CN106986932A - A kind of c Met epitope peptides and its application - Google Patents

Abstract

The invention discloses a kind of c Met epitope peptides, its amino acid sequence contains the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.3, or containing to the amino acid sequence addition shown in SEQ ID No.2 or SEQ ID No.3, lack or replace one or more amino acids formed amino acid sequences.The c Met epitope peptides can be used for producing antigen presenting cell, major histocompatibility antigen compound or cytotoxic T lymphocyte derivant, moreover it is possible in the medicine applied to the cancer for preparing treatment such as liver cancer.

Description

A kind of c-Met epitope peptide and its application

technical field

The invention belongs to the field of molecular immunology and relates to antigenic epitope peptides, in particular to epitope peptides recognized by HLA-A0201-restricted c-Met protein cytotoxic T lymphocytes (CTL) and applications thereof.

Background technique

Hepatocellular carcinoma (hereinafter referred to as "liver cancer") is a malignant tumor of the digestive tract with a high degree of malignancy and poor prognosis. It ranks sixth in the incidence of cancer worldwide and ranks second in the death caused by cancer. It is essentially a polygenic abnormality disease, involving the overexpression of oncogenes and the mutation and deletion of tumor suppressor genes, so that tumor cells escape the normal growth regulation mechanism and autonomously proliferate and invade. Although traditional treatment methods (including surgery, chemotherapy, and targeted drug therapy) have improved in recent years, their prognosis is poor, the recurrence rate and metastasis rate of liver cancer are still high, and the overall survival rate of liver cancer patients is still unsatisfactory. At present, it is believed that the root cause of the recurrence of these patients is the existence of minimal residual lesions in the body. Therefore, it is necessary to find an effective method to effectively remove the minimal residual lesions in patients after radical treatment or radiotherapy and chemotherapy.

Immunotherapy is a very suitable option. Some scholars have used idiotype antigens to vaccinate patients with liver cancer for immunotherapy, but the efficacy of this treatment has been disappointing, partly due to the insufficient immunogenicity of this idiotype antigen. Therefore, we urgently need to discover a new type of tumor antigen shared by most patients to improve the efficacy of immunotherapy for liver cancer. An ideal tumor antigen should be expressed on the surface of tumor cells of different origins, low-expressed or not expressed in normal tissues, and indispensable for the growth and survival of tumor cells, so that it is impossible for tumor cells to express this antigen through non-expression or down-regulation. antigens to evade immune attack.

Cytotoxic T cells can recognize peptides through MHC class I molecules expressed on the cell surface, and then bind to target cells to kill target cells. MHC class I molecules and antigen-specific peptides determine the MHC restriction and antigen specificity when CTL cells kill target cells. The expression rate of HLA A site 0201 of MHC class I molecule in Chinese Han population is more than 50%, so this site widely expressed by MHC class I molecule was chosen to study the antigen specificity of CTL cells.

Therefore, those skilled in the art are devoting themselves to developing an epitope peptide recognized by cytotoxic T lymphocytes of HLA-A0201-restricted c-Met protein.

Contents of the invention

In view of the problems of insufficient immunogenicity of tumor-specific antigens in the prior art, the present invention provides a c-Met epitope peptide and its application.

The first aspect of the present invention provides a c-Met epitope peptide selected from any of the following:

1) A peptide consisting of an amino acid sequence containing the amino acid sequence shown in SEQ ID No.2;

2) A peptide consisting of an amino acid sequence containing the amino acid sequence shown in SEQ ID No.3;

3) A peptide composed of an amino acid sequence formed by adding, deleting or replacing one or more amino acids to the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.3, the peptide can form a complex with HLA-A0201 molecules and Recognized or induced by HLA-A0201-restricted cytotoxic T lymphocytes.

Further, the above-mentioned c-Met epitope peptide is a peptide composed of the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.3.

The second aspect of the present invention provides a nucleotide sequence encoding the amino acid sequence of the above-mentioned c-Met epitope peptide.

The third aspect of the present invention provides an antigen-presenting cell pulsed with the above-mentioned c-Met epitope peptide.

The fourth aspect of the present invention provides a major histocompatibility antigen complex, which includes the HLA-A0201 molecule and the above-mentioned c-Met epitope peptide.

The fifth aspect of the present invention provides a cytotoxic T lymphocyte inducer, the active ingredients of which include:

1) c-Met epitope peptide as described above;

2) antigen presenting cells as described above; or

3) Major histocompatibility antigen complex as described above.

The sixth aspect of the present invention provides a cancer vaccine, the active ingredients of the cancer vaccine include:

1) c-Met epitope peptide as described above;

2) antigen presenting cells as described above; or

3) Major histocompatibility antigen complex as described above.

Further, the aforementioned cancer is liver cancer.

The present invention also provides the application of the above-mentioned antigenic epitope peptide in the preparation of medicine for treating cancer.

Preferably, the cancer is liver cancer.

