CN117486999A - Tumor T cell epitope peptide, pMHC, and preparation and application thereof - Google Patents
Tumor T cell epitope peptide, pMHC, and preparation and application thereof Download PDFInfo
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- CN117486999A CN117486999A CN202311435378.XA CN202311435378A CN117486999A CN 117486999 A CN117486999 A CN 117486999A CN 202311435378 A CN202311435378 A CN 202311435378A CN 117486999 A CN117486999 A CN 117486999A
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- cell epitope
- epitope peptide
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- A61K2039/515—Animal cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a tumor T cell epitope peptide, the amino acid sequence of which is shown in SEQ ID NO: 1-SEQ ID NO:3, respectively. The tumor T cell epitope peptide can be used for in-vitro T cell activation experiments, and can be applied to research and development of general tumor vaccines for tumors with high expression of MAGE-A3 and MAGE-A10 antigens, such as melanoma, non-small cell lung cancer, liver cancer, head and neck tumors and the like, diagnosis of tumors and research of TCR-T therapy. The tumor T cell epitope peptide is prepared into a pMHC complex or a direct load guide antigen presenting cell, so that the T cell is activated, and the tumor T cell epitope peptide can be applied to: development and preparation of tumor vaccine, detection of whether the organism has anti-tumor cell immunity function, auxiliary diagnosis and TCR-T cell immunotherapy development.
Description
The application is a divisional application of an invention patent application with the application date of 2022, 8-month and 30-day and the application number of 202211049861X and the invention name of 'a tumor T cell epitope peptide, pMHC and preparation and application'.
Technical Field
The invention relates to the technical field of biology, in particular to a tumor T cell epitope peptide, pMHC, preparation and application thereof.
Background
The incidence and mortality of tumors are in an increasing state worldwide, and the most significant problem that exists today is the lack of effective treatments. Tumor immunotherapy refers to exogenous intervention of the immune system of an organism, restarting and maintaining 'tumor-immune' circulation, recovering and improving the anti-tumor immune response of the organism, enhancing the recognition and killing ability of tumor cells, and thus achieving the therapeutic effect of controlling and even specifically eliminating tumors. Compared with the traditional treatment means such as surgery, radiotherapy, chemotherapy and the like, the tumor immunotherapy has the advantages of strong specificity and small side effect, and currently, the tumor immunotherapy methods which are applied to clinic mainly comprise immune checkpoint inhibitor treatment, adoptive cell immunotherapy, cancer vaccine, some emerging immunotherapy methods and the like. The immunotherapy of cancer is evaluated by the journal of science as the first of ten scientific breakthroughs in 2013, and is also a novel anti-tumor treatment means which is widely researched and developed at present.
The cancer immune cycle includes several steps: antigens produced by cancer cells are released after death of the cancer cells and captured by Dendritic Cells (DCs). Next, DCs present antigens captured on MHC molecules to T cells, thereby eliciting and activating responses of effector T cells to cancer antigens. Activated T cells enter and infiltrate the tumor site under the direction of chemokines. T cells specifically recognize and bind to and kill cancer cells through the interaction of TCRs with antigen-MHC complexes. Thus, T cell immune responses play an important role in anti-tumor immune defenses, occupying an important role in vaccine development. The first step in the cd8+ T cell immune response is the specific recognition of the epitope peptide presented by the cancer cell by the T cell through its surface antigen recognition receptor. Therefore, the epitope peptide is an important key molecule for specifically recognizing tumor cells and playing an immunoprotection role by T cells, and is a key targeting molecule for immunodetection, immunotherapy and vaccine development.
