CN114773468B - A type of polypeptide that specifically binds to PD-L1 and its application - Google Patents

Abstract

The present invention relates to a type of polypeptide that specifically binds to PD-L1 and its application. By examining the inhibitory effect of the polypeptide on PD-1/PD-L1 protein binding in vitro, the anti-tumor effect in vivo and in vitro, and the immune activation effect in vivo, it was verified that the polypeptide can block the PD-1/PD-L1 signaling pathway and activate The immune response of T cells has anti-tumor effects. The polypeptides of the present invention can be prepared by solid phase synthesis methods. Compared with monoclonal antibodies and small molecule compounds, this polypeptide has the advantages of low manufacturing cost, high stability, low immunogenicity, and strong organ or tumor penetration ability.

Description

A type of polypeptide that specifically binds to PD-L1 and its application

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a type of polypeptide that blocks the interaction between the programmed death receptor PD-1 and its target PD-L1, and can be used to prepare drugs for anti-tumor treatment.

Background technique

The immune checkpoint programmed cell death receptor (Programmed cell death 1, PD-1) and its ligand (Programmed cell death 1ligand 1, PD-L1) combine under the influence of the tumor microenvironment and can inhibit the tumor killing function of T cells. . Therefore, blocking the interaction between PD-1 and PD-L1 is an effective means to combat the immunosuppressive effect of tumors and activate tumor immune responses. PD-L1 is highly expressed on the surface of various tumor cells, so it can be used as an ideal immune and anti-tumor target without affecting the normal immune function of T cells. In recent years, the development of PD-1/PD-L1 inhibitors has become a research hotspot. The PD-1/PD-L1 inhibitors currently successfully developed and marketed are all monoclonal antibodies, which have high specificity and can specifically bind to immune checkpoints after administration, thus preventing the immune checkpoints from binding to their ligands. , to avoid immunosuppressive effects. In 2014, the first PD-1 inhibitor Pembrolizumab was approved by the FDA for the late-stage treatment of melanoma, non-small cell lung cancer, gastric cancer and liver cancer. Currently, a variety of PD-1/PD-L1 inhibitors have been developed and marketed for late-stage treatment of different cancers. Although the PD-L1 monoclonal antibody inhibitors that have been marketed have good efficacy, they are highly specific. Sometimes it can cause serious immune-related adverse reactions, and has the disadvantages of high production cost and poor stability. Compared with monoclonal antibodies, polypeptides have the advantages of low production cost, high stability, low immunogenicity and high tissue permeability. advantage. Therefore, it is of great significance to develop peptide inhibitors that can avoid the above defects. In the present invention, based on the characteristics of immune checkpoints, new polypeptide inhibitors that block the PD-1/PD-L1 interaction are designed.

Contents of the invention

The present invention provides a polypeptide inhibitor that can specifically bind to PD-L1 to block PD-1/PD-L1 interaction, regulate the immune system, and have anti-tumor effects.

In order to achieve the above purpose, the present invention provides a class of polypeptide inhibitors that can block the PD-1/PD-L1 interaction. The polypeptide has amphipathic α-helical properties. The amino acid sequence of the polypeptide is as follows and has 85 Any one of %-99% identical sequences:

Peptide: H-G-D-W-F-K-A-Y-Y-D-K-V-A-E-G-OH(H-Gly-Asp-Trp-Phe-Lys-Ala-Tyr-Tyr-Asp-Lys-Val-Ala-Glu-Gly-OH)

The structural formula of the polypeptide is:

Further, the polypeptide is used to block the interaction of PD-1/PD-L1 and overcome immune evasion.

Application of the polypeptide in the preparation of drugs for treating cancer:

The anti-PD-L1 polypeptide of the present invention can be used to prepare anti-tumor drugs or for anti-tumor treatment. Preferably, the tumor can be selected from breast cancer, non-small cell lung cancer, prostate cancer, kidney cancer, melanoma, liver cancer, etc. But it is not limited to anti-tumor drugs or anti-tumor treatments.

The technical solution of the present invention is as follows:

In this invention, polypeptides are synthesized by solid-phase peptide method, and the resin of the connected first amino acid is swollen and then deprotected, and the next amino acid is condensed, and so on, the resin peptide is obtained and cleaved to obtain the polypeptide. The synthesized polypeptide has high binding affinity to PD-L1. Homogeneous Time-resolved Fluorescence (HTRF) technology shows that the polypeptide inhibitor of the present invention can effectively block the PD-1/PD-L1 interaction.

