LY6K antigen epitope peptide and application thereof in tumor diagnosis and treatment

Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to LY6K antigen epitope peptide and application thereof in tumor diagnosis and treatment. The technical problem to be solved by the invention is to provide a new method for tumor treatment or clinical detection of high-expression LY6K. According to the technical scheme, the LY6K antigen epitope peptide has an amino acid sequence as shown in SEQ ID No.13. The invention further discloses a preparation method of the LY6K antigen epitope peptide. The invention provides an LY6K antigen epitope peptide. The antigen epitope peptide is used for preparing a pMHC compound or directly loaded antigen presenting cells, and T cells can be activated. Therefore, the epitope peptide can be applied to treatment or diagnosis of tumors with high expression of the LY6K antigen.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy, and specifically relates to LY6K antigen epitope peptides and their application in tumor diagnosis and treatment. Background Art

[0002] LY6K (Lymphocyte Antigen 6 Family Member K) is significantly overexpressed in a variety of epithelial malignancies, including non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma (such as oral and laryngeal cancer), esophageal squamous cell carcinoma, gynecological cancers (cervical and ovarian cancer), urological cancers (bladder and prostate cancer), and digestive system cancers (gastric and colorectal cancer). LY6K is a glycosylphosphatidylinositol-anchored protein with a molecular weight of approximately 15 kDa and a member of the LY6 / uPAR superfamily. Its expression is limited in normal tissues but is specifically overexpressed in a variety of malignant tumors.

[0003] In these tumor microenvironments, CD8+ T cell-mediated specific immune responses are the core of anti-tumor immunity, which starts with the precise recognition of the epitope peptide presented by the tumor cell major histocompatibility complex class I (MHC I) molecules by the T cell receptor (TCR) on the surface of T cells. When tumor cells abnormally express LY6K antigen, the specific epitope peptide fragments produced by the protein product after proteasome degradation are combined with MHC class I molecules through the transporter associated with antigen processing (TAP) in the endoplasmic reticulum to form stable peptide-MHC complex (pMHC), and are expressed on the surface of tumor cells. During this process, CD8+ T cells recognize these pMHC complexes through their TCR receptors in a highly specific manner. This recognition not only requires sufficient binding affinity between the epitope peptide and the MHC molecule, but also requires the formation of a complex with the appropriate conformation to effectively activate the signal transduction pathway in the T cell. Successful recognition will trigger a cascade of reactions including CD3 zeta chain phosphorylation and ZAP70 kinase activation, ultimately leading to the clonal expansion of cytotoxic T lymphocytes (CTLs) and the exertion of effector functions. However, in clinical practice, LY6K highly expressed tumors often inhibit this process through various immune escape mechanisms, including the immunosuppressive effect of regulatory T cells in the tumor microenvironment, the abnormal up-regulation of immune checkpoint molecules such as PD-L1, and the functional defects of antigen presentation mechanisms. Therefore, by providing exogenous optimized LY6K antigen epitope peptides or pre-formed pMHC complexes, these immune suppression barriers can be effectively broken, and the body's inherent anti-tumor immune response ability can be reactivated and enhanced, which provides an important theoretical basis and practical direction for the development of new tumor immunotherapy strategies.

