, and their application and development in tumor immunotherapy have attracted widespread attention. During the tumor-immunity cycle, the binding of PD-L1 on tumor cells to the immune checkpoint PD1 on immune cells helps tumors evade immune cell capture, leading to tumor progression. Therefore, immunotherapy targeting PD-L1 can effectively block tumor cell immune evasion. However, in clinical practice, only a subset of patients demonstrate ultimate clinical benefit, and some even develop acquired resistance after experiencing initial benefit. It is generally believed that abnormally low expression of MHC class I is one of the primary mechanisms underlying the low benefit rate and acquired resistance associated with PD-L1 therapy. However, despite numerous studies investigating increasing MHC class I expression, these studies have yielded disappointing results and questions about biosafety, resulting in virtually no approved drugs.

[0004] Studies have confirmed that simultaneously reducing PD-L1 and promoting MHC-I expression is beneficial for tumor immunotherapy and can achieve clinical benefits. However, currently, drugs that regulate the tumor immune microenvironment in a single direction are predominant, and there are no drugs that can both reduce PD-L1 and promote MHC-I expression. If this treatment approach is to be used, a combination drug approach is required. However, the shortcomings of this combination drug administration may lead to poor patient compliance due to high dosages and concerns about toxic side effects, thus affecting the ultimate clinical treatment effect. Therefore, to further break through the bottleneck of tumor immunotherapy and increase future patient compliance, there is an urgent need to develop a drug that can bidirectionally regulate the tumor immune microenvironment. Summary of the Invention

[0005] The purpose of the present invention is to provide a polypeptide with the function of bidirectionally regulating the tumor immune microenvironment. The drug prepared based on the polypeptide is an anti-tumor drug. This type of drug can reduce PD-L1 on the surface of tumor cells and promote the expression of MHC-I on the surface of tumor cells, thereby achieving bidirectional regulation of the tumor immune microenvironment.

[0006] The present invention is achieved through the following technical solutions:

[0007] A polypeptide with the function of bidirectionally regulating the tumor immune microenvironment, the amino acid sequence of the polypeptide is shown in SEQ ID NO.1: CSNFYMPLGGGSK.

[0008] The structural formula of the above polypeptide is:

[0009]

[0010] A use of the polypeptide in preparing a drug for improving the tumor immune microenvironment.

[0011] Preferably, the drug can reduce the expression of PD-L1 and promote the expression of MHC-I.

[0012] Preferably, the concentration of the polypeptide is 19-160 μg / mL.

[0013] Preferably, the drug is prepared by using the polypeptide alone or in combination with excipients.

[0014] Preferably, the polypeptide is prepared as an injection preparation when used alone.

[0015] Preferably, the excipient is an additive commonly used in pharmacy;

[0016] The polypeptide is prepared into a pharmaceutical composition with commonly used pharmaceutical additives.

[0017] Preferably, the tumor is a solid tumor.

[0018] Compared with the prior art, the present invention has at least the following technical effects:

[0019] (1) The present invention provides a polypeptide with the function of bidirectionally regulating the tumor immune microenvironment. The drug prepared based on the polypeptide is an anti-tumor drug. This type of drug can reduce PD-L1 on the surface of tumor cells and promote the expression of MHC-I on the surface of tumor cells, thereby achieving bidirectional regulation of the tumor immune microenvironment.

[0020] (II) Compared with the current PD-L1 monoclonal antibodies on the market, the excellent effects of this peptide drug are as follows:

[0021] 1. Fewer amino acid sequences and lower molecular weight facilitate drug entry into tumor tissues, and the preparation method is simpler and less expensive.

[0022] 2. Start by inhibiting the upstream pathway of PD-L1 and fundamentally reduce the expression of PD-L1;

[0023] 3. It can both reduce PD-L1 and promote MHC-I expression, reducing tumor cell immune escape while increasing antigen presentation, thereby promoting immune cell capture of tumor cells. This innovative approach can significantly enhance the effectiveness of tumor immunotherapy;

[0024] 4. A drug can both reduce PD-L1 and promote MHC-I expression, producing bidirectional regulation on the tumor immune microenvironment, which can increase patient compliance and help achieve ideal clinical treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the expression results of PD-L1 and MHC-I proteins in the cell model after peptide treatment compared with the blank group in Experimental Example 1;

[0026] Figure 2 Schematic diagram of the results of immunofluorescence staining and flow cytometry detection of PD-L1 and MHC-Ⅰ expression in tumor tissues of the animal tumor model after treatment in Experimental Example 1;

[0027] Figure 3 This is a schematic diagram of the expression results of core proteins EGFR, P38, pP38, PD-L1 and MHC-Ⅰ in the MAPK pathway detected by western blot based on the transcriptomics results in Experimental Example 1;

