Ly6k epitope peptide and its application in tumor diagnosis and treatment

Abstract

The application belongs to the technical field of immunotherapy, and particularly relates to a LY6K antigen epitope peptide and application thereof in tumor diagnosis and treatment. The application aims to provide a new method for treating or clinically detecting tumors with high expression of LY6K. The technical scheme of the application is a LY6K antigen epitope peptide, and the amino acid sequence of the LY6K antigen epitope peptide is shown as SEQ ID No. 13. The application provides a LY6K antigen epitope peptide, and a pMHC complex prepared by using the antigen epitope peptide or an antigen presenting cell directly loaded with the antigen epitope peptide can activate T cells. Therefore, the antigen epitope peptide can be applied to treatment or diagnosis of tumors with high expression of the LY6K antigen.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of immunotherapy, and particularly relates to a LY6K antigen epitope peptide and application thereof in tumor diagnosis and treatment. BACKGROUND

[0002] In various epithelial-derived malignant tumors, including 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), urinary system tumors (bladder cancer and prostate cancer), and digestive system tumors (gastric cancer and colorectal cancer), etc., LY6K (Lymphocyte Antigen 6 Family Member K) antigen presents a significant high expression characteristic. LY6K is a glycosylphosphatidylinositol-anchored protein with a molecular weight of about 15 kDa, belonging to the LY6 / uPAR superfamily member, and its expression in normal tissues is limited but specifically highly expressed in various 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 characteristics of LY6K antigen, LY6K antigen shows two important application directions in the field of tumor immunotherapy: first, in the aspect of vaccine development, the high immunogenicity LY6K MHC restricted epitope peptide screened out through modern bioinformatics prediction combined with experimental verification can be directly used as a polypeptide vaccine, or can be used to construct a nucleic acid vaccine or prepare a dendritic cell vaccine, and preclinical studies have confirmed that the LY6K vaccine can effectively induce epitope-specific CTL response and show significant tumor growth inhibition in animal models, and the LY6K-dendritic cell (DC) vaccine for head and neck cancer has entered the clinical trial stage; second, in the field of T cell receptor-engineered T cell therapy (TCR-T) cell therapy, by isolating the TCR sequence of LY6K-specific T cells or using phage display technology to screen high-affinity TCR, a TCR-T therapy targeting LY6K can be developed, and this therapy has the advantage of recognizing intracellular antigens compared with CAR-T, which is particularly suitable for LY6K and other non-surface antigens, and experimental studies have shown that TCR-T cells targeting specific LY6K epitopes show significant anti-tumor effect in esophageal cancer models. In addition to direct treatment applications, LY6K-related immune reagents also have important value in tumor diagnosis and immune monitoring, including evaluating vaccine effectiveness by detecting epitope-specific CTL in peripheral blood using MHC multimer technology, and predicting patient clinical outcomes as a prognostic marker. With the development of individualized medicine, the combination of LY6K-based immunotherapy strategies with existing treatment methods will open up new avenues for solid tumor treatment, and solving technical challenges such as HLA typing coverage of epitope peptides and improving the stability of pMHC complexes will further enhance the clinical translation value and application prospects of such immunotherapy products. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a new method for treating or clinically detecting tumors with high expression of LY6K.

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

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

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

[0009] Further, the pMHC complex is obtained by renaturation of an 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:

[0025] The application provides a LY6K antigen epitope peptide. A pMHC complex or an antigen presenting cell directly loaded with the antigen epitope peptide can activate T cells. Therefore, the antigen epitope peptide can be applied to the research and development of a tumor vaccine, the diagnosis of a tumor, and the research of TCR-T therapy, which is used for tumors with high expression of LY6K antigens, 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), urinary system tumors (bladder cancer and prostate cancer), and digestive system tumors (gastric cancer and colorectal cancer). Specifically as follows:

[0026] (1) The research and development of a tumor vaccine is based on a specific immune response mechanism induced by a tumor antigen. The tumor T cell antigen epitope peptide identified in the application can effectively activate the immune response of an effector T cell to a tumor antigen through the antigen presentation of T2-A2 cells. Experiments prove that the antigen epitope peptide can induce the body to produce antigen-specific T cell clonal expansion and form immune memory. Therefore, the LY6K antigen epitope peptide has the potential to be used as a candidate antigen of a broad-spectrum tumor vaccine, which can stimulate a specific cellular immune response against a tumor through a T2-A2 cell-mediated antigen presentation pathway, and provides a new antigen selection for the development of a universal tumor vaccine.

