Antigen epitope peptide of tumor cell high expression antigen ly6k and application thereof

Abstract

The application belongs to the technical field of immunotherapy, and particularly relates to an antigen epitope peptide of a tumor cell high-expression antigen LY6K and application thereof. The technical problem to be solved by the application is to provide a new method for treating or clinically detecting a tumor high in LY6K expression. The technical scheme of the application is an antigen epitope peptide of a tumor cell high-expression antigen LY6K, the amino acid sequence of which is shown in SEQ ID No. 4. The application provides a LY6K antigen epitope peptide, and 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 treatment or diagnosis of a tumor high in LY6K antigen expression.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology, specifically relating to the antigenic epitope peptide of tumor cells that highly expresses the antigen LY6K and its application. Background Technology

[0002] In various epithelial-derived 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 tumors (cervical and ovarian cancer), urinary system tumors (bladder and prostate cancer), and digestive system tumors (gastric and colorectal cancer), the LY6K antigen (Lymphocyte Antigen 6 Family Member K) exhibits significantly high expression. LY6K is a glycosylphosphatidylinositol-anchored protein with a molecular weight of approximately 15 kDa, belonging to the LY6 / uPAR superfamily. Its expression is restricted in normal tissues but is 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. This begins with the precise recognition of antigenic epitope peptides presented by major histocompatibility complex class I (MHC I) molecules on the T cell surface by the T-cell receptor (TCR). When tumor cells abnormally express the LY6K antigen, its protein products, after proteasome degradation, produce specific epitope peptide fragments that bind to MHC I molecules via transporters associated with anti-antigen processing (TAPs) in the endoplasmic reticulum, forming stable peptide-major histocompatibility complexes (pMHC) and expressing them on the tumor cell surface. During this process, CD8+ T cells recognize these pMHC complexes with high specificity through their TCR receptors. This recognition requires not only sufficient binding affinity between the epitope peptide and the MHC molecule but also the formation of a complex with an appropriate conformation to effectively activate intracellular signaling pathways within the T cell. Successful recognition triggers a cascade of reactions, including CD3ζ chain phosphorylation and ZAP70 kinase activation, ultimately leading to the clonal expansion of cytotoxic T lymphocytes (CTLs) and the exertion of their effector functions. However, in clinical practice, tumors with high LY6K expression often suppress this process through various immune escape mechanisms, including the immunosuppressive effects of regulatory T cells in the tumor microenvironment, abnormal upregulation of immune checkpoint molecules such as PD-L1, and functional defects in antigen presentation mechanisms. Therefore, by providing exogenously optimized LY6K antigen epitope peptides or pre-formed pMHC complexes, these immunosuppressive barriers can be effectively overcome, reactivating and enhancing the body's inherent anti-tumor immune response. This provides an important theoretical basis and practical direction for developing novel tumor immunotherapy strategies.

[0004] Based on the unique tumor-specific expression pattern and good immunogenicity of the LY6K antigen, it has shown two important applications in the field of tumor immunotherapy: First, in vaccine development, highly immunogenic LY6K MHC-restricted epitope peptides, selected through modern bioinformatics prediction combined with experimental verification, can be used directly as peptide vaccines, or used to construct nucleic acid vaccines or 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-modified T-cell therapy... In the field of TCR-T cell therapy, TCR-T therapies targeting LY6K 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 therapy has the advantage of recognizing intracellular antigens, making it particularly suitable for non-surface antigens like LY6K. Experimental studies have shown that TCR-T cells targeting specific LY6K epitopes exhibit significant anti-tumor effects in esophageal cancer models. Besides direct therapeutic applications, LY6K-related immunotherapies also have significant value in tumor diagnosis and immune monitoring, including assessing vaccine efficacy by detecting epitope-specific CTLs in peripheral blood using MHC multimer technology, and serving as prognostic biomarkers to predict patient clinical outcomes. With the development of personalized medicine, the combined application of LY6K-based immunotherapy strategies with existing treatments will open new avenues for solid tumor treatment. Overcoming 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 immunotherapies. Summary of the Invention

[0005] The technical problem to be solved by this 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 an antigenic epitope peptide of tumor cells that highly expresses the antigen LY6K, and its amino acid sequence is shown in SEQ ID No. 4.

