CN110746493A

CN110746493A - PD-L1 antagonist polypeptide and application thereof

Info

Publication number: CN110746493A
Application number: CN201910842112.4A
Authority: CN
Inventors: 徐寒梅; 胡加亮; 林炳静; 刘晨
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-09-06
Filing date: 2019-09-06
Publication date: 2020-02-04
Also published as: WO2021043339A1

Abstract

The invention discloses a PD-L1 antagonist polypeptide and application thereof, belonging to the field of biological medicine. The polypeptides of the invention have the amino acid sequence IYLCGAISLHPKAKIEECPGA (C-C) or pharmaceutically acceptable salts thereof. The polypeptide can specifically recognize PD-L1 on the cell surface, mediate the specific killing of PD-L1 positive cells, simultaneously can prevent an immunosuppressive signal path of PD-1/PD-L1, eliminate the immunosuppressive action of tumors, and thus achieve the specific killing action.

Description

PD-L1 antagonist polypeptide and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a polypeptide with immune checkpoint antagonistic activity and application thereof.

Background

The tumor immunotherapy is divided into a cell immunotherapy and an immune checkpoint inhibitor, the effect of the monoclonal antibody drug and the chimeric antigen receptor T (CAR-T) cell immunotherapy is gradually accepted, and the concept of cancer immunotherapy is more and more deep. The former is to modify the immune cells in the body of the patient in vitro, so that the immune cells have more effective and accurate immune capacity to the tumor cells, and the modified immune cells are transfused back to the tumor patient to play a role in directionally killing the tumor cells, and the therapy comprises LAK, DC, CAR-T, NK, CAR-NK therapy and the like. The immune checkpoint (immune Check-point) is also called immune checkpoint, the most important aspect of the current tumor immune drug therapy is tumor immune checkpoint inhibitor, and the immune checkpoint inhibitor relieves the tolerance/shielding effect of tumor on immunity, so that immune cells can know tumor cells again and attack the tumor cells. Current research has focused primarily on CTLA-4, PD-1 and PD-L1 molecules.

Throughout the course of the immune response, dual signaling pathways are required for T cell activation. Firstly, recognizing antigen presenting cell APC or MHC molecule antigen peptide complex on the surface of tumor cell through T cell receptor TCR specificity on the surface of T cell; second, co-stimulatory molecules on the APC or tumor cell bind to co-stimulatory receptors on the T cell, activating or inhibiting the T lymphocyte. Costimulatory molecules on T cells mainly include the coactivator 4-1BB, CD27, CD28, OX40, ICOS, and the coactivator CTLA4, PD-1. Programmed Death receptor 1 (PD-1) is an important immunosuppressive transmembrane protein expressed predominantly on activated T cells, 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 2B 4.11. Other members of the family, such as CTLA-4 and BTLA, were found to be differentially expressed 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, and the extracellular domain is two immunoglobulin constant regions (IgC) and IgV-like domains, and is mainly expressed on the membrane surface of mature hematopoietic cells such as CD4+ T cells, CD8+ T cells, monocytes, macrophages, B cells, dendritic cells, and some non-hematopoietic cells such as endothelial cells, pancreatic islet cells, mast cells, etc. PD-L2 is a transmembrane protein composed of 274 amino acid residues, has high similarity with PD-L1, but has certain difference, such as the in vivo tissue distribution of PD-L2 has limitation, and is only expressed on the membrane surface of macrophages, dendritic cells and some B cell subsets. In addition to its inducible expression by proinflammatory 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, and the like, and can enhance the metastatic potential of tumors, leading to increased patient mortality. Binding to PD-1, which is highly expressed on tumor-infiltrating T lymphocytes, indicates that the highly expressed PD-1/PD-L1 signaling pathway mediates immunosuppression of tumors. The binding of these two ligands to PD-1 results in tyrosine phosphorylation of the intracellular structure of PD-1 and recruitment of the tyrosine phosphatase SHP-2, thereby reducing phosphorylation of the TCR signaling pathway, reducing activation signals downstream of the TCR pathway and activation and proliferation of T cells and production of regulatory cytokines, and simultaneously, blocking the cells in the G0/G1 phase to inhibit T cell proliferation, prevent their differentiation into plasma cells, and induce apoptosis of T cells. This avoids the immortalization of T cells and maintains them in homeostasis. The negative feedback regulation effect of the T cells involved in the PD-1/PD-L1 signal channel plays a crucial role in clearing antigens and maintaining the balance of the organism. Inhibition of the PD-1 and PD-L1 pathways thus accelerates and potentiates autoimmunity.

