WO2021043339A1 - Pd-l1 antagonist polypeptide and application thereof - Google Patents

Abstract

Disclosed are a PD-L1 antagonist polypeptide and an application thereof, belonging to the field of biomedicine. The PD-L1 antagonist is a polypeptide having the amino acid sequence IYLCGAISLHPKAKIEECPGA (C-C), or a pharmaceutically acceptable salt thereof. The polypeptide specifically recognizes PD-L1 on the surface of cells and mediates the specific killing of PD-L1 positive cells. In addition, the polypeptide blocks the immunosuppressive signal pathway of PD-1/PD-L1 and eliminates the immunosuppressive effect of tumors, thereby achieving a specific killing effect.

Description

A PD-L1 antagonist polypeptide and its application Technical field

The invention belongs to the field of biomedicine, and more specifically, relates to a polypeptide with immune checkpoint antagonistic activity and its application.

Background technique

Tumor immunotherapy is divided into cellular immunotherapy and immune checkpoint inhibitors. The effects of monoclonal antibody drugs and chimeric antigen receptor T (CAR-T) cellular immunotherapy are gradually recognized, and the concept of cancer immunotherapy is also increasing. Deep into the hearts of the people. The former is to take the patient’s immune cells outside the body for modification, so that it has a more effective and precise immunity to tumor cells. After modification, it is injected back into the tumor patient to play a targeted role in killing tumor cells. This therapy includes LAK. , DC, CAR-T, NK, CAR-NK therapy, etc. Immune checkpoint (immune Check-point) is also called immune card control point. At present, the most important aspect of tumor immune drug treatment is tumor immune checkpoint inhibitors, and immune checkpoint inhibitors relieve tumors’ tolerance/shield from immunity. The function allows immune cells to re-recognize tumor cells and attack tumor cells. The current research mainly focuses on the three molecules of CTLA-4, PD-1 and PD-L1.

During the entire immune response process, T cell activation needs to be mediated by dual signaling pathways. First, the T cell receptor TCR on the surface of T cells specifically recognizes the antigen-presenting cell APC or the MHC molecule antigen peptide complex on the surface of tumor cells; secondly, the costimulatory molecules on APC or tumor cells and the costimulatory receptors on T cells Binding, activating or inhibiting T lymphocytes. The costimulatory molecules on T cells mainly include co-activating molecules 4-1BB, CD27, CD28, OX40, ICOS, and co-inhibitory molecules CTLA4, PD-1. Programmed Death Receptor 1 (PD-1) is an important immunosuppressive transmembrane protein mainly expressed on activated T cells, and is a member of the CD28 superfamily. It was originally discovered by screening for differential expression in apoptotic cells, specifically cloned from apoptotic mouse T hybridoma 2B4.11. Other members of the family, such as CTLA-4 and BTLA, were found by screening differential expression in cytotoxic T lymphocytes and TH1 cells, respectively.

PD-1 contains two ligands: PD-L1 (also known as CD274 or B7-H1) and PD-L2 (also known as CD273 or B7-DC). PD-L1 is a transmembrane protein composed of 290 amino acid subunits. The extracellular segment is composed of two immunoglobulin constant regions (IgC) and IgV-like domains. It is mainly expressed in mature CD4+ T cells and CD8+ T cells. , Monocytes, macrophages, B cells, dendritic cells and other hematopoietic cells, as well as some non-hematopoietic cells, such as endothelial cells, pancreatic islet cells, mast cells and other membrane surface. PD-L2 is a transmembrane protein composed of 274 amino acid residues. It has a high similarity with PD-L1, but the two also have certain differences. For example, the distribution of PD-L2 in vivo has limitations. It is expressed on the membrane surface of macrophages, dendritic cells and some subclasses of B cells. In addition to the expression induced by pro-inflammatory cytokines in hematopoietic and non-hematopoietic cells, PD-L1 has also been found to be overexpressed in many solid tumors, such as melanoma, non-small cell lung cancer, renal cell carcinoma, etc., and can enhance tumor Transfer ability, leading to an increase in patient mortality. Combined with PD-1 that is highly expressed in tumor-infiltrating T lymphocytes, it indicates that the highly expressed PD-1/PD-L1 signaling pathway mediates tumor immunosuppression. The combination of these two ligands with PD-1 will cause the tyrosine phosphorylation of the intracellular structure of PD-1 and recruit the tyrosine phosphatase SHP-2, thereby reducing the phosphorylation of the TCR signaling pathway and lowering the TCR The activation signal downstream of the pathway, the activation and proliferation of T cells and the production of regulatory cytokines, at the same time, block cells in the G0/G1 phase to inhibit the proliferation of T cells, hinder their differentiation into plasma cells, and induce the growth of T cells Apoptosis. In this way, the infinite proliferation of T cells is avoided and the dynamic balance is maintained. The negative feedback regulation of T cells involved in the PD-1/PD-L1 signaling pathway plays a vital role in the elimination of antigens and the maintenance of the body's balance. Therefore, inhibition of PD-1 and PD-L1 pathways will accelerate and strengthen autoimmunity.