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

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

[0009] Furthermore, the pMHC complex is obtained by refolding an MHC monomer and the antigenic epitope peptide.

[0010] In this mixture, MHC monomers and antigenic epitope peptides are mixed in equal volumes.

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

[0012] The present invention further provides an antigen epitope peptide-antigen presenting cell complex, which is an antigen presenting cell with the antigen epitope peptide or antigen epitope peptide composition loaded on its surface.

[0013] The antigen-presenting cells are CD8+ T cells.

[0014] Preferably, the CD8+ T cells are T2-A2 cells.

[0015] The present invention also provides the use of the above-mentioned antigenic epitope peptide, the nucleic acid molecule encoding the antigenic epitope peptide, the pMHC complex and / or the antigenic peptide-antigen presenting cell complex in the preparation of tumor drugs with high expression of LY6K.

[0016] This invention also provides the application of the above-mentioned antigenic epitope peptide, the nucleic acid molecule encoding the antigenic epitope peptide, the pMHC complex and / or the antigenic peptide-antigen presenting cell complex in screening tumor drugs that highly express LY6K.

[0017] The present invention further provides the application of the above-mentioned antigenic epitope peptide, the nucleic acid molecule encoding the antigenic epitope peptide, the pMHC complex and / or the antigenic peptide-antigen presenting cell complex in the preparation of a tumor vaccine with high expression of LY6K.

[0018] The present invention further provides the application of the above-mentioned antigenic epitope peptide, the nucleic acid molecule encoding the antigenic epitope peptide, the pMHC complex and / or the antigenic peptide-antigen presenting cell complex in evaluating the efficacy of tumor drugs with high LY6K expression.

[0019] Specifically, the tumors that highly express LY6K are 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.

[0020] Among them, the head and neck squamous cell carcinoma is oral cancer or laryngeal cancer.

[0021] The gynecological tumors mentioned are cervical cancer or ovarian cancer.

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

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

[0024] The beneficial effects of this invention are:

[0025] This invention provides an LY6K antigenic epitope peptide. Using these epitope peptides to prepare pMHC complexes or directly load antigen-presenting cells can activate T cells. Therefore, the epitope peptides can be applied to the development of universal vaccines for tumors with high LY6K antigen expression, such as non-small cell lung cancer, breast cancer, head and neck squamous cell carcinoma (e.g., oral and laryngeal cancer), esophageal squamous cell carcinoma, gynecological tumors (cervical and ovarian cancer), urinary system tumors (bladder and prostate cancer), and digestive system tumors (gastric and colorectal cancer), as well as to tumor diagnosis and TCR-T therapy research. Specifically:

[0026] (1) The development of tumor vaccines is based on the specific immune response mechanism induced by tumor antigens. The tumor T cell antigenic epitope peptide identified in this invention can effectively activate the immune response of effector T cells to tumor antigens through antigen presentation by T2-A2 cells. Experiments have confirmed that these antigenic epitope peptides can induce the body to produce antigen-specific T cell clonal expansion and form immune memory. Therefore, the LY6K antigenic epitope peptide described in this invention has the potential to be a candidate antigen for a broad-spectrum tumor vaccine. Through the antigen presentation pathway mediated by T2-A2 cells, it can stimulate a specific cellular immune response against tumors, providing a new antigen selection for the development of universal tumor vaccines.

[0027] (2) The principle of detecting the body's anti-tumor cellular immune function is as follows: When specific T cells against a specific tumor antigen are detected in the peripheral blood or tumor-infiltrating lymphocytes of the examinee, it indicates that the individual has successfully established an adaptive cellular immune response against that antigen. By refolding the antigen epitope peptide of the present invention with MHC molecules 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 strength of the existing anti-tumor immune response in the examinee, providing an important basis for assessing the patient's immune status. The detection results can not only be used to determine whether the body has generated an effective anti-tumor immune response, but also provide a reference for the formulation of subsequent immunotherapy plans.

