Active small peptides targeting and inhibiting exosome pd-l1 abundance and application thereof

Abstract

The application belongs to the technical field of biological medicine, and more particularly relates to an active small peptide for targeted inhibition of the abundance of exosome PD-L1 and application thereof. The active small peptide, multimer or fusion polypeptide provided in the application can target and inhibit the abundance of exosome PD-L1, and can be used as a targeted blocking inhibitor for tumor exosome PD-L1. In addition, the application can also improve the tumor microenvironment, enhance the number and activity of immune cells, has excellent anti-tumor effect, has no obvious toxic side effects, has good biological safety, has important development and application value in the preparation of anti-tumor drugs, and can be used to develop anti-tumor polypeptide drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, more specifically, relates to an active small peptide for targeted inhibition of exosome PD-L1 abundance and application thereof. BACKGROUND

[0002] Tumor is a common malignant disease, and its morbidity and mortality are increasing year by year, which has become one of the main diseases threatening human health. At present, the main treatment methods for tumor are surgical resection, radiotherapy, chemotherapy and targeted therapy. However, due to the stubbornness, complexity and concealment of tumor disease, in addition to early patients who can be cured by surgical resection, patients with advanced tumor are difficult to be treated effectively, and because cytotoxic anticancer agents not only affect cancer cells but also affect normal cells, there are problems such as drug resistance, large toxic side effects, and unsatisfactory treatment effect. Therefore, finding new treatment methods and treatment drugs has always been a hot spot of global drug research.

[0003] Compared with traditional treatment methods, tumor immunotherapy can activate or induce tumor patients to establish specific immune response to tumor antigens, clear primary tumor cells, and establish immune memory to prevent tumor recurrence. However, during the immunotherapy, tumor cells can use immune checkpoint protein molecules to induce immune tolerance and escape, which eventually leads to poor treatment effect. Studies have shown that the immune checkpoint molecule PD-L1 on the surface of tumor cells can inhibit the activation and proliferation of T cells, leading to immune tolerance and tumor immune escape, and tumor cells can also release PD-L1 through tumor exosomes. After the tumor exosomes carrying PD-L1 enter the body circulation, they can further inhibit the killing effect of immune cells on tumors. Using PD-L1 blockers as tumor immunotherapy drugs has good application prospect and safety, but there is still a lack of good PD-L1 blocker products, especially those suitable for blocking and inhibiting tumor exosome PD-L1. SUMMARY

[0004] In view of the defects of the prior art, the purpose of the present application is to provide an active small peptide for targeted inhibition of exosome PD-L1 abundance and application thereof. The active small peptide can target and inhibit the abundance of exosome PD-L1, has good antitumor effect, can improve the tumor microenvironment, enhance the activity of immune cells, and has no obvious toxic side effects and good biological safety.

[0005] To achieve the above purpose, in a first aspect, the present application provides an active small peptide for targeted inhibition of exosome PD-L1 abundance, wherein the amino acid sequence of the active small peptide is selected from at least one of (a) to (d):

[0006] (a): a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1 and / or SEQ ID NO. 2;

[0007] (b): a polypeptide having 90% or greater identity to (a);

[0008] (c): a polypeptide in which one or more of substitution, deletion, insertion of one or more amino acids are made to the amino acid sequence shown in (a);

[0009] (d): a polypeptide in which the amino acid sequence shown in (a) is chemically modified.

[0010] Preferably, the chemical modification comprises one or more of cyclization, N-methylation, acetylation, phosphorylation, succinylation, myristoylation, palmitoylation, glycosylation, prenylation, ubiquitination, biotinylation, polyethylene glycol modification, and fluorescent labeling.

[0011] In a second aspect, the present application provides a multimer, which is polymerized by two or more polypeptide monomers, wherein at least one polypeptide monomer is the active small peptide as described above.

[0012] Preferably, the polypeptide monomers in the multimer are covalently linked.

[0013] Preferably, the multimer is a homodimer or a heterodimer.

[0014] In a third aspect, the present application provides a fusion polypeptide, which comprises the active small peptide as described above, or the multimer as described above; and further comprises a cell-penetrating peptide.

[0015] Preferably, the cell-penetrating peptide comprises one or more of TAT peptide, R8 peptide, penetratin peptide, MAP peptide, and low molecular weight protamine (LMWP).

[0016] Preferably, the amino acid sequence of the fusion polypeptide is selected from at least one of the amino acid sequences shown in SEQ ID NO. 3~6.