So far, by inhibiting the PD-1/PD-L1 signaling pathway for tumor treatment, good results have also been achieved in the clinical stage. PD-L1 antibodies include atezolizumab from Roche/Genentech, durvalumab from AstraZeneca, and avelumab from EMD serono/Pfizer. It has been approved by the FDA for the treatment of non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma, etc. Other clinical studies include BMS’s BMS936559, domestic PD-1 antibodies such as Hengrui’s SHR-1210, BeiGene’s BGB-A317, Taizhou Junshi’s JS001, etc., which inhibit the PD-1/PD-L1 signaling pathway in tumors. Great potential in immunotherapy.

Summary of the invention

1. The problem to be solved

The present invention provides a PD-L1 antagonist polypeptide and its application. The polypeptide of the present invention acts on PD-L1 to inhibit the PD-1/PD-L1 pathway. It has been verified by various experimental models and has a good anti-tumor effect. Development prospects.

2. Technical solution

In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

A PD-L1 antagonist polypeptide, characterized in that: the amino acid sequence of the polypeptide includes IYLCGAISLHPKAKIEECPGA (CC), or one or more amino acids have been deleted, replaced, or added on the basis of a polypeptide that still has an immunosuppressive effect , Or a pharmaceutically acceptable salt of the polypeptide.

Said PD-L1 antagonist polypeptide is characterized in that the amino acid sequence of said polypeptide is IYLCGAISLHPKAKIEECPGA (C-C), or a pharmaceutically acceptable salt thereof.

The application of the polypeptide in the preparation of drugs for preventing or treating cancer.

The application is characterized in that: the application is realized by the specific binding of the polypeptide and PD-L1.

The application is characterized in that the cancer is renal cell carcinoma, ovarian cancer, head and neck cancer, prostate cancer, breast cancer, colon cancer, non-small cell lung cancer, urothelial cancer, liver cancer, lymphoma, osteosarcoma, Brain tumor, bladder cancer, pancreatic cancer, cervical cancer, myeloma, thyroid cancer, gallbladder cancer, salivary gland cancer, testicular cancer, or melanoma.

A compound characterized in that one or more pharmaceutically acceptable excipients are added to the polypeptide of claim 1 or 2.

The compound is characterized in that the auxiliary materials include conventional diluents, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants and stabilizers in the pharmaceutical field.

The medicament of the present invention can be treated by various administration methods including subcutaneous or intramuscular injection, intravenous injection or intravenous drip such as injection, dry powder injection, oral administration such as pills, capsules, etc., nasal spray.

At this stage, the main clinical immune checkpoint inhibitors are monoclonal antibodies, but there are problems such as expensive treatment costs during the use of antibodies. Therefore, the present invention uses computer three-dimensional simulation technology, based on the analysis of a large number of traditional structures and pharmacological experiments, and the independently designed polypeptide structure has a good anti-tumor effect. The present invention uses a three-dimensional structure to independently design an immune checkpoint antagonistic polypeptide with a brand-new structure that acts on PD-L1.

3. Beneficial effects

Compared with the prior art, the beneficial effects of the present invention are:

(1) The polypeptide of the present invention has a simple structure, is easy to synthesize, separate and purify, effectively inhibits the PD-1/PD-L1 pathway, and eliminates the effect of immunosuppression;

(2) The polypeptide of the present invention has weak adverse reactions and toxic and side effects;

(3) The immunosuppressive elimination effect of the polypeptide according to the present invention is good in in vivo and in vitro models. The specific effect is to significantly increase the activity of T cells to eliminate the immunosuppressive effect and prevent the escape of tumor cells to produce tumor suppressive effects.

Description of the drawings

Figure 1. Flow cytometry to detect the binding of HT-29 and FITC-anti-PD-L1 polypeptide; A is a negative control, B, C, D are HT-29 and 15nM, 150nM, 1.5μM FITC-conjugated anti-PD -L1 polypeptide binding rate; 26.3%, 58.0% and 80.4% respectively;

Figure 2. Fluorescence microscopy to detect the binding of HT-29 and FITC-anti-PD-L1 polypeptide; A: cell membrane; B: FITC-anti-PD-L1 polypeptide; C: Merge;

Figure 3 Secretion of IFN-γ in the co-incubation system

G1: T cells; G2: co-incubation ratio of 100:1; G3: co-incubation ratio of 80:1; G4: co-incubation ratio of 60:1; G5: co-incubation ratio of 40:1; G6: co-incubation ratio It is 20:1; G7: the co-incubation ratio is 10:1. Compared with the negative group, *P<0.05, **P<0.01, ***P<0.001;

Figure 4 IL-2 secretion in the co-incubation system

G1: T cells; G2: co-incubation ratio of 100:1; G3: co-incubation ratio of 80:1; G4: co-incubation ratio of 60:1; G5: co-incubation ratio of 40:1; G6: co-incubation ratio It is 20:1; G7: the co-incubation ratio is 10:1. Compared with the negative group, *P<0.05, **P<0.01, ***P<0.001;