[0028] (3) The LY6K-specific T cell antigen epitope peptides and their compositions described in this invention have important application value in the auxiliary diagnosis of tumors. By detecting the specific T cell immune response to these epitope peptides in patient samples, the following diagnostic functions can be achieved: First, the specific cytokines (such as IFN-γ) or T cell proliferation response generated after stimulation by the epitope peptides can significantly improve the specificity of tumor diagnosis; second, based on pMHC multimer technology, LY6K antigen-specific T cells can be accurately identified and quantified, providing an efficient and reliable detection method for screening LY6K-positive tumors. This detection method based on specific T cell responses can not only quickly identify LY6K-highly expressed tumor patients, but also provide an important basis for the selection of subsequent individualized immunotherapy strategies, thereby realizing an integrated precision medicine solution from diagnosis to treatment.

[0029] (4) Development of TCR-T cell immunotherapy. By identifying HLA-restricted epitope peptides derived from LY6K, high-affinity T cell receptors (TCRs) can be screened, and TCR-T cells can be constructed. These engineered T cells can specifically recognize and kill LY6K-positive tumor cells, while reducing off-target toxicity due to the low expression of LY6K in normal tissues, making them ideal targets for TCR-T cell therapy. Attached Figure Description

[0030] Figure 1 A: Statistical chart of T2-A2 antigen presentation of 15 candidate LY6K antigen epitope peptides; B: Statistical chart of detection of pMHC complex formation of 15 candidate tumor T cell antigen epitope peptides.

[0031] Figure 2 A: Activation of CD8+ T cells by 15 candidate LY6K antigen epitope peptides; B: Statistical graph of CD8+ T cell activation corresponding to LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13.

[0032] Figure 3 A: Flow cytometry results of CD8+ T cells specific to the LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 antigen epitopes in HLA-A2-positive healthy individuals and NSCLC patients; B: Statistical graph of the results of detecting CD8+ T cells in NSCLC patients and healthy individuals using pMHC complexes prepared from LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13.

[0033] Figure 4The cytotoxic effects of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 peptides on target cells; assessment of epitope-specific CD8+ T cell-mediated cytotoxicity after 7 days of co-culturing CD8+ T cells with T2-A2 cells loaded with different histones. A: After 7 days of culture with CD8+ T cells, the proportions of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 peptide antigens in CFSE+Annexin V+T2A2 cells served as an indicator of epitope-stimulated T cell-mediated T2-A2 apoptosis. B: Statistical graph of apoptosis in target cells mediated by LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 peptides.

[0034] Figure 5 The killing effects of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 peptides on target cells (PC9 cells in non-small cell lung cancer); the cytotoxicity of epitope-specific CD8+ T cells mediated by CD8+ T cells after co-culturing with PC9 cells loaded with different histones for 7 days. A: The proportions of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 peptide antigens in CFSE+Annexin V+T2A2 cells after 7 days of culture with CD8+ T cells, serving as an indicator of epitope-stimulated T cell-mediated apoptosis in PC9 cells. B: Statistical graph of apoptosis mediated by LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 peptides in PC9 cells. Detailed Implementation

[0035] The purpose of this invention is to provide a new method for the treatment or clinical detection of tumors with high LY6K expression.

[0036] The applicant screened 15 candidate antigenic epitope peptides of LY6K using bioinformatics methods and synthesized them artificially; subsequently, a series of identification and functional verification experiments were conducted on these 15 candidate antigenic epitope peptides of LY6K.

[0037] This invention utilizes the T2-A2 cell line as an artificial antigen presentation system, which stably expresses the human HLA-A*02:01 molecule through gene recombination technology. Because T2-A2 cells have a defect in processing endogenous antigens, when provided with exogenous effective epitope peptides, these peptides can be loaded with MHC class I molecules to form a stable pMHC complex, thus serving as an ideal platform for T cell activation.