So far, the inhibition of the PD-1/PD-L1 signal pathway is used for realizing tumor treatment, and good effect is obtained in clinical stage. The antibodies to PD-L1 include atezolizumab, Roche/Takken gene, durvalumab, Aslicon, and avelumab, EMD serono/Pfizer. Has been approved by the FDA for treating non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma, etc. Other clinical studies include BMS936559 in BMS, SHR-1210 in Henry for the antibody against PD-1 made in China, BGB-A317 in Baiji, JS001 in Junshi in Taizhou, and the like, which have great potential in tumor immunotherapy for inhibiting the PD-1/PD-L1 signaling pathway.

Disclosure of Invention

1. Problems to be solved

The polypeptide acts on PD-L1 to inhibit a PD-1/PD-L1 pathway, and has good tumor inhibition effect and great development prospect through verification of various experimental models.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

a PD-L1 antagonist polypeptide, wherein: the amino acid sequence of the polypeptide comprises IYLCGAISLHPKAKIEECPGA (C-C), or a polypeptide which has the effect of relieving immunosuppression after one or more amino acids are deleted, replaced or added on the basis of the amino acid sequence, or a pharmaceutically acceptable salt of the polypeptide.

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

The application of the polypeptide in preparing a medicament 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 use 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 complex comprising the polypeptide of claim 1 or 2 and one or more pharmaceutically acceptable excipients.

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

The medicine of the invention can be used for treating various drug delivery modes including subcutaneous or intramuscular injection, intravenous injection or intravenous drip such as injection and dry powder injection, oral drug delivery such as pills, capsules and the like, and nasal spray.

In the present clinical application, the immune checkpoint inhibitor is mainly monoclonal antibody, but the antibody has the problems of high treatment cost and the like in the using process. Therefore, the invention adopts the computer three-dimensional simulation technology, and the independently designed polypeptide structure has good tumor inhibition effect on the basis of analyzing a large number of traditional structures and performing pharmacological experiments. The invention utilizes a three-dimensional structure to independently design the immune checkpoint antagonistic polypeptide which has a brand-new structure and acts on PD-L1.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

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

(2) the adverse reaction and toxic and side reaction of the polypeptide are weak;

(3) the polypeptide has good effect of eliminating immunosuppression in vivo and in vitro models, and has the specific effects of obviously improving the activity of T cells, eliminating immunosuppression, preventing tumor cells from escaping and further generating tumor inhibition effect.

Drawings

FIG. 1, flow cytometry detection of binding of HT-29 to FITC-anti-PD-L1 polypeptide; a is a negative control, B, C, D is the binding rate of HT-29 to 15nM, 150nM, 1.5. mu.M FITC-conjugated anti-PD-L1 polypeptide; 26.3%, 58.0% and 80.4%, respectively;

FIG. 2, fluorescence microscopy of binding of HT-29 to FITC-anti-PD-L1 polypeptide; a: a cell membrane; b: FITC-anti-PD-L1 polypeptide; c: merge;

FIG. 3 secretion of IFN-. gamma.in Co-incubation System

G1: a T cell; g2: the co-incubation ratio is 100: 1; g3: the co-incubation ratio is 80: 1; g4: the co-incubation ratio was 60: 1; g5: the co-incubation ratio is 40: 1; g6: the co-incubation ratio was 20: 1; g7: the co-incubation ratio was 10: 1. P <0.05, P < 0.01, P < 0.001;

FIG. 4 secretion of IL-2 in Co-incubation System

FIG. 5 Effect of the Polypeptides on IFN-. gamma.in Co-incubation systems

G1: a control group; g2: a model group; g3: 100nM anti-PD-L1 polypeptide; g4: 200nM anti-PD-L1 polypeptide; g5: 400nM anti-PD-L1 polypeptide; g6: 800nM anti-PD-L1 polypeptide; g7: 1.6 μ Μ anti PD-L1 polypeptide. P <0.05, P < 0.01, P < 0.001;

FIG. 6 Effect of the Polypeptides on IL-2 in Co-incubation systems

FIG. 7 shows that the first group is a negative control group for detecting the inhibition of tumor cell growth by anti-PD-L1 polypeptide through bioluminescence; the second group was given with HT-29 cells alone, and anti-PD-L1 polypeptide; the third group was the subcutaneous injection of anti-PD-L1 antibody durvalumab (0.1mg/kg) after the simultaneous injection of human T cells and human HT-29 cells; the fourth group was a subcutaneous injection of anti-PD-L1 polypeptide (4mg/kg) following simultaneous injection of human T cells and human HT-29 cells;

FIG. 8 shows that the first group is a negative control group for detecting the inhibition of tumor cell growth by anti-PD-L1 polypeptide by tumor volume; the second group was given with HT-29 cells alone, and anti-PD-L1 polypeptide; the third group was the subcutaneous injection of anti-PD-L1 antibody durvalumab (0.1mg/kg) after the simultaneous injection of human T cells and human HT-29 cells; the fourth group was a subcutaneous injection of anti-PD-L1 polypeptide (4mg/kg) following simultaneous injection of human T cells and human HT-29 cells.

