A polypeptide and its application in the preparation of drugs for preventing and treating tumors

Abstract

The present invention discloses the use of a polypeptide that specifically interrupts the binding of lactate and GLUT10 in the tumor microenvironment in the preparation of a drug for preventing and treating tumors. The amino acid sequence of the polypeptide is as shown in the amino acid sequence of SEQ ID No.1 in the sequence listing; the polypeptide derivative is a chimeric peptide formed by connecting the polypeptide with a cell-penetrating peptide and / or a dosage form formed by combining the polypeptide with a pharmaceutical excipient. The polypeptide and polypeptide derivative of the present invention can specifically bind to GLUT10, thereby blocking the interaction between lactate and GLUT10 protein in the tumor microenvironment and restoring the sugar uptake ability and killing function of CD8+ T cells in the tumor microenvironment. Therefore, it is applied to the preparation of drugs for improving immunotherapy to prevent and treat tumors. The prepared drug has the advantages of significant curative effect, few toxic and side effects, and safe use in the treatment of tumor diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a polypeptide and its application in the preparation of a drug for preventing and treating tumors. Background Art

[0002] Tumor immunotherapy inhibits tumor growth and eliminates tumor cells by activating immune cells in the body and enhancing the body's anti-tumor immune response. CD8+ T cells are the main immune cells for the body's immune system to exert tumor-killing functions. CD8+ T cells are activated by recognizing MHC class I and antigen complexes on the surface of tumor cells, and then exert tumor-killing functions.

[0003] In the tumor microenvironment, the growth and proliferation of tumor cells depend on their high-intensity metabolism to meet their biosynthetic needs. High-intensity aerobic glycolysis is an important feature of tumor cell metabolism. The high-intensity glycolysis of tumor cells will consume a large amount of glucose in the microenvironment and produce a large amount of lactic acid, which is discharged into the tumor microenvironment, resulting in the characteristics of low glucose and high acid in the tumor microenvironment. On the one hand, in the tumor microenvironment, due to the lack of glucose, T cells cannot fully uptake glucose, ultimately affecting T cell activation. On the other hand, a large amount of lactic acid accumulated in the tumor microenvironment can directly affect the proliferation and effector functions of CD8+ T cells. In addition, regulatory T cells (Tregs) in the tumor microenvironment can use lactic acid as an energy source and increase the expression of PD-1 on the surface of Tregs, blocking the effect of anti-PD-1 monoclonal antibodies on CD8+ T cells. Our previous research results found that in the tumor microenvironment, CD8+ T cells mainly transport glucose through the GLUT10 transporter, and lactic acid inhibits the function of GLUT10 to transport glucose, thereby inhibiting the function of CD8+ T cells to kill tumors. Therefore, developing compounds or polypeptide drugs that target the interaction between GLUT10 and lactic acid can, on the one hand, enhance the tumor-killing function of CD8+ T cells, and on the other hand, have strong targeting, small toxic and side effects, and have good prospects for drug development in inhibiting tumor occurrence and development.

[0004] So far, tumor immunotherapy such as anti-PD-1 / PD-L1 monoclonal antibodies has achieved great success in the treatment of tumors, but non-antibody inhibitors such as small molecules, polypeptides or DNA aptamers have rarely entered clinical research. Due to the limitations of monoclonal antibody drugs, such as high drug production costs and large immunogenic side effects, their role in tumor immunotherapy is restricted. Therefore, the development of polypeptide drugs has great development space and provides new opportunities for tumor immunotherapy. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a polypeptide PG10 that interrupts the binding of GLUT10 to lactate for use in the preparation of drugs for treating, preventing, and / or controlling tumors, in view of the current lack of inhibitors with effective lactate-inhibiting functions and activities.

[0006] The polypeptide PG10 of the present invention has an amino acid sequence as shown in SEQ ID No.1 or as follows:

[0007] His-Leu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Leu-Ser-Glu-Ile-Tyr-Pro-Val-Glu-Ile-Arg-Gly-Arg.

[0008] The second technical problem to be solved by the present invention is to provide the application of a drug containing the polypeptide PG10 in treating, preventing, and / or controlling tumors.

[0009] Through in-depth research and repeated experiments, the inventors of the present invention obtained a polypeptide PG10 that can bind to GLUT10 and interrupt the binding of GLUT10 to lactate (the amino acid sequence is shown in Sequence Listing SEQ ID No.1), and the polypeptide PG10 has good stability and biological activity.