Chimeric antigen receptor T cell technology (CAR-T) and T cell receptor chimeric T cells (TCR-T) are currently receiving extensive attention and research as two most recent immune cell technologies. CAR-T cell therapy has achieved better efficacy in hematological tumors, but the cytokine storm and neurotoxic side effects it causes can be life threatening. TCR-T cell therapy is one of the effective immune cell therapies for solid tumors, and TCR alpha and beta chain genes capable of recognizing tumor-specific antigens are transfected into T cells, which are modified to express the tumor-antigen-specific TCR antigen-binding regions, thereby specifically recognizing the corresponding tumor antigens. Then the T lymphocyte expressing the tumor antigen specific TCR can recognize the HLA-antigen peptide complex on the surface of the tumor cell by in vitro amplification and reinfusion into human body, thereby triggering the immune effect of the T cell and achieving the purpose of killing the tumor cell. Tumor vaccine is prepared by inducing organism to generate tumor specific immune response by using tumor related antigens (TAAs), tumor polypeptide or tumor cell lysate, etc., protecting organism from tumor cell invasion, and preventing and treating tumor. Including two major classes, prophylactic tumor vaccines and therapeutic tumor vaccines. The therapeutic tumor vaccine mainly aims at tumor patients, and the purpose of treating tumors is achieved by generating specific antibodies, effector cells and specific immune memory cells in the body of the patients through immune induction. Clinical trials have demonstrated that tumor whole cell vaccines, tumor specific protein or polypeptide vaccines, and tumor nucleic acid vaccines exhibit great potential for use in tumor therapy.
The MAGE family is a proto-cancer antigen, one of the members of the cancer testis (cancer/testes) antigen superfamily. More than 60 members of the MAGE family are present in the same MAGE homology domain. Two main subclasses are: MAGE-I antigens and MAGE-II antigens. The expression of MAGE-II antigens is commonly found in normal cells, while the MAGE-I antigens are TAAs, i.e. they are not expressed in tissues of the human body except testis germ cells and placenta trophoblast, but are highly expressed in tumors, which is a hot spot in tumor research. The antigen of cancer testis cannot be recognized by T cells because of the lack of HLA by germ cells, but the simultaneous expression of antigen of cancer testis and HLA molecule can be recognized by T cells in malignant tumor. MAGE-I antigens can be divided into three subclasses: MAGE-A, MAGE-B and MAGE-C. The MAGE-A subfamily has strict expression patterns, wherein MAGE-A3, MAGE-A4, MAGE-A10, MAGE-A12 and the like are antigens with unique characteristics in the MAGE family, and have high specificity expression in patients with melanoma, liver cancer, lung cancer, esophageal cancer and the like. In recent years, two methods have been used for targeting anticancer effects of MAGE-A family genes: (1) vaccinating with MAGE epitope antigen vaccine; (2) TCR-T immune cell therapy.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a tumor T cell epitope peptide, pMHC, and preparation and application thereof.
The first object of the invention is to provide a tumor T cell epitope peptide.
The second object of the invention is to provide a tumor T cell epitope peptide composition.
A third object of the present invention is to provide a pMHC complex.
It is a fourth object of the present invention to provide an antigenic peptide-antigen presenting cell complex.
The fifth object of the present invention is to provide a gene encoding the above tumor T cell epitope peptide.
The sixth object of the present invention is to provide the application of the tumor T cell epitope peptide, the tumor T cell epitope peptide composition, the pMHC complex, the antigen peptide-antigen presenting cell complex and/or the gene in preparing tumor vaccine and/or tumor medicine.
In order to achieve the above object, the present invention is realized by the following means:
T2-A2 is an antigen presenting cell line expressing human MHC class I molecule HLa-A2 by recombinant genetic engineering techniques. Only effective epitope peptides can be presented, so that a stable pMHC complex is formed on the cell surface, and therefore, the antigen peptide can be used as artificial antigen presenting cells for stimulating T cells.
T cell epitope peptides alone cannot work and must be activated in the manner of pMHC complexes or antigen peptide-antigen presenting cell complexes. The invention utilizes MHC monomer and identified novel tumor T cell epitope to carry out joint renaturation, thus preparing the pMHC complex. The identified novel tumor T cell epitope peptide is loaded on the surface of antigen presenting cells (T2-A2 cells), antigen peptide-antigen presenting cell complexes are prepared, then the prepared pMHC complexes are used for labeling the T cells, and the antigen epitope peptide can be found to effectively activate T cells in peripheral blood of healthy people to produce killer cytokines and also can effectively kill target cells carrying tumor MAGE-A3 and MAGE-A10 antigens. The T cell epitope peptide is assembled into a pMHC complex, and can be detected in peripheral blood of a non-small cell lung cancer patient. The newly discovered MAGE-A3 and MAGE-A10T cell epitope peptides can effectively induce T cell immunity, and can be applied to the development of general tumor vaccines and immune cell treatment means, such as TCR-T.