Detailed Description

The invention is further described with reference to specific examples. The following description is only exemplary of the present invention, and is not intended to limit the present invention in any way, and those skilled in the art can modify the present invention by applying the above-described technical disclosure to equivalent embodiments with equivalent modifications. Any simple modifications or equivalent changes made to the following examples according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

In the examples, the activity of the polypeptide (IYLCGAISLHPKAKIEECPGA (C-C)) for preventing cancer and/or inhibiting tumor activity was mainly studied. The polypeptide is synthesized by Gill Biochemical (Shanghai) Co., Ltd, and the purity is more than 95%.

Example 1

Detection assay for specific binding of a polypeptide to a target at the cellular level

The flow cytometry is a technology for exciting a single unidirectional flowing particle by a laser beam, detecting scattered light of the particle and a fluorescent marker carried by the particle, and thus rapidly detecting and analyzing a plurality of physical characteristics of the single particle and sorting cells. Is widely applied to scientific research and clinical medical examination, and is the most advanced cell quantitative analysis technology in the present generation. The invention relates to a polypeptide with cancer prevention and/or tumor inhibition activity, which has a main target point of PD-L1, and the research connects a designed anti-PD-L1 polypeptide with a FITC fluorescent marker, and reflects the binding capacity of the polypeptide with cell surface PD-L1 on a cell level according to the binding rate of cells and a FITC-anti-PD-L1 polypeptide solution.

1.1 Experimental materials

Human colon cancer cells (HT-29) were purchased from ATCC. The anti-PD-L1 polypeptide is synthesized autonomously in the laboratory, and the purity is over 95 percent. BSA blocking solution. CO 2₂Cell incubator, flow cytometer, inverted microscope, liquid nitrogen tank, super clean bench, small centrifuge, electronic balance, pH meter, automatic high pressure steam sterilizer, oven, ultrapure water producing instrument, digital display constant temperature water bath.

1.2 Experimental methods:

HT-29 cells were plated in 6-well plates and harvested by digestion when the cells reached 80% confluency. Washed twice with ice-cold PBS. 1ml of a 1% BSA solution was added, fixed on a rotary mixer, and mixed at 4 ℃ for 30 min. Mu.l of a 1mg/ml FITC-anti-PD-L1 polypeptide solution was added under exclusion of light, fixed on a rotary mixer, and mixed at 4 ℃ for 1h in the dark. After the incubation, the supernatant was discarded after centrifugation at 800rpm for 5 min. Wash once with ice-cold PBS. Adjusting the cell concentration with PBS to adjust the sample concentration1×10⁶cells/ml. 3 replicates per cell were prepared, 0.5ml per sample.

Detecting the combination of the polypeptide and the PD-L1 by using a flow cytometer, and sequentially turning on a stabilized voltage power supply, a transformer, a flow cytometer host, a computer and a printer from left to right. The flow drawer was opened and purified water was added to the sheath fluid bucket until 2/3 was reached. The waste solution was decanted and 200ml of sodium hypochlorite solution containing 10% available chlorine was added. The hydraulic valve is adjusted to the pressure position to remove air bubbles between the flow line and the filter. The sample tube was removed and PRIME function was performed twice, 1ml PBS, HINGRUN 2 min. The measurement is started and the sample is analyzed. After the measurement is completed, the sample holder is moved to the left and 1ml of FACSClean is evacuated. The sample holder was then returned to the positive position for HING RUN 5 min. FACS Clean was changed to purified water, the sample holder was moved to the left, 1ml was extracted under vacuum, the sample holder was returned to the right, HING RUN 10 min. The sample tube was removed and PRIME function was performed twice per Standby. Finally, 1ml of purified water is left in the flow test tube. The flow cytometer was turned off after fan cooling the laser, and was operated for 20min according to Standby. And exiting the program and closing the computer.

1.3 results of the experiment

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

Example 2

Qualitative detection experiment-fluorescence imaging experiment for specific binding of polypeptide to target at cellular level

1.1 Experimental materials

Human colon cancer cells (HT-29) were purchased from ATCC. anti-PD-L1 polypeptide, synthesized autonomously in this laboratory. Dil dye and BSA blocking solution.

1.2 Experimental instruments

CO₂Cell culture case, single channel pipettor, fluorescence microscope, ultrapure water manufacture appearance, inverted microscope, superclean platform, electronic balance, pH meter, full-automatic high pressure steam sterilization pot, oven.