[0010] The present invention also provides a pharmaceutical composition composed of the polypeptide PG10 and its pharmaceutically acceptable carrier or excipient.

[0011] In the pharmaceutical composition of the present invention, the weight ratio of the polypeptide PG10 is 0.01 - 99.99%, and the weight ratio of the pharmaceutically acceptable carrier in the composition is 0.01 - 99.99%.

[0012] The pharmaceutical composition of the present invention further contains other anti-tumor active substances or immune checkpoint inhibitors as active ingredients.

[0013] The pharmaceutical composition of the present invention can be formulated into any pharmaceutically acceptable dosage form.

[0014] The pharmaceutical composition of the present invention is administered by injection or orally.

[0015] For the pharmaceutical composition of the present invention, preferred injection routes include intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, subcutaneous injection, etc.

[0016] In the present invention, the carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient, preferably selected from one or more of chitosan and its derivatives, carbomer, and liposomes. The pharmaceutical composition is various dosage forms as conventionally described in the art, preferably one or more of gels, emulsions, films, microspheres, and nanospheres.

[0017] The preferred route of administration of the pharmaceutical composition of the present invention is parenteral administration or oral administration. The parenteral administration preferably includes routes such as intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is various dosage forms as conventionally described in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, a non-aqueous solution, or a suspension, more preferably tablets, capsules, granules, injections, or infusions, etc.

[0018] Preferably, the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents, etc. More preferably, the pharmaceutical composition includes 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of the pharmaceutical carrier, and the percentages are by mass of the pharmaceutical composition.

[0019] Preferably, the dosage of the pharmaceutical composition is an effective amount, and the effective amount is an amount capable of alleviating or delaying the progression of a disease, degenerative, or traumatic condition. The effective amount can be determined on an individual basis and will be partly based on consideration of the symptoms to be treated and the result sought.

[0020] On the basis of conforming to common general knowledge in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain various preferred examples of the present invention.

[0021] Another object of the present invention is to provide the use of polypeptide PG10 in the preparation of drugs for targeted treatment, prevention, and / or control of tumors related to GLUT10.

[0022] The use according to the present invention is characterized in that the tumor is colorectal cancer, liver cancer, melanoma, lung cancer, breast cancer.

[0023] The use of polypeptide PG10 according to the present invention in the preparation of drugs for targeted treatment, prevention, and / or control of tumors related to GLUT10.

[0024] In the present invention, the tumor mentioned may be a conventional tumor in the art. Preferably, it is colorectal cancer, liver cancer, melanoma, lung cancer, breast cancer, etc. Among them, the liver cancer mentioned may be a conventional liver cancer in the art, preferably primary liver cancer or secondary liver cancer; the lung cancer mentioned may be a conventional lung cancer in the art, preferably small cell lung cancer or non-small cell lung cancer; the breast cancer mentioned may be a conventional breast cancer in the art, preferably non-invasive breast cancer, early invasive breast cancer, invasive special type breast cancer or invasive non-special type breast cancer; the colorectal cancer mentioned may be a conventional colorectal cancer in the art, preferably colon cancer or rectal cancer. The melanoma is a conventional melanoma in the art;

[0025] The prevention and treatment mentioned may be conventional concepts in the art, that is, including prevention and / or treatment. The prevention may be conventional prevention in the art, preferably referring to preventing or reducing the occurrence of tumors after use when there are possible tumor factors. The treatment may be conventional treatment in the art, preferably referring to reducing the degree of tumors, or curing tumors to make them normal, or slowing down the progression of tumors.

[0026] The active ingredient refers to a compound with the function of preventing or treating tumors. In the pharmaceutical composition, the polypeptide targeting to promote the interruption of the binding between GLUT10 and lactate can be used alone as the active ingredient or together with other antitumor active compounds or immune checkpoint inhibitor PD-1, etc. as the active ingredient.

[0027] The reagents and raw materials used in the present invention are all commercially available.

[0028] The positive and progressive effects of the present invention are as follows: The polypeptide of the present invention can target and bind to GLUT10, inhibit the binding between GLUT10 and lactate, and restore the killing function of CD8+ T cells, so as to be applied to the preparation of antitumor drugs. The polypeptide and its derivatives have shown the ability to significantly inhibit tumor growth in the in vivo and in vitro experiments for treating tumor diseases, especially for liver cancer, colon cancer and melanoma, and in the in vitro experiment for breast cancer, with remarkable curative effects, and the polypeptide and its derivatives have the advantages of less toxic and side effects and safe use. Description of the Drawings

[0029] Figure 1 . Effect of polypeptide PG10 on the binding ability between lactate and GLUT10.

