CN110669108A - A kind of polypeptide and its application - Google Patents

Abstract

The invention discloses a polypeptide and its application, which belong to the field of biomedicine. The series of polypeptides of the present invention respectively have the amino acid sequence LNWCRLSPSNQTEKCA (C-C), or a pharmaceutically acceptable salt thereof. Specifically, the peptide can specifically recognize PD-L1 on the cell surface, mediate the specific killing of PD-L1-positive cells, and at the same time, it can block the immunosuppressive signaling pathway of PD-1/PD-L1 and eliminate the immunosuppressive effect, thereby achieving specificity. Sexual killing effect.

Description

A kind of polypeptide and its application

technical field

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

Background technique

Tumor immunotherapy is divided into two types: cellular immunotherapy and immune checkpoint inhibitors. The effects of monoclonal antibody drugs and chimeric antigen receptor T (CAR-T) cell immunotherapy are gradually recognized, and the concept of cancer immunotherapy is becoming more and more deeply rooted in the hearts of the people. The former is to take the patient's immune cells out of the body for transformation, so that they have more effective and more accurate immunity to tumor cells. After transformation, they are returned to the tumor patient to play the role of targeted killing of tumor cells. This therapy includes LAK. , DC, CAR-T, NK, CAR-NK therapy, etc. Immune checkpoint (immune Check-point) is also known as immune card control point. Currently, the most important aspect of tumor immunotherapy is tumor immune checkpoint inhibitor, and immune checkpoint inhibitor is to relieve the tumor's immune tolerance/shield. It allows immune cells to re-recognize tumor cells and attack tumor cells. Current research focuses on three molecules, CTLA-4, PD-1 and PD-L1.

During the entire immune response, T cell activation requires dual signaling pathways. First, the T cell receptor TCR on the surface of T cells specifically recognizes 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 Binds, activates or inhibits T lymphocytes. The co-stimulatory molecules on T cells mainly include co-activating molecules 4-1BB, CD27, CD28, OX40, ICOS, and co-inhibiting molecules CTLA4 and PD-1. Programmed Death 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 initially found by screening for differential expression in apoptotic cells, specifically cloned from apoptotic mouse T hybridoma 2B4.11. Other members of its family, such as CTLA-4 and BTLA, were screened for 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 with two immunoglobulin constant regions (IgC) and IgV-like domains in the extracellular segment. It is mainly expressed in mature CD4+ T cells, CD8+ T cells , monocytes, macrophages, B cells, dendritic cells and other hematopoietic cells, as well as some non-hematopoietic cells, such as endothelial cells, islet cells, mast cells and other membrane surfaces. PD-L2 is a transmembrane protein composed of 274 amino acid residues. It has high similarity with PD-L1, but the two also have certain differences. For example, the tissue distribution of PD-L2 in vivo is limited, only in Membrane surface expression on macrophages, dendritic cells and some B cell subsets. In addition to its inducible expression 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 progression ability to metastasize, resulting in increased patient mortality. Combined with the highly expressed PD-1 in tumor-infiltrating T lymphocytes, indicating that the highly expressed PD-1/PD-L1 signaling pathway mediates tumor immunosuppression. Binding of these two ligands to PD-1 leads to tyrosine phosphorylation of the intracellular structure of PD-1 and recruits tyrosine phosphatase SHP-2, thereby reducing the phosphorylation of TCR signaling and reducing 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 the cells in the G0/G1 phase to inhibit the proliferation of T cells, hinder their differentiation into plasma cells, and induce T cell proliferation. apoptosis. This avoids the immortal proliferation of T cells and keeps them in homeostasis. The negative feedback regulation of T cells involved in the PD-1/PD-L1 signaling pathway plays a crucial role in the clearance of antigens and in maintaining the balance of the body. Therefore, inhibition of PD-1 and PD-L1 pathways will accelerate and strengthen autoimmunity.

So far, the inhibition of PD-1/PD-L1 signaling pathway has been used to achieve tumor therapy, and good results have also been achieved in the clinical stage. Antibodies for PD-L1 include Roche/Genentech's atezolizumab, AstraZeneca's durvalumab, and EMD serono/Pfizer's avelumab. 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. Others in clinical research include BMS’s BMS936559, domestic PD-1 antibodies include 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 for immunotherapy.