[0038] T cell antigenic epitope peptides cannot function alone; they must be activated via pMHC complexes or antigen peptide-antigen-presenting cell complexes. The LY6K antigenic epitope peptide alone is not immunogenic; it must bind to MHC molecules to form a pMHC complex or be presented by antigen-presenting cells to activate a T cell response.

[0039] This invention successfully prepared a functional pMHC complex through MHC monomer recombination technology and the combined refolding of a novel LY6K epitope peptide. Experiments demonstrated that loading the identified LY6K epitope peptide onto the surface of T2-A2 cells effectively activated peripheral blood T cells from healthy individuals to produce cytotoxic factors and specifically killed target cells expressing the LY6K antigen. Using pMHC multimer technology, the presence of LY6K-specific T cells was detected in the peripheral blood of non-small cell lung cancer patients. These results indicate that the newly discovered LY6K T cell epitope peptide can effectively induce antigen-specific immune responses and has the potential to develop universal tumor vaccines and TCR-T cell therapy products.

[0040] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0041] Example 1: Prediction and Identification of LY6K Antigenic Epitope Peptide

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

[0043] Table 1. 15 candidate LY6K antigenic epitope peptides

[0044] Following the sequences in Table 1, 15 candidate LY6K antigen epitope peptides obtained through artificial synthesis (Nanjing Genscript Biotech Co., Ltd.) were each prepared into 10 mM stock solutions using DMSO. Logarithmic growth phase T2-A2 cells (Zhongyuan Biotechnology Co., Ltd.) were seeded into 96-well plates (2 × 10⁶ cells / wells). 5The wells were divided into blank control, negative control, positive control, and various LY6K antigenic epitope peptide groups, with three replicates for each group and a final volume of 200 μL per well. The blank control group contained only T2-A2 cells; the negative control group was co-incubated with T2-A2 cells and a negative peptide (EB virus peptide, amino acid sequence SEQ ID No. 16, IVTDFSVIK); the positive control group was co-incubated with T2-A2 cells and a positive peptide (influenza A M1 peptide, amino acid sequence SEQ ID No. 17, GILGFVEFTL); and 15 LY6K antigenic epitope peptide groups were set up, each co-incubated with 15 different synthetically produced candidate LY6K antigenic epitope peptides. After incubating the 96-well plates at 37°C for 4 hours, centrifuged to remove the supernatant, and washed twice. The cell pellet was then incubated with FITC-labeled human HLA-A2 antibody (β2m) (BioLegend) at 4°C in the dark for 30 min. Flow cytometry was then used to analyze the cells in each group. The above procedure was repeated three times in parallel.

[0045] The results of the identification are as follows Figure 1 As shown in Figure A, among the 15 predicted LY6K antigenic epitope peptides, 5 (LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13) can be effectively presented to T cells by antigen-presenting cells. That is, these 5 LY6K antigenic epitope peptides are immunogenic antigenic peptides.

[0046] Example 2: Detection of pMHC tetramer complex formed by tumor T cell antigen epitope peptides

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

[0048] The LY6K antigenic epitope peptide stock solution (10 mM) obtained in Example 1 was diluted to 400 μM with PBS to obtain the diluted stock solution, which was then placed on ice for later use. 20 μL of the diluted stock solution and 20 μL of MHC monomer (200 μg / mL) were added to a 96-well U-shaped plate, mixed by pipetting, and the seal was removed. The 96-well U-shaped plate was placed on ice and irradiated with a UV lamp for 30 min. The seal was then replaced, and the plate was incubated at 37°C in the dark for 30 min to obtain 15 candidate LY6K antigenic epitope peptides and MHC (pMHC complexes composed of α-chain and β2 microglobulin (β2m)). After biotin labeling, these peptides could further bind with fluorescently labeled streptavidin to form tetramers composed of four pMHC complexes, which were then used as the experimental group. The pMHC complex formed by influenza A M1 peptide (amino acid sequence: GILGFVEFTL) and MHC monomer was set as the positive control group (Pos ctrl); the pMHC complex formed by EB virus peptide (amino acid sequence: IVTDFSVIK) and MHC monomer was set as the negative control group (Neg ctrl); and the pMHC monomer formed by PBS and MHC monomer was set as the UV control group.