Immunotherapy has always been a research hotspot in the medical field. Many researchers have found many therapeutic targets for tumors, such as EBV-C-MET and LMP2a in liver cancer, but these targets have their own limitations, which requires us to Constantly looking for new therapeutic targets for liver cancer to finally achieve the goal of individualized treatment: for each patient, detect the expression of various tumor antigens in the body, and select the appropriate target according to its strength to achieve the best therapeutic effect , this kind of individualized and differentiated treatment may be the development direction of tumor immunotherapy in the future.

c-Met, also known as hepatocyte growth factor receptor, is a protein with tyrosine kinase activity encoded by the MET gene. The phosphorylation activation of c-Met can cause the activation of its downstream pathway targets, such as PI3K/Akt and MAPK/Erk pathways. By activating these pathways, c-Met participates in processes including cell proliferation, apoptosis, cytoskeleton rearrangement, and differentiation. In malignant tumors, c-Met can promote the proliferation and metastasis of tumor cells. c-Met is associated with poor prognosis of various malignant tumors and can be used as a therapeutic target for these tumors, including lung cancer, gastric cancer, lymphoma, etc. In liver cancer, the expression level of c-Met is significantly higher than that in adjacent tissues, which is closely related to the proliferation and metastasis of liver cancer. By using c-Met inhibitor or RNA interference technology to down-regulate the expression of c-Met, it can be observed that the proliferation and metastasis of liver cancer are significantly reduced in vivo and in vitro.

It is speculated that c-Met protein, which is widely expressed in liver cancer, actually acts as an anti-apoptotic molecule in tumors, especially liver cancer cells. c-Met protein is also an ideal direct target for immunotherapy of malignant tumors including liver cancer.

The present invention obtains two high-integration peptides aa1211 and aa948 through online screening, and then further screens out the peptide aa1211 with high affinity and stability through T2 cell affinity and stability experiments. After the shortness of breath pentamer was synthesized, different levels of c-Met-CTL were detected in the peripheral blood of healthy donors and patients, proving that the epitope peptide exists naturally under physiological and pathological conditions, which is the basis for follow-up experiments. Necessary conditions are provided.

Through the above antigenic epitope peptide, with the help of autocratic antigen-presenting cells (such as DC), using the characteristics of CTL cells, it will be able to block immune tolerance and immune neglect by acting on c-Met protein, and improve its immunogenicity. sex. Established c-Met protein-specific cytotoxic T cells from healthy donors, whether these cytotoxic T cells can effectively kill liver cancer cells, and have no killing effect on normal hematopoietic cells. It is speculated that after c-Met-CTL cells down-regulate the expression of c-Met in tumor cells, the normal activated T lymphocytes will be greatly restored to eliminate tumors.

Description of drawings

Fig. 1 is a graph showing the results of T2 cell affinity experiment in one embodiment of the present invention. Among them, the negative control FLU-matrix is influenza matrix protein, the positive control is HIV pol, and aa1211 and aa948 are two predicted peptides.

Fig. 2 is a graph showing the results of a T2 cell binding stability experiment in an embodiment of the present invention.

Fig. 3 is a graph showing the results of c-Met-CTL cell phenotype determination in an embodiment of the present invention.

Fig. 4 is a diagram showing the results of a c-Met-CTL proliferation experiment in an embodiment of the present invention.

Fig. 5 is a graph showing the results of a c-Met-CTL killing experiment according to an embodiment of the present invention.

Fig. 6 is a graph showing the results of c-Met-CTL cell function experiments in an embodiment of the present invention.

detailed description

The present invention will be further described below in conjunction with examples, and it should be understood that these examples are only for the purpose of illustration and are not intended to limit the protection scope of the present invention.

The reagents used in the following specific embodiments can be purchased directly unless otherwise specified.

The flow cytometer was purchased from BECKMAN COULTER, model Navios.

T2 cells were purchased from ADCC.

FITC-labeled HLA-A0201 monoclonal antibody BB7.2 was purchased from ebioscience.

FITC-labeled CD8+ monoclonal antibody was purchased from ebioscience.

Polypeptide synthesis can be carried out by any method in the prior art, such as solid-phase peptide synthesis and the like.

Amino acid substitution: Mutant peptides with similar or equivalent activities can be obtained by amino acid substitution, which can be performed with amino acids that have similar charge, solubility, hydrophilicity/hydrophobicity, and polarity to the amino acid before the substitution.

The principle of T2 cell affinity and stability analysis: T2 cells lack the necessary antigen processing-related transporters in the presentation of endogenous antigen peptides, and only a small amount of empty HLA-A0201 molecules are expressed on their surface and are extremely unstable. However, when MHC class I molecules on the surface of HLA-A0201 molecules are combined with peptides with strong binding force to HLA-A0201 molecules, the expression of HLA-A0201 will be enhanced and stabilized. Moreover, the stronger the binding force between HLA-A0201 molecules and peptides, the less degradation of HLA-A0201 molecules on the surface of T2 cells, and the higher the expression level of HLA-A0201 molecules. Therefore, the strength of HLA-A0201 molecule expression on the surface of T2 cells can be directly used to reflect the binding ability of the predicted epitope peptide to HLA-A0201.

