Active small peptide for targeted inhibition of exosome PD-L1 abundance and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an active small peptide for targeted inhibition of exosome PD-L1 abundance and application thereof. The active small peptide, the polymer or the fusion polypeptide provided by the invention can inhibit the abundance of the exosome PD-L1 in a targeted manner, and can be used as a targeted blocking inhibitor of the tumor exosome PD-L1. In addition, the polypeptide can improve the tumor microenvironment and enhance the number and activity of immune cells, has an excellent anti-tumor effect, is free of obvious toxic and side effects, is good in biological safety, has important development and application values in preparation of anti-tumor drugs, and can be used for developing anti-tumor polypeptide drugs.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and more specifically, relates to an active small peptide that targets and inhibits the abundance of exosome PD-L1 and its application. Background Art

[0002] Tumor is a common malignant disease, and its morbidity and mortality rate are rising year by year, and it has become one of the major diseases threatening human health. At present, the main treatment methods for tumors are surgical resection, radiotherapy, chemotherapy and targeted therapy. However, due to the stubbornness, complexity and hiddenness of tumor diseases, except for early patients who can be cured by surgical resection, it is difficult to carry out effective radical treatment for patients in the middle and late stages of tumors. In addition, because cytotoxic anticancer agents affect not only cancer cells but also normal cells, there are problems such as drug resistance, large toxic side effects, and unsatisfactory treatment effects. Therefore, finding new treatment methods and therapeutic drugs has always been a hot topic in global drug research.

[0003] Compared with traditional treatment methods, tumor immunotherapy can activate or induce tumor patients to establish specific immune responses to tumor antigens, eliminate primary tumor cells, and establish immune memory to prevent tumor recurrence. However, during immunotherapy, tumor cells can use immune checkpoint protein molecules to achieve immune tolerance and escape, which ultimately leads to poor efficacy. Studies have confirmed that the immune checkpoint molecule PD-L1 on the surface of tumor cells can inhibit the activation and proliferation of T cells, thereby leading to immune tolerance and tumor immune escape. At the same time, tumor cells can also release PD-L1 through tumor exosomes. After tumor exosomes carrying PD-L1 enter the body's circulation, they can further inhibit the killing effect of immune cells on tumors. The use of PD-L1 blockers as tumor immunotherapy drugs has good application prospects and safety, but there is currently a lack of good PD-L1 blocker products, especially suitable for use as tumor exosome PD-L1 blocking inhibitors. Summary of the invention

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

[0005] To achieve the above objectives, in a first aspect, the present application provides an active small peptide that targets and inhibits the abundance of exosome PD-L1, wherein the amino acid sequence of the active small peptide is selected from at least one of (a)-(d): (a): a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and / or SEQ ID NO.2; (b): a polypeptide having more than 90% identity with (a); (c): a polypeptide in which the amino acid sequence shown in (a) has undergone one or more of the following substitutions, deletions, and insertions of one or more amino acids; (d): A polypeptide whose amino acid sequence is chemically modified as shown in (a).

[0006] Preferably, the chemical modification comprises one or more of cyclization, N-methylation, acetylation, phosphorylation, succinylation, myristoylation, palmitoylation, glycosylation, isoprenylation, ubiquitination, biotinylation, polyethylene glycol modification and fluorescent labeling.

[0007] In a second aspect, the present application provides a polymer, which is formed by the polymerization of two or more polypeptide monomers, wherein at least one polypeptide monomer is the above-mentioned active small peptide.

[0008] Preferably, the polypeptide monomers in the above-mentioned multimer are covalently linked.

[0009] Preferably, the multimer is a homodimer or a heterodimer.

[0010] In a third aspect, the present application provides a fusion polypeptide, which includes the above-mentioned active small peptide, or the above-mentioned polymer; and also includes a cell-penetrating peptide.

[0011] Preferably, the cell-penetrating peptides include one or more of TAT peptide, R8 peptide, penetratin peptide, MAP peptide and low molecular weight protamine LMWP.

[0012] Preferably, the amino acid sequence of the fusion polypeptide is selected from at least one of the amino acid sequences shown in SEQ ID NOs. 3 to 6.

[0013] In a fourth aspect, the present application provides the use of the above-mentioned active small peptide, the above-mentioned polymer or the above-mentioned fusion polypeptide as or in the preparation of an anti-tumor drug.