SUMMARY OF THE INVENTION

2. Technical solutions

In order to solve the above problems, the technical scheme adopted in the present invention is as follows:

A polypeptide, characterized in that: the polypeptide sequence is LNWCRLSPSNQTEKCA (C-C), and one or more amino acids are deleted, substituted or added on the basis and still have a polypeptide that relieves immunosuppression, or the polypeptide is pharmaceutically acceptable Accepted salt.

The polypeptide and application thereof are characterized in that: the polypeptide is the amino acid sequence LNWCRLSPSNQTEKCA (C-C) (here refers to a cyclic peptide, connected by C-C), or a pharmaceutically acceptable salt thereof.

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

The application is characterized in that: the application is realized by the specific binding of the polypeptide to 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 complex is characterized in that one or more pharmaceutically acceptable adjuvants can also be added to the polypeptide.

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

At present, the main immune checkpoint inhibitors in clinical practice are monoclonal antibodies, but there are problems such as high treatment costs in the use of antibodies. Therefore, the present invention adopts the computer three-dimensional simulation technology, and based on the analysis of a large number of traditional structures and pharmacological experiments, the self-designed polypeptide structure has a good tumor suppressing effect. The present invention independently designs an immune checkpoint antagonistic polypeptide acting on PD-L1 with a brand-new structure by utilizing the three-dimensional structure.

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, isolate and purify, effectively inhibits the PD-1/PD-L1 pathway, and eliminates the effect of immunosuppression;

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

(3) The polypeptide involved in the present invention has a good effect of eliminating immunosuppression in in vitro and in vivo models, and the specific effect is to significantly increase the activity of T cells, so that it can eliminate the immunosuppressive effect, prevent the escape of tumor cells and produce a tumor suppressing effect.

Description of drawings

Figure 1 Flow cytometry detection of the binding of HT-29 to FITC-anti-PD-L1 peptide; A: negative control; B: 15nM peptide; C: 150nM peptide; D: 1.5μM peptide;

Figure 2 Fluorescence microscope detection of the binding of HT-29 to FITC-anti-PD-L1 polypeptide; A: cell membrane (green); B: FITC-anti-PD-L1 polypeptide (red); C: Merge (yellow)

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 20:1; G7: co-incubation ratio is 10:1. Compared with the negative group, *P<0.05, **P<0.01, ***P<0.001;

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 20:1; G7: co-incubation ratio is 10:1. Compared with the negative group, *P<0.05, **P<0.01, ***P<0.001;

Figure 5 Effect of polypeptide on IFN-γ in 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 Effect of polypeptide on IL-2 in 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;

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

Detailed ways

The present invention will be further described below with reference to 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 contents disclosed above to change into equivalent embodiments with equivalent changes. Any simple modifications or equivalent changes made to the following examples according to the technical essence of the present invention without departing from the content of the solution of the present invention fall within the protection scope of the present invention.

In the examples, the polypeptide (LNWCRLSPSNQTEKCA (C-C)) was mainly used as the object to study its cancer-preventing and/or tumor-inhibiting activity. The polypeptide is synthesized by Gill Biochemical (Shanghai) Co., Ltd., and the purity is over 95%.

Example 1

Detection Experiment of the Specific Binding of Polypeptides to Targets at the Cell Level

Flow cytometry is a method that excites a single unidirectional flow of particles by a laser beam, and detects the scattered light of the particles and the fluorescent markers carried by them, so as to complete the rapid detection and analysis of multiple physical properties of a single particle and cell sorting. technology. Widely used in scientific research and clinical medical testing, it is the most advanced cell quantitative analysis technology. The main target of a polypeptide with cancer-preventing and/or tumor-inhibiting activity involved in the present invention is PD-L1. In this study, the designed anti-PD-L1 polypeptide is linked to a FITC fluorescent marker, and the cells are combined with FITC-anti-PD. The binding rate of the -L1 polypeptide solution reflects the binding ability of the polypeptide to the cell surface PD-L1 at the cellular level.

1.1 Experimental materials

Human colon cancer cells (HT-29) were purchased from ATCC. The anti-PD-L1 peptide was independently synthesized by our laboratory with a purity of more than 95%. BSA blocking solution. CO ₂ cell incubator, flow cytometer, inverted microscope, liquid nitrogen tank, ultra-clean bench, small centrifuge, electronic balance, pH meter, automatic high pressure steam sterilizer, oven, ultrapure water maker, digital display Constant temperature water bath.