Figure 5 The effect of peptides on IFN-γ in the co-incubation system

G1: control group; G2: model group; G3: 100 nM anti-PD-L1 polypeptide; G4: 200 nM anti-PD-L1 polypeptide; G5: 400 nM anti-PD-L1 polypeptide; G6: 800 nM anti-PD-L1 polypeptide; G7: 1.6 μM Anti-PD-L1 polypeptide. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001;

Figure 6 The effect of peptides on IL-2 in the co-incubation system

G1: control group; G2: model group; G3: 100 nM anti-PD-L1 polypeptide; G4: 200 nM anti-PD-L1 polypeptide; G5: 400 nM anti-PD-L1 polypeptide; G6: 800 nM anti-PD-L1 polypeptide; G7: 1.6 μM Anti-PD-L1 polypeptide. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001;

Figure 7 Bioluminescence detection of the inhibitory effect of anti-PD-L1 polypeptide on tumor cell growth

The first group is the negative control group; the second group is injected with only HT-29 cells and anti-PD-L1 polypeptides at the same time; the third group is injected with human T cells and human HT-29 cells at the same time, followed by subcutaneous injection of anti-PD-L1 Antibody durvalumab (0.1mg/kg); The fourth group is after the simultaneous injection of human T cells and human HT-29 cells, followed by subcutaneous injection of anti-PD-L1 polypeptide (4mg/kg);

Figure 8 Detect the inhibitory effect of anti-PD-L1 polypeptide on tumor cell growth by tumor volume

The first group is the negative control group; the second group is injected with only HT-29 cells and anti-PD-L1 polypeptides at the same time; the third group is injected with human T cells and human HT-29 cells at the same time, followed by subcutaneous injection of anti-PD-L1 Antibody durvalumab (0.1mg/kg); the fourth group is after the simultaneous injection of human T cells and human HT-29 cells, followed by subcutaneous injection of anti-PD-L1 polypeptide (4mg/kg).

detailed description

The present invention will be further described below in conjunction with specific embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to change into equivalent embodiments with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the solution of the present invention falls within the protection scope of the present invention.

In the examples, the polypeptide (IYLCGAISLHPKAKIEECPGA (C-C)) was mainly used as the object to study its activity in preventing cancer and/or tumor suppressor activity. The polypeptide is synthesized by Gil Biochemical (Shanghai) Co., Ltd., and the purity is more than 95%.

Example 1

Detection experiment of the specific binding of peptides to the target at the cellular level

Flow cytometry is a kind of single unidirectional flow particle excited by a laser beam, and the scattered light of the particle and the fluorescent marker carried by it are detected, so as to complete the rapid detection and analysis of multiple physical characteristics of a single particle and cell sorting. technology. It is widely used in scientific research and clinical medical examination, and it is the most advanced cell quantitative analysis technology. The main target of a polypeptide with anti-cancer and/or anti-tumor activity involved in the present invention is PD-L1. In this study, the designed anti-PD-L1 polypeptide was connected to FITC fluorescent markers to connect cells with FITC-anti-PD The binding rate of -L1 polypeptide solution reflects the ability of the polypeptide to bind to PD-L1 on the cell surface at the cellular level.

1.1 Experimental materials

Human colon cancer cells (HT-29) were purchased from ATCC. Anti-PD-L1 polypeptide, synthesized by our laboratory, the purity is more than 95%. BSA blocking solution. CO ₂ cell incubator, flow cytometer, inverted microscope, liquid nitrogen tank, ultra-clean table, small centrifuge, electronic balance, pH meter, automatic high-pressure steam sterilizer, oven, ultra-pure water maker, digital display Constant temperature water bath.

1.2 Experimental method:

The HT-29 cells were plated into a 6-well plate, and when the cells reached 80% confluency, they were digested and collected. Wash twice with ice-cold PBS. Add 1ml of 1% BSA solution, fix it on a rotary mixer, and mix for 30min at 4°C. Add 10μl of 1mg/ml FITC-anti-PD-L1 peptide solution under dark conditions, fix it on a rotary mixer, and mix at 4℃ in the dark for 1h. After incubating the drug, centrifuge at 800 rpm for 5 min and discard the supernatant. Wash once with ice-cold PBS. Adjust the cell concentration with PBS, and adjust the sample concentration to 1×10 ⁶ cells/ml. Make 3 parallels for each cell, and prepare 0.5ml for each sample.

Use a flow cytometer to detect the binding of peptides to PD-L1, turn on the stabilized power supply, transformer, flow cytometer host, computer, and printer in turn from left to right. Open the liquid flow drawer and add pure water to the sheath liquid bucket until it reaches 2/3. Pour out the waste liquid and add 200ml of sodium hypochlorite solution containing 10% effective chlorine concentration. Adjust the hydraulic valve to the pressurize position to remove air bubbles between the liquid flow line and the filter. Remove the sample tube, perform the PRIME function twice, 1ml PBS, HING RUN for 2 minutes. Start measuring and analyzing the sample. After the measurement is completed, move the sample support frame to the left, and vacuum 1ml FACS Clean. Then return the sample support to the normal position and HING RUN 5 minutes. Change the FACS Clean to pure water, move the sample support rack to the left, vacuum 1ml, return the sample rack to the normal position, and HING RUN for 10 minutes. Press Standby, remove the sample tube, and perform the PRIME function twice. Finally, leave 1ml of purified water in a flow test tube. Press Standby and let it work for 20 minutes. After the fan cools the laser, turn off the flow cytometer. Exit the program and shut down the computer.