[0014] In a fifth aspect, the present application provides an anti-tumor drug, which includes the above-mentioned active small peptide, the above-mentioned polymer or the above-mentioned fusion polypeptide.

[0015] Preferably, the above-mentioned tumor includes 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 cancer, 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.

[0016] Preferably, the anti-tumor drug further comprises pharmaceutical excipients, which include one or more of excipients, diluents, isotonic agents, buffers, flavoring agents, fillers, binders, disintegrants and lubricants.

[0017] In general, the above technical solutions conceived by this application have the following technical advantages compared with the prior art: (1) The active small peptides, polymers or fusion polypeptides provided in the present application can inhibit the abundance of exosomal PD-L1 without affecting the abundance of other exosomal cargo proteins, such as ALIX, CD63 and CD81, and can be used as targeted blocking inhibitors of tumor exosomal PD-L1.

[0018] (2) The active small peptides, polymers or fusion polypeptides provided in this application can improve the tumor microenvironment, enhance the number and activity of immune cells, relieve the inhibitory effect of the tumor microenvironment with high expression of PD-L1 in exosomes on the anti-tumor immune response, have excellent anti-tumor effects, can significantly inhibit tumor growth, and have no significant toxic side effects and good biosafety. The active small peptides, polymers or fusion polypeptides provided in this application have 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 THE DRAWINGS

[0019] Figure 1 This is a mass spectrometry identification diagram of the fusion polypeptide-1 prepared in the example of the present application; Figure 2 This is a mass spectrometry identification diagram of the fusion polypeptide-3 prepared in the example of the present application; Figure 3 It is a protein immunoblot of the secretion amount of exosomes when SUM159 breast cancer cells and the fusion polypeptide prepared in the present application were co-incubated for 24 hours; Figure 4 This is a graph showing the changes in tumor volume in mice during the process of intraperitoneal administration of the fusion polypeptide prepared in the present application to 4T1 orthotopic breast cancer-bearing mice; Figure 5 This is a picture of the tumor mass when the fusion polypeptide prepared in the present application was intraperitoneally administered to 4T1 orthotopic breast cancer-bearing mice for 21 days; Figure 6 The weight of the tumor mass after intraperitoneal administration of the fusion polypeptide prepared in the present application to 4T1 orthotopic breast cancer-bearing mice for 21 days; Figure 7 The weight change of mice during the process of intraperitoneal administration of the fusion polypeptide prepared in the present application to 4T1 orthotopic breast cancer-bearing mice; Figure 8 It is the content of multiple blood routine indicators in the serum of mice when the fusion polypeptide prepared in the present application is intraperitoneally administered to 4T1 orthotopic breast cancer-bearing mice for 21 days, wherein content A to content C are the content of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) in the serum respectively; Fig. 9It is the content of multiple blood biochemical indicators in the serum of mice when the fusion polypeptide prepared in the present application is intraperitoneally administered to 4T1 orthotopic breast cancer-bearing mice for 21 days, wherein content A to content E are the content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CREA), and creatine kinase (CK), respectively; Fig.10 This is an H&E staining image of the sections of the main organs (heart, liver, spleen, lung, and kidney) after intraperitoneal administration of the fusion polypeptide prepared in the present application to 4T1 orthotopic breast cancer-bearing mice for 21 days; Fig.11 The fusion polypeptide prepared in the present application is intraperitoneally administered to 4T1 orthotopic breast cancer-bearing mice for 21 days, and the number of immune T cells and the expression level of granzyme B in the tumor tissue are measured, wherein content A and content B are statistical graphs of the proportion of the number of CD4-positive T cells and CD8-positive T cells, respectively, content C is a representative flow contour graph of CD4-positive T cells and granzyme B, content D is a representative flow contour graph of CD8-positive T cells and granzyme B, and content E and content F are statistical graphs of the expression level of granzyme B in CD4-positive T cells and CD8-positive T cells, respectively. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0021] In the description of this application, it should be understood that the term "and / or" is a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The symbol " / " herein indicates that the associated objects are in an or relationship, for example, A / B means A or B.

[0022] In the description of the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0023] In the description of the embodiments of the present application, unless otherwise specified, “plurality” means two or more than two.

[0024] The term "identity" refers to the relatedness between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by aligning and comparing the sequences.