Isotype Control: Use immunoglobulin from the same source, label, dose, and subtype as the fluorescently labeled antibody to eliminate background staining due to nonspecific binding of the antibody to the cell surface.

Example 1: Prediction and synthesis of HLA-A0201-restricted c-Met protein CTL epitope peptide

(1) Determination of the amino acid sequence of c-Met protein:

By searching the c-Met protein sequence in the Genbank database, the c-Met protein with the Genbank accession number: NP_001002963.1 was found, which consists of 1382 amino acid residues, as shown in SEQ ID No.1.

(2) Online prediction of c-Met protein epitope peptide:

Using the Epitope Peptide Prediction Database

a: https://www-bimas.cit.nih.gov/cgi-bin/molbio/ken_parker_comboform and

b: http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm)

The service provided is to independently predict the epitope peptides of the HLA-A0201 restricted c-Met protein, select several epitope peptides with the highest scores, and then use the super-motif and quantitative motif scheme to analyze the initially predicted CTL expression Peptides were modified to further improve integration. The two epitope peptides with the highest scores after modification are shown in Table 1:

Table 1 Prediction score of c-Met protein epitope peptide and human HLA-A0201 affinity

(Score I ^a and Score II ^b represent the respective predicted scores using the a and b sites above, respectively.

(3) Synthesis, purification and identification of peptides:

Two peptides aa1211 and aa948 in Table 1 were synthesized, purified and identified by means of biosynthesis (Shanghai Botai Biotechnology Co., Ltd.).

(4) Control peptide:

HLA A0201 positive and negative peptides reported in the literature were selected as controls. The two peptides are:

Positive peptide HIV pol: ILKEPVHGV (SEQ ID No.4);

Negative peptide: influenza virus matrix protein peptide (FLU-matrix peptide): GILGFVFTL (SEQ ID No.5).

Example 2: T2 cell epitope peptide binding affinity and stability experiments

The affinity and stability of HLA-A0201 and peptides were determined with the help of the characteristics of T2 cells.

(1) T2 cell affinity experiment

Collect T2 cells, centrifuge and wash three times with sterile PBS at 4°C, add serum-free 1640 medium, inoculate 2×10 ⁵ /well cells in a 24-well cell culture plate, set up an experimental group and a control group, and repeat three times for each group Secondary wells, T2 cells stimulated without peptides were used as blank control, and corresponding experimental peptides (aa1211 and aa948) and control peptides were added to each well (positive control: HIV pol peptide; negative control: influenza virus matrix protein peptide FLU-matrix) (final concentration 50 μM), while adding β2 microglobulin (final concentration 2.5 μg/ml). Place the cells in a 37°C, 5% CO ₂ incubator and incubate for 18h, collect the cells again, wash three times with PBS at 4°C, add FITC-labeled HLA-A0201 monoclonal antibody BB7.2 (2 μl/well), and protect from light at 4°C Incubate for 15 minutes, wash 3 times with PBS at 4°C, and measure the average fluorescence intensity by flow cytometry.

Fluorescence coefficient (FI) is used as an indicator of affinity. Epitope peptides with a fluorescence coefficient >1 are considered to have high affinity with HLA-A0201 molecules. The fluorescence coefficient is calculated by the following formula:

Fluorescence coefficient (FI)=(average fluorescence intensity of sample-average fluorescence intensity of blank control)/average fluorescence intensity of blank control.

The results of the affinity experiment are shown in Figure 1. The fluorescence coefficient (FI) of aa1211 is greater than 1, which indicates that aa1211 has a high affinity with HLA-A0201 molecules; The affinity of -A0201 molecule is weaker than that of aa1211 and HLA-A0201 molecule, but still has a higher affinity compared with the negative control FLU-matrix.

(2) T2 cell epitope peptide binding stability experiment

T2 cells were collected, 1×10 ⁵ /well was inoculated in a 96-well cell culture plate, and serum-free 1640 medium, β2 microglobulin and corresponding experimental peptides (aa1211 and aa948) were added, and three auxiliary wells were set up for the experimental group. Place the cells in a 5% CO ₂ incubator and incubate for 18 hours, collect the cells again, add serum-free 1640 medium and extracellular brefeldin after washing and incubate for 1 hour, collect the cells, add extracellular brefeldin after washing Dermectin (0.5 μg/ml) in serum-free 1640 medium was incubated in an incubator, and cells were collected at different time points (0, 2, 4, 8, 12, 24 h). Add FITC-labeled HLA-A0201 monoclonal antibody BB7.2 (10 μl/well), detect the average fluorescence intensity by flow cytometry, calculate the FI at each time point, and use this to measure the HLA-A0201 induced by the epitope peptide. Express the situation.