[0017] In a fourth aspect, the present application provides use of the active small peptide as described above, the multimer as described above, or the fusion polypeptide as described above, in the manufacture of, or as, an anti-tumor drug.

[0018] In a fifth aspect, the present application provides an anti-tumor drug, which comprises the active small peptide as described above, the multimer as described above, or the fusion polypeptide as described above.

[0019] Preferably, the tumor comprises one or more of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, colon cancer, gastric cancer, breast cancer, hepatocellular carcinoma, urothelial carcinoma, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, melanoma, brain glioma, pleural mesothelioma, lymphoma, and myeloma.

[0020] Preferably, the above-mentioned antitumor drugs further comprise pharmaceutical excipients, and the above-mentioned pharmaceutical excipients comprise one or more of excipients, diluents, isotonic agents, buffers, flavoring agents, fillers, binders, disintegrants and lubricants.

[0021] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages:

[0022] (1) The active small peptide, multimer or fusion polypeptide provided by the present application can inhibit the abundance of exosome PD-L1, while not affecting the abundance of other cargo proteins of exosomes such as ALIX, CD63 and CD81, and can be used as a tumor exosome PD-L1 targeted blocking inhibitor.

[0023] (2) The active small peptide, multimer or fusion polypeptide provided by the present application can improve the tumor microenvironment, enhance the number and activity of immune cells, relieve the inhibition of the tumor microenvironment with high expression of PD-L1 on exosomes on the anti-tumor immune response, has excellent anti-tumor effect, can significantly inhibit the growth of tumors, and has no significant toxic side effects and good biological safety. The active small peptide, multimer or fusion polypeptide provided by the present application has important development and application value in the preparation of anti-tumor drugs, and can be used to develop anti-tumor polypeptide drugs. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the mass spectrum identification diagram of the fusion polypeptide-1 prepared by the embodiments of the present application;

[0025] Figure 2 is the mass spectrum identification diagram of the fusion polypeptide-3 prepared by the embodiments of the present application;

[0026] Figure 3 is the Western blot diagram of the exosome secretion amount when SUM159 breast cancer cells and the fusion polypeptide prepared by the present application are co-incubated for 24h;

[0027] Figure 4 is the diagram of the change of tumor volume of mice during the process of intraperitoneal administration of the fusion polypeptide prepared by the present application to 4T1 orthotopic breast cancer tumor-bearing mice;

[0028] Figure 5 is the tumor mass picture when the fusion polypeptide prepared by the present application is intraperitoneally administered to 4T1 orthotopic breast cancer tumor-bearing mice for 21 days;

[0029] Figure 6 is the tumor mass weight when the fusion polypeptide prepared by the present application is intraperitoneally administered to 4T1 orthotopic breast cancer tumor-bearing mice for 21 days;

[0030] Figure 7 is the change of body weight of mice during the process of intraperitoneal administration of the fusion polypeptide prepared by the present application to 4T1 orthotopic breast cancer tumor-bearing mice;

[0031] Figure 8 Content A~Content C are the contents of white blood cells (WBC), red blood cells (RBC) and platelets (PLT) in serum, respectively.

[0032] Figure 9 Content A~Content E are the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CREA) and creatine kinase (CK), respectively.

[0033] Figure 10 Content A~Content C are the contents of white blood cells (WBC), red blood cells (RBC) and platelets (PLT) in serum, respectively.

[0034] Figure 11 Content A~Content E are the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CREA) and creatine kinase (CK), respectively. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0036] In the description of the present application, it should be understood that the term "and / or" is a description of the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can represent: A exists alone, A and B exist together, and B exists alone. The symbol " / " in this paper represents the relationship of or in the associated objects, for example, A / B represents A or B.

[0037] In the description of the embodiments of the present application, the words "exemplary" and "for example" are used to mean serving as an example, instance, or illustration, at 99% accuracy or greater. Any embodiment or design described as "exemplary" or "for example" is not necessarily to be construed as preferred or advantageous over other embodiments or designs.

[0038] In the description of the embodiments of the present application, "a plurality of" means two or more, unless otherwise specified.

[0039] The term "identity" refers to the relatedness between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing and aligning the sequences.