[0030] Figure 2 . Effect of polypeptide PG10 on the glucose uptake ability of CD8+ T cells. Figure (A) Effect of different concentrations of PG10 on the glucose uptake ability of human CD8+ T cells. Figure (B) Effect of different concentrations of PG10 on the glucose uptake ability of mouse CD8+ T cells.

[0031] Figure 3. Effects of polypeptides PG10 at different concentrations on apoptosis of tumor cells and functions of CD8+ T cells in the co-culture system. Figure (A) Effects of polypeptides PG10 at different concentrations on the apoptosis ability of tumor cells in the co-culture system. Figure (B) Effects of polypeptides PG10 at different concentrations on glucose uptake ability and killing function of CD8+ T cells in the co-culture system.

[0032] Figure 4 . Effects of polypeptide PG10 on the growth of melanoma cells B16. Figure (A) Flow chart for evaluating the therapeutic effect of PG10 on the subcutaneous melanoma model. Figure (B) Subcutaneous melanoma model, with PBS control or PG10 polypeptide administered, tumor growth curve. Figure (C) Effects of PBS control or PG10 polypeptide on glucose uptake ability of CD8+ T cells. Figure (D) Effects of PBS control or PG10 polypeptide on killing ability of CD8+ T cells. Figure (E) Effects of PBS control or PG10 polypeptide on proliferation activity of CD8+ T cells.

[0033] Figure 5 . Effects of polypeptide PG10 on the growth of liver cancer PDX mice. Figure (A) Flow chart showing the evaluation of the therapeutic effect of polypeptide PG10 on the liver cancer PDX model. Figure (B) Growth of liver cancer PDX. Figure (C) Effects of polypeptide PG10 on glucose uptake ability and killing function of CD8+ T cells.

[0034] Figure 6 . Effects of the combination of PG10 and anti-PD-1 antibody on the growth of melanoma cells. Figure (A) Flow chart showing the evaluation of the therapeutic effect of the combination of polypeptide and anti-PD-1 antibody on the mouse melanoma model. Figure (B) Effects of PG10 and anti-PD-1 antibody on melanoma growth. Figure (C) Effects of PG10 and anti-PD-1 antibody on glucose uptake ability, killing ability and proliferation activity of CD8+ T cells. Detailed implementation manners

[0035] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0036] Unless otherwise specified, the PBS solution mentioned in the examples refers to a phosphate buffer solution with a concentration of 0.1 M and a pH value of 7.2.

[0037] The room temperature mentioned in the examples is the conventional room temperature in the art, preferably 15 - 30 °C. The experimental results are expressed as mean ± standard error. After parametric or non-parametric variance tests, a p < 0.05 is considered to have a significant difference, and a p < 0.01 is considered to have an extremely significant difference.

[0038] Synthesis of the polypeptide in Example 1

[0039] The amino acid sequence of polypeptide PG10 is shown as SEQ ID No.1 in the sequence listing. Polypeptide PG10 was synthesized and purified by Anhui Guoping Pharmaceutical Co., Ltd.

[0040] SEQ ID No.1:

[0041] His-Leu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Leu-Ser-Glu-Ile-Tyr-Pro-Val-Glu-Ile-Arg-Gly-Arg;

[0042] Example 2 Immunoprecipitation verification that polypeptide PG10 can disrupt the binding of lactate to GLUT10.

[0043] The immunoprecipitation reagents are as follows:

[0044] Lysis buffer solution A: 0.6057 g Tris base, 1.7532 g NaCl, 0.1017 g MgCl2·6H2O, 0.0742 g EDTA, 10 mL glycerol, 10 mL 10% NP40, add deionized water to 150 mL, adjust the pH value to 7.6 with HCl, make up the volume to 191 mL, mix well, filter with a 0.45 μm filter membrane, and store at 4°C.

[0045] Lysis buffer solution B: 200 μL 2M β-glycerophosphate, 4 mL 2.5M NaF, 2 mL 100 mM NaVO3, 2 mL 100 mM PMSF, 200 μL 1M DTT, 200 μL each of 1 mg / mL Leu, Pep, and Apr, with a total volume of 9 mL. The mother liquor is stored at -20°C. Before use, thaw the mother liquors of each component in solution B, and add them to solution A according to the above composition ratio and mix well.

[0046] Biotin magnetic beads were purchased from Bolemai Company.