The polypeptide of the present invention can inhibit the interaction of PD-1/PD-L1 in an in vitro cell co-culture system. In the co-culture system of tumor cells MDA-MB-231 and Jurkat cells, the surface of tumor cells MDA-MB-231 expresses PD-L1, and the surface of Jurkat cells expresses PD-1. The interaction of PD-L1/PD-1 inhibits Jurkat cells. Cellular secretion of cytokines such as IL-2. The polypeptide inhibitor of the present invention has a significant promoting effect on the secretion of IL-2 by Jurkat cells, indicating that the polypeptide of the present invention can block the PD-1/PD-L1 interaction, restore the immune effect of Jurkat cells, and thus exert its effect on tumor cells MDA- Attack of MB-231.

The present invention constructs a 4T1 tumor-bearing mouse model, induces tumor PD-L1 expression through mouse-derived IFN-γ, and monitors the mouse body weight and tumor changes during drug administration and treatment. After the administration, the tumors of mice treated with the polypeptide were significantly smaller than those in the BMS-202 positive control group, indicating that the polypeptide has an anti-tumor effect. The concentrations of IFN-γ and IL-4 in mouse serum were significantly increased, and immunohistochemistry technology showed that CD8 ⁺ , GZMB and IFN-γ were significantly expressed in mouse tumor tissues, indicating that the polypeptide has the ability to regulate tumor immunity. The role of the system.

The advantages of the present invention are:

1. The polypeptide of the present invention can effectively block the PD-1/PD-L1 interaction and performs well in in vivo and in vitro models. The specific effect is to significantly increase the activity of T cells, eliminate the immunosuppressive effect, and prevent the growth of tumor cells. Immune evasion in turn produces tumor suppressor effects.

2. Compared with monoclonal antibodies, the polypeptide of the present invention has better tumor tissue penetration and can reduce immunogenicity.

3. The polypeptide of the present invention has high biocompatibility and will not have other effects on normal cells and the body.

4. The polypeptide of the present invention can be prepared by solid-phase synthesis, which is simple, easy and low-cost.

Description of the drawings

Figure 1 shows the use of HTRF technology to detect the blocking effect of the polypeptide on PD-1/PD-L1 interaction in vitro.

Figure 2 shows the experimental results of the polypeptide and positive control BMS-202 promoting the secretion of IL-2 by Jurkat cells within a certain range of concentrations when Jurkat cells and MDA-MB-231 cells were co-cultured.

Figure 3 is a diagram showing the tumor inhibitory effect of the polypeptide on 4T1-tumor-bearing mice after two weeks of treatment.

Figure 4 is a graph showing mouse tumor growth curve during the treatment of 4T1-tumor-bearing mice with the polypeptide.

Figure 5 is a graph showing changes in mouse body weight during the treatment of 4T1-tumor-bearing mice with the polypeptide.

Figure 6 is a graph showing the use of the Elisa method to detect that the polypeptide promotes the expression of the cytokine IL-4 in the serum of 4T1-tumor-bearing mice.

Figure 7 is a graph showing the use of the Elisa method to detect that the polypeptide promotes the expression of cytokine IFN-γ in the serum of 4T1-tumor-bearing mice.

Figure 8 shows the detection of GZMB, CD8, and IFN-γ contents in tumor tissues of 4T1-tumor-bearing mice using IHC method.

Detailed ways

The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

Experiment on blocking PD-1/PD-L1 interaction in vitro by the polypeptide described in Example 1

1. Drug handling

Before the experiment, dissolve the peptide to be tested and the positive control drug (BMS-202) in Diluent Buffer and DMSO respectively to prepare a mother solution with a concentration of 100mM, and then dilute them with Diluent Buffer to 10mM, 1000μM, 100μM, 10μM, and 1μM respectively. The test solution should be frozen at -20°C. Dilute the Tag1-PD-L1 (0.1mg/mL, 1.845μM, 35μL) and Tag2-PD-1 (0.5mg/Ml, 27.8μM, 25μL) protein working solutions in the HTRF kit to the final concentration with Diluent Buffer. 25nM and 250nM, aliquot into sample tubes. Dilute the energy donor Anti-Tag1-Eu ³⁺ (25 μL) and energy donor Anti-Tag2-XL665 (100 μL) stock solutions in the HTRF kit with Detection Buffer 100 times and 25 times respectively, and distribute them into sample tubes. , uniformly stored at -80°C, aliquoted reagents for immediate use, avoid repeated freezing and thawing.