[0040] The present application provides an active small peptide for targeting and inhibiting the abundance of exosome PD-L1, and the amino acid sequence of the active small peptide is selected from at least one of (a) to (d):

[0041] (a): a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1 and / or SEQ ID NO. 2;

[0042] (b): a polypeptide with 90% or more identity to (a);

[0043] (c): a polypeptide with the amino acid sequence shown in (a) having one or more of substitution, deletion, and insertion of one or more amino acids;

[0044] (d): a polypeptide with the amino acid sequence shown in (a) being chemically modified.

[0045] It is well known to those skilled in the art that in certain regions of a polypeptide, such as non-essential regions, changing a few amino acid residues will not substantially change the biological activity. It should be noted that if the amino acid sequence of a polypeptide has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or more identity to the amino acid sequence shown in the active small peptide SEQ ID NO. 1 and / or SEQ ID NO. 2, and the polypeptide also has the effect of targeting and inhibiting the abundance of exosome PD-L1 (the inhibitory effect is comparable to or slightly decreased or slightly increased or greatly increased compared to the active small peptide of the present application, etc.), it is also within the scope of the present application.

[0046] In some embodiments, the chemical modification includes one or more of cyclization, N-methylation, acetylation, phosphorylation, succinylation, myristoylation, palmitoylation, glycosylation, isoprenylation, ubiquitination, biotinylation, polyethylene glycol modification, and fluorescent labeling.

[0047] In another aspect, the present application also provides a multimer, which is polymerized by two or more polypeptide monomers, wherein at least one polypeptide monomer is the active small peptide.

[0048] In some embodiments, the polypeptide monomers in the multimer are covalently linked.

[0049] In some embodiments, the multimer is a homodimer or a heterodimer.

[0050] The active small peptide or the multimer provided by the present application can also be combined with various targeting polypeptides, so that the active small peptide or the multimer has better targeting and cell penetration. Based on this, the present application also provides a fusion polypeptide, which comprises the active small peptide or the multimer; and further comprises a cell-penetrating peptide. The fusion polypeptide provided by the present application can be used as an exosome PD-L1 blocking inhibitor.

[0051] The cell-penetrating peptide in the present application can make the active small peptide or the multimer effectively enter tumor cells, improve the bioavailability of the active small peptide or the multimer, and thus better play the anti-tumor effect. It can be understood that the present application does not limit the type of the cell-penetrating peptide, and any cell-penetrating peptide reported in the prior art is suitable for the present application. In some embodiments, the cell-penetrating peptide includes but is not limited to TAT peptide (amino acid sequence YGRKKRRQRRR), R8 peptide (amino acid sequence RRRRRRRRC), Penetratin peptide (amino acid sequence RQIKWFQNRRMKWKK), MAP peptide (amino acid sequence KLALKALKALKAALKA), and low molecular weight protamine LMWP (amino acid sequence VSRRRRRRGGRRRRC), etc.

[0052] It is known in the art that the connection order of the polypeptides in the fusion polypeptide does not affect the effect of the fusion polypeptide on inhibiting the expression of exosome PD-L1, and therefore the present application does not limit the connection order of the active small peptide / multimer and the cell-penetrating peptide. The active small peptide / multimer can be connected to the cell-penetrating peptide, or the cell-penetrating peptide can be connected to the active small peptide / multimer. After the active small peptide / multimer is coupled with the cell-penetrating peptide, the active small peptide / multimer can penetrate the cell membrane under the guidance of the cell-penetrating peptide, enter the cell, and better play the effect.

[0053] In some embodiments, the amino acid sequence of the fusion polypeptide is selected from at least one of the amino acid sequences shown in SEQ ID NO. 3-6.

[0054] The present application also provides the use of the active small peptide, the multimer or the fusion polypeptide in the preparation of an anti-tumor drug.

[0055] Based on this, the present application provides an anti-tumor drug, which comprises the active small peptide, the polymer or the fusion polypeptide.

[0056] The expression of PD-L1 has been found in many human cancers such as lung squamous, ovarian cancer, colon cancer, melanoma and myeloma, and the high expression of PD-L1 in tumor cells can inhibit the anti-tumor immune response by increasing the apoptosis of T cells, thereby playing an important role in the immune escape of tumors. By inhibiting the expression of PD-L1, the growth of tumors can be effectively inhibited, and therefore the present application also provides the use of the active small peptide, the polymer or the fusion polypeptide in the preparation of an anti-tumor drug for tumors related to PD-L1.

