Polypeptide with function of bidirectionally regulating tumor immune microenvironment and application thereof

Abstract

The invention discloses a polypeptide with a function of bidirectionally regulating a tumor immune microenvironment and application of the polypeptide, and belongs to the technical field of tumor immunity. The medicine prepared on the basis of the polypeptide is an anti-tumor medicine, and the medicine can reduce PD-L1 on the surfaces of tumor cells and promote MHC-I expression on the surfaces of the tumor cells, so that the tumor immune microenvironment is regulated bidirectionally. The polypeptide drug has the excellent effects that the amino acid sequences are fewer, the molecular weight is lower, the drug can enter tumor tissues conveniently, the preparation method is simpler, and the cost is lower; according to the present invention, with the application of the polypeptide, the PD-L1 expression can be reduced, the MHC-I expression can be promoted, the antigen presentation can be increased while the tumor cell immune escape is reduced so as to promote the tumor cell capture by the immune cells, such that the tumor immunotherapy effect can be significantly enhanced; the tumor immune microenvironment is bidirectionally adjusted, so that the compliance of a patient can be increased, and an ideal clinical treatment effect is favorably obtained.

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 tumor immune microenvironment and its application. Background Art

[0002] The tumor immune microenvironment includes tumor cells, immune cells, cytokines, etc. These components can be divided into tumor suppressor factors and tumor promoter factors according to their specific functions. Their strength distribution in the immune microenvironment determines the trend of tumor immunity. Although the human immune system can theoretically eliminate tumors, tumors evade immune surveillance and hunting by forming an immunosuppressive microenvironment, which is manifested by the increase of PD-L1 on the surface of tumor cells to evade immune surveillance and the decrease of MHC-I class substance expression to destroy antigen presentation and thus evade immune surveillance.

[0003] At present, monoclonal antibody drugs targeting PD-L1 have been approved for marketing in China, and their application and development in tumor immunotherapy have attracted widespread attention. In the tumor immune cycle, the combination of PD-L1 of tumor cells and the immune checkpoint PD1 of immune cells can help tumors escape the capture of immune cells, thereby leading to tumor progression. Therefore, immunotherapy targeting PD-L1 can effectively block the immune escape of tumor cells. However, in clinical practice, only some patients show the final clinical benefit, and some patients even develop acquired resistance after obtaining the initial benefit. It is generally believed that the abnormal low expression of MHC-I substances is one of the main mechanisms for the low benefit rate of PD-L1 treatment and the generation of acquired resistance. However, there are endless studies on improving MHC-I substances, but the experimental results are unsatisfactory and biosafety cannot be guaranteed, which leads to almost zero drugs approved for marketing.

[0004] Studies have confirmed that reducing PD-L1 and promoting MHC-Ⅰ expression at the same time is beneficial to tumor immunotherapy and can achieve clinical benefits. However, currently, drugs that regulate the tumor immune microenvironment in a single direction are the main ones, and there are no drugs that can both reduce PD-L1 and promote MHC-Ⅰ expression. If this treatment regimen is to be used, a combination of drugs must be used. However, the shortcomings of this combination of drugs may lead to poor compliance of patients due to high dosage and concerns about toxic side effects, thus affecting the final clinical treatment effect. Therefore, in order to further break through the bottleneck of tumor immunotherapy and increase the compliance of future patients, it is urgent 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 having the function of bidirectionally regulating tumor immune microenvironment, wherein 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 a polypeptide alone or in combination with an excipient.

[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 composition drug with additives commonly used in pharmacy.

[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] (I) 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 make it easier for drugs to enter tumor tissues. The preparation method is also simpler and the cost is lower.

[0022] 2. Start from regulating the upstream pathway of PD-L1 to fundamentally reduce the expression of PD-L1;

[0023] 3. It can both reduce PD-L1 and promote MHC-Ⅰ expression, thereby reducing tumor cell immune escape and increasing antigen presentation, thereby promoting immune cell capture of tumor cells. This innovative move can significantly enhance the effect 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 in comparison with the blank group in Experimental Example 1;

[0026] Figure 2 This is a schematic diagram of the results of immunofluorescence staining and digestion of tumor masses into single cell suspensions for flow cytometry detection of PD-L1 and MHC-Ⅰ expression in tumor tissues after the animal tumor model in Experimental Example 1 was treated;