The results of the binding stability experiment are shown in Figure 2. After 24 hours of incubation, the peptide aa1211 can still maintain a good affinity with the HLA-A0201 molecule, which shows that the binding stability of the peptide aa1211 is good; the peptide aa948 was incubated At 12 hours, it can still maintain its best affinity, but after 24 hours of incubation, the affinity decreases, indicating that its binding stability at 24 hours becomes weaker. Therefore, the affinity and binding stability of peptide aa948 are slightly worse than that of peptide aa1211.

Example 3: Detection of pentamer-positive cytotoxic T cells corresponding to peptide aa1211

According to the results in Example 2, the affinity and binding stability of the peptide aa1211 are relatively good, therefore, further experiments were conducted on the peptide aa1211.

The corresponding pentamer of peptide aa1211 is based on the principle of double recognition of T cell activation. MHC class I molecular heavy chain α and β2 microglobulin are assembled in vitro by bioengineering technology, and combined with the antigenic epitope peptide to form a TCR specific Sexually bound monomers, and then five monomers are assembled together to form a pentamer.

A healthy donor and seven patients with liver cancer who were diagnosed with no remission were selected, and the number of CD8 ⁺ cells positive for c-Met epitope peptide pentamer in their peripheral blood was detected. Collect 5ml of peripheral blood from healthy donors and patients diagnosed with liver cancer without remission, and extract peripheral blood mononuclear cells with lymphocyte separation medium. After counting the cells, take 1× ¹⁰⁶ cells, dissolve them in 100 μl of PBS, and add 2 μl of peptides The PE-labeled pentamer corresponding to aa1211 and the isotype control were incubated at room temperature in the dark for 40 minutes, placed on ice for 1 minute, added FITC-labeled CD8+ monoclonal antibody, kept in the dark for 15 minutes at 4°C, taken out and washed with PBS at 4°C 3 times, resuspended with 500 μl PBS and detected by flow cytometry.

The interpretation of flow cytometry results is shown in Table 2, indicating that peptide aa1211 is a natural product of c-Met protein in vivo or intracellular cleavage, an epitope peptide fragment produced under physiological conditions, and c-Met in healthy donors -CTL levels were significantly lower than those in liver cancer patients.

Table 2 The detection results of the number of CD8 ⁺ cells positive for pentamer of aa1211

Example 4: In vitro verification of c-Met protein-specific cytotoxic T cells on liver cancer cell lysis and study of its characteristics

1) Induce the generation of cytotoxic T cells:

Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy donors with stable HLA-A0201+, and DC cells and cytotoxic T cells (CTL) were induced and cultured. After 4 weeks, c-Met-CTL with a CD8+ positive and pentamer positive cell ratio of more than 10% was obtained through flow cytometry detection to complete subsequent experiments. The detection results are shown in Table 3.

1.1) Obtain peripheral blood mononuclear cells from HLA-A0201 ⁺ healthy donors

(1). Take about 20ml of peripheral blood from HLA-A0201 ⁺ healthy donors and anticoagulate with heparin sodium.

(2). Put the peripheral blood into a 50ml sterile centrifuge tube, and add an equal volume of sterile PBS.

(3). Divide into two 50ml centrifuge tubes, and slowly add about 40ml of diluted peripheral blood to 40ml of lymphocyte separation medium, so that the two are divided into two layers (operate in two tubes).

(4). Centrifuge at 400g for 18min (equivalent to 1500rpm/min), and select slow acceleration and slow deceleration programs for the centrifuge.

(5). Aspirate the misty cell layer (that is, the mononuclear cell layer) at the junction of the liquid phase and put it into another 10ml centrifuge tube.

(6). Resuspend the mononuclear cells with sterile PBS, 300g for 10min (use about 10ml of washing solution each time).

(7). Discard the supernatant after centrifugation, resuspend with 4ml of erythrocyte lysate stored on ice, mix and pipette, place in the refrigerator at 4°C for 10 minutes, then centrifuge at 300g for 10 minutes, and discard the supernatant.

(8). Wash the mononuclear cells twice with sterile PBS, 300 g for 10 min (use about 10 ml of washing solution each time).

(9). Resuspend the mononuclear cells with 8 ml of 1640 medium containing 10% FBS.

1.2) Induction of DC cells

(1). Add 1×10 ⁶ extracted mononuclear cells into a 24-well plate, and place them in a 5% carbon dioxide incubator at 37° C. for 2 hours to make the mononuclear cells adhere to the wall.

(2). Aspirate the cell suspension in the 24-well plate, and gently wash each well with 0.5ml of 1640 medium containing 10% FBS, repeat three times, and decide whether to collect the aspirated cell suspension (which contains a single nucleus) cell).

(3). Add 1 ml of 1640 medium containing 10% FBS to each well, and add 10 ng/ml of GM-CSF and 10 ng/ml of IL-4.

(4). Change the medium at half volume every 3 days, add GM-CSF 10ng/ml and IL-410ng/ml when changing the medium, change the medium at half volume on the fifth day, add GM-CSF 10ng/ml, IL-4 10ng/ml ml and TNF-α10ng/ml, DC cells matured after 48h.

(5). Add aa27 or aa670 peptide at a concentration of 50 ng/ml, and let stand in a 5% carbon dioxide incubator at 37° C. for 2-4 hours.