[0025] The present application provides an active small peptide that targets and inhibits the abundance of exosome PD-L1, wherein the amino acid sequence of the active small peptide is selected from at least one of (a)-(d): (a): a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and / or SEQ ID NO.2; (b): a polypeptide having more than 90% identity with (a); (c): a polypeptide in which the amino acid sequence shown in (a) has undergone one or more of the following substitutions, deletions, and insertions of one or more amino acids; (d): A polypeptide whose amino acid sequence is chemically modified as shown in (a).

[0026] It is well known to those skilled in the art that changing a few amino acid residues in certain regions of a polypeptide, such as non-important regions, 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 with the amino acid sequence of the above-mentioned active small peptide SEQ ID NO.1 and SEQ ID NO.2, and the polypeptide also has the effect of targeted inhibition of the abundance of exosome PD-L1 (the inhibitory effect is equivalent to or slightly decreased or slightly increased or greatly increased compared with the active small peptide of the present application), it is also within the scope of protection of the present application.

[0027] In some embodiments, the chemical modification comprises one or more of cyclization, N-methylation, acetylation, phosphorylation, succinylation, myristoylation, palmitoylation, glycosylation, isoprenylation, ubiquitination, biotinylation, polyethylene glycol modification, and fluorescent labeling.

[0028] On the other hand, the present application also provides a polymer, which is polymerized by two or more polypeptide monomers, wherein at least one polypeptide monomer is the above-mentioned active small peptide.

[0029] In some embodiments, the polypeptide monomers in the above-mentioned multimer are covalently linked.

[0030] In some embodiments, the multimer is a homodimer or a heterodimer.

[0031] The above-mentioned active small peptide or the above-mentioned multimer provided in the present application can also be combined with various targeting polypeptides, so that the active small peptide or multimer has better targeting and cell permeability. Based on this, the present application also provides a fusion polypeptide, which includes the above-mentioned active small peptide or the above-mentioned multimer; and also includes a cell-penetrating peptide. The fusion polypeptide provided in the present application can be used as an exosome PD-L1 blocking inhibitor.

[0032] In the present application, the above-mentioned cell-penetrating peptides can effectively enter tumor cells with active small peptides or polymers, improve the bioavailability of active small peptides or polymers, and thus better exert anti-tumor effects. It is understandable that the present application does not limit the types of the above-mentioned cell-penetrating peptides, and any cell-penetrating peptides reported in the prior art are applicable to the present application. In some embodiments, the above-mentioned cell-penetrating peptides include but are not limited to TAT peptide (amino acid sequence is YGRKKRRQRRR), R8 peptide (amino acid sequence is RRRRRRRRC), Penetratin peptide (amino acid sequence is RQIKWFQNRRMKWKK), MAP peptide (amino acid sequence is KLALKALKALKAALKA) and low molecular weight protamine LMWP (amino acid sequence is VSRRRRRGGRRRRC), etc.

[0033] It is well known in the art that the connection order between the polypeptides in the fusion polypeptide will not affect the effect of the fusion polypeptide in inhibiting the expression of exosomal PD-L1. Therefore, the present application does not limit the connection order of the active small peptide / polymer and the cell-penetrating peptide. It can be the active small peptide / polymer connected to the cell-penetrating peptide, or the cell-penetrating peptide connected to the active small peptide / polymer. After coupling the active small peptide / polymer with the cell-penetrating peptide, the active small peptide / polymer can pass through the cell membrane under the guidance of the cell-penetrating peptide and enter the cell to better exert the effect.

[0034] In some embodiments, the amino acid sequence of the above fusion polypeptide is selected from at least one of the amino acid sequences shown in SEQ ID NO.3~6.

[0035] The present application also provides the use of the above-mentioned active small peptide, the above-mentioned polymer or the above-mentioned fusion polypeptide as or in the preparation of anti-tumor drugs.

[0036] Based on this, the present application provides an anti-tumor drug, which includes the above-mentioned active small peptide, the above-mentioned polymer or the above-mentioned fusion polypeptide.

[0037] The expression of PD-L1 has been found in a variety of human cancers, including squamous cell lung cancer, ovarian cancer, colon cancer, melanoma, and myeloma. PD-L1 highly expressed in tumor cells can inhibit anti-tumor immune responses by increasing T cell apoptosis, thus playing an important role in tumor immune escape. Tumor growth can be effectively inhibited by inhibiting the expression of PD-L1. Therefore, the present application also provides the use of the above-mentioned active small peptides, polymers, or fusion polypeptides in the use or preparation of anti-tumor drugs for tumors associated with PD-L1.