1.3 Experimental methods

Plating HT-29 cells into 25cm²After the cells reached 80% confluency in the cell culture flask, they were collected by digestion and the sample concentration was adjusted to 1X 10⁵cells/ml were plated on 24-well plates. Washed twice with ice-cold PBS. 1ml of a 1% BSA solution was added thereto, and mixed at 4 ℃ for 30 min. 10ml of 1mg/ml FITC-anti-PD-L1 polypeptide solution was added under exclusion of light and mixed at 4 ℃ for 1 hour in the dark. After incubation with the drug was complete, one wash with ice-cold PBS. Dil dye was added and washed twice with ice-cold PBS.

Detecting the binding of the polypeptide and PD-L1 by using a fluorescence microscope, fixing parameters, respectively shooting in proper channels, superposing fluorescence images of different channels, and judging the binding of the polypeptide and PD-L1.

1.4 results of the experiment

The results are shown in FIG. 2, which again demonstrates that the anti-PD-L1 polypeptide binds to HT-29 cells and to the cell membrane surface by fluorescence microscopy.

Example 3

Detecting the effect of anti-PD-L1 polypeptide on cell factor in co-incubation system

1.1 Experimental materials

Human colon cancer cells were purchased from (HT-29). IFN-gamma detection kit, IL-2 detection kit, Ficoll reagent, sorting buffer, IL-2 cell factor and injector.

1.2 Experimental instruments

Horizontal centrifuge, ELIASA, magnetic bead sorting column, magnetic bead sorter, inverted microscope, small centrifuge, electronic balance, pH meter, blood count board, superclean bench.

1.3 Experimental methods

HT-29 tumor cell line adopts DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. The effect of blocking the PD-L1 pathway of lymphocyte effector cells was demonstrated by changes in cytokines in the co-incubation system. Under inspectionThe optimal incubation ratio needs to be screened first before the effect of the polypeptide on the co-incubation system can be determined. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells were cultured, each gradient was set by equal dilution, and the cells were cultured in a 37 ℃ cell incubator for 3 days. Three groups are set according to the screened proportion, namely a control group, a model group and a polypeptide experimental group under the condition of co-incubation, cell supernatant is taken out from each culture after 3 days of co-incubation, and the secretion conditions of IFN-gamma and IL-2 of the experimental groups of the two groups are measured. Statistical analysis was performed using SPSS19.0(SPSS inc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 marked statistical differences in differences<0.01，***P<0.001 is a very significant statistical difference.

1.4 results of the experiment

1. FIG. 3 secretion of IFN-. gamma.in the Co-incubation System:

g1: a T cell; g2: the co-incubation ratio is 100: 1; g3: the co-incubation ratio is 80: 1; g4: the co-incubation ratio was 60: 1; g5: the co-incubation ratio is 40: 1; g6: the co-incubation ratio was 20: 1; g7: the co-incubation ratio was 10: 1. P <0.05, P < 0.01, P <0.001, to negative groups.

The co-incubation ratio was screened by detecting IFN-. gamma.secretion in the co-incubation system. The co-incubation ratio is selected to be 40:1 under comprehensive consideration, and the activity test of the polypeptide is carried out. The experimental results have statistical significance.

2. FIG. 4 secretion of IL-2 in the Co-incubation System:

The co-incubation ratio was screened by detecting the secretion of IL-2 in the co-incubation system. The co-incubation ratio is selected to be 40:1 under comprehensive consideration, and the activity test of the polypeptide is carried out. The experimental results have statistical significance.

3. FIG. 5 Effect of the polypeptide on IFN-. gamma.in Co-incubation systems:

g1: a control group; g2: a model group; g3: 100nM anti-PD-L1 polypeptide; g4: 200nM anti-PD-L1 polypeptide; g5: 400nM anti-PD-L1 polypeptide; g6: 800nM anti-PD-L1 polypeptide; g7: 1.6 μ Μ anti PD-L1 polypeptide. P <0.05, P < 0.01, P <0.001, compared to model group.

The change of IFN-P gamma in the co-incubation system proves that the polypeptide can activate T cells. The model group and the control group have very significant difference, which indicates that the molding is successful. The very significant difference between the experimental and model groups indicates that the polypeptide was able to successfully activate T cells in the co-incubation system.

4. FIG. 6 Effect of the Polypeptides on IL-2 in Co-incubation systems

Changes in IL-2 in the co-incubation system demonstrated that the polypeptide was able to activate T cells. The model group and the control group have very significant difference, which indicates that the molding is successful. The very significant difference between the experimental and model groups indicates that the polypeptide was able to successfully activate T cells in the co-incubation system.