(6).30Gy gamma ray irradiated mature DC cells.

(7). Aspirate all the supernatant and prepare to add lymphocytes or induced CTL cells.

1.3). Generation of PBMC-derived CTLs

(1). When lymphocytes and DC cells are co-cultured for the first time, the cell suspension in step 2.1.2.b is collected and centrifuged at 1000 rpm for 10 minutes, and then mixed with IL-2 50IU/ml, IL-7 2.5ng/ml and IL-155ng/ml were resuspended in 1640 medium with 10% FBS, counted lymphocytes and mixed with mature DC cells at a ratio of 10:1.

(2). Change the medium once every 2-3 days, and add IL-2 50IU/ml, IL-7 2.5ng/ml and IL-155ng/ml at the same time.

(3). After the new DC matured on the 7th day, the lymphocytes were pipetted and aspirated, and transferred into the newly generated DC wells and co-cultured with them.

(4). Before each co-cultivation with new DC cells, it is necessary to take a sample to test the specificity, and generally co-culture with DC 4 times.

1.4) Detection of double positive rate of CD8 and pentamer in CTL cells

(1) Take 5×10 ⁵ cultured CTL cells (7 days after co-culture with DC each time), add 5ml of sterile PBS, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and resuspend in 100μl sterile PBS .

(2) Add 5 μl of fluorescently labeled MHC/peptide polymer (Epimer) to the cell suspension, and react at room temperature for 15 minutes in the dark. Place cells on ice and incubate for 1 minute. Add anti-CD8-FITC. Continue the reaction in the dark for 20 minutes on ice.

(3) Wash the cells 3 times with PBS washing solution, 400×g for 5 minutes, and resuspend the cell pellet in an appropriate amount of PBS washing solution.

(4) Cell flow cytometry analysis, the analysis results are shown in Table 3. After 4 weeks, the proportions of CD8 ⁺ positive and pentamer positive cells were 22% and 12.1% respectively through the detection of flow cytometry. More than 10% c-Met-CTL can be used to complete follow-up experiments.

Table 3 Pentamer positive rate of c-Met-CTL cells

the first week the fourth week CD8 ⁺ 15% twenty two% c-Met-ctls 4.7% 12.1%

2) Determination of CTL cell phenotype:

The phenotype of antigen-specific CTL cells should be different from that of naive T cells. The CTL cells induced by CD45RO and CD45RA detection should be different from the peripheral blood phenotype of healthy donors from the same source. It is generally believed that the specific CTL cells in CD8+ CD45RO was highly expressed and CD45RA was low; correspondingly, CD8+ cells highly expressed CD45RA and lowly expressed CD45RO in peripheral blood PBMCs from healthy donors who had not been co-cultured with DC cells.

1. Take 3×10 ⁶ cultured CTL cells (5 days after the fourth co-cultivation with DC), add 5 ml of sterile PBS, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and resuspend in 100 μl of sterile PBS. At the same time, peripheral blood PBMCs that had not been co-cultured with DC were taken as the control group.

2. Add 5 μl of fluorescently labeled MHC/peptide polymer (Epimer) to the cell suspension, and react for 15 minutes at room temperature in the dark. Place cells on ice and incubate for 1 minute. Anti-CD8-FITC and anti-CD45RA-APC or anti-CD45RO-APC were added. Continue the reaction in the dark for 20 minutes on ice.

3. Wash the cells 3 times with PBS washing solution, 400xg for 5 minutes, and resuspend the cell pellet in an appropriate amount of PBS washing solution.

4. Cytometry analysis. The results are shown in Figure 3, indicating that among the induced specific CTL cells, CD8+ cells highly express CD45RO and low express CD45RA, and are functional CTL cells.

3) CTL cell proliferation experiment:

In the flow cytometry method, CTL cells were labeled with CFSE and co-cultured with irradiated HLA-A0201+ liver cancer cell lines to detect whether C-MET-CTL would exhibit specific cell proliferation. It is generally believed that only when co-cultured with HLA-A0201+ liver cancer cell lines, c-Met-CTL cells can significantly proliferate. In the absence of any antigen stimulation, c-Met-CTL cells do not proliferate. In matched cases, c-Met-CTL cells also did not proliferate. Through this experiment, it will be demonstrated whether the effect of c-Met-CTL cells on liver cancer cells is limited by antigen specificity and MHC class I molecules.

The CTLs cells co-cultured with DC cells for four weeks were used for experiments.

CCK-8 kit method, take the CTLs cells that have been co-cultured with DC cells for four weeks for the experiment:

(1). To obtain DC cells, refer to Methodology 2.1.2 of this article for details, and induce maturation of DC cells in a 24-well plate.

(2). The mature DC cells were divided into the experimental group and the control group: the DC cells in the experimental group were impacted with the selected peptide aa1211 (50 ng/ml), and the DC cells in the control group were not added with the peptide.