1.3 Experimental results

See Figure 1, the binding of HT-29 and FITC-conjugated anti-PD-L1 polypeptide, where A is a negative control, B, C, and D are HT-29 and 15nM, 150nM, 1.5μM FITC-conjugated anti-PD- The binding rate of L1 polypeptide is 26.3%, 58.0% and 80.4%, respectively.

Example 2

Qualitative detection experiment of the specific binding of peptides to the target at the cellular level-fluorescence imaging experiment

1.1 Experimental materials

Human colon cancer cells (HT-29) were purchased from ATCC. Anti-PD-L1 polypeptide, synthesized by our laboratory. Dil dye and BSA blocking solution.

1.2 Experimental equipment

CO ₂ cell incubator, single-channel pipette, fluorescence microscope, ultrapure water maker, inverted microscope, ultra-clean table, electronic balance, pH meter, automatic high-pressure steam sterilizer, oven.

1.3 Experimental method

The HT-29 cells were plated into a 25cm ² cell culture flask. When the cells reached 80% confluency, they were digested and collected. The sample concentration was adjusted to 1×10 ⁵ cells/ml and plated on a 24-well plate. Wash twice with ice-cold PBS. Add 1ml of 1% BSA solution and mix for 30min at 4°C. Add 10ml of 1mg/ml FITC-anti-PD-L1 polypeptide solution under dark conditions and mix for 1h at 4°C in the dark. After the incubation of the drug is completed, wash once with ice-cold PBS. Add Dil dye and wash twice with ice-cold PBS.

Use a fluorescence microscope to detect the binding of the polypeptide to PD-L1, fix the parameters, and shoot in the appropriate channel respectively, superimpose the fluorescence images of different channels, and judge the binding of the polypeptide to PD-L1.

1.4 Experimental results

The results are shown in Figure 2. The fluorescence microscopy method verified that the anti-PD-L1 polypeptide can bind to HT-29 cells and bind to the cell membrane surface.

Example 3

Detection of the effect of anti-PD-L1 polypeptide on cytokines in the co-incubation system

1.1 Experimental materials

Human colon cancer cells were purchased from (HT-29). IFN-γ detection kit and IL-2 detection kit, Ficoll reagent, sorting buffer, IL-2 cytokine, syringe.

1.2 Experimental equipment

Horizontal centrifuge, microplate reader, magnetic bead sorting column, magnetic bead sorter, inverted microscope, small centrifuge, electronic balance, pH meter, hemocytometer, ultra-clean table.

1.3 Experimental method

The HT-29 tumor cell line adopts DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{at 37°C and 5% CO 2} Cultivate to the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The effect of blocking the PD-L1 pathway of lymphocyte effector cells was demonstrated by the changes of cytokines in the co-incubation system. Before detecting the influence of peptides on the co-incubation system, it is necessary to screen the optimal incubation ratio first. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the gradients were set by equal ratio dilution, and cultured in a cell incubator at 37°C for 3 days. Use the screened ratio to set up three groups, namely the control group, the model group, and the co-incubation experiment group with peptides added. After 3 days of co-incubation, the cell supernatant was removed from each culture and the IFN of the two groups was measured. -γ, the secretion of IL-2. Statistical analysis was performed using SPSS19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, *P<0.05 means that the difference is statistically significant, **P<0.01, ***P<0.001 is that the difference is extremely significant.

1.4 Experimental results

1. Figure 3 The secretion of IFN-γ in the co-incubation system:

G1: T cells; G2: co-incubation ratio of 100:1; G3: co-incubation ratio of 80:1; G4: co-incubation ratio of 60:1; G5: co-incubation ratio of 40:1; G6: co-incubation ratio It is 20:1; G7: the co-incubation ratio is 10:1. Compared with the negative group, *P<0.05, **P<0.01, ***P<0.001.

Screen the co-incubation ratio by detecting the secretion of IFN-γ in the co-incubation system. Under comprehensive consideration, the co-incubation ratio is selected as 40:1 to detect peptide activity experiment. The experimental results are statistically significant.

2. Figure 4 Secretion of IL-2 in the co-incubation system:

G1: T cells; G2: co-incubation ratio of 100:1; G3: co-incubation ratio of 80:1; G4: co-incubation ratio of 60:1; G5: co-incubation ratio of 40:1; G6: co-incubation ratio It is 20:1; G7: the co-incubation ratio is 10:1. Compared with the negative group, *P<0.05, **P<0.01, ***P<0.001.

Screen the co-incubation ratio by detecting the secretion of IL-2 in the co-incubation system. Under comprehensive consideration, the co-incubation ratio is selected as 40:1 to detect peptide activity experiment. The experimental results are statistically significant.