2. Experimental steps

(1) Take a brand new 96 shallow well plate, preset the administration group, negative group, positive group and positive control group. Each group has three duplicate wells and number them.

(2) Take out the working solutions of Tag2-PD-1, Tagl-PD-L1, Anti-Tagl-Eu ³⁺ and Anti-Tag2-XL665 from the -80°C refrigerator and thaw them on ice.

(3) Use a pipette to add 4μL Tag1-PD-L1 working solution, 4μL Tag2-PD-1 working solution and 2μL peptide solution to the administration group; use a pipette to add 4μL Tag1-PD-L1 working solution to the positive control group solution, 4 μL Tag2-PD-1 working solution and 2 μL BMS-202 solution; use a pipette to add 4 μL Tag1-PD-L1 working solution, 4 μL Tag2-PD-1 working solution and 2 μL Diluent Buffer solution to the positive group; use a pipette to Use the liquid gun to add 4 μL Tag1-PD-L1 working solution and 6 μL Diluent Buffer solution to the negative group. Mix by pipetting and incubate for 15 minutes.

(4) Use a pipette to add 5 μL of Anti-Tag1-Eu ³⁺ and 5 μL of Anti-Tag2-XL665 stock solution to each well. After mixing by pipetting, seal the membrane and incubate in the dark for 2 hours.

(5) After the incubation, use a BioTek multifunctional microplate reader to stimulate at 320nm, emit at 665nm and 620nm, and measure the emission signal value of each well.

(6) According to the following formula, calculate the emission signal ratio and suppression rate, and repeatedly calculate the average value and standard deviation (SD).

Emission Ratio(ER)＝665nm Emission signal/620nm Emission signal

Rejection rate＝(ERpositive-ERsample)/(ERpositive-ERnegative)×100%

HTRF results (Figure 1) show that compound BMS-202 inhibits the formation of PD-1/PD-L1 complex by inducing PD-L1 dimerization, effectively blocking the interaction between PD-1 and PD-L1. . Although the polypeptides do not exhibit the same level of inhibitory activity as BMS-202, they have the ability to significantly block PD-1/PD-L1 interaction in a dose-dependent manner. The _IC50 value of the polypeptide is 0.206 μM. According to the IC ₅₀ value, it can be seen that the peptide has the ability to block tumor PD-1/PD-L1 binding, thereby activating CD8 ⁺ T cells to secrete cytokines and inhibit tumor growth.

Example 2 Jurkat cells and MDA-MB-231 cells were co-cultured, and Elisa method was used to detect IL-2 secretion in the cell supernatant.

1. Culture medium preparation: Dissolve 20 μg human IFN-γ in 1 mL ddH ₂ O, prepare a 20 μg/mL stock solution, distribute it into multiple EP tubes, and freeze it in a -80°C ultra-low temperature refrigerator. Use DMEM complete medium to prepare the human IFN-γ stock solution into DMEM complete medium containing 10ng/mL human IFN-γ, aliquot and freeze. In the same way, use RPMI-1640 complete medium to prepare Phorbol 12-myristate 13-acetate (PMA) and Phytohemagglutinin (PHA) to contain 50ng /L PMA and 1μg/mL PHA in RPMI-1640 complete medium, aliquot and freeze.

2. Cell culture: Breast cancer MDA-MB-231 cells were cultured in DMEM complete medium containing 10ng/mL human IFN-γ for 24 hours to induce MDA-MB-231 cells to highly express PD-L1. Jurkat cells were Jurkat cell differentiation was promoted by culturing in RPMI-1640 complete medium containing 50ng/L PMA and 1μg/mL PHA for 24 hours.

3. Cell co-culture: Add 1×10 ⁴ MDA-MB-231 cells/well into a 96-well plate and incubate for 4 hours to allow cells to adhere. Then discard the culture supernatant and add 4 × ¹⁰ Jurkat cells to each well. The co-culture system was cultured in an incubator at 37°C for 48 h.