(3). After cultured in a 37°C 5% carbon dioxide incubator for 2-4 hours, the DC cells of the experimental group and the control group were taken out of the incubator, the supernatant was discarded, and the DC cells were resuspended with a small amount of medium and counted.

(4). The CTLs cells co-cultured for four weeks were taken out, resuspended by pipetting and counted.

(5). The DC cells of the experimental group and the control group were mixed and co-cultured with CTLs cells according to the gradient, and three gradients were set up: the ratio of DC cells to CTLs cells was 4:1, 20:1 and 100:1 respectively.

(6). The mixed groups of cells were counted again, and 1×10 ⁵ cells were inoculated in each well of a 96-well plate. % carbon dioxide incubator for 12 hours. At this time, the cells should be divided into the following groups: the experimental group, the control group and the blank control group, with 4 replicate wells in each group, and 10 μl of CCK-8 reagent was added to each well.

(7). After 12 hours, take out the 96-well plate, shake it for 10 minutes, and read the absorbance (OD) value at 450nm wavelength with a dual-wavelength microplate reader, and subtract the OD value of the blank control group.

(8). According to the OD value measured at the 12th hour time point, the cell proliferation in each group was plotted.

The results are shown in Figure 4, only DC cells loaded with peptides can stimulate the proliferation of CTL cells and form specific c-Met-CTL cells. After the addition of peptide aa1211 for shock, T cells significantly proliferated.

4) CTL cell killing assay:

The CTLs cells co-cultured with DC cells for four weeks were used for experiments.

The specific lysis of c-Met-CTL cells to HLA-A0201± primary liver cancer cells and liver cancer cell lines was detected by flow cytometry target cell PI and CFSE double-labeled staining.

(1). Take ¹⁰ ×106 cultured CTL cells, add 5ml of sterile PBS, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and resuspend with 10ml of sterile PBS to make it reach ¹ ×106 per 1ml cell.

(2). Add CFSE to make the final concentration 3μM, put it in a 37°C carbon dioxide incubator and incubate for 15 minutes, and shake the cells every 5 minutes.

(3). After taking out the cells, add an equal amount of fetal bovine serum and put them in a refrigerator at 4°C for 10 minutes to stop.

(4). Wash three times with sterile PBS, each time at 1000 rpm for 10 minutes.

(5). Resuspend with 1640 containing 10% FBS to make the cell concentration reach 1×10 ⁶ /ml.

(6). Transfer the cells into 24-well culture plates in groups, with 1×10 ⁶ CTL cells per well. In each experimental group, CTL cells were added to HLA-A0201 ⁺ liver cancer cell line or primary cell, HLA-A0201 ^- liver cancer cell line or primary cell at a ratio of 10:1), and 3 replicate wells were set for each group.

(7). After the effector target cells were mixed and cultured for 4 hours, the cells of each group were taken out and collected, resuspended in PBS and centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded;

(8). Resuspend in 100 μl PBS, add PI in the dark, 15 minutes at 4° C. in the dark, and then run it on a flow cytometer for detection.

The result is shown in Figure 5. Among them, HepG2 is an HLA A2 liver cancer cell line; LM3 and Hep3B are non-HLA A2 liver cancer cell lines; P1 and P2 are HLA A2 patients, and the CTL cells cultured on the surface can only kill HLA A2 cell lines and primary cells, but cannot kill HLA non-HLA cells. A2 cells.

5) c-Met-CTLs cell function assay:

In order to measure the efficacy of c-Met-CTL cells in killing liver cancer cell lines, we will measure the expression of CD107a on the surface of c-Met-CTL cells to evaluate the degranulation effect of the cells in killing liver cancer cells. Degranulation is the release of CD107a in CTL cells Cytotoxic granules are transiently expressed on the surface of CTL cell membranes.

(1). Take 1×10 ⁶ cultured CTLs cells, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and resuspend with 1640 containing 10% FBS to make the volume 333 μl.

(2). Take 2×10 ⁶ cultured liver cancer cell lines (HLA-A0201 ^- and HLA-A0201 ⁺ each group) from the two groups respectively, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and wash with a medium containing 10% FBS. 1640 to resuspend to a volume of 667 μl.

(3). Mix 333 μl of CTL cells with 667 μl of target cells (HLA-A0201 ^- and HLA-A0201 ⁺ each group), 1 ml in total.

(4). Put the effector-target cell mixture into a V-bottom 96-well culture plate, 100 μl per well, 10 wells in total.

(5). Put the 96-well plate with the cells in a 5% carbon dioxide incubator at 37° C. for 4 hours (centrifuge the 96-well plate at 1000 rpm for 5 minutes).

(6). After culturing for 4 hours, take out the culture plate, blow evenly and suck out the cell suspension in each well, and mix the cells in 10 wells.

(7). Add 5 ml of sterile PBS to the target cell mixture, mix well and centrifuge at 1000 rpm for 10 minutes, discard the supernatant. Resuspend the target cell mixture in 100 μl sterile PBS, add 2 μl anti-CD8-FITC and 10 μl anti-CD107a-PC5 and react on ice for 20 minutes in the dark.

