CN106117338B - HLA-A0201-restricted CTL epitope of cytokeratin 19 - Google Patents
HLA-A0201-restricted CTL epitope of cytokeratin 19 Download PDFInfo
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Abstract
The invention discloses an HLA-A0201 restrictive CTL epitope of specific tumor antigen cytokeratin 19, which is nonapeptide, and the amino acid sequence of the nonapeptide is Gly-Ala-Thr-Ile-Glu-Asn-Ser-Arg-Ile. The invention also discloses application of the epitope peptide in preparing a tumor therapeutic polypeptide vaccine expressed by cytokeratin 19. The invention also discloses a method for preparing corresponding DC cells and DC-CIK cells by using the epitope peptide. The invention also discloses application of the DC cell and the DC-CIK cell.
Description
Technical Field
The invention relates to the technical field of epitope peptide, in particular to HLA-A0201 restrictive CTL recognition epitope peptide of specific tumor antigen cytokeratin 19 and application thereof, a preparation method and application of a tumor antigen cytokeratin 19 specific DC cell, and a preparation method and application of a tumor antigen cytokeratin 19 specific DC-CIK cell.
Background
Cytokeratins are intermediate filaments of the cell body, are mainly distributed in epithelial cells, and play an important role in maintaining the integrity and continuity of epithelial tissues. The differentiation of cytokeratins has tissue specificity, and is divided into 20 types according to different properties, which are closely related to the proliferation and differentiation of epithelial cells. Wherein the cytokeratin 19 is an acidic protein with the relative molecular weight of 40000 and the isoelectric point of 5.2, is mainly distributed in monolayer and pseudo-multilayer epithelial cells, and the abnormal expression of the cytokeratin can appear in various tumors. When cells become cancerous, a large number of keratin fragments in dead cells are degraded and become soluble substances which enter the blood, so that the blood content is increased. In clinic, cytokeratin 19 is the first choice marker of non-small cell lung cancer, especially the tumor marker of squamous cell carcinoma which is the first choice at present.
Traditional malignant tumor treatment methods such as surgery, radiotherapy and chemotherapy play an irreplaceable role in the process of antitumor treatment, but have limitations of the traditional malignant tumor treatment methods, so that new treatment measures need to be developed as auxiliary treatment methods.
Malignant tumor cells can have genetic and epigenetic heterogeneity and instability by adjusting the expression of self-antigens, and the like, thereby obtaining the selective growth and adaptability of the clone avoiding the external pressure; in contrast, the immune system is in a relatively stable state. In recent decades, with the continuous and intensive research on the intrinsic mechanism of tumorigenesis and development and the external microenvironment, it is theorized that the ultimate disruption of tumors requires immunotherapeutic approaches. The application of immunotherapy, especially cellular immunotherapy, in tumor therapy gradually moves from the back to the front, becoming a fourth tumor therapy approach.
The immune cell therapy has the characteristics of breaking through the genetics and cell line mechanism of tumor drug resistance, targeting tumor cells and simultaneously extremely little damaging normal tissues. The immune cell therapy comprises CIK, DC-CIK and the like, the DC-CIK shows better targeting property and specificity, is more effective in treating tumors, has no toxic or side effect, has obvious effects of improving the life quality and prolonging the life cycle, is one of the best means for whole-body treatment of tumors at present, and has huge clinical potential.
The invention discloses an anti-tumor CTL epitope peptide derived from cytokeratin 19 and a method for preparing tumor therapeutic DC-CIK by using the peptide, which have potential value for treating various tumors related to the cytokeratin 19.
Disclosure of Invention
Based on the technical problems of the background art, the present invention aims to provide an HLA-A0201-restricted CTL epitope peptide specific for tumor antigen cytokeratin 19.
The invention also aims to provide the application of the HLA-A0201 restrictive CTL recognition epitope peptide of the specific tumor antigen cytokeratin 19.
The invention also aims to provide a preparation method of the tumor antigen cytokeratin 19 specific DC cell.
The invention also aims to provide a preparation method of the tumor antigen cytokeratin 19 specific DC-CIK cell.
The invention also aims to provide application of the DC cell and the DC-CIK cell with the specificity of the tumor antigen cytokeratin 19.
In order to achieve the purpose, the invention adopts the following technical scheme:
the HLA-A0201 restrictive CTL recognition epitope peptide of specific tumor antigen cytokeratin 19 provided by the invention is nonapeptide, and the amino acid sequence of the CTL recognition epitope peptide is as follows: Gly-Ala-Thr-Ile-Glu-Asn-Ser-Arg-Ile.