1.2 Experimental method:

HT-29 cells were plated into 6-well plates, and when the cells reached 80% confluence, they were digested and collected. Wash twice with ice-cold PBS. Add 1 ml of 1% BSA solution, fix it on a rotary mixer, and mix at 4°C for 30 min. 10 μl of 1 mg/ml FITC-anti-PD-L1 polypeptide solution was added in the dark, fixed on a rotary mixer, and mixed at 4°C for 1 h in the dark. After incubating the drug, centrifuge at 800 rpm for 5 min and discard the supernatant. Wash once with ice-cold PBS. The cell concentration was adjusted with PBS, and the sample concentration was adjusted to 1×10 ⁶ cells/ml. Do 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 regulated power supply, transformer, flow cytometer host, computer and printer in turn from left to right. Open the fluid flow drawer and add purified water to the sheath fluid bucket until it reaches 2/3. The waste liquid was poured out, and 200 ml of sodium hypochlorite solution containing 10% available chlorine concentration was added. Adjust the hydraulic valve to the pressurize position to remove air bubbles between the fluid line and the filter. Remove the sample tube, perform the PRIME function twice, 1ml PBS, HINGRUN 2min. Start measuring and analyze the sample. After the measurement is complete, move the sample holder to the left and vacuum 1ml of FACSClean. Then return the sample support frame to the normal position, and HING RUN for 5min. Change the FACS Clean to pure water, move the sample support rack to the left, extract 1 ml of vacuum, return the sample rack to the positive position, and HING RUN for 10 minutes. Press Standby, remove the sample tube, and perform the PRIME function twice. Finally, 1 ml of purified water was left in the flow test tube. Press Standby to make it work for 20min, after the fan cools the laser, turn off the flow cytometer. Exit the program and shut down the computer.

1.3 Experimental results

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

Example 2

Qualitative Detection Experiment of the Specific Binding of Polypeptides to Targets at the Cell Level-Fluorescence Imaging Experiment

1.1 Experimental materials

Human colon cancer cells (HT-29) were purchased from ATCC. The anti-PD-L1 peptide was independently synthesized by our laboratory. Dil dye and BSA blocking solution.

1.2 Experimental instruments

CO ₂ cell incubator, single-channel pipette, fluorescence microscope, ultrapure water maker, inverted microscope, ultra-clean bench, electronic balance, pH meter, automatic autoclave, oven.

1.3 Experimental method

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

Fluorescence microscopy was used to detect the binding of peptides to PD-L1, and the parameters were fixed, respectively, and photographed in appropriate channels, and the fluorescence images of different channels were superimposed to judge the binding of peptides to PD-L1.

1.4 Experimental results

Figure 2 shows that the anti-PD-L1 polypeptides can be combined with HT-29 cells and bound to the cell membrane surface by fluorescence microscopy.

Example 3

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

1.1 Experimental materials

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

1.2 Experimental instruments

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

1.3 Experimental method

The HT-29 tumor cell line was grown in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% _CO2 Culture to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The effect of blocking the PD-L1 pathway of lymphocyte effector cells was demonstrated by changes in cytokines in the co-incubation system. Before testing the effect of peptides on the co-incubation system, it is necessary to first screen the optimal incubation ratio. Peripheral blood lymphocytes are extracted from blood, and T cells are obtained by further extraction. At the same time, the tumor cells were cultured, and each gradient was set up by equal dilution, and cultured in a 37°C cell incubator for 3 days. Three groups were set up using the screened ratios, namely the control group, the model group, and the experimental group in which the polypeptide was added under the condition of co-incubation. -γ, secretion of IL-2. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 was a significant statistical difference, **P<0.01, ***p<0.001 was a very significant statistical difference.

1.4 Experimental results

1. As shown in Figure 4, 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 20:1; G7: co-incubation ratio is 10:1. Compared with the negative group, *P<0.05, **P<0.01, ***P<0.001.

The co-incubation ratio was screened by detecting the secretion of IFN-γ in the co-incubation system. Under comprehensive consideration, the co-incubation ratio was selected to be 40:1 for the detection of peptide activity. The experimental results are statistically significant.

2. As shown in Figure 5, the 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 20:1; G7: co-incubation ratio is 10:1. Compared with the negative group, *P<0.05, **P<0.01, ***P<0.001.

The co-incubation ratio was screened by detecting the secretion of IL-2 in the co-incubation system. Under comprehensive consideration, the co-incubation ratio was selected to be 40:1 for the detection of peptide activity. The experimental results are statistically significant.