[0028] Figure 4 This is a schematic diagram of the tumor changes in the experimental group mice after treatment in Experimental Example 1, compared with the blank group;

[0029] Figure 5 Schematic diagram of immunofluorescence detection of cell apoptosis in tumor tissue in Experimental Example 2;

[0030] Figure 6 Schematic diagram of digesting the tumor mass into a single cell suspension and then sending it to flow cytometry to detect apoptotic cells in Experimental Example 2;

[0031] Figure 7 The apoptosis data verified by flow cytometry in Experiment 3 is statistically significant

[0032] Figure 8 Schematic diagram of the biosafety of the polypeptide detected by HE staining of blood routine, liver function, kidney function, etc. in Experimental Example 3. DETAILED DESCRIPTION

[0033] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0034] The technical solution of a specific embodiment of the present invention is:

[0035] A polypeptide with the function of bidirectionally regulating the tumor immune microenvironment, the amino acid sequence of the polypeptide is shown in SEQ ID NO.1: CSNFYMPLGGGSK.

[0036] Test Example 1: The above-mentioned peptide drug can reduce PD-L1 and promote MHC-Ⅰ expression

[0037] 1.1 Tumor Cell Model: Cells were divided into a control group and an experimental group. When human osteosarcoma cells (143B) were in logarithmic growth phase, the control group was given serum-free culture medium, while the experimental group was given serum-free culture medium containing 19 μg / mL of peptide. The total volume of culture medium in each T75 cell culture flask was approximately 6 mL. After 24 hours of incubation in a cell culture incubator, the expression of PD-L1 and MHC-I proteins in each group was detected by western blot.

[0038] Figure 1 Schematic diagram of a comparative experiment showing that peptide drugs can reduce PD-L1 and promote MHC-Ⅰ expression in tumor cell models.

[0039] The results are as follows Figure 1 As shown in Figures A and B, compared with the control group, the expression of PD-L1 protein in the experimental group was significantly decreased, and the expression of MHC-Ⅰ protein was significantly increased, and the data were statistically significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0040] At the same time, the same cell grouping and culture method was used, and Real Time PCR was used to detect the mRNA expression of PD-L1 and MHC-Ⅰ after 24 hours of treatment.

[0041] The results are as follows Figure 1 As shown in Figure C, the expression of PD-L1 was significantly decreased, and the expression of MHC-I substances (MHC-I substances of human cells specifically include HLA-A, HLA-B, and HLA-C) was significantly increased, and the data were statistically significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0042] 1.2 Animal Tumor Model: Immunocompetent mice were inoculated with the mouse osteosarcoma cell line K7M2. After tumors reached a certain volume, the mice were divided into a control group and an experimental group. The control group received 200 μL of normal saline daily, while the experimental group received 200 μL of normal saline containing 160 μg / mL of the peptide via tail vein injection once daily for 5 consecutive days. Tumors in the mice were regularly observed and measured during treatment. One week after treatment, the mice were sacrificed and tumor masses were removed.

[0043] Immunofluorescence assay was used to detect the expressions of PD-L1 and MHC-Ⅰ in tumor tissues.

[0044] Figure 2 Schematic diagram of a comparative experiment showing that peptide drugs can reduce PD-L1 and promote MHC-Ⅰ expression in animal tumor models.

[0045] Specifically, the results are as follows Figure 2 Figure A shows the expression of PD-L1 and MHC class I proteins. Compared with the control group, the expression of PD-L1 protein in the experimental group was significantly decreased, while the expression of MHC class I protein was significantly increased (DiI represents PD-L1, and FITC represents MHC class I).

[0046] At the same time, the tumor mass was digested into a single cell suspension and sent to flow cytometry to detect the expression of PD-L1 and MHC-Ⅰ.

[0047] The results are as follows Figure 2 As shown in Figures B and C, compared with the control group, the expression of PD-L1 protein in the experimental group was significantly decreased, and the expression of MHC-I protein was significantly increased, and the data were statistically significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0048] The above results show that peptide drugs can reduce PD-L1 and promote MHC-Ⅰ expression.

[0049] Test Example 2: The mechanism by which the above-mentioned peptide drug reduces PD-L1 and promotes MHC-I expression

[0050] 143B cells were divided into a control group and an experimental group. When the cells reached logarithmic growth phase, the control group was given serum-free medium, while the experimental group was given serum-free medium containing 19 μg / mL of peptide. Approximately 6 mL of medium was placed in each T75 cell culture flask. After 24 hours of incubation in a cell culture incubator, cells were harvested and sent for transcriptomics analysis to generate pathway enrichment maps.