(8). Wash the cells 3 times with sterile PBS washing solution at 400×g for 5 minutes, and resuspend the cell pellet in 500 μl of PBS. Detection by flow cytometry.

The results are shown in Figure 6, wherein, HepG2 refers to the HLA A2 cell line; LM3 refers to the HLA A2 cell line. The results show that HLA A2 tumor cells can stimulate the proliferation of cultured CTL cells, and highly express CD107a, indicating that the function is good. Non-A2 cells cannot stimulate CTL to express CD107a. Cannot express CD107a.

sequence listing

<110> Haikou People's Hospital

<120> A kind of c-Met epitope peptide and its application

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Met Lys Ala Pro Ala Val Leu Ala Pro Gly Ile Leu Val Leu Leu Phe

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Glu Asp Asn Ile Asn Met Ala Leu Leu Val Asp Thr Tyr Tyr Asp Asp

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Asp His Ser Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Gln

210 215 220

Asp Gly Phe Lys Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro

225 230 235 240

Glu Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala Phe Glu Ser

245 250 255

Asn His Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asp Ala

260 265 270

Gln Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser Val Asp Ser Gly

275 280 285

Leu His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys

290 295 300

Arg Arg Lys Arg Ser Thr Arg Glu Glu Val Phe Asn Ile Leu Gln Ala

305 310 315 320

Ala Tyr Val Ser Lys Pro Gly Ala His Leu Ala Lys Gln Ile Gly Ala

325 330 335

Asn Leu Asn Asp Asp Ile Leu Tyr Gly Val Phe Ala Gln Ser Lys Pro

340 345 350

Asp Ser Ala Glu Pro Met Asn Arg Ser Ala Val Cys Ala Phe Pro Ile

355 360 365

Lys Tyr Val Asn Glu Phe Phe Asn Lys Ile Val Asn Lys Asn Asn Val

370 375 380

Arg Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn

385 390 395 400

Arg Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Asn Asp Glu

405 410 415

Tyr Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met

420 425 430

Gly Gln Phe Asn Gln Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys

435 440 445

Gly Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Ser Glu Gly Arg Phe Met

450 455 460

Gln Val Val Val Ser Arg Ser Gly Leu Ser Thr Pro His Val Asn Phe

465 470 475 480

Arg Leu Asp Ser His Pro Val Ser Pro Glu Ala Ile Val Glu His Pro

485 490 495

Leu Asn Gln Asn Gly Tyr Thr Leu Val Val Thr Gly Lys Lys Ile Thr

500 505 510

Arg Ile Pro Leu Asn Gly Leu Gly Cys Glu His Phe Gln Ser Cys Ser

515 520 525

Gln Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp

530 535 540

Arg Cys Val His Leu Glu Glu Cys Pro Thr Gly Ala Trp Thr Gln Glu

545 550 555 560

Val Cys Leu Pro Ala Ile Tyr Glu Val Phe Pro Thr Ser Ala Pro Leu

565 570 575

Glu Gly Gly Thr Val Leu Thr Val Cys Gly Trp Asp Phe Gly Phe Arg

580 585 590

Arg Asn Asn Lys Phe Asp Leu Lys Lys Thr Lys Val Phe Leu Gly Asn

595 600 605

Glu Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Thr Asn Met Leu Lys

610 615 620

Cys Thr Val Gly Pro Ala Val Asn Glu His Phe Asn Ile Ser Ile Ile

625 630 635 640

Ile Ser Asn Gly Arg Gly Thr Ala Gln Tyr Ser Thr Phe Ser Tyr Val

645 650 655

Asp Pro Ile Ile Thr Ser Ile Ser Pro Ser Tyr Gly Pro Lys Asn Gly

660 665 670

Gly Thr Leu Leu Thr Leu Thr Gly Lys Tyr Leu Asn Ser Gly Asn Ser

675 680 685

Arg His Ile Ser Met Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser

690 695 700

Asp Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Ala Thr Ala Thr Glu

705 710 715 720

Phe Pro Ile Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Met Asn Ser

725 730 735

Phe Ser Tyr Gln Glu Asp Pro Ile Val Tyr Ala Ile His Pro Thr Lys

740 745 750

Ser Phe Ile Ser Gly Gly Ser Thr Ile Thr Ala Val Gly Lys Asn Leu

755 760 765

Asn Ser Val Ser Val Leu Arg Met Val Ile Asp Val His Glu Thr Arg

770 775 780

Arg Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile

785 790 795 800

Cys Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu

805 810 815

Lys Thr Lys Ala Phe Phe Met Leu Asp Gly Ile His Ser Lys Tyr Phe

820 825 830

Asp Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys Pro

835 840 845

Val Met Ile Ser Ile Gly Asn Glu Asn Val Leu Glu Ile Lys Gly Asn

850 855 860

Asp Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly Asn

865 870 875 880

Lys Ser Cys Glu Thr Ile Tyr Ser Asp Ser Lys Ala Val Leu Cys Lys

885 890 895

Val Pro Asn Asp Leu Leu Lys Leu Asn Asn Glu Leu Asn Ile Glu Trp

900 905 910

Lys Gln Ala Val Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro

915 920 925

Asp Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Ile Ser Ile Ser Thr

930 935 940

Ile Val Leu Leu Leu Leu Leu Gly Leu Phe Leu Trp Leu Lys Arg Lys Lys

945 950 955 960

Gln Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg Val