The HLA-A0201-restricted CTL epitope peptide of the specific tumor antigen cytokeratin 19 is synthesized by a solid phase synthesis method. The basic flow is as follows: firstly, connecting an amino acid with an amino group protected by Fmoc group on Wang resin of an insoluble solid phase carrier, and then removing the protecting group of the amino group, wherein the first amino acid is connected to the solid phase carrier; secondly, activating the second amino group by a carboxyl condensing agent of amino acid protected by Fmoc group, and reacting the activated second amino acid carboxyl with the amino group of the first amino acid connected with the solid phase carrier to form a peptide bond, thus generating the dipeptide with the protecting group on the solid phase carrier. Repeating the above peptide bond formation reaction to make peptide chain grow from C end to N end until reaching the required peptide chain length, finally cutting to obtain target peptide, and purifying by HPLC to obtain purity higher than 90%.
The invention also provides the application of the HLA-A0201 restrictive CTL recognition epitope peptide of the specific tumor antigen cytokeratin 19 in preparing a tumor therapeutic polypeptide vaccine expressed by the cytokeratin 19.
Preferably, the HLA-A0201 restrictive CTL recognition epitope peptide of the specific tumor antigen cytokeratin 19 is applied to preparing a lung cancer therapeutic polypeptide vaccine, and particularly has lethality to non-small cell lung cancer cells L78.
The invention also provides a preparation method of the DC cell with the specificity of the tumor antigen cytokeratin 19, which is obtained by adding the HLA-A0201 restrictive CTL recognition epitope peptide of the tumor antigen cytokeratin 19 into the DC cell for culture.
The invention also provides application of the specific DC cell obtained by the preparation method of the tumor antigen cytokeratin 19 specific DC cell in preparing a medicament for treating lung cancer.
The invention also provides a preparation method of the tumor antigen cytokeratin 19 specific DC-CIK cell, which is obtained by adopting HLA-A0201 restrictive CTL recognition epitope peptide of the specific tumor antigen cytokeratin 19 for induction.
The invention also provides application of the specific DC-CIK cell obtained by the preparation method of the tumor antigen cytokeratin 19 specific DC-CIK cell in preparing a medicament for treating lung cancer.
The invention skillfully utilizes cytokeratin 19 as a tumor-associated antigen with stronger specificity, presents high expression in lung cancer, breast cancer and digestive tumor, and has low or trace expression in normal tissues, thereby screening the epitope peptide of the tumor-associated antigen by adopting a method combining theory and experiment, the obtained epitope peptide is not reported in documents, providing a theoretical basis for developing tumor vaccines or tumor specific CTL cells based on the cytokeratin 19, and laying a foundation for the construction of subsequent multivalent antigen peptide vaccines.
Drawings
FIG. 1 is a diagram showing mass spectrometry analysis of an HLA-A0201-restricted CTL-recognizing epitope peptide of cytokeratin 19, which is a specific tumor antigen, according to the present invention.
FIG. 2 is a graph showing a comparison of INF-. gamma.secretion ability of specific CTL cells induced by P1, P2, and P3 … … P20 obtained in example 1 of the present invention.
FIG. 3 is a graph showing the comparison of the killing ability of specific CTL cells induced by P7, P9, P16 and P17 obtained in example 1 of the present invention against lung cancer cells L78.
FIG. 4 is a diagram showing the statistical analysis of flow cytometry detection data of specific CTL cell immune cell populations prepared from an HLA-A0201-restricted CTL epitope peptide of specific tumor antigen cytokeratin 19 according to the present invention.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1: synthetic epitope peptides
By adopting a method combining theory and practice, according to the primary structure of the antigen, an immunoinformatics method is comprehensively applied, SYFPEITHI, BIMAS, NetCTL, WAPP and EpiJen are comprehensively applied to carry out prediction analysis on the antigen CTL epitope of the cytokeratin 19, and the polypeptide sequence 20 before scoring is selected for experimental screening, wherein the polypeptide sequences are named as P1, P2 and P3 … … P20.