Therefore, the invention claims a tumor T cell epitope peptide, the amino acid sequence of which is shown in SEQ ID NO: 1-SEQ ID NO:3, respectively.
MAGE-A3-Mp4(SEQ ID NO：1)：LVFGIELMEV；
MAGE-A10-Mp30(SEQ ID NO：2)：VIWEALNMM；
MAGE-A10-Mp32(SEQ ID NO：3)：SLLKFLAKV。
The invention also claims a tumor T cell epitope peptide composition, wherein the tumor T cell epitope peptide contains an amino acid sequence shown in SEQ ID NO: 1-SEQ ID NO:3, and one or more of the tumor T cell epitope peptides.
The invention also claims a pMHC complex, which contains the tumor T cell epitope peptide and/or the tumor T cell epitope peptide composition.
Preferably, the preparation method of the pMHC complex comprises the following steps: renaturation of HLA-A2 heavy chain, HLA-A2 light chain beta 2m and the tumor T cell epitope peptide is carried out.
The invention also claims an antigen peptide-antigen presenting cell complex, which is an antigen presenting cell with the tumor T cell antigen epitope peptide and/or the tumor T cell antigen epitope peptide composition on the surface.
Preferably, the antigen presenting cells are T2-A2 cells.
More preferably, the T2-A2 cells are T2 cells that overexpress HLa-A2.
More preferably, the preparation method of the antigen peptide-antigen presenting cell complex comprises the following steps: and mixing and incubating the tumor T cell epitope peptide and the antigen presenting cell to obtain an antigen peptide-antigen presenting cell complex.
The invention also discloses a gene for encoding the tumor T cell epitope peptide.
The invention also claims application of the tumor T cell epitope peptide, the tumor T cell epitope peptide composition, the pMHC complex, the antigen peptide-antigen presenting cell complex and/or the gene in preparing tumor vaccine and/or tumor medicine; the tumor is lung cancer.
Preferably, the tumor also includes a tumor with high expression of MAGE-A3 and/or MAGE-A10.
More preferably, the tumor in which MAGE-A3 and/or MAGE-A10 is highly expressed is melanoma, liver cancer, and/or esophageal cancer.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers 3 tumor T cell epitope peptides, and can be used for in vitro T cell activation experiments. The tumor T cell epitope peptide can be applied to research and development of general tumor vaccines for tumors such as melanoma, non-small cell lung cancer, liver cancer, head and neck tumors and the like, diagnosis of tumors and research of TCR-T therapy aiming at tumors with high expression of MAGE-A3 and MAGE-A10 antigens.
The tumor T cell epitope peptide is prepared into a pMHC complex or a direct load guide antigen presenting cell, so that the T cell is activated, and the tumor T cell epitope peptide can be applied to:
(1) Development and preparation of tumor vaccine: the tumor antigen is released after the tumor cells die, DC presents the antigen captured on MHC molecules to T cells, so that the response of effector T cells to cancer antigens is initiated and activated.
(2) Detecting whether the organism has anti-tumor cell immunity function: the tumor antigen specific T cells are detected in the body of the subject, which represents that the body has generated T cell immune function, and the proportion of the antigen specific CD8T marked by the pMHC complex prepared by the antigen epitope peptide is used for reflecting the intensity of the anti-tumor immune reaction of the subject.
(3) Auxiliary diagnosis: the tumor T cell epitope peptide or the tumor T cell epitope peptide composition can detect specific immunoreaction components, thereby improving the diagnosis specificity and rapidly screening tumor patients positive to MAGE-A3 or MAGE-A10 antigen.
(4) TCR-T cell immunotherapy development.
