HLA-A*11:01 renal cell carcinoma tumor antigen short peptide derived from long noncoding RNA LINC02717

Abstract

The application discloses an HLA-A*11:01 renal cell carcinoma tumor antigen short peptide derived from a long-chain non-coding RNA LINC02717. The application provides a tumor antigen peptide, and the amino acid sequence of the tumor antigen peptide is shown as SEQ ID No. 1. The application finds that the complex formed by the short peptide and a corresponding HLA molecule (HLA-A*11:01) can be recognized by T cells, and an immune response is activated. The application provides a potential target for renal cell carcinoma treatment, and provides a new target screening direction for subsequent tumor antigen treatment of renal cell carcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to HLA-A*11:01 renal cell carcinoma tumor antigen short peptide derived from long non-coding RNA LINC02717. BACKGROUND

[0002] According to the 2020 Global Cancer Data Observation Website Globocan Registry, renal carcinoma ranks 12th among the 12 most common cancers in the world, with a total of 1.2 million reported cases in 5 years, and its global incidence is still rising, with more than 400,000 new reported cases and more than 175,000 deaths each year. Among the newly reported cases, about 25% of patients have metastasis at the time of diagnosis, and 30% of renal carcinoma patients have distant metastasis after radical nephrectomy, and the 5-year survival rate of these patients is only 10-18%. The common treatment for renal cell carcinoma is surgical resection. When the tumor volume is too large or metastasis occurs, immunological checkpoint and targeted combination therapy is generally used, but the treatment effect is limited by various factors such as the patient's immune microenvironment. Therefore, it is necessary to develop new drugs for treating renal carcinoma.

[0003] Tumor antigen is an antigen produced by tumor cells that can trigger T cell immune response. Tumor antigen is generally a short peptide of 8-12 amino acids derived from intracellular degradation of proteins. Long non-coding RNA (lncRNA) is a non-coding RNA with a length of more than 200 nucleotides. Traditional theory believes that long non-coding RNA (lncRNA) does not have coding ability and mainly plays a role in epigenetic, RNA transcription and post-transcriptional regulation. However, in recent years, more and more evidence has confirmed that lncRNA does not completely lack amino acid coding ability, although lncRNA does not have a start codon and cannot encode a complete macromolecular protein, but lncRNA can encode short amino acid peptide segments through small open reading frames (sORF). The polypeptide encoded by lncRNA also plays an important role in the biological process of tumors, such as some tumor-specific lncRNA polypeptides that can be recognized by T cells as tumor antigens and cause tumor immune response. Through the study of non-coding antigens represented by lncRNA, we can improve the possibility of finding tumor-specific and patient-shared antigens in tumors.

[0004] For tumors, T cells can only exert immune function by recognizing the antigens presented on tumor cells. The antigen presentation process of tumor cells starts from the endoplasmic reticulum in the cytoplasm. Endogenous proteins in tumor cells are degraded in the cytoplasm to form short peptides, and a polypeptide library formed by the decomposition of various proteins is generated in the process. Part of these peptides is transferred to the endoplasmic reticulum through a transporter associated with antigen processing (TAP) that is specifically associated with antigen processing. Once the short peptides enter the endoplasmic reticulum, they are assembled and folded into complexes with human leukocyte antigens (HLA) by internal enzymes, and then finally presented to the cell surface. Those skilled in the art are committed to discovering and determining these polypeptide fragments presented to the surface of target cells. SUMMARY

[0005] The purpose of the present application is to provide an HLA-A*11:01 renal cell carcinoma tumor antigen short peptide derived from long-chain non-coding RNA LINC02717.

[0006] In a first aspect, the present application claims a tumor antigen peptide.

[0007] The amino acid sequence of the tumor antigen peptide claimed in the present application is shown as SEQ ID No. 1. It is derived from the abnormal translation of lncRNA LINC02717.

[0008] In a second aspect, the present application claims a multi-epitope peptide connected by multiple epitope peptides.

[0009] The multi-epitope peptide claimed in the present application comprises the tumor antigen peptide described in the first aspect above.

[0010] Further, the multi-epitope peptide can be connected by 2-12 epitope peptides.