3. Figure 5: The effect of peptides on IFN-γ in the co-incubation system:

G1: control group; G2: model group; G3: 100 nM anti-PD-L1 polypeptide; G4: 200 nM anti-PD-L1 polypeptide; G5: 400 nM anti-PD-L1 polypeptide; G6: 800 nM anti-PD-L1 polypeptide; G7: 1.6 μM Anti-PD-L1 polypeptide. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

The change of IFN-Pγ in the co-incubation system proved that the polypeptide can activate T cells. There is a very significant difference between the model group and the control group, indicating that the model is successful. The significant difference between the experimental group and the model group indicates that the polypeptide can successfully activate T cells in the co-incubation system.

4. Figure 6 The effect of peptides on IL-2 in the co-incubation system

G1: control group; G2: model group; G3: 100 nM anti-PD-L1 polypeptide; G4: 200 nM anti-PD-L1 polypeptide; G5: 400 nM anti-PD-L1 polypeptide; G6: 800 nM anti-PD-L1 polypeptide; G7: 1.6 μM Anti-PD-L1 polypeptide. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

The change of IL-2 in the co-incubation system proved that the polypeptide can activate T cells. There is a very significant difference between the model group and the control group, indicating that the model is successful. The significant difference between the experimental group and the model group indicates that the polypeptide can successfully activate T cells in the co-incubation system.

Example 4

Detection of the effect of anti-PD-L1 polypeptide on the apoptosis of HT-29 cells in the co-incubation system

1.1 Experimental materials

Human colon cancer cells (HT-29) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The HT-29 tumor cell line adopts DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{at 37°C and 5% CO 2} Cultivate to the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. The LDH secretion of the two groups was measured from each culture. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 1 The effect of peptides on the apoptosis of HT-29 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	12.28±0.39
Low-dose peptide group	100nM	17.19±0.25*
Peptide mid-dose group	400nM	20.74±0.30*
High-dose peptide group	1.6μΜ	24.31±0.27*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of HT-29 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 5

Detection of the effect of anti-PD-L1 polypeptide on the apoptosis of BT474 cells in the co-incubation system

1.1 Experimental materials

Breast cancer (BT474) was purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The BT474 tumor cell line was cultured in 1640 culture medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator _{containing 5% CO 2 to} Logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 2 The effect of peptides on the apoptosis of BT474 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	9.05±0.56
Low-dose peptide group	100nM	17.31±1.68*
Peptide mid-dose group	400nM	20.04±0.26*
High-dose peptide group	1.6μΜ	26.73±0.97*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of BT474 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 6

To detect the effect of anti-PD-L1 polypeptide on SKmel100 cell apoptosis in co-incubation system

1.1 Experimental materials

Melanoma cells (SKmel100) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The BT474 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C.} Logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 3 The effect of peptides on the apoptosis of SKmel100 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	6.74±0.84
Low-dose peptide group	100nM	19.25±0.18*
Peptide mid-dose group	400nM	20.68±0.75*
High-dose peptide group	1.6μΜ	27.27±0.39*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of SKmel100 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 7

Detection of the effect of anti-PD-L1 polypeptide on the apoptosis of 769-P cells in the co-incubation system

1.1 Experimental materials

Renal cell carcinoma (769-P) was purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The BT474 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C.} Logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 4 The effect of peptides on the apoptosis of 769-P cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	6.47±0.85

Low-dose peptide group	100nM	15.24±0.20*
Peptide mid-dose group	400nM	24.33±0.27*
High-dose peptide group	1.6μΜ	27.26±0.56*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of 769-P cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 8

Detection of the effect of anti-PD-L1 polypeptide on the apoptosis of H1299 cells in the co-incubation system

1.1 Experimental materials

Non-small cell lung cancer (H1299) was purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The H1299 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C.} Logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 5 The effect of peptides on the apoptosis of H1299 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	13.37±0.17
Low-dose peptide group	100nM	18.49±0.40*
Peptide mid-dose group	400nM	25.13±0.56*
High-dose peptide group	1.6μΜ	29.11±0.72*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of H1299 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 9

To detect the effect of anti-PD-L1 polypeptide on A2740 cell apoptosis in co-incubation system

1.1 Experimental materials

Ovarian cancer (A2780) was purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The H1299 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C.} Logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 6 The effect of peptides on A2740 cell apoptosis in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	13.38±0.14
Low-dose peptide group	100nM	16.84±0.63*
Peptide mid-dose group	400nM	19.57±0.59*
High-dose peptide group	1.6μΜ	22.37±0.96*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on A2780 cell apoptosis in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 10

Detection of the effect of anti-PD-L1 polypeptide on the apoptosis of DU-145 cells in the co-incubation system

1.1 Experimental materials

Prostate cancer (DU-145) was purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

DU-145 tumor cell line adopts 1640 culture medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{at 37°C and 5% CO 2} Cultivate to the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were incubated for 3 days in a 37°C cell incubator according to the previously screened ratio. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 7 The effect of peptides on the apoptosis of DU-145 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	16.86±0.37
Low-dose peptide group	100nM	19.22±0.85*
Peptide mid-dose group	400nM	24.62±0.77*

High-dose peptide group

1.6μΜ

27.54±0.59*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of DU-145 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 11

Detection of the effect of anti-PD-L1 polypeptide on the apoptosis of SCC-4 cells in the co-incubation system