Drawings
FIG. 1 is an assembled exemplary diagram of a pMHC complex;
FIG. 2 is an identification of T2-A2 antigen presentation of 31 candidate tumor T cell epitope peptides; a: identification test results of T2-A2 antigen presentation of 31 candidate tumor T cell epitope peptides; b: a statistical plot of plot a;
FIG. 3 is a diagram showing the detection of the formation of pMHC complexes by 31 candidate tumor T cell epitope peptides;
FIG. 4 is a graph showing activation of CD8+ T cells by 31 candidate tumor T cell epitope peptides; a: a flow cytometer detection map; b: CD8+ T cell activation bar graphs corresponding to MAGE-A3-MP4, MAGE-A10-MP30 and MAGE-A10-MP 32;
FIG. 5 is a graph showing the release assays of IFN-. Gamma.and GZMB from CD8+ T cells; a: flow cytometry detected GZMB fraction map of cd8+ T cell release; b: flow cytometry detects IFN-gamma ratio maps of CD8+ T cell release;
FIG. 6 is a graph showing the detection results of Annexin V-APC;
FIG. 7 is a graph showing specific CD8+ T cell fractions in non-small cell cancer patients and healthy humans; a: results of flow cytometry detection of MAGE-A3-Mp4, MAGE-A10-Mp30 and MAGE-A10-Mp32 epitope specific CD8+ T cells in HLA-A2 positive NSCLC patients and healthy humans; b: results for detection of CD8+ T cells in NSCLC patients and healthy humans by pMHC complexes prepared by MAGE-A3-Mp4, MAGE-A10-Mp30 and MAGE-A10-Mp 32.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 prediction and identification of tumor T cell epitope peptides
An example of the assembly of pMHC complexes is shown in figure 1.
1. Experimental method
(1) Prediction of tumor T cell epitope peptides
CD8T cell epitope prediction was performed on tumor antigens MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10 and MAGE-A12 protein sequences using the "MHC I Binding" tool in the IEDB Recommended 2.22 (http:// www.iedb.org /) online website.
MHC alleles were selected as HLA-A 02:01, yielding 31 candidate tumor T cell epitope peptides, see Table 1.
(2) Identification of tumor T cell epitope peptides
According to the sequences of Table 1, 31 candidate tumor T cell epitope peptides obtained in step (1) were artificially synthesized, and each was prepared as a stock solution at a concentration of 10mM with DMSO.
Taking logarithmic phase T2-A2 cells and inoculating into 96-well plate (2×10) 5 Well), a blank group, a negative control group, a positive control group and a respective tumor T cell epitope peptide group were respectively set, 3 duplicate wells per group, and the final volume of each well was 200. Mu.L.
Blank groups are only T2-A2 cells in the wells;
the Negative control group is the co-incubation of T2-A2 cells and Negative peptides (EB virus peptide, amino acid sequence IVTDFSVIK, shown as SEQ ID NO: 4);
the Positive control group is the co-incubation of T2-A2 cells and Positive peptides (influenza A M1 peptide, amino acid sequence GILGFVEFTL, shown as SEQ ID NO: 5);
31 tumor T cell epitope peptide groups are arranged, and 31 different candidate tumor T cell epitope peptides and T2-A2 cells which are artificially synthesized are respectively incubated together.
After incubating the 96-well plate at 37 ℃ for 4 hours, centrifuging to remove supernatant, washing twice, incubating cell precipitates with FITC anti-human HLA-A2 (beta 2 m) antibody at 4 ℃ for 30 minutes in a dark place, and detecting each group of cells by a flow cytometer.
The above operation was repeated for three parallel tests.
2. Experimental results
Specific information for predicting 31 candidate tumor T cell epitope peptides is shown in table 1.
Table 1:31 candidate tumor T cell epitope peptides
The identification results of the tumor T cell epitope peptide are shown in fig. 2, and the results show that: the 31 tumor T cell epitope peptides predicted in the step (1) have 29 tumor T cell epitope peptides (except MAGE-A4-Mp14 and MAGE-A6-Mp 24) which can be effectively presented to T cells by antigen presenting cells.
The results illustrate: the 29 tumor T cell epitope peptides are T cell epitope peptides.
Example 2 detection of tumor T cell epitope peptide formation of pMHC tetramer complex
1. Experimental method
(1) Preparation of pMHC complex monomer of tumor T cell epitope peptide
The mother liquor (10 mM) obtained in step (2) of example 1 was diluted to 400. Mu.M with PBS to obtain a diluted mother liquor, which was placed on ice for use.