Example 4

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

1.1 Experimental materials

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

1.2 Experimental instruments

Horizontal centrifuge, single channel pipettor, ELIASA, magnetic bead sorting rack, small centrifuge, electronic balance, pH meter, cell counter, and cell culture box.

1.3 Experimental methods

HT-29 tumor cell line adopts DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. Adjusting the cell concentration to the desired concentrationAnd seeded in 96-well plates. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. The effect of blocking the PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis was demonstrated using a co-incubation reaction. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(SPSS inc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 1 Effect of Polypeptides on apoptosis of HT-29 cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	12.28±0.39
Polypeptide low dose group	100nM	17.19±0.25*
			Polypeptide medium dose group	400nM	20.74±0.30*
High dose group of polypeptides	1.6μΜ	24.31±0.27*

Note: p <0.05 compared to control.

Effect of LDH changes in the Co-incubation System on 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 the T cells on the tumor cells.

Example 5

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

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

BT474 tumor cell line is cultured in 1640 culture medium containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured, respectively, as a control group, an experimental group and a Triton X positive group, and two experimental groups were taken out from each cultureLDH secretion profile of the group. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 2 Effect of Polypeptides on apoptosis of BT474 cells in Co-incubation System

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	9.05±0.56
Polypeptide low dose group	100nM	17.31±1.68*
			Polypeptide medium dose group	400nM	20.04±0.26*
High dose group of polypeptides	1.6μΜ	26.73±0.97*

Note: p <0.05 compared to control.

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

Example 6

Detecting influence 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 cell factor.

1.2 Experimental instruments

1.3 Experimental methods

BT474 tumor cell line is prepared by culturing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) in DMEM at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 3 Effect of the Polypeptides on apoptosis of SKmel100 cells in a Co-incubation System

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	6.74±0.84
Polypeptide low dose group	100nM	19.25±0.18*
			Polypeptide medium dose group	400nM	20.68±0.75*
High dose group of polypeptides	1.6μΜ	27.27±0.39*

Note: p <0.05 compared to control.

And (3) the change of the LDH in the co-incubation system reflects the influence of the polypeptide on the apoptosis of SKmel100 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 the T cells on the tumor cells.

Example 7

Detecting the effect of anti-PD-L1 polypeptide on 769-P cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

1.4 results of the experiment

TABLE 4 Effect of Polypeptides on apoptosis of 769-P cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	6.47±0.85
Polypeptide low dose group	100nM	15.24±0.20*
			Polypeptide medium dose group	400nM	24.33±0.27*
High dose group of polypeptides	1.6μΜ	27.26±0.56*

Note: p <0.05 compared to control.

Effect of LDH changes in Co-incubation systems on 769-P apoptosis in Co-incubation systems. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of the T cells on the tumor cells.

Example 8

Detecting the effect of anti-PD-L1 polypeptide on H1299 cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

The H1299 tumor cell line adopts DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) and contains 5% CO at 37 DEG C₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 5 Effect of polypeptides on H1299 cell apoptosis in Co-incubation systems

Note: p <0.05 compared to control.

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

Example 9

Detecting the influence of anti-PD-L1 polypeptide on apoptosis of A2740 cells in a co-incubation system

1.1 Experimental materials

Ovarian cancer (a2780) was purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cell factor.

1.2 Experimental instruments

1.3 Experimental methods

1.4 results of the experiment

TABLE 6 Effect of Polypeptides on apoptosis of A2740 cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	13.38±0.14
Polypeptide low dose group	100nM	16.84±0.63*
			Polypeptide medium dose group	400nM	19.57±0.59*
High dose group of polypeptides	1.6μΜ	22.37±0.96*

Note: p <0.05 compared to control.

The change of LDH in the co-incubation system reflects the effect of the polypeptide on apoptosis of A2780 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 the T cells on the tumor cells.

Example 10

Detecting the influence of anti-PD-L1 polypeptide on the apoptosis of DU-145 cells in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

DU-145 tumor cell line was obtained by culturing 1640 medium containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Extracting peripheral blood from bloodAnd (5) further extracting the lymphocytes to obtain T cells. Meanwhile, tumor cells are cultured, and the cells are incubated in a cell culture box at 37 ℃ for static culture for 3 days according to the proportion screened out before. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 7 Effect of the Polypeptides on apoptosis of DU-145 cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	16.86±0.37
Polypeptide low dose group	100nM	19.22±0.85*
			Polypeptide medium dose group	400nM	24.62±0.77*
High dose group of polypeptides	1.6μΜ	27.54±0.59*

Note: p <0.05 compared to control.

The change of LDH in the co-incubation system reflects the influence 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 the T cells on the tumor cells.