The basic flow is as follows: firstly, connecting an amino acid with an amino group protected by Fmoc group on Wang resin of an insoluble solid phase carrier, and then removing the protecting group of the amino group, wherein the first amino acid is connected to the solid phase carrier; secondly, activating the second amino group by a carboxyl condensing agent of amino acid protected by Fmoc group, and reacting the activated second amino acid carboxyl with the amino group of the first amino acid connected with the solid phase carrier to form a peptide bond, thus generating the dipeptide with the protecting group on the solid phase carrier. Repeating the peptide bond forming reaction to enable the peptide chain to grow from the C end to the N end until the required peptide chain length is reached, and finally cutting to obtain the target epitope peptide crude product. And (3) purifying the crude product of the target epitope peptide by HPLC to obtain the target epitope peptide refined peptide, wherein the purity of the target epitope peptide refined peptide is more than 90%, and the molecular weight of the target epitope peptide refined peptide is proved to accord with a theoretical value by mass spectrometry.
The HLA-A0201 restrictive CTL recognition epitope peptide of specific tumor antigen cytokeratin 19 provided by the invention is synthesized by adopting an Fmoc solid phase synthesis method, the CTL recognition epitope peptide is nonapeptide with the number of P17, and the amino acid sequence is as follows: Gly-Ala-Thr-Ile-Glu-Asn-Ser-Arg-Ile. Mass spectrometry of P16 was carried out, and the results are shown in FIG. 1, which confirmed that the molecular weight was 960.04326g/mol, which is in line with the theoretical value.
Example 2: polypeptide function detection
The P17 and the rest 19 epitope peptides obtained above can be used for preparing a polypeptide vaccine for treating tumors with cytokeratin 19 expression.
1. The CTL recognition epitope peptide induces specific CTL cells and IFN-gamma secretion and carries out killing experiment detection on tumor target cells: extracting peripheral blood of a patient, carrying out density gradient centrifugal separation to obtain PBMCs, adding cell factors to culture DC cells and CTL cells, further adopting the DC cells to load the cytokeratin 19 epitope peptide disclosed by the invention, carrying out coculture with CTL to stimulate specific CTL to amplify, and further adopting ELISA and LDH experiments in vitro to detect the secretion of INF-gamma of the specific CTL under the stimulation of specific antigen and the killing effect on lung cancer cells L78.
The specific method comprises the following steps:
(I) and PBMC separation and induction:
1) centrifuging 50mL of anticoagulated peripheral blood at 2000rpm for 10 min;
2) the upper plasma was collected and frozen, and the remaining blood cells were diluted with PBS (pH 7.4);
3) adding the diluted blood cells to the liquid surface of the equal volume of the lymph separation liquid;
4) centrifuging at 20 deg.C for 20min, and closing the centrifuge to brake;
5) centrifuging, dividing into four layers, and sucking the white membrane layer (i.e. the second layer) with a suction nozzle glass dropper;
6) the white film layer taken out is washed twice by PBS;
7) the cells are treated at 2-5X 106The cells are inoculated into a 6-well plate in a concentration of one milliliter (mL) mode, and after 2 hours, nonadherent cells are recovered and activated and cultured by using a culture plate pre-coated with anti-CD3 IgG and anti-CD28 IgG;
8) adding GM-CSF and IL-4 into adherent cells, stimulating and culturing for 5 days to induce DC cells, and changing the fluid for half the third day;
9) on the 5 th day, DC cells were collected, 10. mu.g of the epitope peptide propeptide of interest obtained in example 1 was added to the DC cells, and after 1 hour, the DC cells were co-cultured with activated T cells, while IL2 and IL-15 were added;
10) after further culturing for 5 days, the antigen specific CTL cell is obtained to carry out cytokine secretion and killing experiment on the tumor cell.
(II) IFN-gamma cytokine secretion detection: human IFN-gamma Platinum ELISA (IFN-gamma ELISA detection kit, eBioscience) detects IFN-gamma secreted by CTL cells, and the steps are as follows:
1) removing cytokines from CTL cells, culturing for 24h, and inoculating the CTL cells into a 96-well plate;
2) adding polypeptide corresponding to stimulation into the cells, stimulating for 24h again, centrifuging to remove the cells, and collecting cell supernatant;
3) and (3) detecting the expression of IFN-gamma in the supernatant by adopting ELISA, and selecting CTL epitope peptide with more IFN-gamma secretion for carrying out killing experiment.
The results are shown in fig. 2, P17 and P7, P9 and P16 loaded DC cells can induce specific CTL cells well and secrete high amounts of IFN- γ.