[0038] In some embodiments, the above-mentioned tumors include 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 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.

[0039] In some embodiments, the anti-tumor drug further comprises pharmaceutical excipients, and the pharmaceutical excipients comprise one or more of excipients, diluents, isotonic agents, buffers, flavoring agents, fillers, binders, disintegrants and lubricants.

[0040] The present application does not specifically limit the dosage form of the above anti-tumor drug, which may be, but is not limited to, emulsions, suspensions or injections, etc. Those skilled in the art may select a suitable dosage form according to the actual application scenario.

[0041] It should be understood that materials of the same or similar type, model, quality, nature or function as 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 sources unless otherwise specified.

[0042] The following are examples: Example 1 Preparation of fusion polypeptide The fusion polypeptide provided in this embodiment is synthesized by Nanjing Yuanpeptide Biotechnology Co., Ltd., and is formed 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 is 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 is 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 embodiment is shown in Table 1, and all fusion polypeptides are purified by high performance liquid chromatography with a purity greater than 95%, and stored at -20°C for standby use.

[0043] Table 1 Amino acid sequence of fusion polypeptide

[0044] 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 Figure 2 shown.

[0045] Example 2 Effect of fusion polypeptide in inhibiting the abundance of exosome PD-L1 1. Experimental Materials SUM159 breast cancer cells were purchased from the American Type Culture Collection (ATCC).

[0046] 2. Experimental steps The fusion polypeptide-1 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 group drug (represented by P-pep). The control polypeptide-1 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 group drug (represented by S-pep). Breast cancer cells (SUM159 cells) were cultured at a rate of 1×10 7 / dish were plated in a cell culture dish with a diameter of 15 cm and divided into an experimental group and a control group, with 10 dishes in each group. Each group of cells was incubated in a carbon dioxide incubator (37°C, 5% carbon dioxide) for 12 hours. After the cells in each group were completely attached to the wall, the culture medium was replaced with a serum-free culture medium, and a peptide drug with a final concentration of 10µg / mL was added. After incubation in a carbon dioxide incubator (37°C, 5% carbon dioxide) for 24 hours, the cell culture medium was collected, exosomes were separated by ultracentrifugation, and the abundance of exosome PD-L1 was detected by immunoblotting.

[0047] 3. Experimental results Depend on Figure 3 It can be seen that compared with the control group, the polypeptide drug in the experimental group significantly inhibited the abundance of exosomal PD-L1, while not affecting the abundance of other exosomal cargo proteins ALIX, CD63 and CD81, indicating that the polypeptide drug prepared in the present application can target and inhibit the abundance of exosomal PD-L1.

[0048] Example 3 Antitumor activity of fusion polypeptide 1. Experimental Materials Mouse 4T1 breast cancer cells were purchased from the American Type Culture Collection (ATCC), and female BALA / c mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0049] 2. Experimental steps: (1) Construction of mouse breast cancer orthotopic model: The concentration of mouse 4T1 breast cancer cells was adjusted to 1×10 7 Then 30 μL of cell suspension (cell number 3×10 5 ) were inoculated into the left mammary pad of each female BALA / c mouse, and the growth of the orthotopic tumor was continuously observed. When obvious tumors were formed in the mammary glands of mice 9 days after inoculation of 4T1 breast cancer cells, the mouse orthotopic breast cancer model was successfully established. The breast cancer mice were then randomly divided into an experimental group and a control group, with 9 mice in each group.

[0050] (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 group drug (denoted by 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 group drug (denoted by S-pep).

[0051] (3) Anti-tumor experiment: Starting from the 9th day after the inoculation of 4T1 breast cancer cells, the above-mentioned peptide drug was administered by intraperitoneal injection, once every 2 days, with an injection dose of 100µL / mouse each time, for a total of 6 times. During the experiment, the mice were free to eat and drink water. The weight of the mice and the long diameter (a) and short diameter (b) of the tumor were observed and measured 9 days after the inoculation of 4T1 breast cancer cells; after the intraperitoneal injection of the peptide drug, the weight of the mice and the long diameter (a) and short diameter (b) of the tumor were measured every two days. According to the tumor volume = (a×b×b) / 2, the tumor volume was calculated and the tumor growth curve was drawn.