[0011] Further, in the multi-epitope peptide, the other epitope peptides can be other renal cell carcinoma tumor-specific antigen peptides other than the polypeptide shown as SEQ ID No. 1. The combined use of multi-epitope peptides can further improve the effectiveness of renal cell carcinoma detection and treatment.

[0012] In a third aspect, the present application claims a biological material related to the tumor antigen peptide described in the first aspect above or the multi-epitope peptide described in the second aspect above:

[0013] (A1) a nucleic acid molecule encoding the tumor antigen peptide described in the first aspect above or the multi-epitope peptide described in the second aspect above;

[0014] (A2) an expression cassette containing the nucleic acid molecule of (A1);

[0015] (A3) a recombinant vector containing the nucleic acid molecule of (A1);

[0016] (A4) a recombinant bacterium containing the nucleic acid molecule of (A1);

[0017] (A5) a recombinant cell containing the nucleic acid molecule of (A1).

[0018] The expression cassette refers to a DNA capable of expressing the tumor antigen peptide of the first aspect or the polytope peptide of the second aspect in a host cell. The DNA can include a promoter to initiate transcription of the coding gene and a termination sequence to terminate transcription. Further, the expression cassette can include an enhancer sequence. The recombinant vector can be a recombinant plasmid carrying the expression cassette. The recombinant bacterium (such as a prokaryotic cell like E. coli or a yeast) and the recombinant cell (such as an animal cell line or a human cell line) can carry the recombinant vector.

[0019] In a fourth aspect, the present application claims a complex formed by the tumor antigen peptide of the first aspect and the HLA-A*11:01 molecule.

[0020] In a fifth aspect, the present application claims a method for preparing an antigen presenting cell.

[0021] The method for preparing an antigen presenting cell claimed by the present application can include the following steps: loading the tumor antigen peptide of the first aspect or the polytope peptide of the second aspect on a cell positive for the HLA-A*11:01 molecule in vitro to obtain an antigen presenting cell with the tumor antigen peptide presented on the cell surface.

[0022] The loading of the tumor antigen peptide of the first aspect or the polytope peptide of the second aspect on a cell positive for the HLA-A*11:01 molecule can be achieved by incubating the tumor antigen peptide or the polytope peptide with the cell positive for the HLA-A*11:01 molecule.

[0023] In an embodiment of the present application, the tumor antigen peptide of the first aspect is incubated with T2-HLA-A*11:01 cells (incubated in a cell incubator at 37°C for 12 hours).

[0024] The cell can be a mammalian cell, preferably an immune system cell, and preferably a professional antigen presenting cell such as a dendritic cell or a B cell, and other preferred cells include T2 cells. The cell can be isolated, preferably in the form of a cell population, or provided in a relatively pure form. The cell can not naturally present the complex of the application (i.e. the complex formed by the tumor antigen peptide and the HLA-A*11:01 molecule), or the cell can present the complex at a level higher than that in the natural state. Such a cell can be obtained by pulsing the cell with the tumor antigen peptide or the polytope peptide of the application. Pulsing involves incubating the cell with the tumor antigen peptide or the polytope peptide for several hours, preferably at a concentration of 25 μM, to further induce presentation of the peptide.

[0025] In a sixth aspect, the present application claims an antigen presenting cell prepared by the method of the fifth aspect above.

[0026] In a seventh aspect, the present application claims an effector T cell inducer.

[0027] The effector T cell inducer claimed by the present application comprises the tumor antigen peptide of the first aspect above, or the polytope peptide of the second aspect above, or the antigen presenting cell of the sixth aspect above.

[0028] In an eighth aspect, the present application claims a method for preparing an effector T cell.

[0029] The method for preparing an effector T cell claimed by the present application can comprise the following steps (B1) or (B2):

[0030] (B1) stimulating PBMC positive for HLA-A*11:01 molecules with the tumor antigen peptide of the first aspect above or the polytope peptide of the second aspect above in vitro, and then obtaining effector T cells from the stimulated cells;

[0031] (B2) stimulating T cells with the antigen presenting cell of the sixth aspect above in vitro, and then obtaining effector T cells.