(III) tumor cell killing experiment: cell killing ability was measured by Lactate Dehydrogenase (LDH) release Assay using a CytoTox 96Non-Radioactive cytoxicity Assay (Cytotoxicity Assay kit, Promega). The method comprises the following steps:
1) setting up a detection culture plate (100 mu L/hole)
a. Experimental groups were set up: the lung cancer cells L78 showing cytokeratin 19 positivity and HLA-A0201 positivity are used as target cells, and CTL cells with the characteristics are added according to the ratio of effector cells to target cells of 5:1, 10:1 and 20:1
b. Establishment of effector cell spontaneous Release group
c. Set up target cell spontaneous release group
d. Setting the maximum release group of target cells
e. Setting up a background control group
2) Cell lysis and supernatant harvest
a.37℃5％CO2Co-culturing for 5h
b. Adding lysis solution into the group with maximum release of target cells, centrifuging all the wells at 250g/min after 45min, and collecting supernatant
3) LDH detection
a. Transfer 50. mu.L of supernatant to another 96-well plate
b. Add 50. mu.L of diluted substrate mixture to each well and incubate in the dark at room temperature for 30min
c. Add 50. mu.L of stop solution per well
d. Detection of the absorbance OD at 490nm
The cell killing rate calculation formula is as follows:
percent of destruction (%) [ (OD)Experimental group-ODEffector cell spontaneous release group-ODTarget cell spontaneous release group)/(ODMaximum release group of target cells-ODTarget cell spontaneous release group)]×100％
The results are shown in FIG. 3, in which FIG. 3 is a graph comparing the killing ability of specific CTL cells induced by P7, P9, P16 and P17 obtained in example 1 of the present invention to lung cancer cells L78; as shown in fig. 3, the CTL cells induced by P17 were most effective in killing lung cancer cells L78.
2. The percentage of each immune cell in the specific CTLs obtained by flow analysis is shown in fig. 4. As shown in FIG. 4, the immune cell population prepared from P17 of the present invention contains a large number of CTL cells and a certain proportion of NKT cells, i.e., the immune cell population has good immunocompetence and strong ability to kill specific tumor cells.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. An HLA-A0201 restrictive CTL recognition epitope peptide of specific tumor antigen cytokeratin 19, which is nonapeptide, and the amino acid sequence of the CTL recognition epitope peptide is Gly-Ala-Thr-Ile-Glu-Asn-Ser-Arg-Ile.
2. Use of the HLA-a 0201-restricted CTL epitope peptide of specific tumor antigen cytokeratin 19 according to claim 1 for the preparation of a tumor therapeutic polypeptide vaccine having cytokeratin 19 expression.
3. Use of the HLA-A0201-restricted CTL (cytotoxic T lymphocyte) recognition epitope peptide of the specific tumor antigen cytokeratin 19 as defined in claim 1 for preparing a lung cancer therapeutic polypeptide vaccine.
4. A method for producing DC cells specific to the tumor antigen cytokeratin 19, which comprises adding the HLA-A0201-restricted CTL epitope-recognizing peptide of the specific tumor antigen cytokeratin 19 according to claim 1 to DC cells and culturing the cells in vitro.
5. The use of the specific DC cell obtained by the method for preparing a tumor antigen cytokeratin 19 specific DC cell according to claim 4 in the preparation of a medicament for treating lung cancer.
6. A method for producing DC-CIK cells specific to the tumor antigen cytokeratin 19, which comprises inducing in vitro the HLA-A0201-restricted CTL-recognizing epitope peptide of the specific tumor antigen cytokeratin 19 according to claim 1.
7. The use of the specific DC-CIK cell obtained by the method for preparing the tumor antigen cytokeratin 19 specific DC-CIK cell according to claim 6 in the preparation of a medicament for treating lung cancer.
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| CN101891815A (en) * | 2009-05-22 | 2010-11-24 | 中国科学院上海生命科学研究院 | CK19 monoclonal antibody, and preparation method and application thereof |
| CN104830782A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | CK19 antigen based DC cell and targeting immune cell population, and preparation method and application thereof |
| US9163276B2 (en) * | 2011-07-22 | 2015-10-20 | Tohoku University | Method for fabricating stable-isotope-labeled target peptide fragment in mass spectrometry |
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| CN101891815A (en) * | 2009-05-22 | 2010-11-24 | 中国科学院上海生命科学研究院 | CK19 monoclonal antibody, and preparation method and application thereof |
| US9163276B2 (en) * | 2011-07-22 | 2015-10-20 | Tohoku University | Method for fabricating stable-isotope-labeled target peptide fragment in mass spectrometry |
| CN104830782A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | CK19 antigen based DC cell and targeting immune cell population, and preparation method and application thereof |
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