[0027] Figure 3 This is a schematic diagram of the expression results of the core proteins EGFR, P38, pP38, PD-L1 and MHC-Ⅰ in the MAPK pathway detected by western blot according to 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 the animal model was treated in comparison with the blank group in Experimental Example 1;

[0029] Figure 5 Schematic diagram of detecting apoptosis of cells in tumor tissue by immunofluorescence method 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 This is a 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 in conjunction with 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. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0034] A technical solution of a specific implementation of the present invention is:

[0035] A polypeptide having the function of bidirectionally regulating tumor immune microenvironment, wherein 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: The cells were divided into a control group and an experimental group. When human osteosarcoma cells (143B) were growing in the logarithmic phase, the control group was given serum-free medium, and the experimental group was given serum-free medium containing 19 μg / mL peptide. The total amount of culture medium in each T75 cell culture flask was about 6 mL. After incubation in a cell culture incubator for 24 hours, western blot was used to detect the expression of PD-L1 and MHC-Ⅰ proteins in each group.

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

[0039] The results are as follows Figure 1 As shown in 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 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 mouse osteosarcoma cell line K7M2, and the mice were divided into control group and experimental group after the tumor grew to a certain volume. The control group was given 200 μL / day of normal saline, and the experimental group was given 200 μL of normal saline containing 160 μg / mL peptide, by tail vein injection, once a day for 5 consecutive days. The tumors of the mice were regularly observed and measured during the treatment, and the mice were killed 1 week after treatment to obtain the tumor mass.

[0043] Immunofluorescence was used to detect the expression 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 A in the figure shows the expression of PD-L1 protein and MHC-Ⅰ protein. Compared with the control group, the expression of PD-L1 protein in the experimental group was significantly reduced, and the expression of MHC-Ⅰ protein was significantly increased (DiI is PD-L1, FITC is MHC-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 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 were in logarithmic growth, the control group was given serum-free medium, and the experimental group was given serum-free medium containing 19 μg / mL peptide. The total amount of medium in each T75 cell culture flask was about 6 mL. After incubation in a cell culture incubator for 24 hours, the cells were collected and sent to transcriptomics to obtain the pathway enrichment map.

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

[0052] like Figure 3As shown in B, it 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 A, based on the pathway enrichment map, the inventors selected the MAPK pathway with the highest tumor relevance for further verification.

[0054] The above-mentioned 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 B, compared with the control group, the expression of PD-L1 protein in the experimental group was significantly reduced, and the expression of MHC-Ⅰ protein was significantly increased. The related pathway proteins EGFR and pP38 were also significantly reduced.

[0056] The above results show 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 mouse osteosarcoma cell line K7M2, and the mice were divided into control group and experimental group after the tumor grew to a certain volume. The control group was given 200μL / day of saline solution, and the experimental group was given 200μL of saline solution containing 160μg / mL peptide, by tail vein injection, once a day for 5 consecutive days. The tumors of the mice were regularly observed and measured during the treatment, and photos were taken 1 week after treatment.

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

[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 was live cell staining, and green was 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] Mouse osteosarcoma cell line K7M2 was inoculated in immune-competent mice, and the mice were divided into control group and experimental group after the tumor grew to a certain volume. The control group was given 200 μL / day of saline solution, and the experimental group was given 200L of saline solution containing 160 μg / mL of peptide, by tail vein injection, once a day for 5 consecutive days.

[0067] One week after treatment, blood was collected from the eye sockets for routine blood tests, liver function tests, kidney function tests, etc. to detect the effects of the drugs on the blood system.

[0068] The results are as follows Figure 8 As shown in A, all blood indices 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 B, no obvious abnormal changes were found in all organs. This was taken under ordinary white light of a microscope, and the scale bar is 200μm.

[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 protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide having the function 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.

3. Use of a polypeptide according to claim 2 in the preparation of a drug for improving tumor immune microenvironment, characterized in that: The drug can reduce the expression of PD-L1 and promote the expression of MHC-I.

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

5. 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 using a polypeptide alone or a preparation using a polypeptide combined with an auxiliary material.

6. Use of a polypeptide according to claim 5 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.

7. Use of a polypeptide according to claim 5 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 composition drug with additives commonly used in pharmacy.

8. Use of a polypeptide according to claim 7 in the preparation of a drug for improving tumor immune microenvironment, characterized in that: The tumor is a solid tumor.

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