[0052] (4) Toxic side effects: After 6 intraperitoneal injections of the peptide drug, blood was collected from the mouse eye sockets for blood biochemistry and routine blood tests. The mice were then killed, the tumors were removed and weighed, and the main organs (heart, liver, spleen, lung, and kidney) were removed for H&E staining.

[0053] 3. Experimental results Depend on Figure 4 , Figure 5 , Figure 6 It can be seen that the tumor growth rate, tumor weight and volume of the experimental group 4T1 orthotopic breast cancer tumor-bearing mice were significantly lower than those of the control group, which indicates that intraperitoneal injection of the fusion polypeptide provided by the present application has a good anti-tumor effect.

[0054] Depend on Figure 7 It can be seen that the volume of mice in the experimental group and the control group was normal, with no significant difference.

[0055] like Figure 8 Content A~ Figure 8 As shown in Content C, the results of routine blood tests showed that there was no significant difference in the content of red blood cells (RBC) and platelets (PLT) in the serum of the experimental group and the control group; the white blood cell (WBC) content in the control group was higher than the normal level and higher than that in the experimental group. The reason for this may be that the degree of development of breast cancer tumors in the control group was significantly higher than that in the experimental group, thereby increasing the number of white blood cells to fight the inflammatory response; while the number of white blood cells in the experimental group was lower, indicating that the inflammatory response in the experimental group was weak. This finding is consistent with the above conclusion, and also proves that the fusion polypeptide provided in the present application can exert an anti-tumor effect and reduce the inflammatory response.

[0056] like Fig. 9 Content A~ Fig. 9 As shown in Content E, the results of blood biochemical tests showed that there were no significant differences in various blood biochemical indicators between the experimental group and the control group.

[0057] like Fig.10 As shown, H&E staining of heart, liver, spleen, lung, and kidney tissue sections in the experimental and control groups showed no abnormalities.

[0058] In summary, the fusion polypeptide provided in the present application has a good anti-tumor effect on 4T1 breast cancer mouse carcinoma in situ, has no obvious toxic side effects, and has good biological safety.

[0059] Example 4 Fusion polypeptide improves tumor microenvironment and enhances immune cell activity 1. Experimental Materials Fixable Viability Stain 575V, Leukocyte Activation Cocktail, CD6 / CD32 antibody, CD45-APC-Cy7 antibody, D3-BV42 antibody, CD4-Alexa Fluor 700 antibody, and CD8-Percp-Cy5.5 antibody were all purchased from BD Biosciences; Granzyme B-FITC antibody was purchased from Invitrogen.

[0060] 2. Experimental steps The tumor tissue blocks of 4T1 breast cancer mice in Example 3 were taken, and the samples were prepared according to the following steps, and the number and activity of immune T cells in the tumor tissue blocks were detected by flow cytometry: (1) Tumor block digestion: Add 3 mL of digestion solution (serum-free 1640 containing 1 mg / mL collagenase D and 0.2 mg / mL DNaseI) to each tumor block and digest at 37°C for 1 h.

[0061] (2) Preparation of single-cell suspension: The digested sample suspension was filtered through a 200-mesh sieve, and then centrifuged at 500 g for 5 min to collect the cells. The supernatant was discarded and 2 mL of red blood cell lysis buffer was added to resuspend the cells. After incubation at room temperature in the dark for 2 min, 5 mL of PBS was immediately added and centrifuged at 500 g for 5 min. The supernatant was discarded and the cell pellet was resuspended in 10 mL of PBS. The pellet was filtered through a 200-mesh sieve and centrifuged at 500 g for 5 min. The supernatant was discarded and the cells were resuspended in 100 µL of PBS to obtain a single-cell suspension of tumor tissue.

[0062] (3) Sample stimulation: The single-cell suspension of the above tumor tissue was placed in 1 mL of complete culture medium 1640 (containing 10% fetal bovine serum and 1 / 1000 Leukocyte Activation Cocktail) and stimulated in a carbon dioxide incubator (37°C, 5% carbon dioxide) for 6 h. The suspension was then centrifuged at 500 g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 µL PBS.

[0063] (4) Dead cell staining: Fixable Viability Stain 575V (dilution ratio 1:1000) and stimulated cell samples were incubated at 4°C for 10 min for staining to distinguish dead cells from live cells. Then 1 mL of PBS was added and centrifuged at 500 g for 5 min for washing. After centrifugation, the supernatant was discarded and the cells were resuspended in 100 µL PBS.