[0051] like Figure 3 A in the middle shows a pathway enrichment map generated based on transcriptomics results.

[0052] like Figure 3Shown in B is a comparison chart of the expression of core proteins EGFR, P38, pP38, PD-L1 and MHC-Ⅰ in the MAPK pathway detected by western blot.

[0053] The results are as follows Figure 3 As shown in middle A, based on the pathway enrichment map, the inventors selected the MAPK pathway with the highest correlation with tumors for further verification.

[0054] The above cell grouping and culture methods were also used, and western blot was used to detect the expression of core proteins EGFR, P38, pP38, PD-L1 and MHC-Ⅰ in the MAPK pathway.

[0055] The results are as follows Figure 3 As shown in Figure B, compared with the control group, the expression of PD-L1 protein in the experimental group was significantly reduced, while the expression of MHC class I protein was significantly increased. The related pathway proteins EGFR and pP38 were also significantly reduced.

[0056] The above results indicate that the polypeptide of the present invention regulates the expression of PD-L1 and MHC-Ⅰ through the MAPK pathway of EGFR-P38.

[0057] Test Example 3: The therapeutic effect of the above-mentioned peptide drug in bidirectionally regulating the tumor immune microenvironment

[0058] Immunocompetent mice were inoculated with the mouse osteosarcoma cell line K7M2. After tumors reached a certain size, they were divided into a control group and an experimental group. The control group received 200 μL of saline daily, while the experimental group received 200 μL of saline containing 160 μg / mL of the peptide, via tail vein injection, once daily for five consecutive days. Tumors in the mice were regularly observed and measured during treatment, and photographs were taken one week after treatment.

[0059] The results are as follows Figure 4 As shown in the figure, compared with the control group, the tumors in the experimental group mice were significantly reduced or even disappeared. After taking pictures, the mice were killed and the tumors were removed.

[0060] Immunofluorescence was used to detect cell apoptosis in tumor tissues.

[0061] The results are as follows Figure 5 As shown, compared with the control group, the number of apoptotic cells in the experimental group was greater (blue is live cell staining, green is apoptotic cell staining).

[0062] At the same time, the tumor mass was digested into a single cell suspension and then sent to flow cytometry to detect apoptotic cells.

[0063] The results are as follows Figure 6 As shown, the number of apoptotic cells in the experimental group was greater than that in the control group.

[0064] The results are as follows Figure 7 As shown, Figure 6 The apoptosis data verified by flow cytometry were statistically significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0065] Test Example 4: Biosafety of Peptide Drugs

[0066] Immunocompetent mice were inoculated with the mouse osteosarcoma cell line K7M2. After tumors reached a certain size, the mice were divided into a control group and an experimental group. The control group received 200 μL of normal saline per day, while the experimental group received 200 μL of normal saline containing 160 μg / mL of the peptide, via tail vein injection, once daily for 5 consecutive days.

[0067] One week after treatment, blood was collected from the orbit and sent for routine blood tests, liver function tests, kidney function tests, etc. to detect the effects of the drug on the blood system.

[0068] The results are as follows Figure 8 As shown in Figure A, all blood parameters were within normal range.

[0069] After killing the mice, the heart, liver, spleen, lung and kidney were collected for HE staining. Figure 8 As shown in Figure B, no obvious abnormalities were observed in any of the organs. This image was taken using a microscope under ordinary white light. The scale bar is 200 μm.

[0070] In summary, the invented polypeptide drug has good biosafety.

[0071] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A polypeptide capable of bidirectionally regulating the tumor immune microenvironment, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1: CSNFYMPLGGGSK.

2. Use of the polypeptide according to claim 1 in the preparation of a drug for improving the tumor immune microenvironment, characterized in that: Improving the tumor immune microenvironment means reducing the expression of PD-L1 and promoting the expression of MHC-I. The tumor is a solid tumor.

3. Use of a polypeptide according to claim 2 in the preparation of a drug for improving the tumor immune microenvironment, characterized in that: The concentration of the polypeptide is 19-160 μg / mL.

4. Use of a polypeptide according to claim 2 in the preparation of a drug for improving tumor immune microenvironment, characterized in that: The medicine includes a preparation of a polypeptide alone or a preparation of a polypeptide combined with an excipient.

5. Use of a polypeptide according to claim 4 in the preparation of a drug for improving tumor immune microenvironment, characterized in that: When the polypeptide is used alone, it is prepared into an injection preparation.

6. Use of a polypeptide according to claim 4 in the preparation of a drug for improving tumor immune microenvironment, characterized in that: The excipients are commonly used additives in pharmacy; The polypeptide is prepared into a pharmaceutical composition with commonly used pharmaceutical additives.

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