CTL recognition epitope peptide of tumor antigen MAGE3 and application thereof
Technical Field
The invention relates to the technical field of epitope peptides, in particular to HLA-A2 restrictive CTL recognition epitope peptide of a specific tumor antigen MAGE3 and application thereof, a preparation method and application of a tumor antigen MAGE3 specific DC cell, and a preparation method and application of a tumor antigen MAGE3 specific DC-CIK cell.
Background
The MAGEs (melanoma antigen genes) gene family was first discovered by the T.Boon research group in the melanoma cell line MZ 2-MEL. The MAGE3 is one of MAGES members, is identified by Gaugler separation in 1994, is expressed in melanoma, breast cancer, lung cancer and digestive tumors, belongs to a tumor-testis antigen, is not expressed in normal tissues except testis and placenta, and is a tumor-associated antigen with strong specificity. Meanwhile, the antigen can cause the organism to generate cellular immunity and humoral immunity, and is considered to be one of tumor antigens with stronger immunogenicity.
In the current clinical tumor treatment strategies, local treatments such as surgery, radiotherapy and the like have better curative effect on local tumors, and the treatment on systemic, metastatic and micro-focus mainly depends on chemotherapy and immunotherapy. Recent studies have shown that some tumor cells metastasize to other tissues or organs at an early stage of tumor development, and remain latent temporarily, which may be the main reason for recurrence and metastasis of many tumors after treatment. Therefore, the treatment of tumor needs to be performed as a systemic disease, and it is possible to actually and effectively improve the cure rate and survival time of tumor patients by not only removing the tumor of local focus, but also preventing the recurrence and metastasis of tumor and the invasion to viscera. However, although the traditional chemotherapy drugs make a certain progress in the treatment of some tumors, the 5-year survival rate of tumor patients is not improved obviously, and meanwhile, the chemotherapy drugs are easy to have acquired multi-drug resistance and toxic and side effects, so that the clinical use of the chemotherapy drugs is limited, and therefore, a better means for systemic treatment is needed.
Immune cell therapy is developed in the last two decades, including 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 remarkable 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 great clinical potential.
However, there is still a need for improvement in inducing antigens of specific DC-CIK cells, and CTL epitope peptide polypeptide antigens capable of efficiently stimulating tumor-specific DC-CIK are lacking.
Disclosure of Invention
Based on the technical problems of the background art, the present invention aims to provide an HLA-A2 restricted CTL recognition epitope peptide of specific tumor antigen MAGE 3.
The invention also aims to provide application of the HLA-A2 restricted CTL epitope-recognizing peptide of the specific tumor antigen MAGE 3.
The invention also aims to provide a preparation method of the tumor antigen MAGE3 specific DC cell.
The invention also aims to provide a preparation method of the tumor antigen MAGE3 specific DC-CIK cell.
The invention also aims to provide application of the tumor antigen MAGE3 specific DC cell and DC-CIK cell.
In order to achieve the purpose, the invention adopts the following technical scheme:
the HLA-A2 restrictive CTL recognition epitope peptide of the specific tumor antigen MAGE3 provided by the invention is nonapeptide, and the amino acid sequence of the CTL recognition epitope peptide is as follows:
Ile-Leu-Gly-Asp-Pro-Lys-Lys-Leu-Leu。
the HLA-A2-restricted CTL-recognition epitope peptide of the specific tumor antigen MAGE3 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 application of the HLA-A2 restricted CTL recognition epitope peptide of the specific tumor antigen MAGE3 in preparing a tumor therapeutic polypeptide vaccine expressed by MAGE 3.
Preferably, the HLA-A2 restricted CTL epitope recognition peptide of the specific tumor antigen MAGE3 is applied to preparing a breast cancer therapeutic polypeptide vaccine, and particularly has lethality to breast cancer cells MCF-7.
The invention also provides a preparation method of the tumor antigen MAGE3 specific DC cell, which is obtained by adding the HLA-A2 restricted CTL recognition epitope peptide of the specific tumor antigen MAGE3 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 MAGE3 specific DC cell in preparation of a medicine for treating breast cancer.
The invention also provides a preparation method of the tumor antigen MAGE3 specific DC-CIK cell, which is obtained by inducing HLA-A2 restricted CTL recognition epitope peptide of the specific tumor antigen MAGE 3.
The invention also provides application of the specific DC-CIK cell obtained by the preparation method of the tumor antigen MAGE3 specific DC-CIK cell in preparation of a medicine for treating breast cancer.
The invention skillfully utilizes MAGE3 as a tumor-associated antigen with stronger specificity, which shows high expression in melanoma, breast cancer, lung cancer and digestive tumor, but does not express in normal tissues, thereby screening the epitope peptide of the tumor-associated antigen by adopting a method combining theory and experiment, and the obtained epitope peptide is not reported in documents, thereby providing a theoretical basis for developing tumor vaccines or tumor-specific CTL cells based on the MAGE3, and laying a foundation for the construction of subsequent multivalent antigen peptide vaccines.
Drawings
FIG. 1 is a diagram showing mass spectrometry analysis of HLA-A2-restricted CTL-recognizing epitope peptide of a specific tumor antigen MAGE3 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 comparison of killing ability of specific CTL cells induced by P2, P7, P16 and P19 obtained in example 1 of the present invention against breast cancer MCF-7 cells.
FIG. 4 is a diagram showing the statistical analysis of flow cytometry results of specific CTL cell immune cell populations prepared from the HLA-A2-restricted CTL epitope peptide of specific tumor antigen MAGE3 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 predictive analysis on the antigen CTL epitope of MAGE3, and the polypeptide sequences before scoring 20 are 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-A2 restrictive CTL recognition epitope peptide of the specific tumor antigen MAGE3 is synthesized by adopting an Fmoc solid phase synthesis method, the CTL recognition epitope peptide is nonapeptide with the serial number of P16, and the amino acid sequence is as follows: Ile-Leu-Gly-Asp-Pro-Lys-Lys-Leu-Leu. Mass spectrometry of P16 was carried out, and the results are shown in FIG. 1, which confirmed that the molecular weight was 996.26g/mol, which is in line with the theoretical value.
Example 2: the CTL recognition epitope peptide induces specific CTL cells and IFN-gamma secretion and killing experiment 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 MAGE3 epitope peptide of the invention, carrying out co-culture 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 breast cancer cells MCF-7.
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 10
6The cells are inoculated into a 6-well plate in a volume of one milliliter (mL) mode, after 2 hours, nonadherent cells are recovered, and activated and cultured by using a culture plate pre-coated with anti-CD3IgG 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, P16 and P2, P7 and P19 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 CytoTox96Non-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 breast cancer cells MCF-7 with MAGE3 positive and HLA-A2 positive 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%CO
2Co-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-OD
Effector cell spontaneous release group-OD
Target cell spontaneous release group)/(OD
Maximum release group of target cells-OD
Target cell spontaneous release group)]×100%
The results are shown in FIG. 3, FIG. 3 is a graph comparing the killing ability of specific CTL cells induced by P2, P7, P16 and P19 obtained in example 1 of the present invention to breast cancer MCF-7 cells; as shown in FIG. 3, P16-induced CTL cells were most effective in killing MCF-7 tumor cells.
Example 3: analysis of various immune cell compositions in specific CTL
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 P16 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.