965 970 975

His Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser Val Ser Pro

980 985 990

Thr Thr Glu Met Val Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe

995 1000 1005

Pro Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg

1010 1015 1020

Gln Val Gln Tyr Pro Leu Thr Asp Leu Ser Pro Met Leu Thr Ser

1025 1030 1035

Gly Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His

1040 1045 1050

Ile Asp Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln

1055 1060 1065

His Val Val Ile Gly Pro Ser Ser Leu Ile Val His Phe Asn Glu

1070 1075 1080

Val Ile Gly Arg Gly His Phe Gly Cys Val Tyr His Gly Thr Leu

1085 1090 1095

Leu Asp Asn Asp Asp Lys Lys Ile His Cys Ala Val Lys Ser Leu

1100 1105 1110

Asn Arg Ile Thr Asp Ile Gly Glu Val Ser Gln Phe Leu Thr Glu

1115 1120 1125

Gly Ile Ile Met Lys Asp Phe Ser His Pro Asn Val Leu Ser Leu

1130 1135 1140

Leu Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu

1145 1150 1155

Pro Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu

1160 1165 1170

Thr His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln

1175 1180 1185

Val Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Lys Phe Val His

1190 1195 1200

Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Asp Glu Lys Phe Thr

1205 1210 1215

Val Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys

1220 1225 1230

Glu Tyr Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro Val

1235 1240 1245

Lys Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr

1250 1255 1260

Lys Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Leu Met

1265 1270 1275

Thr Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp Ile

1280 1285 1290

Thr Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro Glu Tyr

1295 1300 1305

Cys Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys Trp His Pro

1310 1315 1320

Arg Ala Glu Leu Arg Pro Ser Phe Ser Glu Leu Val Ser Arg Ile

1325 1330 1335

Ser Ala Ile Phe Ser Thr Phe Ile Gly Glu His Tyr Val His Val

1340 1345 1350

Asn Ala Thr Tyr Val Asn Val Lys Cys Val Ala Pro Tyr Pro Ser

1355 1360 1365

Leu Leu Ser Ser Gln Asp Asn Ile Asp Gly Glu Gly Asp Thr

1370 1375 1380

<210> 2

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> aa1211

<400> 2

Cys Met Leu Asp Glu Lys Phe Thr Val

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> aa948

<400> 3

Leu Leu Leu Gly Leu Phe Leu Trp Leu

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HIV pol

<400> 4

Ile Leu Lys Glu Pro Val His Gly Val

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> FLU-matrix

<400> 5

Gly Ile Leu Gly Phe Val Phe Thr Leu

1 5

Claims (10)

1. A c-Met epitope peptide, characterized in that it is selected from any of the following: 1) A peptide consisting of an amino acid sequence containing the amino acid sequence shown in SEQ ID No.2; 2) A peptide consisting of an amino acid sequence containing the amino acid sequence shown in SEQ ID No.3; 3) A peptide composed of an amino acid sequence formed by adding, deleting or replacing one or more amino acids to the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.3, and the peptide can form a complex with HLA-A0201 molecules And be recognized by HLA-A0201 restricted cytotoxic T lymphocytes or induce HLA-A0201 restricted cytotoxic T lymphocytes. 2. The c-Met epitope peptide according to claim 1, wherein the c-Met epitope peptide is a peptide composed of the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.3. 3. A nucleotide sequence, characterized in that the nucleotide sequence encodes the amino acid sequence of the c-Met epitope peptide as claimed in claim 1. 4. An antigen-presenting cell, characterized in that the antigen-presenting cell is pulsed with the c-Met epitope peptide according to claim 1 or 2. 5. A major histocompatibility antigen complex, characterized by comprising HLA-A0201 molecules and the c-Met epitope peptide according to claim 1 or 2. 6. A cytotoxic T lymphocyte inducer, characterized in that the active ingredient of the cytotoxic T lymphocyte inducer comprises: 1) c-Met epitope peptide as claimed in claim 1 or 2; 2) the antigen presenting cell as claimed in claim 4; or 3) The major histocompatibility antigen complex as claimed in claim 5. 7. A cancer vaccine, characterized in that the active ingredient of the cancer vaccine comprises: 1) c-Met epitope peptide as claimed in claim 1 or 2; 2) the antigen presenting cell as claimed in claim 4; or 3) The major histocompatibility antigen complex as claimed in claim 5. 8. The cancer vaccine according to claim 5, wherein the cancer is liver cancer. 9. Use of the c-Met epitope peptide as claimed in claim 1 or 2 in the preparation of medicines for treating cancer. 10. The use according to claim 9, wherein the cancer is liver cancer.