[0032] In (B1), since PBMC contains a large number of T cells and a small number of cells with antigen presenting ability, such as DC cells, when the tumor antigen peptide or the polytope peptide is co-incubated with PBMC positive for HLA-A*11:01 molecules, the HLA-A*11:01 molecules on the antigen presenting cells bind to the tumor antigen peptide and form a ternary complex with the TCR on the T cells, thereby activating the T cells and obtaining effector T cells.

[0033] In (B2), since the HLA-A*11:01 molecule on the antigen presenting cell used binds the tumor antigen peptide, when it is co-cultured with the T cell, the HLA-A*11:01 molecule, the tumor antigen peptide and the TCR on the T cell form a ternary complex, thereby activating the T cell to obtain the effector T cell.

[0034] Further, in the method, a step of adding IL-2, IL-7 and IL-5 to the stimulation system is further included.

[0035] In an embodiment of the present application, the effector T cell is an activated CD8 + T cell.

[0036] In a ninth aspect, the present application claims an effector T cell prepared by the method of the eighth aspect above.

[0037] In a tenth aspect, the present application claims a medicament.

[0038] The effective component of the medicament claimed in the present application includes the tumor antigen peptide of the first aspect above, the polyepitope peptide of the second aspect above, the biomaterial of the third aspect above, the complex of the fourth aspect above, the antigen presenting cell of the sixth aspect above or the effector T cell of the ninth aspect above.

[0039] In an eleventh aspect, the present application claims a detection reagent for detecting the effector T cell of the ninth aspect above.

[0040] The detection reagent claimed in the present application for detecting the effector T cell includes the complex of the fourth aspect above.

[0041] In a twelfth aspect, the present application claims any of the following applications:

[0042] (C1) The use of a cell positive for HLA-A*11:01 molecule in the preparation of a product for detecting the tumor antigen peptide of the first aspect above.

[0043] (C2) The use of the tumor antigen peptide of the first aspect above in the preparation of a product for detecting a cell positive for HLA-A*11:01 molecule.

[0044] In an embodiment of the present application, the cell positive for HLA-A*11:01 molecule is a T2 cell expressing HLA-A*11:01 molecule.

[0045] (C3) The use of the effector T cell described in the ninth aspect above in killing target cells in vitro; the target cells are cells (HLA-A*11:01 molecule positive) that present the tumor antigen peptide described in the first aspect above on the surface.

[0046] (C4) The use of the tumor antigen peptide described in the first aspect above or the polytope peptide described in the second aspect above or the biomaterial described in the third aspect above or the complex described in the fourth aspect above or the antigen presenting cell described in the sixth aspect above or the effector T cell described in the ninth aspect above in the preparation of a drug for killing target cells; the target cells are cells (HLA-A*11:01 molecule positive) that present the tumor antigen peptide described in the first aspect above on the surface.

[0047] Among them, the target cells can be all tumor cells positive for HLA-A*11:01, such as renal cancer cells.

[0048] In an embodiment of the present application, the target cells are T2 cells expressing HLA-A*11:01 molecules stimulated by the tumor antigen peptide described in the first aspect above.

[0049] (C5) The use of the tumor antigen peptide described in the first aspect above or the polytope peptide described in the second aspect above or the biomaterial described in the third aspect above or the complex described in the fourth aspect above or the antigen presenting cell described in the sixth aspect above or the effector T cell described in the ninth aspect above in the preparation of a drug for treating and / or preventing renal cancer.

[0050] (C6) The use of the complex described in the fourth aspect above in detecting the effector T cell described in the ninth aspect above.

[0051] The present application relates to a newly discovered long-chain non-coding RNA derived from renal cell carcinoma tissue, an antigen short peptide (AVILPQPPK), which can be recognized by T cells and activate immune response when forming a complex with the corresponding HLA molecule (HLA-A*11:01). The present application provides a potential target for renal cell carcinoma treatment, and provides a new target screening direction for subsequent tumor antigen therapy of renal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 A representative mass spectrum of the short peptide of the present application identified by mass spectrometry.