Example 11

Detection of Effect of anti-PD-L1 Polypeptides on SCC-4 apoptosis in Co-incubation systems

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

The SCC-4 tumor cell line adopts 1640 culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 8 Effect of Polypeptides on SCC-4 cell apoptosis in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	13.46±0.84
Polypeptide low dose group	100nM	17.64±0.73*
			Polypeptide medium dose group	400nM	21.06±0.79*
High dose group of polypeptides	1.6μΜ	27.16±0.95*

Note: p <0.05 compared to control.

Effect of LDH Change in Co-incubation on SCC-4 apoptosis in Co-incubation systems. The significant difference between the experimental group and the control group indicates that the polypeptide can promote the killing effect of the T cells on the tumor cells.

Example 12

Detecting the influence of anti-PD-L1 polypeptide on T24 cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

The T24 tumor cell line is prepared from DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 9 Effect of Polypeptides on apoptosis of T24 cells in Co-incubation System

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	15.52±0.18
Polypeptide low dose group	100nM	18.44±0.25*
			Polypeptide medium dose group	400nM	22.45±0.79*
High dose group of polypeptides	1.6μΜ	25.34±0.75*

Note: p <0.05 compared to control.

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

Example 13

Detecting the influence of anti-PD-L1 polypeptide on HepG2 cell apoptosis in a co-incubation system

1.1 Experimental materials

Liver cancer cells (HepG2) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cell factor.

1.2 Experimental instruments

1.3 Experimental methods

HepG2 tumor cell line was prepared by culturing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) in DMEM at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 10 Effect of Polypeptides on apoptosis of HepG2 cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	14.56±0.92
Polypeptide low dose group	100nM	19.74±0.86*
			Polypeptide medium dose group	400nM	26.46±1.32*
High dose group of polypeptides	1.6μΜ	29.21±0.74*

Note: p <0.05 compared to control.

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

Example 14

Detecting the effect of anti-PD-L1 polypeptide on L428 cell apoptosis in a co-incubation system

1.1 Experimental materials

Hodgkin lymphoma cells among lymphoma cells (L428) were purchased from ATCC. LDH detection kit, sorting buffer, IL-2 cell factor.

1.2 Experimental instruments

1.3 Experimental methods

The L428 tumor cell line is prepared from DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate 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,t cells are obtained by further extraction. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 11 Effect of Polypeptides on L428 cell apoptosis in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	13.64±0.74
Polypeptide low dose group	100nM	17.53±0.96*
			Polypeptide medium dose group	400nM	19.24±2.64*
High dose group of polypeptides	1.6μΜ	23.64±2.78*

Note: p <0.05 compared to control.

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

Example 15

Detecting the influence of anti-PD-L1 polypeptide on MG63 cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

MG63 tumor cell line is prepared from DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100MG/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 12 Effect of Polypeptides on apoptosis of MG63 cells in Co-incubation System

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	5.68±0.06
Polypeptide low dose group	100nM	11.12±0.35*
			Polypeptide medium dose group	400nM	16.35±0.83*
High dose group of polypeptides	1.6μΜ	19.48±1.48*

Note: p <0.05 compared to control.

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

Example 16

Detecting the influence of anti-PD-L1 polypeptide on SF17 cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

1.4 results of the experiment

TABLE 13 Effect of Polypeptides on apoptosis of SF17 cells in Co-incubation System

Note: p <0.05 compared to control.

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

Example 17

Detecting the influence of anti-PD-L1 polypeptide on HTB-9 cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

HTB-9 tumor cell line is prepared from DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 14 Effect of Polypeptides on apoptosis of HTB-9 cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	15.63±1.86
Polypeptide low dose group	100nM	18.17±0.69*
			Polypeptide medium dose group	400nM	20.57±0.94*
High dose group of polypeptides	1.6μΜ	24.15±1.46*

Note: p <0.05 compared to control.

The effect of LDH changes in the co-incubation system 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 the T cells on the tumor cells.

Example 18

Detecting the influence of anti-PD-L1 polypeptide on AsPc1 cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

The AsPc1 tumor cell line is prepared from DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 15 Effect of Polypeptides on apoptosis of AsPc1 cells in Co-incubation System

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	11.26±0.78
Polypeptide low dose group	100nM	14.47±0.85*
			Polypeptide medium dose group	400nM	19.25±0.69*
High dose group of polypeptides	1.6μΜ	23.24±0.97*

Note: p <0.05 compared to control.

The change of LDH in the co-incubation system reflects the effect of polypeptide on the apoptosis of AsPc1 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 the T cells on the tumor cells.