20 mu L of diluted mother solution and 20 mu L of Flex-T monomer (200 mu g/mL) are added into a 96-well U-shaped plate, and the mixture is blown and mixed uniformly, and a sealing plate is taken off for mold; placing the 96-hole U-shaped plate on ice, and irradiating for 30min by using a UV lamp (the distance between the UV lamp and a sample is kept between 2 and 5 cm); covering a sealing plate, and incubating for 30min at 37 ℃ in a dry and dark place to obtain 31 pMHC complex monomers formed by 31 candidate tumor T cell epitope peptides and Flex-T monomers respectively, and setting the 31 pMHC complex monomers as an experimental group.
The pMHC complex formed by influenza A M1 peptide (amino acid sequence: GILGFVEFTL, shown as SEQ ID NO: 5) and Flex-T monomer was set as positive control group Pos;
the pMHC complex formed by EB virus peptide (amino acid sequence: IVTDFSVIK, shown as SEQ ID NO: 4) and Flex-T monomer was set as negative control group Neg;
pMHC monomers formed by PBS and Flex-T monomers were set as UV control;
(2) ELISA detects the ability of epitope peptides to form pMHC tetramer complexes
ELISA is adopted to detect whether 31 candidate tumor T cell epitope peptides synthesized in the step (2) of the example 1 can form pMHC tetramers, and the specific method is as follows:
100. Mu.L of 0.5. Mu.g/mL streptavidin (Streptavidin solution) was added to the 96-well plate at room temperature and incubated overnight (16-18 h), followed by 3 washes with 300. Mu.L of 1 XWash Buffer and 1 XDiluon Buffer (1M NaCl,0.5M Tris,1%BSA (w/v), 0.2% Tween 20 (w/v), pH=8.0) and blocking at room temperature for 30min.
The pMHC complex monomers of step (1) (positive control Pos, negative control Neg, UV control and experimental) were diluted 1200-fold with 1x Dilution Buffer, respectively.
Simultaneously setting a Monomer group: equally diluting Flex-T monomer; blank group: only 1 Xvolume Buffer was added in equal amounts to the other groups.
Taking positive control Pos as an example:
the 96-well plate was discarded, the solution was dried on filter paper, and 100. Mu.L of diluted pMHC complex monomer was added to the 96-well plate.
Negative control Neg, UV control, experimental, monomer and Blank groups were equally treated and added to 96-well plates.
Cover the plate and incubate for 1h at 37 ℃. After the incubation, the 96-well plate was washed 3 times with washing buffer, followed by addition of 100. Mu.L of diluted HRP-anti-. Beta.2M (antibody BioLegend, cat#280303, U.S. Pat. No.), further incubation at 37℃for 1h, and washing after the incubation was completed.
To each well was then added 100. Mu.L of substrate solution (10.34 mL of deionized water, 1.2mL of citric acid monohydrate/trisodium citrate dihydrate at pH 4.0,0.1M, 240. Mu.L of 40nM ABTS, 120. Mu.L of hydrogen peroxide solution) and developed for 8min with shaking (400-500 rpm) in the dry dark at room temperature (18-25 ℃).
The reaction was stopped using 50. Mu.L Stop Solution (2% oxalic acid dihydrate, w/v). Absorbance (OD) was measured at 414nm using a microplate reader over 30min.
2. Experimental results
The relative OD values of the UV control group, the positive control group Pos, the influence control group Neg and the experimental group are calculated by taking the OD value of the Monomer group as 100%, and the ratio value represents the formation of pMHC tetramer of each tumor antigen epitope peptide. If the relative OD value is greater than that of the UV control group, it is judged that pMHC tetramers can be formed.
The results are shown in fig. 3, which shows: the relative OD values of the MAGE-A4-Mp14 and MAGE-A4-Mp22 complex groups were significantly reduced compared to the UV control group, and the remaining groups were significantly elevated (P < 0.001). The results illustrate: the 31 candidate tumor T cell epitope peptides can form pMHC tetramer complexes except MAGE-A4-Mp14 and MAGE-A4-Mp22, and the rest 29 candidate tumor T cell epitope peptides are potential candidate epitope peptides which can cause specific immune response.