[0053] Figure 2Figure 2A is the identification of T2-HLA-A*11:01 cell line and the T2 cell presentation map of the polypeptide of the lncRNA LINC02717 of the present application (SEQ ID No. 1: AVILPQPPK) and the corresponding HLA molecule (HLA-A*11:01) (SEQ ID No. 3) and other non-corresponding HLA molecule (HLA-A*24:02) (SEQ ID No. 4). Wherein, A is the identification of T2-HLA-A*11:01 cell line and T2-HLA-A*24:02 cell line (Western blotting detects the expression of HLA-A*11:01 protein / HLA-A*24:02 protein). B is the T2 cell presentation map of the polypeptide of the lncRNA LINC02717 of the present application (SEQ ID No. 1: AVILPQPPK) and the corresponding HLA molecule (HLA-A*11:01) and irrelevant HLA molecule (HLA-A*24:02). In the figure, the T2-HLA-A*11:01 control group is the T2-HLA-A*11:01 cell line without any polypeptide stimulation. The HLA-I average fluorescence intensity represents the presentation efficiency of the T2 cell to the polypeptide, and the higher the value, the stronger the affinity of the polypeptide to the corresponding HLA. In the figure, **** represents P<0.0001, the difference is extremely significant.

[0054] Figure 3 Figure 2D is the detection of CD137 activation after the polypeptide of the lncRNA LINC02717 of the present application (SEQ ID No. 1: AVILPQPPK) and the corresponding HLA-A*11:01 patient PBMC were co-cultured. In the figure, ** represents P<0.01, the difference is extremely significant; * represents P<0.05, the difference is significant.

[0055] Figure 4 Figure 2E is the detection of IFNγ ELISPOT secretion after the polypeptide of the lncRNA LINC02717 of the present application (SEQ ID No. 1: AVILPQPPK) and the corresponding HLA-A*11:01 patient PBMC were co-cultured. In the figure, *** represents P<0.001, the difference is extremely significant.

[0056] Figure 5 Figure 2F is the killing detection after the polypeptide of the lncRNA LINC02717 of the present application (SEQ ID No. 1: AVILPQPPK) and the corresponding HLA-A*11:01 patient PBMC were co-cultured and co-cultured with T2 cells presenting polypeptides. In the figure, *** represents P<0.001, the difference is extremely significant; ** represents P<0.01, the difference is extremely significant. DETAILED DESCRIPTION

[0057] The application will be described in further detail below with specific reference to the embodiments. The examples given are only intended to illustrate the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0058] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0059] Example 1, Human long non-coding RNA polypeptide retrieval

[0060] (1) After resection, the surgical tissues (including tumor tissues at the lesion and para-cancer tissues of renal cell carcinoma patients) were quickly preserved in the sample preservation solution, transported to the laboratory via cold chain within 2 hours, and weighed and recorded.

[0061] The specific formulation of the sample preservation solution (100 mL) is shown in Table 1.

[0062] Table 1, Sample preservation solution (100 mL)

[0063]

[0064] After the sample preservation solution is prepared, it is divided into 15 mL centrifuge tubes, each containing 5 mL. After division, it can be stored at 4°C for 1 month.

[0065] (2) Before laboratory processing, the surgical tissues were washed with PBS containing penicillin and streptomycin for at least five times, each time for 1-2 minutes. Then the sample was divided into 100 mm 3 small pieces

[0066] (3) The renal cancer and para-cancer tissues were divided into 1.5 mL centrifuge tubes and cut into small pieces in the tubes. 500 μL of lysis solution was added to each centrifuge tube, and the tissues were ground in a low-temperature tissue grinder for 2 min, followed by tissue lysis.

[0067] (4) The lysed sample was centrifuged at 4°C, 12000g for 30 min, and the supernatant was taken. Then the impurities in the supernatant were filtered out using a 0.45 μm filter. The filtered supernatant was placed in a clean 15 mL centrifuge tube for standby.

[0068] (5) HLA-A antibody (Proteintech, 15240-1-AP) and protein-A beads (Invitrogen, 101041) were added to the tissue lysate for immunoprecipitation, and incubated at 4°C overnight.

[0069] (6) Discard the supernatant and retain the beads pellet.