As shown in Figure 5, the effect of polypeptide on IFN-γ in co-incubation system; G1: control group; G2: model group; G3: 100nM anti-PD-L1 polypeptide; G4: 200nM anti-PD-L1 polypeptide; G5: 400nM 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 changes of IFN-γ in the co-incubation system proved that the polypeptide can activate T cells. There was a very significant difference between the model group and the control group, indicating that the modeling was successful. There is a very significant difference between the experimental group and the model group, indicating that the polypeptide can successfully activate the T cells in the co-incubation system.

3. The effect of the polypeptide shown in Figure 6 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 changes of IL-2 in the co-incubation system proved that the polypeptide can activate T cells. There was a very significant difference between the model group and the control group, indicating that the modeling was successful. There is a very significant difference between the experimental group and the model group, indicating that the polypeptide can successfully activate the T cells in the co-incubation system.

Example 4

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of HT-29 cells in 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 instruments

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 was grown in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% _CO2 Culture to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was used to demonstrate the effect of blocking the PD-L1 pathway of lymphocyte effector cells on tumor cell apoptosis. Peripheral blood lymphocytes are extracted from blood, and T cells are obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 1 Effects of polypeptides on apoptosis of HT-29 cells in co-incubation system

group dose Cytotoxicity (%) control group / 15.62±0.34 Low-dose peptide group 100nM 19.15±0.68* Peptide medium dose group 400nM 22.34±0.30* Peptide high-dose group 1.6 μM 25.26±0.29*

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. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating 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 apoptosis of BT474 cells in 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 instruments

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 medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% _CO2 until logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 2 Effects of polypeptides on apoptosis of BT474 cells in co-incubation system

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

The changes of LDH in the co-incubation system reflect the effect of peptides on the apoptosis of BT474 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 6

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of SKmel100 cells 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 instruments

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) at 37°C in an incubator containing 5% _CO2 until logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 3 Effects of polypeptides on apoptosis of SKmel100 cells in co-incubation system

group dose Cytotoxicity (%) control group / 8.54±0.37 Low-dose peptide group 100nM 11.75±0.96* Peptide medium dose group 400nM 17.38±0.59* Peptide high-dose group 1.6 μM 20.64±0.86*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of SKmel100 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating 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 apoptosis of 769-P cells in 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 instruments

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) at 37°C in an incubator containing 5% _CO2 until logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 4 Effects of polypeptides on apoptosis of 769-P cells in co-incubation system

group dose Cytotoxicity (%) control group / 9.12±0.58 Low-dose peptide group 100nM 11.64±0.29* Peptide medium dose group 400nM 16.96±0.25* Peptide high-dose group 1.6 μM 22.91±0.62*

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. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating 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 apoptosis of H1299 cells in 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 instruments

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

H1299 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% CO ₂ until logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 5 Effects of polypeptides on apoptosis of H1299 cells in co-incubation system

group dose Cytotoxicity (%) control group / 11.47±0.73 Low-dose peptide group 100nM 12.97±0.48* Peptide medium dose group 400nM 16.73±0.51* Peptide high-dose group 1.6 μM 19.78±0.69*

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. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 9

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of A2740 cells 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 instruments

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

H1299 tumor cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% CO ₂ until logarithmic growth period. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 6 Effects of polypeptides on apoptosis of A2740 cells in co-incubation system

group dose Cytotoxicity (%) control group / 11.27±0.68 Low-dose peptide group 100nM 14.14±0.93* Peptide medium dose group 400nM 18.47±0.85* Peptide high-dose group 1.6 μM 22.95±0.75*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of A2780 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating 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 apoptosis of DU-145 cells in 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 instruments

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 DU-145 tumor cell line was cultured in 1640 medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% _CO2 Culture to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated in a 37°C cell culture incubator for 3 days according to the ratio previously screened. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SP SS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 7 Effects of polypeptides on apoptosis of DU-145 cells in co-incubation system

group dose Cytotoxicity (%) control group / 12.86±0.27 Low-dose peptide group 100nM 17.49±0.25* Peptide medium dose group 400nM 19.06±038* Peptide high-dose group 1.6 μM 22.17±0.85*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of DU-145 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating 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 apoptosis of SCC-4 cells in co-incubation system

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 cytokine.