EXAMPLE 3 tumor T cell epitope peptide-antigen presenting cell activating T cells
The antigen peptide/MHC complex on the surface of an APC or target cell binds to a TCR providing a first signal of T cell activation; the B7 molecule on the APC surface binds to the CD28 molecule on the T cell, providing a second signal; IL-2, etc. provide a costimulatory signal. The ability of vaccinated tumor antigen-loaded T2A2 to induce activation of CD8T cells was analyzed by co-culturing tumor antigen-epitope peptides capable of binding to HLA molecules with CD8T cells after loading them with T2A2, detecting the proportion of activated CD8T cells by tetramers, and flow-analyzing the antigen-specific T cell proportion producing gamma-interferon (IFN-gamma) and Granzyme (GZMB).
1. Experimental method
Mononuclear lymphocytes (PBMCs) from peripheral venous blood of healthy volunteers were isolated and cd8+ T cells were further isolated. T2-A2 cells were labeled with CFSE, followed by treatment with mitomycin at 20. Mu.g/mL for 30min, and incubated with 31 candidate tumor T cell epitope peptides of step (1) of example 1, respectively.
mu.L of the pMHC complex monomer of the tumor T cell epitope peptide obtained in the step (1) of the example 2 was taken out into a 1.5mL EP tube, 3.3 mu.L of streptavidin (BioLegend Cat#405203, US) was added, the mixture was blown and mixed with a gun head, and incubated at 4℃for 30min in the absence of light.
After the incubation was completed, 2.4. Mu.L of blocking solution (1.6 ul,50mM D-biotin (Thermo Fisher, cat#B20656, US), 6ul of 10% (w/v) NaN3 and 192.4ul PBS) was added to the EP tube and the reaction was stopped by pipetting. Incubating overnight at 4-8 ℃ to obtain the pMHC tetramer complex.
The Mixed peps group is set to: will be 0.5X10 6 Cd8+ T cells of (2) and 0.5x10 6 T2-A2 cell on-culture loaded with 31 candidate tumor T cell epitope peptidesCo-culturing in culture medium.
And co-stimulated with 1. Mu.g/mL of anti-human CD28 antibody core 50IU/mL of IL-2. And (3) supplementing 50IU/mL of IL-2 and 20 mu M of candidate tumor T cell epitope peptide into the culture medium every two days in the culture process.
After 7 days of culture, specific cd8+ T cells were labeled with pMHC tetramer complex, specific cd8+ T proportion and antigen specific cd8+ T cell release IFN- γ and GZMB were detected, and T2-A2 apoptosis marker Annexin V-APC percentage was simultaneously detected.
Meanwhile, a positive control group (T2-A2 cell-loaded influenza A M1 peptide), a negative control group (T2-A2 cell-loaded EB virus peptide) and a UV control group (pMHC complex formed by PBS and Flex-T monomer) are arranged for equivalent treatment detection.
2. Experimental results
The results of activation of cd8+ T cells by different tumor T cell epitope peptides are shown in fig. 4; the results show that: the 31 candidate tumor T cell epitope peptides have 3 tumor T cell epitope peptides which can activate T cells, namely MAGE-A3-Mp4 (SEQ ID NO: 1), MAGE-A10-Mp30 (SEQ ID NO: 2) and MAGE-A10-Mp32 (SEQ ID NO: 3), and the proportion of activated CD8+ T cells is 5.20%,0.55% and 5.21%, respectively.
The release of IFN-gamma and GZMB by CD8+ T cells is shown in FIG. 5; the results show that: the efficiency of activated CD8+ T cells in the mixed peps group to release IFN-. Gamma.and GZMB was significantly increased (P < 0.001) compared to the negative control group, neg ctrl; the results illustrate: specific CD8+ T cells activated by MAGE-A3-MP4 (SEQ ID NO: 1), MAGE-A10-MP30 (SEQ ID NO: 2) and MAGE-A10-MP32 (SEQ ID NO: 3) epitopes can release IFN-gamma and GZMB.