1.1 Experimental materials

Oral squamous cell carcinoma (SCC-4) among head and neck squamous cell carcinomas was purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

SCC-4 tumor cell line adopts 1640 culture medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{at 37°C and 5% CO 2} Cultivate to the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 8 The effect of peptides on the apoptosis of SCC-4 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	13.46±0.84
Low-dose peptide group	100nM	17.64±0.73*
Peptide mid-dose group	400nM	21.06±0.79*
High-dose peptide group	1.6μΜ	27.16±0.95*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of SCC-4 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 12

To detect the effect of anti-PD-L1 polypeptide on the apoptosis of T24 cells in the co-incubation system

1.1 Experimental materials

Bladder cancer cells (T24) in urothelial carcinoma were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The T24 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C to} Several growth periods. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured, respectively, the control group, the experimental group and the Triton X positive group. The LDH secretion of the two experimental groups was measured from each culture. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 9 The effect of peptides on the apoptosis of T24 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	15.52±0.18
Low-dose peptide group	100nM	18.44±0.25*
Peptide mid-dose group	400nM	22.45±0.79*
High-dose peptide group	1.6μΜ	25.34±0.75*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of T24 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 13

To detect the effect of anti-PD-L1 polypeptide on HepG2 cell apoptosis in co-incubation system

1.1 Experimental materials

Hepatocellular carcinoma cells (HepG2) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The HepG2 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C to} Several growth periods. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 10 The effect of peptides on the apoptosis of HepG2 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	14.56±0.92
Low-dose peptide group	100nM	19.74±0.86*
Peptide mid-dose group	400nM	26.46±1.32*
High-dose peptide group	1.6μΜ	29.21±0.74*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of HepG2 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 14

Detection of the effect of anti-PD-L1 polypeptide on the apoptosis of L428 cells in the co-incubation system

1.1 Experimental materials

Among the lymphoma cells, Hodgkin's lymphoma cells (L428) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The L428 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C to} Several growth periods. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 11 The effect of peptides on the apoptosis of L428 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	13.64±0.74
Low-dose peptide group	100nM	17.53±0.96*
Peptide mid-dose group	400nM	19.24±2.64*
High-dose peptide group	1.6μΜ	23.64±2.78*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of L428 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 15

To detect the effect of anti-PD-L1 polypeptide on the apoptosis of MG63 cells in the co-incubation system

1.1 Experimental materials

Osteosarcoma cells (MG63) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The MG63 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C.} Several growth periods. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured, respectively, the control group, the experimental group and the Triton X positive group. The LDH secretion of the two experimental groups was measured from each culture. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 12 The effect of peptides on the apoptosis of MG63 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	5.68±0.06
Low-dose peptide group	100nM	11.12±0.35*
Peptide mid-dose group	400nM	16.35±0.83*
High-dose peptide group	1.6μΜ	19.48±1.48*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of MG63 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 16

To detect the effect of anti-PD-L1 polypeptide on the apoptosis of SF17 cells in the co-incubation system

1.1 Experimental materials

Brain tumor cells (SF17) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The MG63 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C.} Several growth periods. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 13 The effect of peptides on the apoptosis of SF17 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	5.73±0.56
Low-dose peptide group	100nM	11.36±1.08*
Peptide mid-dose group	400nM	14.75±0.03*
High-dose peptide group	1.6μΜ	17.58±0.35*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of SF17 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 17

To detect the effect of anti-PD-L1 polypeptide on the apoptosis of HTB-9 cells in the co-incubation system

1.1 Experimental materials

Bladder cancer cells (HTB-9) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The HTB-9 tumor cell line is cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C} To the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 14 The effect of peptides on the apoptosis of HTB-9 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	15.63±1.86
Low-dose peptide group	100nM	18.17±0.69*
Peptide mid-dose group	400nM	20.57±0.94*
High-dose peptide group	1.6μΜ	24.15±1.46*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of HTB-9 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 18

To detect the effect of anti-PD-L1 polypeptide on AsPc1 cell apoptosis in co-incubation system

1.1 Experimental materials

Pancreatic cancer cells (AsPc1) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The AsPc1 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C to} Several growth periods. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 15 The effect of peptides on the apoptosis of AsPc1 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	11.26±0.78
Low-dose peptide group	100nM	14.47±0.85*
Peptide mid-dose group	400nM	19.25±0.69*
High-dose peptide group	1.6μΜ	23.24±0.97*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of AsPc1 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 19

Detection of the effect of anti-PD-L1 polypeptide on the apoptosis of MS751 cells in the co-incubation system

1.1 Experimental materials

Cervical cancer cells (MS751) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The MS751 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) in an incubator _{containing 5% CO 2 at 37°C to} Several growth periods. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 16 The effect of peptides on the apoptosis of MS751 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	25.43±1.85
Low-dose peptide group	100nM	39.45±1.18*
Peptide mid-dose group	400nM	53.13±3.72*
High-dose peptide group	1.6μΜ	59.25±2.56*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of MS751 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 20

Detection of the effect of anti-PD-L1 polypeptide on U266 cell apoptosis in co-incubation system