1.2 Experimental instruments

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 SCC-4 tumor cell line was cultured in 1640 medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% _CO2 Culture to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 8 Effects of polypeptides on apoptosis of SCC-4 cells in co-incubation system

group dose Cytotoxicity (%) control group / 16.75±0.86 Low-dose peptide group 100nM 19.24±0.58* Peptide medium dose group 400nM 24.05±0.58* Peptide high-dose group 1.6 μM 26.33±0.96*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of SCC-4 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 12

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of T24 cells in 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 instruments

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) at 37°C in an incubator containing 5% CO ₂ until the number of growing seasons. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 9 Effects of polypeptides on apoptosis of T24 cells in co-incubation system

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

The effect of the change of LDH in the co-incubation system on the apoptosis of T24 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 13

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of HepG2 cells in co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

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) at 37°C in an incubator containing 5% CO ₂ until the number of growing seasons. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 10 Effects of polypeptides on apoptosis of HepG2 cells in co-incubation system

group dose Cytotoxicity (%) control group / 11.26±0.83 Low-dose peptide group 100nM 14.85±0.47* Peptide medium dose group 400nM 18.95±1.02* Peptide high-dose group 1.6 μM 22.85±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 HepG2 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating 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 apoptosis of L428 cells in co-incubation system

1.1 Experimental materials

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

1.2 Experimental instruments

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) at 37°C in an incubator containing 5% CO ₂ until the number of growing seasons. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 11 Effects of polypeptides on apoptosis of L428 cells in co-incubation system

group dose Cytotoxicity (%) control group / 6.65±0.58 Low-dose peptide group 100nM 11.43±0.99* Peptide medium dose group 400nM 15.24±2.64* Peptide high-dose group 1.6 μM 19.95±1.68*

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

The changes of LDH in the co-incubation system reflect the effect of peptides on the apoptosis of L428 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 15

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of MG63 cells in 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 instruments

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) at 37°C in an incubator containing 5% CO ₂ until the number of growing seasons. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SP SS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 12 Effects of polypeptides on apoptosis of MG63 cells in co-incubation system

group dose Cytotoxicity (%) control group / 16.68±0.96 Low-dose peptide group 100nM 19.72±0.65* Peptide medium dose group 400nM 22.15±0.43* Peptide high-dose group 1.6 μM 26.48±1.08*

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

The changes of LDH in the co-incubation system reflect the effect of peptides on the apoptosis of MG63 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 16

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of SF17 cells in 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 instruments

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) at 37°C in an incubator containing 5% CO ₂ until the number of growing seasons. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SP SS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 13 Effects of polypeptides on apoptosis of SF17 cells in co-incubation system

group dose Cytotoxicity (%) control group / 8.73±0.46 Low-dose peptide group 100nM 14.83±1.27* Peptide medium dose group 400nM 17.96±0.26* Peptide high-dose group 1.6 μM 19.15±0.32*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of SF17 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 17

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of HTB-9 cells in 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 instruments

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

HTB-9 tumor cell line was cultured in DMEM 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 the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group, and the LDH secretion of the experimental group was measured from each culture. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 14 Effects of polypeptides on apoptosis of HTB-9 cells in co-incubation system

group dose Cytotoxicity (%) control group / 11.45±1.04 Low-dose peptide group 100nM 14.15±0.68* Peptide medium dose group 400nM 19.37±0.54* Peptide high-dose group 1.6 μM 23.17±1.96*

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

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

Example 18

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of AsPc1 cells 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 instruments

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) at 37°C in an incubator containing 5% CO ₂ until the number of growing seasons. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 15 Effects of polypeptides on apoptosis of AsPc1 cells in co-incubation system

group dose Cytotoxicity (%) control group / 10.74±0.39 Low-dose peptide group 100nM 13.27±0.54* Peptide medium dose group 400nM 17.16±0.63* Peptide high-dose group 1.6 μM 22.85±0.37*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of AsPc1 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating 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 apoptosis of MS751 cells in 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 instruments

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) at 37°C in an incubator containing 5% CO ₂ until appropriate number of growing seasons. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 16 Effects of polypeptides on apoptosis of MS751 cells in co-incubation system

group dose Cytotoxicity (%) control group / 16.04±1.46 Low-dose peptide group 100nM 19.45±1.08* Peptide medium dose group 400nM 22.85±0.68* Peptide high-dose group 1.6 μM 26.55±2.17*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of MS751 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating 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 apoptosis of U266 cells 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 instruments

