CN110669108A - Polypeptide and application thereof - Google Patents
Polypeptide and application thereof Download PDFInfo
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Abstract
The invention discloses a polypeptide and application thereof, and belongs to the field of biological medicines. The polypeptides of the invention have the amino acid sequence LNWCRLSPSNQTEKCA (C-C) or pharmaceutically acceptable salts thereof. The polypeptide can specifically recognize PD-L1 on the cell surface, mediate specific killing of PD-L1 positive cells, simultaneously can prevent an immunosuppressive signal path of PD-1/PD-L1, and eliminate the immunosuppressive effect, thereby achieving the specific killing effect.
Description
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 and is a member of the CD28 superfamily. It was originally discovered by screening for differential expression in apoptotic cells, specifically cloned from apoptotic mouse T hybridoma 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
2. Technical scheme
In order to solve the problems, the technical scheme adopted by the invention is as follows:
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.
The polypeptide and the application thereof are characterized in that: the polypeptide has an amino acid sequence of LNWCRLSPSNQTEKCA (C-C) (referred to herein as a cyclic peptide, linked via C-C), or a pharmaceutically acceptable salt thereof.
The use of the polypeptide of (1) for the preparation of a medicament for the prevention or treatment of 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 compound, characterized in that one or more pharmaceutically acceptable auxiliary materials can be added into the polypeptide.
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.
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 cytometer detects binding of HT-29 to FITC-anti-PD-L1 polypeptide; a: negative control; b: 15nM polypeptide; c: 150nM polypeptide; d: 1.5 μ M polypeptide;
FIG. 2 fluorescence microscopy of binding of HT-29 to FITC-anti-PD-L1 polypeptide; a: cell membrane (green); b: FITC-anti-PD-L1 polypeptide (red); c: merge (yellow)
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
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. 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
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. 7 bioluminescence assay of inhibition of tumor cell growth by anti-PD-L1 polypeptide;
FIG. 8 detection of the inhibitory Effect of anti-PD-L1 polypeptide on tumor cell growth by tumor volume
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 (LNWCRLSPSNQTEKCA (C-C)) for preventing cancer and/or inhibiting tumor activity was mainly studied as a subject. 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. anti-PD-L1 polypeptide, BenshiThe product is synthesized autonomously in laboratory, and the purity is more than 95%. 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. Cell concentration was adjusted to 1X 10 with PBS6cells/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
Binding of HT-29 to FITC-conjugated anti-PD-L1 polypeptide; in FIG. 1, A is a negative control and B, C, D is the binding rate of HT-29 to a 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. Mu.l of a 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
FIG. 2 shows that the anti-PD-L1 polypeptide was again confirmed to bind 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 (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. The optimal incubation ratio needs to be first screened before the effect of the polypeptide on the co-incubation system can be detected. 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. As shown in FIG. 4, 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. As shown in FIG. 5, secretion of IL-2 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 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.
As shown in fig. 5, the effect of the polypeptide on IFN- γ in the co-incubation system; 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-gamma 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.
3. The effect of the polypeptide on IL-2 in the co-incubation system as shown in FIG. 6;
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.