The detection results of Annexin V-APC are shown in FIG. 6; the results show that: the proportion of mixed peps group Annexin v+cfse+t2a2 cells was extremely significantly increased (P < 0.001) compared to the negative control group Neg ctrl; the results illustrate: specific CD8+ T cells activated by MAGE-A3-Mp4 (SEQ ID NO: 1), MAGE-A10-Mp30 (SEQ ID NO: 2) and MAGE-A10-Mp32 (SEQ ID NO: 3) epitopes can kill target cells T2-A2.
Example 4 detection of peripheral blood epitope peptide-specific cytotoxic T cells in clinical patients
1. Experimental method
Mononuclear lymphocytes (PBMCs) in peripheral venous blood of non-small cell cancer (NSCLC) patients and healthy humans are isolated and HLA subtypes thereof are identified.
Samples of PBMCs in which HLA-A2 was positive were stained with the pMHC tetramer complex and CD8-APC antibody obtained in example 3 and then flow-on-machine observed.
2. Experimental results
The flow type on-machine observation result diagram is shown in fig. 7, and the result shows that the antigen specific CD8+ T cells generated in NSCLC patients can be identified by the pMHC complex of tumor T cell epitope peptides MAGE-A3-MP4 (SEQ ID NO: 1), MAGE-A10-MP30 (SEQ ID NO: 2) and MAGE-A10-MP32 (SEQ ID NO: 3) in 3 with the amino acid sequences shown in SEQ ID NO 1-3, and the antigen specific CD8+ T cells are remarkably increased compared with healthy people; the results illustrate: in NSCLC patients, there are specific killer CD8+ T cells with three antigen epitope peptides of MAGE-A3-Mp4 (SEQ ID NO: 1), MAGE-A10-Mp30 (SEQ ID NO: 2) and MAGE-A10-Mp32 (SEQ ID NO: 3) generating immune responses
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. The tumor T cell epitope peptide is characterized in that the amino acid sequence of the tumor T cell epitope peptide is shown as SEQ ID NO: 2.
2. The tumor T cell epitope peptide composition is characterized in that the tumor T cell epitope peptide contains a polypeptide with an amino acid sequence shown in SEQ ID NO:2, and a tumor T cell epitope peptide shown in the specification.
3. A pMHC complex, characterized in that it contains the tumor T cell epitope peptide of claim 1 and/or the tumor T cell epitope peptide composition of claim 2.
4. The method for producing a pMHC complex according to claim 3, wherein the antigen epitope peptide of the tumor T cell according to claim 1 is obtained by renaturation of the HLA-A2 heavy chain, the HLA-A2 light chain β2m.
5. An antigen peptide-antigen presenting cell complex, wherein the antigen peptide-antigen presenting cell complex is an antigen presenting cell having the tumor T cell epitope peptide of claim 1 and/or the tumor T cell epitope peptide composition of claim 2 on the surface.
6. The antigen peptide-antigen presenting cell complex of claim 5, wherein the antigen presenting cell is a T2-A2 cell.
7. The antigenic peptide-antigen presenting cell complex of claim 6 wherein the T2-A2 cells are T2 cells over-expressing HLa-A2.
8. The method for preparing the antigen peptide-antigen presenting cell complex according to any one of claims 5 to 7, characterized in that the tumor T cell epitope peptide according to claim 1 is mixed with antigen presenting cells for incubation to obtain the antigen peptide-antigen presenting cell complex.
9. A gene encoding the tumor T cell epitope peptide of claim 1.
10. Use of the tumor T cell epitope peptide of claim 1, the tumor T cell epitope peptide composition of claim 2, the pMHC complex of claim 3, the antigen peptide-antigen presenting cell complex of claim 5 and/or the gene of claim 9 in the preparation of tumor vaccines and/or tumor medicaments; the tumor is lung cancer.
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| US20140206574A1 (en) * | 2011-08-31 | 2014-07-24 | Karen Chapman | Methods and Compositons for the Treatment and Diagnosis of Cancer |
| KR101503341B1 (en) * | 2014-03-12 | 2015-03-18 | 국립암센터 | Methods for isolation and proliferation of autologous cancer antigen-specific CD8+ T cells |
| US11723950B2 (en) * | 2019-10-18 | 2023-08-15 | Trustees Of Boston University | CAL-T constructs and uses thereof |
| CN116444631A (en) * | 2022-02-24 | 2023-07-18 | 暨南大学 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
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