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 medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% CO ₂ until appropriate number of growing seasons. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group, and the LDH secretion of the experimental group was measured from each culture. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 17 Effects of polypeptides on apoptosis of U266 cells in co-incubation system

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

The effect of the change of LDH in the co-incubation system on the apoptosis of U266 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 21

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of GBC-SD cells in 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 instruments

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 was cultured in 1640 medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37°C in an incubator containing 5% _CO2 to the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 18 Effects of polypeptides on apoptosis of GBC-SD cells in co-incubation system

group dose Cytotoxicity (%) control group / 7.37±0.48 Low-dose peptide group 100nM 11.25±0.95* Peptide medium dose group 400nM 19.75±0.69* Peptide high-dose group 1.6 μM 22.61±0.85*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of GBC-SD cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 22

Detection of the effect of anti-PD-L1 polypeptide on apoptosis of TPC-1 cells in 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 instruments

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 was cultured in 1640 medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% _CO2 to the logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 19 Effects of polypeptides on apoptosis of TPC-1 cells in co-incubation system

group dose Cytotoxicity (%) control group / 11.54±0.28 Low-dose peptide group 100nM 18.14±0.37* Peptide medium dose group 400nM 24.68±0.48* Peptide high-dose group 1.6 μM 27.97±0.16*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of TPC-1 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 23

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

1.1 Experimental materials

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

1.2 Experimental instruments

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-2cl.D1 tumor cell line was cultured in 1640 medium containing 10% fetal bovine serum (FBS), penicillin (100kU/L), and streptomycin (100mg/L) at 37°C in an incubator containing 5% CO ₂ Medium to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in 96-well plates. Incubate for 24h at 37°C in an incubator with 5% CO ₂ . The co-incubation reaction was 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 obtained by further extraction. At the same time, the tumor cells were cultured, and the cells were co-incubated according to the previously screened ratio, and the cells were cultured statically for 3 days in a 37°C cell incubator. After 3 days, the two groups were co-incubated and measured, respectively, the control group, the experimental group and the Triton X positive group. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.4 Experimental results

Table 20 Effects of polypeptides on apoptosis of NTERA-2cl.D1 cells in co-incubation system

group dose Cytotoxicity (%) control group / 18.16±0.74 low-dose peptide group 100nM 20.16±0.95* Peptide medium dose group 400nM 24.87±0.29* Peptide high-dose group 1.6 μM 28.43±2.71*

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

The effect of the change of LDH in the co-incubation system on the apoptosis of NTERA-2cl.D1 cells in the co-incubation system. Compared with the control group, there is a significant difference between the experimental group and the control group, indicating that the polypeptide can promote the killing effect of T cells on tumor cells.

Example 24

Detection of Toxicity of Anti-PD-L1 Polypeptides to Caenorhabditis elegans

1.1 Experimental materials

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

1.2 Experimental method

OP50 E. coli was cultured and transferred to 150 mL of LB medium every other day. Place Caenorhabditis elegans on the OP50 Escherichia coli bacteria solution, and control the room temperature at 20°C to synchronize the Caenorhabditis elegans, and make the larvae stay in the L1 stage by splitting the adults and controlling nutrition. Three concentrations of 100nM, 400nM and 1.6μM were set, and durvalumab group and blank group (anti-PD-L1 antibody, 68nM) were set as control. 50 μl of OP50 E. coli bacteria solution and 40 L1-stage larvae (50 μL) were added to each well of the 96-well plate. The blank group was added with 100 μL/well of M9 buffer, the positive control group was added with 100 μL/well of durvalumab, and the experimental group was added with 100 μL/well of PD-L1 polypeptide (100nM, 400nM, 1.6μM) according to the dose gradient. There were 4 replicate wells in each group and the final volume of each well was guaranteed to be 200 μL. After the dosing treatment was completed, the number of nematodes per well was first observed in the initial state, recorded as N ₀ , then the nematodes were placed in a 20°C electric thermostatic incubator for 48 hours, and the number of nematodes per well N _t was recorded after 48 hours. Statistical analysis was performed using SPSS 17.0 mathematical statistics software package, mean ± SD. T-test was used for comparison between groups, *P<0.05 has a significant statistical difference.