Example 19

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

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

MS751 tumor cell line is prepared from DMEM culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. Adjusting the cell concentration toAt required concentrations, the cells were plated in 96-well plates. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 16 Effect of Polypeptides on apoptosis of MS751 cells in Co-incubation System

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	25.43±1.85
Polypeptide low dose group	100nM	39.45±1.18*
			Polypeptide medium dose group	400nM	53.13±3.72*
High dose group of polypeptides	1.6μΜ	59.25±2.56*

Note: p <0.05 compared to control.

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

Example 20

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

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

U266 tumor cell line adopts the culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L)1640 at 37 ℃ and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two were incubated and measured, respectively, as a control group, an experimental group and a Triton X-positive group, and two of them were taken out from each cultureLDH secretion in the experimental groups. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 17 Effect of Polypeptides on apoptosis of U266 cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	4.95±0.46
Polypeptide low dose group	100nM	9.85±0.95*
			Polypeptide medium dose group	400nM	13.46±0.68*
High dose group of polypeptides	1.6μΜ	16.86±0.16*

Note: p <0.05 compared to control.

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

Example 21

Detecting the influence of anti-PD-L1 polypeptide on GBC-SD cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

GBC-SD tumor cell line adopts culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L)1640 at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 18 Effect of Polypeptides on apoptosis of GBC-SD cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	11.43±0.68
Polypeptide low dose group	100nM	18.65±1.75*
			Polypeptide medium dose group	400nM	23.58±1.30*
High dose group of polypeptides	1.6μΜ	27.91±0.25*

Note: p <0.05 compared to control.

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

Example 22

Detecting the influence of anti-PD-L1 polypeptide on the apoptosis of TPC-1 cells in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

TPC-1 tumor cell line is prepared from culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L)1640 at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(SPSS inc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 19 Effect of Polypeptides on apoptosis of TPC-1 cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	15.54±0.38
Polypeptide low dose group	100nM	19.24±0.67*
			Polypeptide medium dose group	400nM	20.58±0.95*
High dose group of polypeptides	1.6μΜ	21.43±0.26*

Note: p <0.05 compared to control.

The change of LDH in the co-incubation system reflects the influence 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 the T cells on the tumor cells.

Example 23

Detecting the influence of anti-PD-L1 polypeptide on NTERA-2cl.D1 cell apoptosis in a co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

1.3 Experimental methods

The NTERA-2cl. D1 tumor cell line is prepared from culture medium containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L)1640 at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, containing5％CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were used to demonstrate the effect of blocking the PD-1/PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. Meanwhile, tumor cells are cultured, the cells are co-incubated according to the proportion screened out before, and the cells are statically cultured for 3 days in a cell culture box at 37 ℃. After 3 days, the two groups were incubated and measured for LDH secretion in the control, experimental and Triton X positive groups, respectively, and the two experimental groups were taken out from each culture. Statistical analysis was performed using SPSS19.0(SPSS inc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

1.4 results of the experiment

TABLE 20 Effect of Polypeptides on apoptosis of NTERA-2cl. D1 cells in Co-incubation systems

Group of	Dosage form	Cytotoxicity (%)
			Control group	/	14.56±0.84
Polypeptide low dose group	100nM	17.15±0.18*
			Polypeptide medium dose group	400nM	22.46±0.95*
High dose group of polypeptides	1.6μΜ	25.67±2.32*

Note: p <0.05 compared to control.

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

Example 24

Detection of toxicity of anti-PD-L1 polypeptide on caenorhabditis elegans

1.1 Experimental materials

N2 caenorhabditis elegans, PD-L1 resisting polypeptide, a culture dish, polypeptone, agar powder and sodium hypochlorite.

1.2 Experimental methods

OP50 E.coli was cultured and transferred every other day to 150mL of LB medium for culture. Placing caenorhabditis elegans on OP50 Escherichia coli liquid, controlling room temperature at 20 deg.C for synchronization of caenorhabditis elegans, and allowing larva to stay in L1 stage by cracking into adult and controlling nutrition. Three concentrations of 100nM, 400nM, 1.6. mu.M were set, and a Durvalumab group and a blank group (anti-PD-L1 antibody, 68nM) were set in addition as controls. Each well of the 96-well plate was initially charged with 50. mu.L of OP50 E.coli strain and 40L 1 larvae (50. mu.L). The blank group was added 100. mu.L/well of M9 buffer, the positive control group was added 100. mu.L/well of Durvalumab, and the experimental group was added 100. mu.L/well of anti-PD-L1 polypeptide (100nM, 400nM, 1.6. mu.M) according to a dose gradient. Each set of 4 duplicate wells and ensuring a final volume of 200 μ L per well. After the drug adding treatment is finished, firstly observing the number of nematodes per hole in an initial state, and recording the number as N₀Then placing the nematodes in an electric heating constant temperature incubator at 20 ℃ for culturing for 48h, and recording the number N of the nematodes in each hole after 48h_t. Statistical analysis was performed using the SPSS19.0 mathematical statistics software package, mean + -SD. The comparison between groups was performed by T test, P<0.05 significant systemThe difference was counted.