[0027] (2) The detection principle of the anti-tumor cellular immune function of the body is that when specific T cells against a specific tumor antigen are detected in the peripheral blood or tumor infiltrating lymphocytes of a subject, it indicates that the individual has successfully established an adaptive cellular immune response against the antigen. The pMHC complex (such as a tetramer or a multimer) formed by the antigen epitope peptide of the application and the MHC molecule can specifically label and quantitatively detect the proportion and quantity of antigen-specific CD8+ T cells. This detection index can objectively reflect the strength of the anti-tumor immune response that already exists in the body of the subject, and provides an important basis for evaluating the immune status of the patient. The detection result can not only be used to determine whether the body has produced an effective anti-tumor immune response, but also can provide a reference for the subsequent development of an immunotherapy scheme.

[0028] (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 program from diagnosis to treatment.

[0029] (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

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Figure 4, LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptide on target cell killing effect; after co-cultured with T2-A2 cells loaded with different groups of peptides, CD8+ T cells, after 7 days of culture, the 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.

[0034] Figure 5 , LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 peptide on target cell killing effect; after co-cultured with T2-A2 cells loaded with different groups of peptides, CD8+ T cells, after 7 days of culture, the 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

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Example 1 Prediction and identification of LY6K antigen epitope peptides

[0042] The "MHC I Binding" tool in the IEDB Recommended 2.22 (http: / / www.iedb.org / ) online website was used to predict CD8+ T cell epitopes of tumor antigen LY6K protein sequence. The MHC allele selected was HLA-A*02:01, and 15 candidate LY6K antigen epitope peptides were obtained, as shown in Table 1.

[0043] Table 1 15 candidate LY6K antigen epitope peptides

[0044] According to the sequences in Table 1, 15 candidate LY6K antigen epitope peptides were artificially synthesized (Nanjing Kingsriver Biotechnology Co., Ltd.) according to the steps, each of which was respectively configured into a mother liquor with a concentration of 10 mM with DMSO. Logarithmic growth phase T2-A2 cells (Zhongyuan Biotechnology 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.

[0045] 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.

[0046] Example 2: Detection of tumor T cell antigen epitope peptide forming pMHC tetramer complex

[0047] (1) Preparation of pMHC complex monomer of LY6K antigen epitope peptide

[0048] The mother liquor of LY6K antigen epitope peptide obtained in Example 1 (10 mM) was diluted to 400 μM with PBS 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 the sealing plate was removed; 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 were further combined with fluorescently labeled streptavidin to form a tetramer with four pMHC complex components, which were 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 Epstein-Barr virus peptide (amino acid sequence: IVTDFSVIK) and the MHC monomer was set as the negative control group Neg ctrl; and the pMHC monomer formed by PBS and the MHC monomer was set as the UV control group.

[0049] (2) ELISA detection of the ability of epitope peptides to form pMHC tetramer complexes

[0050] 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 tetramers, 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 3 times with 300 μL of 1× Wash Buffer, 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-fold with 1× Dilution Buffer. At the same time, the MHC group was set: the MHC monomer was diluted equally; the Blank group: only the same amount of 1× Dilution Buffer as other groups was added.