4.IL-2 content detection:

(1) Collect the cell supernatant, place it in a high-speed centrifuge and centrifuge at 3000 rpm for 20 minutes, and draw the supernatant for later use.

(2) Preparation of washing buffer: dilute the concentrated washing buffer 30 times with distilled water and set aside. If crystals precipitate in the concentrated washing buffer, heat it in a water bath until it is completely dissolved.

(3) Preparation of IL-2 standard curve solution: The concentration of IL-2 starting stock solution is 480pg/mL. Add 150 μL IL-2 starting stock solution to the first centrifuge tube, pipette evenly with a pipette, and then proceed backward Transfer 150 μL into a centrifuge tube to prepare IL-2 standard solution with concentrations of 320pg/mL, 160pg/mL, 80pg/mL, 40pg/mL, 20pg/mL and 10pg/mL, and IL-2 standard solution dilution buffer. (0pg/mL) can be used as a blank well.

(4) Sample incubation and absorbance measurement

Add sample: Add 50 μL of standard solutions of 6 concentrations per well to the bottom of the wells of the enzyme plate. The sample group is composed of 1 candidate peptide administration group (5 concentration gradients), 1 positive drug group (5 concentration gradients), and 1 normal control group. Add 10 μL of the sample to be tested to each well at the bottom of the well of the enzyme plate. Then dilute 5 times with sample diluent, each group has 3 duplicate holes, set blank holes without adding sample and enzyme label reagent, and operate the same in other steps. After adding the sample, shake gently to mix evenly.

Sample incubation: After shaking gently to mix, seal the 96-well microplate with a new sealing film and incubate at 37°C for 30 minutes.

Wash the plate: Carefully tear off the sealing film, use a pipette to remove the liquid and spin dry, fill each well with washing buffer, let stand for 30 seconds and discard, wash the plate 5 times, and pat the enzyme plate dry.

Add enzyme and incubate: Add 50 μL of enzyme-labeled reagent to each well, seal the 96-microwell plate with a new sealing film, and incubate at 37°C for 30 minutes.

Wash the plate: Carefully tear off the sealing film, use a pipette to remove the liquid and spin dry, fill each well with washing buffer, let stand for 30 seconds and discard, wash the plate 5 times, and pat the enzyme plate dry.

Color development: Add 50 μL of chromogen A and chromogen B to each well, shake gently to mix, wrap the enzyme plate with tin foil, and develop color for 15 minutes at 37°C in the dark.

Termination: Add 50 μL/well stop solution to each well to terminate the reaction, shake gently to mix, and the color in the well changes from blue to yellow.

Detection: Within 15 minutes after adding the stop solution, use a microplate reader to measure the absorbance OD value of each well at a wavelength of 450 nm.

(5)Data processing

With the concentration of the standard substance as the abscissa and the OD value as the ordinate, draw a standard curve and determine the linear regression equation. Substitute the OD value of the sample into the standard curve equation to calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

In order to study the ability of the polypeptide to inhibit PD-1/PD-L1 interaction at the cellular level, the present invention co-cultured Jurkat cells and tumor cells MDA-MB-231 to simulate in vitro PD-1/PD-L1 interaction. . During Jurkat activation, the cytokine IL-2 is mainly released. Therefore, the secreted content of IL-2 can indicate that the polypeptide inhibits the PD-1/PD-L1 interaction, thereby inferring that the polypeptide has the ability to block the immune evasion of tumor cells. ability. As shown in Figure 2, it was observed that the IL-2 level of the polypeptide group was significantly higher than that of the control group within a certain concentration range, indicating that the polypeptide has the ability to inhibit the PD-1/PD-L1 interaction. The polypeptide can restore IL-2 levels to 108.865pg/mL at a concentration of 200 μM. In addition, the positive control group BMS-202 had the best effect in increasing IL-2 levels, proving that the use of Elisa method to measure IL-2 secretion is accurate.