[0004] Based on the unique tumor-specific expression pattern and good immunogenicity of LY6K antigen, LY6K antigen has two important application directions in the field of tumor immunotherapy: First, in vaccine development, highly immunogenic LY6K MHC-restricted epitope peptides screened through modern bioinformatics prediction combined with experimental verification can be used directly as peptide vaccines, or can be used to construct nucleic acid vaccines or to prepare dendritic cell vaccines. Preclinical studies have confirmed that LY6K vaccines can effectively induce epitope-specific CTL responses and show significant tumor growth inhibition in animal models. Currently, LY6K-dendritic cell (DC) vaccines for head and neck cancer have entered the clinical trial stage; second, in T cell receptor engineered T cell therapy (TCR-modified T cell In the field of TCR-T cell therapy, LY6K-targeted TCR-T therapies can be developed by isolating the TCR sequences of LY6K-specific T cells or screening for high-affinity TCRs using techniques such as phage display. Compared to CAR-T, this approach has the advantage of recognizing intracellular antigens and is particularly suitable for non-surface antigens such as LY6K. Experimental studies have shown that TCR-T cells targeting specific LY6K epitopes exhibit significant anti-tumor effects in esophageal cancer models. In addition to direct therapeutic applications, LY6K-related immunotherapies also have significant value in tumor diagnosis and immune monitoring. These include assessing vaccine efficacy by detecting epitope-specific CTLs in peripheral blood using MHC multimer technology and serving as prognostic markers for predicting patient clinical outcomes. With the advancement of personalized medicine, the combination of LY6K-based immunotherapy strategies with existing therapies will open new avenues for the treatment of solid tumors. Addressing technical challenges such as HLA typing coverage of epitope peptides and improving the stability of pMHC complexes will further enhance the clinical translational value and application prospects of these immunotherapeutic products. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new method for the treatment or clinical detection of tumors with high LY6K expression.

[0006] The technical solution of the present invention is a LY6K antigen epitope peptide, the amino acid sequence of which is shown in SEQ ID No.13.

[0007] Furthermore, the present invention also provides a nucleic acid molecule encoding the antigen epitope peptide.

[0008] The present invention also provides a pMHC complex containing the antigen epitope peptide.

[0009] Furthermore, the pMHC complex is obtained by renaturing the MHC monomer and the antigen epitope peptide.

[0010] The MHC monomer and the epitope peptide are mixed in equal volume.

[0011] Further, the concentration of the MHC monomer before mixing is 200 μg / mL, and the concentration of the epitope peptide before mixing is 400 μM.

[0012] The application further provides an antigen epitope peptide-antigen presenting cell complex, which is an antigen presenting cell loaded with the antigen epitope peptide or the antigen epitope peptide composition.

[0013] The antigen presenting cell is a CD8+ T cell.

[0014] Preferably, the CD8+ T cell is a T2-A2 cell.

[0015] The application further provides the use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and / or the antigen peptide-antigen presenting cell complex in the preparation of a LY6K highly-expressed tumor drug.

[0016] The application further provides the use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and / or the antigen peptide-antigen presenting cell complex in the screening of a LY6K highly-expressed tumor drug.

[0017] The application further provides the use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and / or the antigen peptide-antigen presenting cell complex in the preparation of a LY6K highly-expressed tumor vaccine.

[0018] The application further provides the use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and / or the antigen peptide-antigen presenting cell complex in the evaluation of the effect of a LY6K highly-expressed tumor drug.

[0019] Specifically, the LY6K highly-expressed tumor is at least one of non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, gynecological tumor, urinary system tumor or digestive system tumor.

[0020] The head and neck squamous cell carcinoma is oral cancer or laryngeal cancer.

[0021] The gynecological tumor is cervical cancer or ovarian cancer.

[0022] The urinary system tumor is bladder cancer or prostate cancer.

[0023] The digestive system tumor is gastric cancer or colorectal cancer.

[0024] The application has the following beneficial effects: The present invention provides LY6K antigen epitope peptides. These epitope peptides can be used to activate T cells when prepared into pMHC complexes or directly loaded onto antigen-presenting cells. Therefore, these epitope peptides can be applied to the development of universal tumor vaccines, tumor diagnosis, and TCR-T therapy for tumors that highly express the LY6K antigen, such as non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma (such as oral cancer and laryngeal cancer), esophageal squamous cell carcinoma, gynecological tumors (cervical cancer and ovarian cancer), urological tumors (bladder cancer and prostate cancer), and digestive system tumors (gastric cancer and colorectal cancer). The details are as follows: (1) The development of tumor vaccines is based on the specific immune response mechanism induced by tumor antigens. The tumor T cell antigen epitope peptides identified in the present invention can effectively activate the immune response of effector T cells to tumor antigens through the antigen presentation of T2-A2 cells. Experiments have confirmed that these antigen epitope peptides can induce the body to produce antigen-specific T cell clones and expand, and form immune memory. Therefore, the LY6K antigen epitope peptides described in the present invention have the potential to serve as candidate antigens for broad-spectrum tumor vaccines. They can stimulate specific cellular immune responses against tumors through the antigen presentation pathway mediated by T2-A2 cells, providing new antigen options for the development of universal tumor vaccines.