[0047] The specific operation steps are as follows:

[0048] (1) Seed HEK293 cells (Institute of Basic Medicine, Chinese Academy of Medical Sciences) in a 10 mm 2 dish. After 12 hours, transfect with the plasmid of GLUT10, and collect the cells after culturing for 24 hours.

[0049] (2) Lyse the cells with the immunoprecipitation lysis buffer, harvest about 4 - 10 mg of total cell protein, and adjust the protein concentration of each group to the same level. Take 200 μg of each group of protein as Input.

[0050] (3) Add about 5 mg of residual protein per group to 3 mM biotin-labeled lactic acid, and at the same time add 30 μL of Biotin magnetic beads and resuspend well. Slowly rotate and shake at 4 °C. After binding for 4 hours, add different concentrations of PG10 polypeptide (0 μM, 1 μM, and 5 μM), and slowly rotate and shake overnight at 4 °C. Place the sample on a magnetic stand, let it stand for 1 - 2 min, carefully aspirate the supernatant. It is better to leave a small amount of supernatant rather than aspirate the magnetic beads. Add 0.5 mL of immunoprecipitation washing solution, mix well, place it on the magnetic stand, let it stand for 1 - 2 min, and carefully aspirate the supernatant. Repeat the washing 5 times, and let it stand for 3 min for the last time. Carefully aspirate the supernatant, add 30 μL of 2×SDS gel loading buffer, mix well, denature at 95 °C for 3 min, and quickly transfer it to an ice bath for cooling. Centrifuge at 12000 rpm at room temperature for 2 min. The supernatant is the precipitated protein sample, and part or all of it is taken for SDS-polyacrylamide gel electrophoresis.

[0051] The results are as Figure 1 shown that PG10 can block the binding of lactic acid and GLUT10 protein, manifested as the expression of GLUT10 detected by immunoblot hybridization gradually decreases with the increase of PG10 concentration.

[0052] Example 3 Detection of the ability of polypeptide to uptake sugar by CD8+ T cells by flow cytometry

[0053] Detect the ability of polypeptide to uptake sugar by CD8+ T cells by flow cytometry. The specific operation steps are as follows:

[0054] 1. Isolate single CD8+ T cells from mouse spleen using a mouse CD8+ T cell isolation kit or isolate single CD8+ T cells from human peripheral blood using a CD8+ T cell isolation kit. Adjust the cell concentration with 1640 medium (purchased from Invitrogen, USA) to prepare a cell suspension of 1 million cells / mL.

[0055] 2. Add 1 mL of the cell suspension prepared in step 1 to a 12-well plate for culture, and stimulate with 1 μg / ml anti-CD3 and 1 μg / ml anti-CD28 antibodies, and add 15 mM lactic acid and different concentrations of polypeptide PG10.

[0056] 3. After 24 hours, collect the cells, discard the supernatant, and resuspend with the glucose uptake assay working solution (100 μL / 1 million cells; working solution preparation: 2NBDG: buffer = 1:300; Cell Meter TM 2-NBDG Glucose Uptake Assay Kit23500). Incubate in the dark at 37 °C in a 5% CO2 incubator for 15 minutes, then add 1 mL of buffer, centrifuge at 1500 rpm for 5 minutes to wash away the unuptaken dye, and then resuspend with 200 μL of buffer.

[0057] 4. Using a flow cytometer with an excitation wavelength of 465 nm and an emission wavelength of 520 nm, measure the intensity of fluorescence inside the cells, and calculate the percentage of fluorescent cells among the total cells. The results are shown in Table 3. The higher the percentage of fluorescent cells among the total cells, the stronger the sugar uptake ability of CD8+ T cells.

[0058] Figure 2 It shows that after treatment with polypeptides PG10 at different concentrations, the proportion of fluorescent cells is significantly higher than that of the control group. Therefore, PG10 can relieve the inhibition of the sugar uptake function of CD8+ T cells by lactic acid and improve the sugar uptake ability of CD8+ T cells.

[0059] Example 4 Detection of the effect of polypeptides on the tumor killing ability of CD8+ T cells by flow cytometry

[0060] Detection of the effect of polypeptides on the tumor killing ability of CD8+ T cells by flow cytometry. The specific operation steps are as follows:

[0061] 1. Use a human CD8+ T cell isolation kit to isolate single CD8+ T cells from human peripheral blood, and adjust the cell concentration with 1640 medium (purchased from Invitrogen, USA) to prepare a cell suspension with a concentration of 1 million cells / mL.