Example 3 Using the polypeptide as an example to conduct in vivo anti-tumor activity experiments

The in vivo anti-tumor activity of the polypeptide was examined by establishing a 4T1-tumor-bearing mouse model. The methods and results are as follows:

1. Materials and grouping: 15 female balb/c mice aged 6-8 weeks were ordered from the Experimental Animal Center of Jiangsu University. The mice were randomly divided into 3 groups, with 5 mice in each group: peptide group (20 mg/kg), positive control BMS-202 (10 mg/kg), and model group (physiological saline). Dissolve 20 mg of polypeptide in 10 mL of physiological saline to prepare a 2 mg/mL polypeptide solution, aliquot and freeze. Add 1 mL of 25 mg/mL clarified DMSO stock solution to 9 mL of 20% SBE-β-CD physiological saline aqueous solution, mix evenly, and dissolve 10 mg of BMS-202 in the solution to obtain a 1 mg/mL BMS-202 solution. Store frozen. Dissolve 50 μg of rat IFN-γ in 0.5 mL of ultrapure water, dilute to 66.67 mL with physiological saline, aliquot and freeze for later use.

2. Construction of 4T1-tumor-bearing mouse model: Collect 4T1 cells and suspend them in PBS to prepare a cell suspension of 1×10 ⁷ /mL. Breast cancer cells 4T1 were inoculated into the left armpit of the mice, 0.2 mL each.

3. Administration: When the tumor grows to approximately ¹⁰⁰ mm in size, 0.2 mL of a total of 1000 IU mouse IFN-γ will be injected into the tumor for three consecutive days to induce high expression of PD-L1 in the tumor. The next day, each animal was injected with 0.2 mL of drug into the tumor each time, and the drug was administered every other day for 2 weeks. Tumor volume and mouse body weight were measured at the time of administration.

4. Tumor dissection: The mice were killed using carbon dioxide asphyxiation on the second day after the administration. Then, the mice in the administration group and the model group were dissected using surgical instruments, the tumor tissue was completely peeled off, and blood was taken from the eyeballs.

As shown in Figure 3, the mice in the physiological saline group had larger tumor volumes, while the mice in the peptide administration group had smaller tumor volumes. As shown in Figure 4, the volume of mice in the saline group increased nearly 2.3 times after 18 days of treatment. On the contrary, the polypeptide and the positive control BMS-202 had a significant inhibitory effect on tumor growth. The tumor inhibition rate of the positive control BMS-202 group reached 53.14%, while the tumor inhibition rate of the polypeptide group could reach 48.18%, which was slightly lower than the 53.14% tumor inhibition rate of the positive control BMS-202 group. Furthermore, the polypeptide had a negligible effect on body weight in mice during treatment (Fig. 5), indicating that the polypeptide is well tolerated and biocompatible.

The IL-4 and IFN-γ contents in mouse serum were examined using the Elisa method. The methods and results are as follows:

1. Let the blood collected from the eyeballs stand at 4°C overnight. After stratification the next day, centrifuge at 5000 rpm for 10 minutes and aspirate the supernatant for later use.

2. Detect the contents of IL-4 and IFN-γ according to the Elisa detection method in Example 2.

To further characterize the immune response, the secretion levels of IL-4 and IFN-γ in mouse serum were compared, as important indicators of T cell activity. As shown in Figure 6 and Figure 7, the levels of IL-4 and IFN-γ in the polypeptide group were significantly higher than those in the physiological saline group. The experimental results showed that the polypeptide can effectively activate T cells, thereby increasing the anti-tumor ability.

Example 4 Taking the polypeptide as an example, the contents of GZMB, CD8 and IFN-γ in mouse tumor tissues were investigated by immunohistochemistry (IHC).

The methods and results are as follows:

1. Obtaining materials and fixing

Soak fresh mouse tissues completely in 4% paraformaldehyde fixative for 24 hours to denature and coagulate the proteins of the tissues and cells to prevent autolysis after death or bacterial decomposition, thereby maintaining the original morphological structure of the cells.

2. Dehydrated and transparent

Generally, low to high concentration alcohol is used as a dehydrating agent to gradually remove the water from the tissue block. Then soak the tissue block in a 1:1 mixed solution of absolute ethanol and xylene for 20 minutes. After taking it out, continue to soak it in another tank of pure xylene for 10 minutes.

3. Wax embedding

Place the transparent tissue block in melted paraffin and place it in a paraffin box to keep warm. Embedding is carried out after the paraffin is completely immersed in the tissue block: first prepare a container, pour in the melted paraffin, and quickly pick up the tissue block that has been soaked in paraffin and place it into it. Cools and solidifies into blocks.