1.1 Experimental materials

Myeloma cells (U266) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

The U266 tumor cell line was cultured in 1640 culture medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator _{containing 5% CO 2 to} Several growth periods. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured, respectively, the control group, the experimental group and the Triton X positive group. The LDH secretion of the two experimental groups was measured from each culture. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 17 The effect of peptides on the apoptosis of U266 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	4.95±0.46
Low-dose peptide group	100nM	9.85±0.95*
Peptide mid-dose group	400nM	13.46±0.68*
High-dose peptide group	1.6μΜ	16.86±0.16*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on U266 cell apoptosis in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 21

To detect the effect of anti-PD-L1 polypeptide on the apoptosis of GBC-SD cells in the co-incubation system

1.1 Experimental materials

Gallbladder cancer cells (GBC-SD) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

GBC-SD tumor cell line is cultured in 1640 culture medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), streptomycin (100mg/L) at 37℃ and 5% CO ₂ To the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. Each culture was taken out to measure the LDH secretion of the two experimental groups. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 18 The effect of peptides on the apoptosis of GBC-SD cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	11.43±0.68
Low-dose peptide group	100nM	18.65±1.75*
Peptide mid-dose group	400nM	23.58±1.30*
High-dose peptide group	1.6μΜ	27.91±0.25*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of GBC-SD cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 22

To detect the effect of anti-PD-L1 polypeptide on the apoptosis of TPC-1 cells in the co-incubation system

1.1 Experimental materials

Thyroid cancer cells (TPC-1) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

TPC-1 tumor cell line is cultured in 1640 culture medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), streptomycin (100mg/L) at 37℃ and 5% CO ₂ To logarithmic growth period The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. The LDH secretion of the two groups was measured from each culture. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 19 The effect of peptides on the apoptosis of TPC-1 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	15.54±0.38
Low-dose peptide group	100nM	19.24±0.67*
Peptide mid-dose group	400nM	20.58±0.95*
High-dose peptide group	1.6μΜ	21.43±0.26*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of TPC-1 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 23

Detect the effect of anti-PD-L1 polypeptide on NTERA-2 cl.D1 cell apoptosis in co-incubation system

1.1 Experimental materials

Testicular cancer cells (NTERA-2 cl.D1) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cytokine.

1.2 Experimental equipment

Horizontal centrifuge, single channel pipette, microplate reader, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, cell incubator.

1.3 Experimental method

NTERA-2 cl.D1 tumor cell line is cultured with 1640 culture medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), streptomycin (100mg/L) at 37℃ and 5% CO ₂ Cultivate in the box to the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. Incubate for 24 hours at 37°C in an _{incubator containing 5% CO 2.} The co-incubation reaction was used to prove the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from the blood and further extracted to obtain T cells. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and cultured in a 37°C cell incubator for 3 days. After 3 days, the two were incubated together and measured. They were the control group, the experimental group and the Triton X positive group. The LDH secretion of the two groups was measured from each culture. Statistical analysis was performed using SPSS19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.4 Experimental results

Table 20 The effect of peptides on the apoptosis of NTERA-2 cl.D1 cells in the co-incubation system

Group	dose	Cytotoxicity (%)
Control group	/	14.56±0.84
Low-dose peptide group	100nM	17.15±0.18*
Peptide mid-dose group	400nM	22.46±0.95*
High-dose peptide group	1.6μΜ	25.67±2.32*

Note: Compared with the control group, *P<0.05.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of NTERA-2 cl.D1 cells in the co-incubation system. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 24

To detect the toxicity of anti-PD-L1 peptides to C. elegans

1.1 Experimental materials

N2 ambitious C. elegans, anti-PD-L1 polypeptide, petri dish, polytryptone, agar powder, sodium hypochlorite.

1.2 Experimental method

Cultivate OP50 Escherichia coli and transfer it to 150 mL of LB medium for culture the next day. Place C. elegans on the OP50 Escherichia coli bacteria solution, and control the room temperature at 20°C to synchronize the C. elegans, and make the larvae stay in the L1 stage by lysing adults and controlling nutrition. Three concentrations of 100 nM, 400 nM, and 1.6 μM were set, and the durvalumab group and the blank group (anti-PD-L1 antibody, 68 nM) were also set as controls. Add 50 μL OP50 E. coli bacteria solution and 40 L1 larvae (50 μL) to each well of the 96-well plate. The blank group was added with 100μL/well of M9 buffer, the positive control group was added with 100μL/well of durvalumab, and the experimental group was added with 100μL/well of anti-PD-L1 polypeptide (100nM, 400nM, 1.6μM) according to the dose gradient. There are 4 replicate wells in each group and the final volume of each well is guaranteed to be 200μL. After the drug treatment is completed, first observe the number of nematodes per hole in the initial state and record it as N ₀ , then place the nematodes in an electric heating constant temperature incubator at 20°C for 48 hours, and record the number of nematodes per hole N _t at the end of 48 hours. Use SPSS 19.0 mathematical statistics software package for statistical analysis, mean±SD means. T test was used for comparison between groups, and *P<0.05 was statistically significant.