[0062] 2. Add 1 mL of the cell suspension prepared in step 1 to a 12-well plate for culture, and stimulate with 1 μg / ml anti-CD3 and 1 μg / ml anti-CD28 antibodies for 48 hours.

[0063] 3. Co-culture 1 million CD8+ T cells with 500,000 human hepatocellular carcinoma cells HepG2. After 12 hours of co-culture, detect the apoptosis of tumor cells and the killing activity of CD8+ T by flow cytometry.

[0064] Figure 3 It shows that after treatment with polypeptides PG10 at different concentrations, the sugar uptake ability of CD8+ T cells gradually increases, the apoptosis ratio of tumor cells significantly increases, and the ratio of cytotoxic CD8+ T cells significantly increases, indicating that polypeptide PG10 can enhance the killing function of CD8+ T cells against tumor cells.

[0065] Example 5 Tumor subcutaneous growth experiment to study the effect of polypeptide PG10 on the growth of melanoma cells B16.

[0066] The operation steps are as follows:

[0067] 1. Experimental consumables and reagents: Sterilized 1.5 mL EP tubes, 15 mL centrifuge tubes, pipette tips, filter screens (100 mesh), absorbent cotton balls, several pairs of forceps, alcohol cotton balls, sterile 1 mL syringes, 500 mL beakers (sterilized, irradiated with ultraviolet light before use), PBS (filtered), trypsin, and serum.

[0068] 2. Experimental animals and grouping: 20 male C57 mice at 6 - 8 weeks old (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 2 groups: PG10 group and PBS control group, with 10 mice in each group.

[0069] 3. Cell preparation: The adherent - cultured tumor cells were digested with trypsin. After the trypsin digestion time (at this time, the cell state should be single - cell and just adherent without falling off), the trypsin was aspirated. Terminate with PBS containing 1% serum at 2 mL / dish, blow down the cells, transfer them to a 15 mL centrifuge tube, and centrifuge at 1200 rpm for 5 min. Discard the supernatant, resuspend with PBS, and filter through a 100 - mesh filter screen once; count the cells and adjust the final cell concentration to 2.5×10 7 / mL. The tumor cells were melanoma cells B16 in the logarithmic growth phase, directly collected into a 15 mL centrifuge tube, centrifuged at 1200 rpm for 5 min. Discard the supernatant, resuspend with PBS, and filter through a 100 - mesh filter screen once; count the cells and adjust the final cell concentration to 1×10 6 / mL.

[0070] 4. Tumor cell inoculation: Inoculate 2×10 5 tumor cells (200 μL of cell suspension) subcutaneously near the axilla in the upper left abdomen of C57 mice.

[0071] 5. Observation of tumor growth: Two weeks after subcutaneous injection of tumor cells, treat with the polypeptide (5 mg / kg body weight, twice a week), and record the tumor size with a vernier caliper. Tumor volume = (length × width × width) / 2;

[0072] The experimental results were expressed as mean ± SEM, and the t - test was used to examine the differences between the control group and the PG10 group.

[0073] Eighteen days after tumor inoculation, the tumor growth curves of each group of mice were as Figure 4 shown in Figure B. The larger the tumor volume, the faster the tumor growth. The glucose uptake ability, killing function, and proliferation activity of CD8+ T cells were significantly increased. Therefore, the polypeptide PG10 can significantly inhibit the growth of tumor cells in mice and improve the killing activity of CD8+ T cells against tumor cells.

[0074] Example 6: Study on the effect of polypeptide PG10 on the growth of liver cancer cells in a liver cancer PDX model.

[0075] The operation steps are as follows:

[0076] 1. Experimental consumables and reagents: Sterilized 1.5 mL EP tubes, 15 mL centrifuge tubes, pipette tips, surgical scalpels, degreased cotton balls, several pairs of forceps, several pairs of scissors, alcohol cotton balls, sterile 1 mL syringes, 500 mL beakers (sterilized, irradiated with ultraviolet light before use), PBS (filtered), trypsin, and serum.

[0077] 2. Experimental animals and grouping: 20 immunodeficient NSG mice at 6 - 8 weeks of age (purchased from Beijing Biocytogen Co., Ltd.) were randomly divided into 2 groups: PG10 group and PBS control group, with 10 mice in each group.