4. Slicing and patching

Fix the embedded wax block on a microtome and cut into thin slices, usually 5-8 μm thick. The cut slices are often wrinkled and need to be ironed in heated water, then attached to a glass slide and dried in a 45°C constant temperature oven.

5. Dewaxing

Soak the slices in xylene for 3 times, 5 minutes each time. The last time is soaked in fresh xylene and absolute ethanol 2 times, 5 minutes each time. The last time is soaked in fresh absolute ethanol and 95% ethanol once, 5 minutes. , soak once in 90% ethanol for 5 min, soak once in 75% ethanol for 5 min, and wash twice with ddH ₂ O on a shaker, 5 min/time.

6. Antigen retrieval

Soak in antigen retrieval solution (1X) and heat at 100°C for 15 minutes, cool to room temperature naturally, wash twice with ddH ₂ O, 5 minutes each time, add 3% hydrogen peroxide aqueous solution to the slices, and incubate at room temperature for 10 minutes. Immunohistochemical strokes were drawn in circles, and goat serum was blocked and incubated at 37°C for 1 hour. The blocking solution was aspirated, and primary antibody was added and incubated at 4°C overnight (antibody recovery). The next day, after the primary antibody incubation is completed, place it at room temperature, raise the temperature, and incubate at 37°C for 30 minutes. Wash with TBST buffer 3 times, 5 minutes/time. Incubate with the secondary antibody at 37°C for 30 minutes, and wash with TBST buffer 3 times, 5 minutes/time. Second-rate. Add Streptavidin-biotin complex (SABC), incubate at 37°C for 30 minutes, and wash with PBS three times, 5 minutes each time. Use DAB chromogen to stain, incubate at room temperature for 3-10 minutes, and wash away with ddH ₂ O. Counterstain with hematoxylin for 20 seconds, wash away with ddH ₂ O, differentiate with acidic differentiation solution (2-5 seconds) (99 mL of 75% alcohol + 1 mL of hydrochloric acid), and rinse with tap water for 10 minutes.

7. Dehydration

Soak sections in 95% ethanol for 2 minutes and dehydrate in fresh 95% ethanol for 2 minutes; dehydrate in 100% ethanol for 2 minutes and dehydrate in fresh 100% ethanol for 2 minutes; soak in xylene twice, 5 minutes each time, and soak in fresh xylene for 5 minutes.

8. Neutral resin sealing

Drop 2-3 drops of gum solution in the center of the slide, then slowly put the cover glass on it and squeeze it gently. The gum solution will spread outward to the entire lens. Let it air dry for a few hours and then use a cotton ball to glue the transparent Use solvent or turpentine to clean excess gum from the edge of the lens. Then place it in a ventilated place to dry. The sealed slide can be stored at room temperature.

As shown in Figure 8, according to the distribution of infiltrating CD8 ⁺ T cells in the tumor, compared with BMS-202, treatment with the peptide significantly increased the degree of CD8 ⁺ T cell infiltration. GZMB is a cytokine secreted by activated CD8 ⁺ T cells. In the immunohistochemistry results, compared with the normal saline group, the GZMB content in the tumor tissues of mice in the peptide group was significantly increased. Compared with the saline group, the peptide group enhanced the expression of IFN-γ cytokines. These results indicate that treatment with the peptides can lead to a stronger immune response and, in turn, an inhibitory effect on tumor growth.

The above data were analyzed by Oneway ANOVA test of GraphPad software. The significance level is defined as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

sequence list

<110> Jiangsu University

<120> A polypeptide that specifically binds to PD-L1 and its application

<141> 2022-04-06

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 14

<212> PRT

<213> Artificial Sequence

<400> 1

Gly Asp Trp Phe Lys Ala Tyr Tyr Asp Lys Val Ala Glu Gly

1 5 10

Claims (2)

1. The polypeptide specifically combined with PD-L1 is characterized by having amphipathic alpha-helix property, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO.1, and the structural formula of the polypeptide is as follows:

2. the use of a class of polypeptides that specifically bind to PD-L1 according to claim 1, for the preparation of an anti-tumor agent, wherein the tumor is breast cancer, non-small cell lung cancer, prostate cancer, renal cancer, melanoma, or liver cancer.