[0025] (2) The principle of detecting the body's anti-tumor cellular immune function is that when specific T cells targeting a specific tumor antigen are detected in the subject's peripheral blood or tumor-infiltrating lymphocytes, it indicates that the individual has successfully established an adaptive cellular immune response against the antigen. By renaturing the antigen epitope peptide of the present invention with the MHC molecule to form a pMHC complex (such as a tetramer or multimer), the proportion and number of antigen-specific CD8+ T cells can be specifically labeled and quantitatively detected. This detection indicator can objectively reflect the intensity of the anti-tumor immune response already existing in the subject's body and provide an important basis for evaluating the patient's immune status. The test results can not only be used to determine whether the body has produced an effective anti-tumor immune response, but also provide a reference for the formulation of subsequent immunotherapy plans.

[0026] (3) The LY6K specific T cell antigen epitope peptide and the composition thereof have important application value in tumor auxiliary diagnosis. By detecting the specific T cell immune response to the epitope peptide in the patient sample, the following diagnostic functions can be realized: first, the specific cytokine (such as IFN-γ) or T cell proliferation response produced after stimulation of the epitope peptide can significantly improve the specificity of tumor diagnosis; second, based on the pMHC multimer technology, LY6K antigen specific T cells can be accurately identified and quantified, providing an efficient and reliable detection means for LY6K positive tumor screening. This detection method based on specific T cell response can not only quickly identify LY6K high expression tumor patients, but also provide an important basis for subsequent individualized immunotherapy strategy selection, thereby realizing an integrated precision medical scheme from diagnosis to treatment.

[0027] (4) TCR-T cell immunotherapy development. By identifying LY6K derived HLA restricted epitope peptides, high affinity T cell receptors (TCR) can be screened, and then TCR-T cells are constructed. These engineered T cells can specifically recognize and kill LY6K positive tumor cells, while the normal tissue low expression of LY6K reduces off-target toxicity, making it an ideal TCR-T cell treatment target. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 , A: statistical chart of identification of T2-A2 antigen presentation of 15 candidate LY6K antigen epitope peptides; B: detection statistical chart of pMHC complex formed by 15 candidate tumor T cell antigen epitope peptides.

[0029] Figure 2 , 15 candidate LY6K antigen epitope peptides on CD8+T cell activation chart; A: representative chart of flow cytometry detection; B: statistical chart of detection of CD8+T cell activation corresponding to LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13.

[0030] Figure 3 , specific antigen specific CD8+T cells in non-small cell cancer patients and healthy people; A: flow cytometry detection result chart of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 antigen epitope specific CD8+T cells in HLA-A2 positive healthy people and NSCLC patients; B: statistical chart of results of detection of CD8+T cells in NSCLC patients and healthy people by pMHC complex prepared by LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13.

[0031] Figure 4, LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptide on target cell killing effect; after co-culture with T2-A2 cells loaded with different groups of peptides, CD8+ T cells, after 7 days of culture, evaluation of cytotoxicity mediated by epitope-specific CD8+ T cells. A: the proportion of CFSE+ Annexin V+ T2A2 cells presenting LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptide antigens after 7 days of culture with CD8+ T cells, as an indicator of T2-A2 apoptosis mediated by epitope-stimulated T cells. B: LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptide-mediated apoptosis of target cells.

[0032] Figure 5 , LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptide on target cell killing effect; after co-culture with T2-A2 cells loaded with different groups of peptides, CD8+ T cells, after 7 days of culture, evaluation of cytotoxicity mediated by epitope-specific CD8+ T cells. A: the proportion of CFSE+ Annexin V+ T2A2 cells presenting LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptide antigens after 7 days of culture with CD8+ T cells, as an indicator of T2-A2 apoptosis mediated by epitope-stimulated T cells. B: LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptide-mediated apoptosis of target cells. DETAILED DESCRIPTION

[0033] The purpose of the present application is to provide a new method for the treatment or clinical detection of tumors with high expression of LY6K.