[0057] In some embodiments, the tumor comprises one or more of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, head and neck squamous carcinoma, nasopharyngeal carcinoma, esophageal cancer, colon cancer, gastric cancer, breast cancer, hepatocellular carcinoma, urothelial carcinoma, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, melanoma, brain glioma, pleural mesothelioma, and lymphoma.

[0058] In some embodiments, the anti-tumor drug further comprises a pharmaceutical excipient, and the pharmaceutical excipient comprises one or more of excipients, diluents, isotonic agents, buffers, flavoring agents, fillers, binders, disintegrants and lubricants.

[0059] The dosage form of the anti-tumor drug is not particularly limited in the present application, and can be, but is not limited to, emulsion, suspension or injection, etc. Those skilled in the art can select a suitable dosage form according to the actual application scenario.

[0060] It should be understood that materials similar to or the same as the types, models, qualities, properties or functions of the reagents and instruments used in the following examples can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0061] The following are examples:

[0062] Example 1 Preparation of a fusion polypeptide

[0063] The fusion polypeptide provided in this example was synthesized by Nanjing Yuanpeptide Biotechnology Co., Ltd. by coupling any one of the active small peptides SEQ ID NO.1 or SEQ ID NO.2 with the cell-penetrating peptide TAT peptide (amino acid sequence YGRKKRRQRRR). Under the guidance of the cell-penetrating peptide TAT peptide, the active small peptide can pass through the cell membrane and enter the cell. At the same time, the amino acid order of the active small peptides SEQ ID NO.1 and SEQ ID NO.2 was randomly shuffled and then coupled with the cell-penetrating peptide TAT peptide to obtain a random polypeptide as a control polypeptide. The amino acid sequence of the fusion polypeptide prepared in this example is shown in Table 1. All fusion polypeptides were purified by high-performance liquid chromatography with a purity greater than 95% and stored at -20°C for future use.

[0064] Table 1 Amino acid sequence of fusion polypeptide

[0065]

[0066] The mass spectrometry analysis of the fusion polypeptide-1 prepared in this example is shown in FIG. Figure 1 As shown, the mass spectrometry analysis of fusion polypeptide-3 is shown in FIG. Figure 2 shown.

[0067] Example 2 Effect of Fusion Peptide on Inhibiting Exosomal PD-L1 Abundance

[0068] 1. Experimental Materials

[0069] SUM159 breast cancer cells were purchased from the American Type Culture Collection (ATCC).

[0070] 2. Experimental steps

[0071] The fusion polypeptide-1 synthesized in Example 1 was dissolved in ultrapure water to prepare a peptide drug with a concentration of 2 mg / mL, which was used as the experimental group (denoted by P-pep). The control polypeptide-1 synthesized in Example 1 was dissolved in ultrapure water to prepare a peptide drug with a concentration of 2 mg / mL, which was used as the control group (denoted by S-pep). Breast cancer cells (SUM159 cells) were cultured at a concentration of 1×10 7 Cells were plated at a concentration of 100 μg / dish in 15-cm-diameter cell culture dishes and divided into experimental and control groups, with 10 dishes in each group. Each cell group was incubated in a CO2 incubator (37°C, 5% CO2) for 12 hours. After complete attachment of the cells, the culture medium was replaced with serum-free medium and the peptide drug was added at a final concentration of 10 μg / mL. After incubation in a CO2 incubator (37°C, 5% CO2) for 24 hours, the cell culture medium was collected, exosomes were isolated by ultracentrifugation, and the abundance of exosomal PD-L1 was detected by immunoblotting.

[0072] 3. Experimental results

[0073] By Figure 3 It can be seen that, compared with the control group, the polypeptide drug of the experimental group significantly inhibited the abundance of exosome PD-L1, while not affecting the abundance of other cargo proteins ALIX, CD63 and CD81 of the exosome, indicating that the polypeptide drug prepared in the application can target and inhibit the abundance of exosome PD-L1.

[0074] Example 3 Anti-tumor activity of the fusion polypeptide

[0075] 1. Experimental materials

[0076] The mouse 4T1 breast cancer cells were purchased from the American Type Culture Collection (ATCC), and the female BALA / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0077] 2. Experimental steps:

[0078] (1) Construction of mouse breast cancer orthotopic model: the concentration of mouse 4T1 breast cancer cells was adjusted to 1×10 7 cells / mL using PBS to prepare a cell suspension. Then 30 µL of the cell suspension (cell number 3×10 5 cells) was inoculated into the left mammary pad of each female BALA / c mouse, and the growth of the orthotopic tumor was continuously observed. When the mice formed obvious tumors in the breast after 9 days of inoculation of 4T1 breast cancer cells, the mouse breast cancer orthotopic model was successfully constructed. Then the breast cancer mice were randomly divided into an experimental group and a control group, 9 mice in each group.