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. 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 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 | / | 15.62±0.34 |
Polypeptide low dose group | 100nM | 19.15±0.68* |
Polypeptide medium dose group | 400nM | 22.34±0.30* |
High dose group of polypeptides | 1.6μΜ | 25.26±0.29* |
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
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
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 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 2 Effect of Polypeptides on apoptosis of BT474 cells in Co-incubation System
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
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
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(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 3 Effect of the Polypeptides on apoptosis of SKmel100 cells in a Co-incubation System
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 8.54±0.37 |
Polypeptide low dose group | 100nM | 11.75±0.96* |
Polypeptide medium dose group | 400nM | 17.38±0.59* |
High dose group of polypeptides | 1.6μΜ | 20.64±0.86* |
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
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
The BT474 tumor cell strain adopts DMEM culture solution containing 10 percent Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L)At 37 ℃ with 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 4 Effect of Polypeptides on apoptosis of 769-P cells in Co-incubation systems
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 9.12±0.58 |
Polypeptide low dose group | 100nM | 11.64±0.29* |
Polypeptide medium dose group | 400nM | 16.96±0.25* |
High dose group of polypeptides | 1.6μΜ | 22.91±0.62* |
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
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
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 the lapse of 3 days,after co-incubation, the two groups were measured, respectively as control, experimental and Triton X positive groups, and the LDH secretion was measured in the two experimental groups after taking 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 5 Effect of polypeptides on H1299 cell apoptosis in Co-incubation systems
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 11.47±0.73 |
Polypeptide low dose group | 100nM | 12.97±0.48* |
Polypeptide medium dose group | 400nM | 16.73±0.51* |
High dose group of polypeptides | 1.6μΜ | 19.78±0.69* |
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
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
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(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 6 Effect of Polypeptides on apoptosis of A2740 cells in Co-incubation systems
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 11.27±0.68 |
Polypeptide low dose group | 100nM | 14.14±0.93* |
Polypeptide medium dose group | 400nM | 18.47±0.85* |
High dose group of polypeptides | 1.6μΜ | 22.95±0.75* |
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
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
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. Peripheral blood lymphocytes are extracted from blood, and T cells are further extracted. 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 SP SS19.0(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 7 Effect of the Polypeptides on apoptosis of DU-145 cells in Co-incubation systems
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 12.86±0.27 |
Polypeptide low dose group | 100nM | 17.49±0.25* |
Polypeptide medium dose group | 400nM | 19.06±038* |
High dose group of polypeptides | 1.6μΜ | 22.17±0.85* |
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
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
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(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 8 Effect of Polypeptides on SCC-4 cell apoptosis in Co-incubation systems
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 16.75±0.86 |
Polypeptide low dose group | 100nM | 19.24±0.58* |
Polypeptide medium dose group | 400nM | 24.05±0.58* |
High dose group of polypeptides | 1.6μΜ | 26.33±0.96* |
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
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
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 were incubated togetherAfter incubation, control, experimental and Triton X positive groups were measured, and LDH secretion was measured in the two experimental groups taken 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 9 Effect of Polypeptides on apoptosis of T24 cells in Co-incubation System
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
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
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(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 10 Effect of Polypeptides on apoptosis of HepG2 cells in Co-incubation systems
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 11.26±0.83 |
Polypeptide low dose group | 100nM | 14.85±0.47* |
Polypeptide medium dose group | 400nM | 18.95±1.02* |
High dose group of polypeptides | 1.6μΜ | 22.85±0.75* |
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
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
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 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. SPSS19 was used.Statistical analysis was performed with 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 11 Effect of Polypeptides on L428 cell apoptosis in Co-incubation systems
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 6.65±0.58 |
Polypeptide low dose group | 100nM | 11.43±0.99* |
Polypeptide medium dose group | 400nM | 15.24±2.64* |
High dose group of polypeptides | 1.6μΜ | 19.95±1.68* |
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
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
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 SP SS19.0(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 12 Effect of Polypeptides on apoptosis of MG63 cells in Co-incubation System
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 16.68±0.96 |
Polypeptide low dose group | 100nM | 19.72±0.65* |
Polypeptide medium dose group | 400nM | 22.15±0.43* |