1.3 results of the experiment

Effect of the TABLE 21 Polypeptides on the mortality of C.elegans

Group of	Dosage form	Hatching rate (%)
			Blank control group	/	1.55±0.33
Durvalumab group	68nM	16.23±0.85
			Polypeptide low dose group	100nM	1.89±0.43^#
Polypeptide medium dose group	400nM	2.15±0.39^*#
			High dose group of polypeptides	1.6μΜ	2.88±0.63^*#

Note: p <0.05 compared to placebo; compared to the durvalumab group, # P < 0.05.

To investigate the acute toxicity of the anti-PD-L1 polypeptide, C.elegans lethality was evaluated primarily. Table 21 shows significant differences from the durvalumab group at each concentration. The anti-PD-L1 polypeptide showed significant differences only at 1.6 μ M compared to the blank control group. It can be speculated that the anti-PD-L1 polypeptide has no influence on the mortality of the caenorhabditis elegans under the experimental dosage, while the durvalumab can cause certain toxicity to the caenorhabditis elegans.

Example 25

Detecting the effect of anti-PD-L1 polypeptide on the activity of T cells on 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 culture medium and calipers.

1.2 Experimental methods

5-6 weeks of female Balb/c nude mice were used for in vivo testing. HT-29 cells were transfected with plvx-pro/luciferase lentiviral plasmid and screened with 1. mu.g/ml puromycin, establishing a cell line stably expressing luciferase. Human peripheral blood T cells are separated from blood of healthy volunteers, a human peripheral blood mononuclear cell layer is obtained by centrifugation, then T cells are obtained by utilizing a CD3 magnetic bead sorting technology, and IL-2(20ng/ml) and a human T cell activator (CD3/CD28 particles, particles: T cells are 1:1) are added into a culture medium in order to obtain activated T cells. On day 5 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 2X 10⁶HT-29-luc and 5X 10⁵Human T cells, total volume 0.1ml, injection site mouse lateral axilla. anti-PD-L1 polypeptide (4mg/kg), durvalumab (anti-PD-L1 antibody, 0.1mg/kg) were injected subcutaneously every two days. As a control group, mice injected with tumor cells alone and treated with an anti-PD-L1 polypeptide (4mg/kg) were used. Tumor length and width were measured with a caliper, and tumor volume was calculated (tumor volume: 1/2 × a × b)²A represents the length of the tumor and b represents the width of the tumor). Tumor size can also be measured by measuring the bioluminescence at the tumor site using the IVIS luminea II system (Perkinelmer), measured weeklyOnce, 5 times total detection. Statistical analysis was performed using SPSS19.0(spssinc. chicago, IL, USA) software. Results are expressed as mean ± SD. The comparison between groups was performed by T test, P<0.05 marked statistical differences in differences<0.01，***p<0.001 is a very significant statistical difference.

1.3 results of the experiment

As can be seen from FIG. 7, the anti-PD-L1 polypeptide increased the killing effect of human T cells on tumor cells and inhibited tumor growth after simultaneous injection of human T cells and human HT-29 cells. 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.

As can be seen from FIG. 8, the anti-PD-L1 polypeptide increased the killing effect of human T cells on tumor cells and inhibited tumor growth after simultaneous injection of human T cells and human HT-29 cells. 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.

Sequence listing

<110> university of Chinese pharmacy

<120> PD-L1 antagonist polypeptide and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>2

<211>21

<212>PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400>2

Ile Tyr Leu Cys Gly Ala Ile Ser Leu His Pro Lys Ala Lys Ile Glu

1 5 10 15

Glu Cys Pro Gly Ala

20

Claims

1. A PD-L1 antagonist polypeptide, wherein: the amino acid sequence of the polypeptide comprises IYLCGAISLHPKAKIEECPGA (C-C), or a polypeptide which has the effect of relieving immunosuppression after one or more amino acids are deleted, replaced or added on the basis of the amino acid sequence, or a pharmaceutically acceptable salt of the polypeptide.

2. A 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 of claim 1 or 2 for the preparation of a medicament for the prevention or treatment of cancer.

4. Use according to claim 3, characterized in that: the application is realized by the specific binding of the polypeptide and PD-L1.

5. The use according to claim 3 or 4, wherein 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 complex comprising the polypeptide of claim 1 or 2 and one or more pharmaceutically acceptable excipients.

7. A complex according to claim 6, characterized in that said excipients comprise diluents, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants and stabilizers which are conventional in the pharmaceutical field.