[0049] (2) The ability of ELISA to detect epitope peptides forming pMHC tetramer complexes

[0050] The ELISA method was used to detect whether the 15 candidate LY6K antigenic epitope peptides synthesized in Example 1 could form pMHC tetramers. The specific method is as follows: At room temperature, 100 μL of 0.5 μg / mL streptavidin solution was added to a 96-well plate and incubated overnight (16-18 h). Then, the plate was washed three times with 300 μL of 1× Wash Buffer and blocked at room temperature for 30 min with 1× Dilution Buffer (1M NaCl, 0.5M Tris, 1% BSA (w / v), 0.2% Tween 20 (w / v), pH=8.0). The pMHC complex monomers from step (1) (positive control group Pos, negative control group Neg, and experimental group) were diluted 1200 times with 1× Dilution Buffer. At the same time, the MHC group was set up: the MHC monomer was diluted equally; the Blank group was set up: only the same amount of 1× Dilution Buffer as the 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, except for LY6K-P1, the relative OD values ​​of the 15 complex groups were significantly increased (P < 0.001). 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-presenting cells (APCs) or target cells (T2A2 cells and the PC9 non-small cell lung cancer cell line) bind to the TCR, providing the first signal for T cell activation; B7 molecules on the surface of APCs bind to CD28 molecules on T cells, providing the second signal; IL-2 and other substances provide co-stimulatory signals. By loading antigenic peptides onto target cells (T2A2 cells and the PC9 non-small cell lung cancer cell line) and co-culturing them with CD8+ T cells, the proportion of activated CD8+ T cells was detected by tetramer analysis, and the proportion of antigen-specific T cells producing interferon-gamma (IFN-γ) was analyzed by flow cytometry. This analysis examined the ability of T2A2 cells seeded with LY6K antigenic epitope peptides to induce CD8+ T cell activation.

[0056] 1. Experimental Methods

[0057] Peripheral venous blood mononuclear lymphocytes (PBMCs) were isolated from healthy volunteers, and CD8+ T cells were further isolated. T2-A2 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), then treated with 20 μg / mL mitomycin C for 30 min, and incubated with the 15 candidate LY6K antigenic epitope peptides from Example 1. 30 μL of the pMHC complex monomer of the LY6K antigenic 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, the mixture was pipetted and incubated at 4°C in the dark for 30 min. After incubation, 2.4 μL of blocking buffer (1.6 μL, 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. The mixture was incubated overnight at 4–8 °C to obtain the pMHC tetramer complex.

[0058] The Mixed peps group is set to: 0.5 × 10 6 0.5 × 10⁸ CD8+ T cells and 0.5 × 10⁸ 6T2-A2 cells loaded with 15 candidate LY6K antigenic 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 culture, 50 IU / mL IL-2 and 20 μM candidate LY6K antigenic epitope peptides 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 measured. Simultaneously, a positive control group (T2-A2 cells loaded with influenza A M1 peptide), a negative control group (T2-A2 cells loaded with EB virus peptide), and a UV control group (pMHC complex formed by PBS and MHC monomers) were set up, and all were subjected to the same treatment and detection.

[0059] 2. Experimental Results

[0060] The results of different LY6K antigenic epitope peptides activating CD8+ T cells are as follows: Figure 2 As shown; the results showed that 5 of the 15 candidate LY6K antigen epitope peptides could activate T cells, namely 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; LY6K-P13 (SEQ ID No. 13): HMDRPYHAEA.

[0061] Example 4: Detection of specific cytotoxic T cells from peripheral blood antigen epitope peptides in clinical patients

[0062] 1. Experimental Methods

[0063] Mononuclear lymphocytes (PBMCs) were isolated from peripheral venous blood of patients with non-small cell lung cancer (NSCLC) and healthy individuals, and their HLA subtypes were identified. HLA-A2-positive PBMC samples were stained with the pMHC tetramer complex obtained in Example 3 and CD8-APC antibody, and then observed by flow cytometry.