[0078] 3. Construction of immunocompetent mice: Peripheral blood cells from normal humans were subjected to density gradient centrifugation using Ficoll lymphocyte separation solution. The lymphocytes in the middle buffy coat layer were taken, counted, and the cell density was adjusted to 1×10 8 / ml. Each mouse was inoculated with 2×10 7 lymphocytes (200 μl of cell suspension) via the tail vein. Approximately 14 days after inoculation, successful reconstruction was considered when the proportion of human CD45 - positive cells in the peripheral blood of the mice detected by flow cytometry was greater than 1%.

[0079] 4. Hepatocellular carcinoma PDX inoculation: The established PDX tumor tissue blocks were resuspended with Matrigel 354248 and PBS at a ratio of 1:1 and inoculated (200 μL of cell suspension) subcutaneously near the axilla in the upper left abdomen of the established immunocompetent mice.

[0080] 5. Observation of tumor growth: Two weeks after subcutaneous injection of tumor cells, treatment was performed with the polypeptide (5 mg / kg body weight, twice a week), and the tumor size was recorded using a vernier caliper. Tumor volume = (length × width × width) / 2;

[0081] The experimental results were expressed as mean ± SEM, and the t - test was used to examine the differences between the control group and the PG10 group.

[0082] Eighteen days after inoculating the tumor, the tumor growth curves of the mice in each group were as Figure 5 shown in Figure B. The larger the tumor volume, the faster the tumor growth. The glucose uptake ability, killing function, and proliferation activity of CD8+ T cells were significantly increased. Therefore, the polypeptide PG10 could significantly inhibit the growth of tumor cells in mice and enhance the killing activity of CD8+ T cells against tumor cells.

[0083] Step 7: Evaluate the effect of the combination of PG10 and the immune checkpoint inhibitor PD - 1 in a melanoma mouse model.

[0084] The operation steps are as follows:

[0085] 1. Experimental consumables and reagents: Sterilized 1.5 mL EP tubes, 15 mL centrifuge tubes, pipette tips, filter screens (100 meshes), absorbent cotton balls, several pairs of forceps, alcohol cotton balls, sterile 1 mL syringes, 500 mL beakers (sterilized, irradiated with ultraviolet light before use), PBS (filtered), trypsin, and serum.

[0086] 2. Experimental animals and grouping: 40 male C57 mice at 6 - 8 weeks of age (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 4 groups: IgG1 group, PG10 + IgG1 group, anti-PD1 group, and PG10 + anti-PD1 group, with 10 mice in each group.

[0087] 3. Preparation of B16 tumor cells, the method is the same as in Example 3

[0088] 4. Tumor cell inoculation: Inoculate 2×10 5 tumor cells (200 μL of cell suspension) subcutaneously near the axilla in the upper left abdomen of C57 mice.

[0089] 5. Observation of tumor growth: Two weeks after subcutaneous injection of tumor cells, treat with the polypeptide (5 mg / kg body weight, twice a week), and record the tumor size with a vernier caliper. Tumor volume = (length × width × width) / 2;

[0090] The experimental results are expressed as mean ± SEM, and the t test is used to examine the differences between the control group and the PG10 group.

[0091] Eighteen days after tumor inoculation, the tumor growth curves of the mice in each group are as Figure 4 shown in Figure B. The larger the tumor volume, the faster the tumor growth. The glucose uptake ability, killing function, and proliferation activity of CD8+ T cells are significantly increased. Therefore, the combination of polypeptide PG10 and PD-1 antibody can significantly inhibit the growth of tumor cells in mice and enhance the killing of tumors by CD8+ T cells.

Claims

1. A polypeptide, characterized in that, The amino acid sequence is as follows: PG10: His-Leu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Leu-Ser-Glu-Ile-Tyr-Pro-Val-Glu-Ile-Arg-Gly-Arg.

2. A pharmaceutical composition, characterized in that, It consists of the polypeptide as claimed in claim 1 and a pharmaceutically acceptable carrier or excipient.

3. The pharmaceutical composition according to claim 2, characterized in that, In the pharmaceutical composition, the weight ratio of the polypeptide is 0.1% to 99.9%, and the weight ratio of the pharmaceutically acceptable carrier in the composition is 0.1% to 99.9%.

4. The pharmaceutical composition according to claim 2, characterized in that, This pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form.

5. The pharmaceutical composition according to claim 2, wherein The administration method of this pharmaceutical composition is injection or oral administration.

6. The pharmaceutical composition according to claim 2, wherein Injection administration includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

7. Use of the polypeptide according to claim 1 in the preparation of a drug for targeted treatment, prevention and / or control of tumors associated with GLUT10, wherein, The tumors mentioned are liver cancer and melanoma.

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