High dose group of polypeptides | 1.6μΜ | 26.48±1.08* |
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
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
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 SP SS19.0(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 13 Effect of Polypeptides on apoptosis of SF17 cells in Co-incubation System
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 8.73±0.46 |
Polypeptide low dose group | 100nM | 14.83±1.27* |
Polypeptide medium dose group | 400nM | 17.96±0.26* |
High dose group of polypeptides | 1.6μΜ | 19.15±0.32* |
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
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
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. Demonstration of blockade Using Co-incubation reactionsThe effect of 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 14 Effect of Polypeptides on apoptosis of HTB-9 cells in Co-incubation systems
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 11.45±1.04 |
Polypeptide low dose group | 100nM | 14.15±0.68* |
Polypeptide medium dose group | 400nM | 19.37±0.54* |
High dose group of polypeptides | 1.6μΜ | 23.17±1.96* |
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
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
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(SPSS inc. chicago, IL, USA) software. The result is 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 | / | 10.74±0.39 |
Polypeptide low dose group | 100nM | 13.27±0.54* |
Polypeptide medium dose group | 400nM | 17.16±0.63* |
High dose group of polypeptides | 1.6μΜ | 22.85±0.37* |
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
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
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. 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 16 Effect of Polypeptides on apoptosis of MS751 cells in Co-incubation System
Group of | Dosage form | Cytotoxicity (%) |
Control group | / | 16.04±1.46 |
Polypeptide low dose group | 100nM | 19.45±1.08* |
Polypeptide medium dose group | 400nM | 22.85±0.68* |
High dose group of polypeptides | 1.6μΜ | 26.55±2.17* |
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
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
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 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 17 Effect of Polypeptides on apoptosis of U266 cells 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 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
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
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(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 18 Effect of Polypeptides on apoptosis of GBC-SD cells in Co-incubation systems
Group of | Dosage form | Cytotoxicity(%) |
Control group | / | 7.37±0.48 |
Polypeptide low dose group | 100nM | 11.25±0.95* |
Polypeptide medium dose group | 400nM | 19.75±0.69* |
High dose group of polypeptides | 1.6μΜ | 22.61±0.85* |
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
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
TPC-1 tumor cell strain adopting 10% fetal calf bloodClear (FBS), penicillin (100kU/L) and streptomycin (100mg/L)1640 culture solution 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 | / | 11.54±0.28 |
Polypeptide low dose group | 100nM | 18.14±0.37* |
Polypeptide medium dose group | 400nM | 24.68±0.48* |
High dose group of polypeptides | 1.6μΜ | 27.97±0.16* |
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
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
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, 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. Simultaneously culturing tumor cells, and screening the cellsThe cells were co-incubated and incubated at 37 ℃ for 3 days in a cell incubator. 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 | / | 18.16±0.74 |
Polypeptide low dose group | 100nM | 20.16±0.95* |
Polypeptide medium dose group | 400nM | 24.87±0.29* |
High dose group of polypeptides | 1.6μΜ | 28.43±2.71* |
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 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. 50 μm of OP50 E.coli strain and 40L 1 larvae (50 μ L) were added to each well of a 96-well plate. The blank group was added 100. mu.L/well M9 buffer, the positive control group was added 100. mu.L/well durvalumab, and the experimental group was added 100. mu.L/well 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 48ht. Statistical analysis was performed using the SPSS 17.0 mathematical statistics software package, mean + -SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.
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 | / | 1.15±0.26 |
Durvalumab group | 68nM | 11.13±0.77 |
Polypeptide low dose group | 100nM | 1.47±0.69# |
Polypeptide medium dose group | 400nM | 1.96±0.75*# |
High dose group of polypeptides | 1.6μΜ | 2.69±0.16*# |
Note: compared with the blank control group, the ratio of the active ingredients,*P<0.05; in contrast 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), once a week for a total of 5 measurements. 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 first group is a negative control group; 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. The third group injected with anti-PD-L1 antibody Durvalumab and the fourth group injected with 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 first group is a negative control group; 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. 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> polypeptide and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>18
<212>PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<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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CN108503691A (en) * | 2017-02-25 | 2018-09-07 | 复旦大学 | A kind of human PD-L 1 albumen high-affinity peptide and its application |
CN108840923A (en) * | 2018-06-22 | 2018-11-20 | 上海交通大学医学院附属仁济医院 | It is a kind of target PD-L1 polypeptide and its application |
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CN108840923A (en) * | 2018-06-22 | 2018-11-20 | 上海交通大学医学院附属仁济医院 | It is a kind of target PD-L1 polypeptide and its application |
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