[0034] The applicant screened 15 LY6K candidate antigen epitope peptides through bioinformatics means for artificial synthesis; then a series of identification and functional verification tests were carried out on the 15 LY6K candidate antigen epitope peptides.

[0035] The present application uses T2-A2 cell line as an artificial antigen presentation system, which stably expresses human HLA-A*02:01 molecules through genetic recombination technology. Because T2-A2 cells have endogenous antigen processing defects, when providing exogenous effective epitope peptides, they can load exogenous effective epitope peptides with their MHC class I molecules to form stable pMHC complexes, so they can be used as an ideal T cell activation platform.

[0036] T cell epitope peptides alone cannot work, and must be in the form of pMHC complex or antigen peptide-antigen presenting cell complex for T cell activation. LY6K antigen epitope peptides alone are not immunogenic and must be combined with MHC molecules to form pMHC complex or be presented by antigen presenting cells to activate T cell response.

[0037] The present application successfully prepared functional pMHC complex by combining MHC monomer recombination technology and refolding of new LY6K epitope peptides. Experiments have proved that after loading the identified LY6K epitope peptides on the surface of T2-A2 cells, the healthy human peripheral blood T cells can effectively produce cytotoxic factors, and have specific killing effect on target cells expressing LY6K antigen. Through pMHC multimer technology, LY6K specific T cells were detected in the peripheral blood of non-small cell lung cancer patients. These results show that the newly discovered LY6K T cell epitope peptide can effectively induce antigen-specific immune response, and has the potential to develop tumor universal vaccine and TCR-T cell therapy products.

[0038] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object of the application, the specific embodiments, structures, features and effects according to the present application are described in detail as follows in combination with the drawings and preferred embodiments.

[0039] Example 1 Prediction and identification of LY6K antigen epitope peptides The CD8+ T cell epitope of tumor antigen LY6K protein sequence was predicted using the "MHC I Binding" tool in the IEDB Recommended 2.22 (http: / / www.iedb.org / ) online website. The MHC allele selected was HLA-A*02:01, and 15 candidate LY6K antigen epitope peptides were obtained, as shown in Table 1.

[0040] Table 1 15 candidate LY6K antigen epitope peptides According to the sequences in Table 1, 15 candidate LY6K antigen epitope peptides were artificially synthesized (Nanjing Kingsun Biotech Co., Ltd.) and each was prepared into a mother liquor with a concentration of 10 mM using DMSO. Logarithmic growth phase T2-A2 cells (Zhongyuan Biotech Co., Ltd.) were inoculated into 96-well plates (2×10 5The 96-well plate was incubated at 37°C for 4 hours, and then centrifuged to remove the supernatant, washed twice, and the cell pellet was incubated with FITC-labeled human HLA-A2 antibody (β2m) (BioLegend) at 4°C in the dark for 30 min. The cells in each group were detected by flow cytometry. The above operation was repeated three times for parallel detection.

[0041] The identification results are shown in Table 1. Figure 1 As shown in Table 1, among the 15 predicted LY6K antigen epitope peptides, 5 LY6K antigen epitope peptides (LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13) can be effectively presented to T cells by antigen presenting cells. That is, the 5 LY6K antigen epitope peptides are antigen peptides with immunogenicity.

[0042] Example 2 Detection of tumor T cell antigen epitope peptide forming pMHC tetramer complex (1) Preparation of pMHC complex monomer of LY6K antigen epitope peptide The mother liquor of LY6K antigen epitope peptide obtained in Example 1 (10 mM) was diluted with PBS to 400 μM to obtain a diluted mother liquor, which was placed on ice for standby. 20 μL of the diluted mother liquor and 20 μL of MHC monomer (200 μg / mL) were added to a 96-well U-shaped plate, mixed by blowing and removed the sealing plate; the 96-well U-shaped plate was placed on ice and irradiated with a UV lamp for 30 min; the sealing plate was covered and incubated at 37°C in the dark for 30 min, to obtain 15 kinds of candidate LY6K antigen epitope peptides and MHC (α chain and β2 microglobulin (β2m) composed of pMHC complex), which can be further combined with fluorescently labeled streptavidin to form a tetramer with four pMHC complex components, which is set as the experimental group. The pMHC complex formed by the influenza A M1 peptide (amino acid sequence: GILGFVEFTL) and the MHC monomer was set as the positive control group Pos ctrl; the pMHC complex formed by the EB virus peptide (amino acid sequence: IVTDFSVIK) and the MHC monomer was set as the negative control group Neg ctrl; the pMHC monomer formed by PBS and the MHC monomer was set as the UV control group.