[0051] Taking the positive control group (Pos) as an example: Discard the liquid in the 96-well plate, pat dry on filter paper, and add 100 μL of diluted pMHC complex monomer to the 96-well plate. Add the negative control group (Neg), UV control group, experimental group, MHC group, and Blank group to the 96-well plate in the same manner. Cover with a sealing film and incubate at 37°C for 1 h. After incubation, wash the 96-well plate three times with washing buffer, then add 100 μL of diluted HRP-anti-β2M (antibody BioLegend, Cat#280303, US), and continue incubation at 37°C for 1 h. Wash again after incubation. Next, add 100 μL of substrate solution to each well (10.34 mL deionized water, 1.2 mL pH 4.0, 0.1 M citrate monohydrate / trisodium citrate dihydrate, 240 μL 40 nM ABTS, 120 μL hydrogen peroxide solution), and develop the color for 8 min at room temperature (18–25 °C) with shaking (400–500 rpm). Terminate the reaction with 50 μL of Stop Solution (2% oxalate dihydrate, w / v). Measure the absorbance (OD value) at 414 nm using a microplate reader within 30 min.

[0052] Using the OD value of the complex formed in the MHC group as 100%, the relative OD values ​​of the UV control group, positive control group (Pos), negative control group (Neg), and experimental group were calculated. The ratio represents the formation of pMHC tetramers by each tumor antigen epitope peptide. If the relative OD value is greater than that of the UV control group, it is determined that pMHC tetramers can be formed.

[0053] The results are as follows Figure 1 As shown in Figure B, the results indicate that, compared with the UV control group, the relative OD values ​​of the 15 complex groups were significantly increased (P < 0.001), except for LY6K-P1. These results suggest that all 15 LY6K antigenic epitope peptides can form pMHC tetramer complexes, representing potential candidate antigenic epitope peptides that can elicit specific immune responses.

[0054] Example 3: LY6K antigenic epitope peptide-antigen presenting cell-activated T cells

[0055] 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.

[0056] 1. Experimental method

[0057] 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 overnight at 4-8°C to obtain pMHC tetramer complex.

[0058] 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. At the same time, 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 treated and detected in the same way.

[0059] 2. Experimental results

[0060] The results of activating CD8+ T cells by different LY6K antigen epitope peptides are shown in Figure 2 The results show that among the 15 candidate LY6K antigen epitope peptides, 5 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.

[0061] Example 4. Detection of antigen epitope peptide-specific cytotoxic T cells in peripheral blood of clinical patients

[0062] 1. Experimental method

[0063] 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.

[0064] 2. Experimental results

[0065] 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.

[0066] Example 5 Killing effect of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 on target cells T2A2

[0067] 1. Experimental method

[0068] 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 EP tubes, 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, and co-cultured for 7 days. 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 evenly. Finally, the cells were collected for flow cytometry killing detection.

[0069] 2. Experimental results

[0070] 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.

[0071] 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

[0072] 1. Experimental method

[0073] 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 well with vortex, mix the non-small cell lung cancer PC9 and polypeptide mixture 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 the corresponding 20 μM polypeptide and 50 IU / mL IL-2, and is blown evenly. Finally, the cells are collected for flow cytometry killing detection.

[0074] 2. Experimental results

[0075] 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.

[0076] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and any brief introduction, modification, equivalent change and modification of the above embodiments according to the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A LY6K antigenic epitope peptide, characterized in that: Its amino acid sequence is shown in SEQ ID No.

13.

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

3. A pMHC complex, characterized in that: The pMHC complex is obtained by refolding an MHC monomer and the antigenic epitope peptide of claim 1.

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

5. An antigenic epitope peptide-antigen presenting cell complex, characterized in that: The antigen-presenting cells are those that have surface-loaded with the antigen epitope peptide of claim 1; the antigen-presenting cells are T2-A2 cells.

6. The use of the antigenic epitope peptide of claim 1, the nucleic acid molecule of claim 2, the pMHC complex of claim 3 or 4, or the antigenic epitope peptide-antigen presenting cell complex of claim 5 in the preparation of a medicament for treating non-small cell lung cancer.

7. The use of the antigenic epitope peptide of claim 1, the nucleic acid molecule of claim 2, the pMHC complex of claim 3 or 4, or the antigenic epitope peptide-antigen presenting cell complex of claim 5 in the preparation of a non-small cell lung cancer vaccine.

Images