[0064] (5) Sample blocking: Add CD6 / CD32 antibody (dilution ratio of 1:50) to the cell sample stained with Fixable Viability Stain 575V and incubate at room temperature in the dark for 20 min. After incubation, centrifuge at 500 g for 5 min for washing. After centrifugation, discard the supernatant and resuspend the cells in 100 µL PBS.

[0065] (6) Cell surface markers (CD8 + T, CD4 + T) Staining: CD45-APC-Cy7 antibody (dilution ratio of 1:50), CD3-BV42 antibody (dilution ratio of 1:50), CD4-Alexa Fluor 700 antibody (dilution ratio of 1:50), CD8-Percp-Cy5.5 antibody (dilution ratio of 1:50) and the above blocked cell samples were incubated at 4°C for 60 min for staining. After staining, 1 mL PBS was added and the samples were centrifuged at 500 g for 5 min for washing.

[0066] (7) Sample fixation and membrane permeabilization and intracellular marker granzyme B (GzmB) staining: Incubate the membrane fixation and membrane permeabilization reagent and the cell samples that have completed surface marker staining at 4°C for 60 minutes to fix and permeabilize the membrane, then add 1 mL of PBS and centrifuge at 600 g for 5 minutes to wash. After centrifugation, discard the supernatant and resuspend the cells in 100 µL of PBS. Incubate the Granzyme B-FITC (dilution ratio of 1:500) antibody and the above membrane-permeabilized samples at room temperature in the dark for 60 minutes for staining. After staining, add 1 mL of PBS and centrifuge at 500 g for 5 minutes to wash.

[0067] (8) On-machine detection: After completing all staining steps, the cell sample is resuspended in 500 µL PBS, filtered through a 200-mesh sieve, and transferred to a flow cytometer for detection using the CytoFLEX cell flow cytometer.

[0068] 3. Experimental results Depend on Fig.11 Content A. Fig.11 Content B shows that compared with the control group, the number of CD4 + The number of T cells, CD8 + The number of T cells increased significantly. Fig.11 Content C~ Fig.11 Content F shows that CD8 + The expression of GzmB in T cells increased significantly, indicating that the activity of T cells was significantly improved.

[0069] In summary, it is shown that the fusion polypeptide provided in the present application can significantly improve the tumor microenvironment and increase the number and activity of immune cells in the tumor microenvironment.

[0070] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. An active small peptide that targets and inhibits the abundance of exosome PD-L1, characterized in that: The amino acid sequence of the active small peptide is selected from at least one of (a)-(d): (a): a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and / or SEQ ID NO.2; (b): a polypeptide having more than 90% identity with (a); (c): a polypeptide in which the amino acid sequence shown in (a) has undergone one or more of the following substitutions, deletions, and insertions of one or more amino acids; (d): A polypeptide whose amino acid sequence is chemically modified as shown in (a).

2. The active polypeptide according to claim 1, characterized in that 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.

3. A polymer, characterized in that The polymer is formed by polymerization of two or more polypeptide monomers, wherein at least one polypeptide monomer is the active small peptide as described in claim 1 or 2.

4. A fusion polypeptide, characterized in that: It includes the active small peptide as described in claim 1 or 2, or the multimer as described in claim 3; and also includes a cell-penetrating peptide.

5. The fusion polypeptide according to claim 4, characterized in that The cell-penetrating peptides include one or more of TAT peptide, R8 peptide, penetratin peptide, MAP peptide and low molecular weight protamine LMWP.

6. The fusion polypeptide according to claim 4 or 5, characterized in that: The amino acid sequence of the fusion polypeptide is selected from at least one of the amino acid sequences shown in SEQ ID NOs. 3 to 6.

7. Use of the active small peptide according to claim 1 or 2, the multimer according to claim 3, or the fusion polypeptide according to any one of claims 4 to 6 for use as or in the preparation of an anti-tumor drug.

8. An anti-tumor drug, characterized in that: It comprises the active small peptide according to claim 1 or 2, the multimer according to claim 3 or the fusion polypeptide according to any one of claims 4 to 6.

9. The anti-tumor drug according to claim 8, characterized in that: The tumor includes 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 cancer, 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.

10. The anti-tumor drug according to claim 8, characterized in that: The anti-tumor drug also includes pharmaceutical excipients, which include one or more of excipients, diluents, isotonic agents, buffers, flavoring agents, fillers, binders, disintegrants and lubricants.

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