[0043] (2) ELISA detection of the ability of epitope peptides to form pMHC tetramer complexes The ELISA method was used to detect whether the 15 kinds of candidate LY6K antigen epitope peptides artificially synthesized in Example 1 could form pMHC tetramer, and the specific method was as follows: 100 μL of 0.5 μg / mL streptavidin solution was added to a 96-well plate at room temperature and incubated overnight (16-18 h), then washed with 300 μL of 1× Wash Buffer for 3 times, and 1× Dilution Buffer (1M NaCl, 0.5M Tris, 1% BSA (w / v), 0.2% Tween 20 (w / v), pH=8.0) was added to block at room temperature for 30 min. The pMHC complex monomer (positive control group Pos, negative control group Neg and experimental group) of step (1) was diluted 1200 times with 1× Dilution Buffer. At the same time, MHC group was set: the MHC monomer was diluted equally; Blank group: only 1× Dilution Buffer was added in an amount equal to other groups.

[0044] For the positive control group (Pos), discard the liquid from the 96-well plate, pat dry on filter paper, and add 100 μL of diluted pMHC complex monomer to the 96-well plate. Treat the negative control group (Neg), UV control group, experimental group, MHC group, and Blank group identically and add the cells to the 96-well plate. Cover with a sealer and incubate at 37°C for 1 hour. After incubation, wash the 96-well plate three times with wash buffer. Then, add 100 μL of diluted HRP-anti-β2M (BioLegend, Cat# 280303, US) and incubate at 37°C for another 1 hour. Wash the plates after incubation. Next, add 100 μL of substrate solution (10.34 mL of deionized water, 1.2 mL of 0.1 M citric acid monohydrate / trisodium citrate dihydrate, pH 4.0, 240 μL of 40 nM ABTS, and 120 μL of hydrogen peroxide solution) to each well. Incubate in the dark at room temperature (18–25°C) with shaking (400–500 rpm) for 8 minutes to develop color. Terminate the reaction with 50 μL of Stop Solution (2% oxalic acid dihydrate, w / v). Measure the absorbance (OD) at 414 nm using a microplate reader within 30 minutes.

[0045] Taking the OD value of the complex formed by the MHC group as 100%, the relative OD values ​​of the UV control group, the positive control group (Pos), the negative control group (Neg), and the experimental group were calculated. The ratio represents the formation of pMHC tetramers with each tumor antigen epitope peptide. Relative OD values ​​greater than those of the UV control group were considered to be able to form pMHC tetramers.

[0046] The results are as follows Figure 1 As shown in Figure B, the results showed that compared with the UV control group, the relative OD values ​​of the 15 complex groups, except LY6K-P1, were significantly increased (P < 0.001). The results indicate that all 15 LY6K epitope peptides can form pMHC tetramer complexes and are potential candidate epitope peptides that can induce specific immune responses.

[0047] Example 3 LY6K antigen epitope peptide-antigen presenting cells activate T cells Antigen peptide / MHC complex on the surface of antigen-presenting cells (APC) or target cells (T2A2 cells and non-small cell lung cancer PC9 cell lines) binds to TCR, providing the first signal for T cell activation; B7 molecules on the surface of APC bind to CD28 molecules on T cells, providing the second signal; IL-2 and the like provide a costimulatory signal. By loading the antigen peptide on the target cells (T2A2 cells and non-small cell lung cancer PC9 cell lines), and then co-culturing with CD8+T cells, the proportion of activated CD8+T cells is detected by tetramer, the proportion of antigen-specific T cells producing gamma-interferon (IFN-γ) is analyzed by flow cytometry, and the ability of LY6K antigen epitope peptide-loaded T2A2 to induce CD8+T cell activation is analyzed.