1.3 Experimental results

Table 21 Effects of polypeptides on the lethality of C. elegans

group dose Hatching rate (%) Blank control group / 1.15±0.26 durvalumab group 68nM 11.13±0.77 Low-dose peptide group 100nM 1.47±0.69^# Peptide medium dose group 400nM 1.96±0.75^*# Peptide high-dose group 1.6 μM 2.69±0.16^*#

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

To study the acute toxicity of anti-PD-L1 polypeptides, the lethality of C. elegans was mainly evaluated. Table 21 shows that there were significant differences from the durvalumab group at each concentration. Compared with the blank control group, the anti-PD-L1 polypeptide showed a significant difference only at 1.6 μM. It can be speculated that at the experimental dose, the anti-PD-L1 polypeptide has little effect on the lethality of C. elegans, while durvalumab can cause certain toxicity to C. elegans.

Example 25

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

1.1 Experimental materials

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

1.2 Experimental method

5-6 week old female Balb/c nude mice were used for in vivo experiments. HT-29 cells were transfected with plvx-pro/luciferase lentiviral plasmid and screened with 1 μg/ml puromycin to establish a cell line stably expressing luciferase. Human peripheral blood T cells were isolated from the blood of healthy volunteers. First, the human peripheral blood mononuclear cell layer was obtained by centrifugation, and then T cells were obtained by CD3 magnetic bead sorting technology. IL-2 (20ng/ml) and human T cell activator (CD3/CD28 particles, particles:T cells=1:1) were added. On day 5 of tumor cell culture, human T cells were added to HT-29 cells stably expressing luciferase, and co-cultured for 3 days. Then, each mouse was subcutaneously injected with 2×10 ⁶ HT-29-luc and 5×10 ⁵ human T cells in 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. Tumor length and width were measured with calipers, and tumor volume was calculated (tumor volume = 1/2 x a x b ² , a represents the length of the tumor, b represents the width of the tumor). Tumor size can also be measured by detecting bioluminescence at the tumor site using the IVIS Lumina II system (PerkinElmer), once a week for a total of 5 times. Statistical analysis was performed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) software. Results are expressed as mean±SD. T-test was used for comparison between groups, *P<0.05 was a significant statistical difference, **P<0.01, ***P<0.001 was a very significant statistical difference.

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, anti-PD-L1 polypeptide can increase the killing effect of human T cells on tumor cells and inhibit tumor growth. The first group was a negative control group; the second group was injected with only HT-29 cells and anti-PD-L1 polypeptides; the third group was injected subcutaneously with anti-PD-L1 after both human T cells and human HT-29 cells were injected Antibody durvalumab (0.1 mg/kg); the fourth group was subcutaneous injection of anti-PD-L1 polypeptide (4 mg/kg) after simultaneous injection of human T cells and human HT-29 cells. The injection of anti-PD-L1 antibody durvalumab in the third group and the injection of PD-L1 polypeptide (4mg/kg) in the fourth group 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, anti-PD-L1 polypeptide can increase the killing effect of human T cells on tumor cells and inhibit tumor growth. The first group was a negative control group; the second group was injected with only HT-29 cells and anti-PD-L1 polypeptides; the third group was injected subcutaneously with anti-PD-L1 after both human T cells and human HT-29 cells were injected Antibody durvalumab (0.1 mg/kg); the fourth group was subcutaneous injection of anti-PD-L1 polypeptide (4 mg/kg) after simultaneous injection of human T cells and human HT-29 cells. The injection of anti-PD-L1 antibody durvalumab in the third group and the injection of anti-PD-L1 polypeptide (4mg/kg) in the fourth group significantly inhibited the proliferation of HT-29 cells in mice.

sequence listing

<110> China Pharmaceutical University

<120> A kind of polypeptide and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> PRT

<213> Artificial Sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

Leu Asn Trp Cys Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Cys Ala

1 5 10 15

Cys Cys

Claims (7)

1. A polypeptide, characterized by: the polypeptide sequence is LNWCRLSPSNQTEKCA (C-C), and polypeptide with one or more amino acids deleted, substituted or added for relieving immunosuppression, or pharmaceutically acceptable salt of the polypeptide.

2. The polypeptide and the use thereof according to claim 1, wherein: the polypeptide has an amino acid sequence of LNWCRLSPSNQTEKCA (C-C), or a pharmaceutically acceptable salt thereof.

3. Use of a polypeptide according to claim 1 or 2 for the preparation of a medicament for the prophylaxis 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, 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 complex comprising the polypeptide of claim 1 or 2 and one or more pharmaceutically acceptable excipients.

7. The complex according to claim 6, wherein the excipient comprises diluents, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants and stabilizers, which are conventional in the pharmaceutical field.

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