[0079] (2) Preparation of polypeptide drugs: the fusion polypeptide-3 synthesized in Example 1 was dissolved in ultrapure water to prepare a polypeptide drug with a concentration of 2 mg / mL, which was used as the experimental drug (denoted as P-pep). The control polypeptide-2 synthesized in Example 1 was dissolved in ultrapure water to prepare a polypeptide drug with a concentration of 2 mg / mL, which was used as the control drug (denoted as S-pep).

[0080] (3) Anti-tumor experiment: from 9 days after inoculation of 4T1 breast cancer cells, the above polypeptide drugs were administered by intraperitoneal injection, with a dosage of 100 µL per mouse per time, once every 2 days, for a total of 6 times. During the experiment, the mice were allowed to eat and drink freely. The body weight of the mice and the long diameter (a) and short diameter (b) of the tumor were measured at 9 days after inoculation of 4T1 breast cancer cells. After intraperitoneal injection of the polypeptide drugs, the body weight of the mice and the long diameter (a) and short diameter (b) of the tumor were measured every two days. The tumor volume was calculated according to the formula: volume = (a×b×b) / 2, and the tumor growth curve was plotted.

[0081] (4) Toxic side effects: After 6 times of intraperitoneal injection of polypeptide drugs, the mouse eye orbit was taken blood, blood biochemical and blood routine test was carried out, then the mouse was killed, the tumor was taken out and weighed, and the main organs (heart, liver, spleen, lung, kidney) were taken out for H&E staining test.

[0082] 3. Experimental results

[0083] From Figure 4 , Figure 5 , Figure 6 It can be seen that the tumor growth rate, tumor weight and volume of the 4T1 orthotopic breast cancer tumor-bearing mice in the experimental group are significantly smaller than those in the control group, which shows that intraperitoneal injection of the fusion polypeptide provided by the application has good anti-tumor effect.

[0084] From Figure 7 It can be seen that the volume of mice in the experimental group and the control group is normal, and there is no obvious difference.

[0085] As Figure 8 Content A Figure 8 Content C shows that the blood routine test results show that the contents of red blood cells (RBC) and platelets (PLT) in the serum of the experimental group and the control group have no obvious difference; the content of white blood cells (WBC) in the control group is higher than the normal level, and higher than that in the experimental group. The analysis reason is that the development degree of breast cancer tumor in the control group is significantly higher than that in the experimental group, so that the number of white blood cells increases to resist the inflammatory reaction; the number of white blood cells in the experimental group is lower, which shows that the inflammatory reaction in the experimental group is weak. This finding is consistent with the above conclusion, and also proves that the fusion polypeptide provided by the application can play an anti-tumor effect and reduce the inflammatory reaction.

[0086] As Figure 9 Content A Figure 9 Content E shows that the blood biochemical test results show that there is no obvious difference in each blood biochemical index of the experimental group and the control group.

[0087] As Figure 10 shown, H&E staining of heart, liver, spleen, lung and kidney tissue sections of the experimental group and the control group showed no abnormalities.

[0088] In summary, the fusion polypeptide provided by the application has good anti-tumor effect on 4T1 breast cancer mouse orthotopic cancer, and has no obvious toxic side effects, and has good biological safety.

[0089] Example 4 Fusion polypeptide improves tumor microenvironment and enhances immune cell activity

[0090] 1. Experimental materials

[0091] Fixable Viability Stain 575V, Leukocyte Activation Cocktail, CD6 / CD32 antibody, CD45-APC-Cy7 antibody, D3-BV42 antibody, CD4-Alexa Fluor 700 antibody, CD8-Percp-Cy5.5 antibody were purchased from BD Biosciences; Granzyme B-FITC antibody was purchased from invitrogen.

[0092] 2. Experimental procedure

[0093] Take the tumor tissue block of 4T1 breast cancer mice in Example 3, prepare the sample according to the following steps, and detect the number and activity of immune T cells in the tumor tissue block by flow cytometry:

[0094] (1) Tumor block digestion: add 3 mL of digestion solution (containing 1 mg / mL collagenase D and 0.2 mg / mL DNase I in serum-free 1640) to each tumor block, and digest at 37°C for 1 h.