[0048] 1. Experimental method Mononuclear lymphocytes (PBMCs) of peripheral venous blood of healthy volunteers were isolated, and CD8+T cells were further isolated. T2-A2 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), then treated with 20 μg / mL of mitomycin for 30 min, and then incubated with 15 candidate LY6K antigen epitope peptides in Example 1. 30 μL of pMHC complex monomer of LY6K antigen epitope peptide obtained in step (1) of Example 2 was taken into a 1.5 mL EP tube, 3.3 μL of streptavidin (BioLegend Cat#405203, US) was added, and the gun head was blown to mix, and incubated at 4°C in the dark for 30 min. After incubation, 2.4 μL of blocking solution (1.6 μL of 50 mM D-biotin (Thermo Fisher, Cat#B20656, US), 6 μL of 10% (w / v) NaN3 and 192.4 μL of PBS) was added to the EP tube to terminate the reaction. Incubate at 4-8°C overnight to obtain pMHC tetramer complex.

[0049] The mixed peps group was set as follows: 0.5×10 6 CD8+T cells were co-cultured with 0.5×10 6T2-A2 cells loaded with 15 candidate LY6K antigen epitope peptides were co-cultured in culture medium, and co-stimulated with 1 μg / mL anti-human CD28 antibody and 50 IU / mL IL-2. During the culture, 50 IU / mL IL-2 and 20 μM candidate LY6K antigen epitope peptide were added to the culture medium every two days. After 7 days of culture, the proportion of specific CD8+ T cells and the release of IFN-γ by antigen-specific CD8+ T cells were detected. A positive control group (T2-A2 cells loaded with influenza A M1 peptide), a negative control group (T2-A2 cells loaded with EBV peptide) and a UV control group (pMHC complex formed by PBS and MHC monomer) were set up and treated in the same way for detection.

[0050] 2. Experimental results The results of activation of CD8+ T cells by different LY6K antigen epitope peptides are shown in Figure 2 The results show that 5 of the 15 candidate LY6K antigen epitope peptides can activate T cells, which are LY6K-P2 (SEQ ID No. 2): ALLLVVALPRV; LY6K-P3 (SEQ ID No. 3): RVWTDANLTA; LY6K-P4 (SEQ ID No. 4): TTPRPAFPV; LY6K-P7 (SEQ ID No. 7): LVPQLTVHL; and LY6K-P13 (SEQ ID No. 13): HMDRPYHAEA.

[0051] Example 4. Detection of antigen epitope peptide-specific cytotoxic T cells in peripheral blood of clinical patients 1. Experimental method Mononuclear lymphocytes (PBMCs) in peripheral venous blood of non-small cell cancer (NSCLC) patients and healthy people were isolated, and their HLA subtypes were identified. For the PBMCs samples positive for HLA-A2, the pMHC tetramer complex obtained in Example 3 and CD8-APC antibody were used for staining, and then flow cytometry was performed for observation.

[0052] 2. Experimental results The flow cytometry observation results are shown in Figure 3 The pMHC complexes of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 can recognize antigen-specific CD8+ T cells produced in NSCLC patients, and are significantly higher than in healthy people. That is, specific killer CD8+ T cells producing immune response by these 5 antigen epitope peptides.

[0053] Example 5 Killing effect of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 on target cells T2A2 1. Experimental method A blank control group (without any peptide), a negative control group (EBV virus, amino acid sequence: IVTDFSVIK), an experimental group, LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptides were combined with 10 5 T2A2 and CD8T+ cells, 3 replicate wells were set for each polypeptide in the experimental group, 100 μL system / well, the corresponding number of T2-A2 cells was taken and cultured in an EP tube, 20 μM of the corresponding polypeptide was added to the corresponding group, and the mixture was mixed by vortexing. The T2-A2 cells and the polypeptide were seeded into a 96-well plate, 1 μg / mL anti-human CD28 antibody and 50 IU / mL IL-2 were added to each group, and vortexed to mix. During the 7-day co-culture, every two days, 50 μL of supernatant was removed along the well wall, then 50 μL of liquid was added, 50 μL of system contained the corresponding 20 μM polypeptide and 50 IU / mL IL-2, and was blown to mix. Finally, the cells were collected for flow cytometry killing detection.