1.3 Experimental results

Table 21 The effect of peptides on the lethality of C. elegans

Group	dose	Incubation rate (%)
Blank control group	/	1.55±0.33
durvalumab group	68nM	16.23±0.85

Low-dose peptide group	100nM	1.89±0.43 #
Peptide mid-dose group	400nM	2.15±0.39 *#
High-dose peptide group	1.6μΜ	2.88±0.63 *#

Note: Compared with the blank control group, *P<0.05; compared with the durvalumab group, #P<0.05.

In order to study the acute toxicity of anti-PD-L1 polypeptides, mainly to evaluate the lethality of C. elegans. Table 21 shows that there are significant differences from the durvalumab group at each concentration. Compared with the blank control group, the anti-PD-L1 polypeptide only showed a significant difference at 1.6 μM. It can be speculated that at the experimental dose, the anti-PD-L1 polypeptide has basically no effect on the lethality of C. elegans, while durvalumab can cause certain toxicity to C. elegans.

Example 25

Detect the effect of anti-PD-L1 polypeptide on T cell killing tumor cells in vivo

1.1 Experimental materials

Balb/c nude mice, anti-PD-L1 polypeptide, puromycin, T cell sorting magnetic beads, HT-29 human colon cancer cells, IL-2 cytokines, DMEM medium, calipers.

1.2 Experimental method

Female Balb/c nude mice aged 5-6 weeks were used for in vivo experiments. HT-29 cells were transfected with plvx-pro/luciferase lentiviral plasmid and screened with 1μg/ml puromycin to establish a cell line stably expressing luciferase. Human peripheral blood T cells are separated from the blood of healthy volunteers. First, centrifugation is used to separate the human peripheral blood mononuclear cell layer, and then CD3 magnetic bead sorting technology is used to obtain T cells. Add IL-2 (20ng/ml) and human T cell activator (CD3/CD28 particles, particles: T cells=1:1). On the 5th day of tumor cell culture, human T cells were added to HT-29 cells stably expressing luciferase for a total of 3 days. Then, each mouse was injected subcutaneously with 2×10 ⁶ HT-29-luc and 5×10 ⁵ human T cells, with a total volume of 0.1 ml, and the injection site was the lateral armpit of the mouse. Anti-PD-L1 polypeptide (4mg/kg) and durvalumab (anti-PD-L1 antibody, 0.1mg/kg) were injected subcutaneously every two days. Only tumor cells were injected, and mice treated with anti-PD-L1 polypeptide (4 mg/kg) served as a control group. The length and width of the tumor were measured with a caliper, and the tumor volume was calculated (tumor volume = 1/2×a×b ² , a represents the length of the tumor, and b represents the width of the tumor). Tumor size can also be detected by detecting the bioluminescence of the tumor site with the IVIS Lumina II system (PerkinElmer), which is detected once a week for a total of 5 times. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. The results are expressed as mean±SD. T test was used for comparison between groups, *P<0.05 means that the difference is statistically significant, **P<0.01, ***p<0.001 is that the difference is extremely significant.

1.3 Experimental results

It can be seen from Figure 7 that when human T cells and human HT-29 cells are injected at the same time, the anti-PD-L1 polypeptide can increase the killing effect of human T cells on tumor cells and inhibit tumor growth. The third group of injection of anti-PD-L1 antibody durvalumab and the fourth group of injection of anti-PD-L1 polypeptide (4mg/kg) both significantly inhibited the proliferation of HT-29 cells in mice.

It can be seen from Figure 8 that when human T cells and human HT-29 cells are injected at the same time, the anti-PD-L1 polypeptide can increase the killing effect of human T cells on tumor cells and inhibit tumor growth. The third group of injection of anti-PD-L1 antibody durvalumab and the fourth group of injection of anti-PD-L1 polypeptide (4mg/kg) both significantly inhibited the proliferation of HT-29 cells in mice.

Claims (7)

1. A PD-L1 antagonist polypeptide, characterized in that: the amino acid sequence of the polypeptide includes IYLCGAISLHPKAKIEECPGA (CC), or one or more amino acids have been deleted, replaced, or added on the basis of a polypeptide that still has an immunosuppressive effect , Or a pharmaceutically acceptable salt of the polypeptide.
2. The PD-L1 antagonist polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide is IYLCGAISLHPKAKIEECPGA (C-C), or a pharmaceutically acceptable salt thereof.
3. Use of the polypeptide described in claim 1 or 2 in the preparation of a drug for preventing or treating cancer.
4. The application according to claim 3, characterized in that: the application is realized by the specific binding of the polypeptide and PD-L1.
5. The application according to claim 3 or 4, characterized in that the cancer is renal cell carcinoma, ovarian cancer, head and neck cancer, prostate cancer, breast cancer, colon cancer, non-small cell lung cancer, urothelial cancer, liver cancer, Lymphoma, osteosarcoma, brain tumor, bladder cancer, pancreatic cancer, cervical cancer, myeloma, thyroid cancer, gallbladder cancer, salivary gland cancer, testicular cancer, or melanoma.
6. A compound characterized in that one or more pharmaceutically acceptable excipients are added to the polypeptide of claim 1 or 2.
7. The compound according to claim 6, characterized in that the auxiliary materials include conventional diluents, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants and stabilizers in the pharmaceutical field.

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