[0064] 2. Experimental Results

[0065] The results of the flow cytometry observation are shown in the figure below. Figure 3 As shown, the pMHC complex of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 can recognize antigen-specific CD8+ T cells produced in NSCLC patients, and the levels are significantly higher compared to healthy individuals. That is, these five antigenic epitope peptides generate specific cytotoxic CD8+ T cells that trigger an immune response.

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

[0067] 1. Experimental Methods

[0068] The experiment included a blank control group (no peptides added), a negative control group (EBV virus, amino acid sequence: IVTDFSVIK), and an experimental group containing LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 peptides with 10... 5 To bind T2A2 and CD8T+ cells, each peptide was used in triplicate in experimental groups, with 100 μL of the mixture per well. The corresponding number of T2-A2 cells and their culture volume were placed in EP tubes. 20 μM of the peptide was added to the corresponding group, and the mixture was vortexed. The T2-A2 cell-peptide mixture was then seeded into 96-well plates. 1 µg / mL anti-human CD28 antibody and 50 IU / mL IL-2 were added to each group, and the mixture was vortexed. During the 7-day co-culture period, every two days, 50 μL of supernatant was aspirated along the well wall, and 50 μL of the mixture was added to each well. The 50 μL mixture contained the corresponding 20 μM peptide and 50 IU / mL IL-2, and was then thoroughly mixed. Finally, the cells were collected for flow cytometry analysis to detect cell killing.

[0069] 2. Experimental Results

[0070] Streaming results as follows Figure 4 As shown, the results indicate that LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 can kill target cells. The percentage of apoptotic cells in the experimental group was higher than that in the control group, and this was statistically significant.

[0071] Example 6: Killing effects of LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7 and LY6K-P13 on PC9 non-small cell lung cancer cells.

[0072] 1. Experimental Methods

[0073] The experiment included a blank control group (no peptides added), a negative control group (EBV virus, amino acid sequence: IVTDFSVIK), and an experimental group containing LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 peptides with 10... 5To bind T2A2 and CD8T+ cells, each peptide was used in triple replicates in the experimental groups, with a 100 μL system per well. Corresponding non-small cell lung cancer (NSCLC) PC9 cells were cultured in EP tubes. 20 μM of the peptide was added to the corresponding group, and the mixture was vortexed. The NSCLC PC9-peptide mixture was seeded into 96-well plates. 1 µg / mL anti-human CD28 antibody and 50 IU / mL IL-2 were added to each group, and the mixture was vortexed. During the 7-day co-culture period, every two days, 50 μL of supernatant was aspirated along the well wall, and 50 μL of the supernatant was added as a top-up. The 50 μL system contained the corresponding 20 μM peptide and 50 IU / mL IL-2, and was thoroughly mixed. Finally, cells were collected for flow cytometry analysis to detect cell killing.

[0074] 2. Experimental Results

[0075] Streaming results as follows Figure 5 As shown, the results indicated that LY6K-P2, LY6K-P3, LY6K-P4, LY6K-P7, and LY6K-P13 could kill PC9 non-small cell lung cancer cells. The percentage of apoptotic PC9 target cells in the experimental group was higher than that in the control group, and this was statistically significant.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An antigenic epitope peptide of LY6K highly expressed by tumor cells, characterized in that: Its amino acid sequence is shown in SEQ ID No.

4.

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 according to claim 3, characterized in that: The MHC monomer and the antigenic epitope peptide were mixed in equal volumes; before mixing, the concentration of the MHC monomer was 200 μg / mL and the concentration of the antigenic epitope peptide was 400 μM.

5. An antigen epitope peptide-antigen presenting cell complex, characterized in that: For antigen-presenting cells with surface load of the antigen epitope peptide of claim 1.

6. The antigen epitope peptide-antigen presenting cell complex according to claim 5, characterized in that: The antigen-presenting cells are T2-A2 cells.

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, and / or the antigenic peptide-antigen-presenting cell complex of claim 5 or 6 in the preparation of a vaccine for tumors that highly express LY6K, characterized in that: The tumor that highly expresses LY6K is non-small cell lung cancer.

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