[0054] 2. Experimental results The flow cytometry results are shown in Figure 4 The results show that LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 can kill target cells, and the percentage of apoptotic cells in the experimental group is higher than that in the control group, and has statistical significance.

[0055] Example 6 Killing effect of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 on target cells non-small cell lung cancer PC9 1. Experimental method A blank control group (without any peptide), a negative control group (EBV virus, amino acid sequence: IVTDFSVIK), an experimental group, LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptides were combined with 10 5T2A2 and CD8 T+ cells, 3 replicates per polypeptide, 100 μL system per well, take the corresponding non-small cell lung cancer PC9 number and culture in EP tube, add 20 μM polypeptide in the corresponding group, mix with vortex, mix the mixture of non-small cell lung cancer PC9 and polypeptide into a 96-well plate, add 1 μg / mL anti-human CD28 antibody and 50 IU / mL IL-2 in each group, vortex, co-culture for 7 days, every two days, 50 μL supernatant is removed along the wall of the hole, then 50 μL is added, 50 μL system contains corresponding 20 μM polypeptide and 50 IU / mL IL-2, and is blown up. Finally, the cells are collected for flow cytometry killing detection.

[0056] 2. Experimental results The flow cytometry results are shown in Figure 5 LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 can kill non-small cell lung cancer PC9, the percentage of apoptotic cells of target cells non-small cell lung cancer PC9 in the experimental group is higher than that in the control group, and has statistical significance.

[0057] The above is only the preferred embodiment of the present application, not any form of limitation on the present application, although the present application has been disclosed as above, however, not to limit the present application, any person skilled in the art, without departing from the scope of the present application, can make some changes or modifications of the above disclosed technical content as equivalent embodiments, but as long as it does not deviate from the technical solution of the present application, according to the technical essence of the present application, any modification, equivalent change and modification of the above embodiments, still belongs to the scope of the present application.

Claims

1. LY6K antigen epitope peptide, characterized by: Its amino acid sequence is shown in SEQ ID No.

13.

2. A nucleic acid molecule encoding the antigenic epitope peptide according to claim 1.

3. A pMHC complex, characterized in that: The pMHC complex is obtained by renaturing an MHC monomer and the antigen epitope peptide according to claim 1.

4. The pMHC complex according to claim 3, wherein: Equal volumes of MHC monomers and antigen epitope peptides were mixed; before mixing, the concentration of MHC monomers was 200 μg / mL and the concentration of antigen epitope peptides was 400 μM.

5. An antigen epitope peptide-antigen presenting cell complex, characterized in that: An antigen-presenting cell carrying the antigen epitope peptide according to claim 1 on its surface.

6. The antigen epitope peptide-antigen presenting cell complex according to claim 5, characterized in that: The antigen presenting cells are CD8+ T cells.

7. The antigen epitope peptide-antigen presenting cell complex according to claim 6, characterized in that: The CD8+T cells are T2-A2 cells.

8. Use of the antigen epitope peptide according to claim 1, the nucleic acid molecule encoding the antigen epitope peptide according to claim 2, the pMHC complex according to claim 3 or 4, and / or the antigen epitope peptide-antigen presenting cell complex according to any one of claims 5 to 7 in preparing high-LY6K expression tumor drugs, screening high-LY6K expression tumor drugs, preparing high-LY6K expression tumor vaccines, or evaluating the effects of high-LY6K expression tumor drugs.

9. The application according to claim 8, characterized in that: The tumor with high LY6K expression is at least one of non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, gynecological tumors, urinary system tumors or digestive system tumors.

10. The application according to claim 9, characterized in that: One of the following characteristics: The head and neck squamous cell carcinoma is oral cancer or laryngeal cancer; The gynecological tumor is cervical cancer or ovarian cancer; The urinary system tumor is bladder cancer or prostate cancer; The digestive system tumor is gastric cancer or colorectal cancer.

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