[0095] (2) Single cell suspension preparation: After filtering the above digested sample suspension through a 200 mesh screen, centrifuge at 500g for 5 min to collect the cells, discard the supernatant, add 2 mL of red blood cell lysis solution to resuspend the cells, incubate at room temperature for 2 min in the dark, immediately add 5 mL of PBS, centrifuge at 500g for 5 min, discard the supernatant, resuspend the cell pellet with 10 mL of PBS, and filter through a 200 mesh screen, centrifuge at 500g for 5 min, discard the supernatant, resuspend the cells with 100 µL of PBS, and obtain a single cell suspension of the tumor tissue.

[0096] (3) Sample stimulation: Place the above single cell suspension of the tumor tissue in 1 mL of complete culture medium 1640 (containing 10% fetal bovine serum and 1 / 1000 Leukocyte Activation Cocktail), stimulate in a carbon dioxide incubator (37°C, 5% carbon dioxide) for 6 h, centrifuge at 500g for 5 min, discard the supernatant, and resuspend the cells with 100 µL of PBS.

[0097] (4) Dead cell staining: Stain the Fixable Viability Stain 575V (dilution ratio 1:1000) and the stimulated cell sample at 4°C for 10 min to distinguish between dead cells and live cells, then add 1 mL of PBS, centrifuge at 500g for 5 min, discard the supernatant after centrifugation, and resuspend the cells with 100 µL of PBS.

[0098] (5) Sample blocking: Add CD6 / CD32 antibody (dilution ratio 1:50) to the cell sample dyed by the above Fixable Viability Stain 575V, and incubate at room temperature for 20 min in the dark. After incubation, centrifuge at 500g for 5 min to wash, discard the supernatant after centrifugation, and resuspend the cells in 100 μL PBS.

[0099] (6) Cell surface index (CD8 + T, CD4 + T) staining: Add CD45-APC-Cy7 antibody (dilution ratio 1:50), CD3-BV42 antibody (dilution ratio 1:50), CD4-Alexa Fluor 700 antibody (dilution ratio 1:50), CD8-Percp-Cy5.5 antibody (dilution ratio 1:50) and the above blocked cell sample to 4°C for 60 min for staining, and then add 1 mL PBS, centrifuge at 500g for 5 min to wash.

[0100] (7) Sample fixation, membrane rupture, and intracellular index Granzyme B (GzmB) staining: Incubate the fixation and membrane rupture reagent with the above cell sample dyed with surface index at 4°C for 60 min for fixation and membrane rupture, then add 1 mL PBS, centrifuge at 600g for 5 min to wash, discard the supernatant after centrifugation, and resuspend the cells in 100 μL PBS. Incubate Granzyme B-FITC (dilution ratio 1:500) antibody and the above membrane-ruptured sample at room temperature for 60 min for staining, then add 1 mL PBS, centrifuge at 500g for 5 min to wash.

[0101] (8) Detection: Resuspend the cell sample after all staining steps using 500 μL PBS, filter through a 200-mesh screen, and transfer to a flow tube for detection by CytoFLEX cell flow cytometry.

[0102] 3. Experimental results

[0103] From Figure 11 Content A, Figure 11 Content B, it can be seen that compared with the control group, the number of CD4 + T cells and the number of CD8 + T cells in the tumor tissue of the experimental group mice increased significantly. From Figure 11 Content C, Figure 11 Content F, it can be seen that the expression of GzmB in CD8 + T cells in the tumor tissue of the experimental group mice increased significantly, indicating that the activity of T cells was significantly improved.

[0104] In summary, the fusion polypeptide provided in the application can significantly improve the microenvironment of the tumor, and increase the number and activity of immune cells in the tumor microenvironment.

[0105] Those skilled in the art can understand that the above description is only a preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A fusion polypeptide, characterized in that, The amino acid sequence of the fusion polypeptide is SEQ ID NO. 3 or SEQ ID NO.

5.

2. Use of the fusion polypeptide of claim 1 in the preparation of an anti-breast cancer tumor drug.

3. An anti-breast cancer tumor agent, comprising, It comprises the fusion polypeptide of claim 1.

4. The anti-breast tumor drug according to claim 3, characterized in that, The anti-breast cancer tumor drug further comprises a pharmaceutical adjuvant, which comprises one or more of an isotonic agent, a flavoring agent, a filler, a binder, a disintegrant, and a lubricant.

Images