[0070] (7) Add 5 mL of 0.1 M acetic acid solution to the beads to elute the pHLA-I complex in the tumor tissue lysate bound to the anti-HLA-I antibody, which includes the HLA molecules and the peptides presented by the HLA molecules.

[0071] (8) The eluted pHLA-I complex was divided into five 1.5 mL centrifuge tubes and concentrated to 200 μL using a freeze dryer. The concentrated sample was frozen at -80°C until use.

[0072] (9) Place the sample in a 3KD ultrafiltration tube and centrifuge at 12000 rpm for 30 min at 4°C to remove large proteins in the eluate and retain the polypeptide solution.

[0073] (10) After the peptide sample is concentrated, it is injected into the nanoLC-MSMS system for analysis:

[0074] The mass spectrometer used a Dionex Ultimate 3000-Thermo QE Plus system, with DDMS2 analysis. Liquid chromatography employed a precolumn (Thermo) Acclaim 100, 100 μm × 2 cm, nanoViper, C18, 5 μm, 100A, 164564, and an analytical column (Thermo) Acclaim 100, 75 μm × 15 cm, nanoViper, C18, 3 μm, 100A, 164568. Mobile phase A consisted of 98% water, 2% acetonitrile, and 0.1% formic acid, and mobile phase B consisted of 98% acetonitrile, 2% water, and 0.1% formic acid, with a mobile phase gradient from 5% to 50% mobile phase B over 74 minutes. The total run time was 90 minutes.

[0075] (11) Mass spectrometry analysis results: With the help of the search software ProteinPilot and Peaks, the Uniprot database of human proteins was searched. The database predicted that the long non-coding RNA LINC02717 encoded a small peptide sequence (SEQ ID No. 1: AVILPQPPK) in renal cancer tissue. However, this sequence was not found in adjacent tissues. The mass spectrometry results are as follows: Figure 1 shown.

[0076] Example 2: T2-HLA-A*11:01 cell line pulsed with target antigen

[0077] T2 cells are a kind of human lymphocyte hybridoma cells, which lack antigen polypeptide transporters, so that endogenous antigens cannot be located to the endoplasmic reticulum nuclear Golgi body, and thus cannot be degraded into antigen peptides. Therefore, the expression of the target HLA protein in T2 cells can obtain the antigen presenting cell line corresponding to HLA. By co-culturing the target polypeptide with the cell line, the polypeptide can be bound to the surface of T2 cells, and T2 cells are often used to study the antigen presentation process and the mutual recognition of T cells and HLA molecules.

[0078] The T2 cells used in this example are products of the American Type Culture Collection (ATCC), with the number CRL-1992.

[0079] The specific construction method of the T2-HLA-A*11:01 cell line and the T2-HLA-A*24:02 cell line used in this example is as follows: first, obtain the full-length gene sequence of HLA-A*11:01 (SEQ ID No. 3) and the full-length gene sequence of HLA-A*24:02 (SEQ ID No. 4) from the National Center for Biotechnology Information NCBI (https: / / www.ncbi.nlm.nih.gov / ), and synthesize the full-length sequences through GenScript, and clone them into the HindIII and NotI enzyme cutting sites of the eukaryotic cell expression vector pcDNA3.1-2A-GFP (the full sequence of the vector is shown in SEQ ID No. 5), to obtain pcDNA3.1-HLA-A*11:01-GFP plasmid and pcDNA3.1-HLA-A*24:02-GFP plasmid. Then, transfect the pcDNA3.1-HLA-A*11:01-GFP plasmid and the pcDNA3.1-HLA-A*24:02-GFP plasmid into the T2 cell line, respectively, to obtain the T2-HLA-A*11:01 cell line and the T2-HLA-A*24:02 cell line, and detect the expression of HLA-A*11:01 protein in the T2-HLA-A*11:01 cell line and the expression of HLA-A*24:02 protein in the T2-HLA-A*24:02 cell line by Western blotting, and the results are shown in Figure 2 As shown in FIG. 1A, HLA-A*11:01 protein and HLA-A*24:02 protein are highly expressed in T2 cells, and the protein size is 40 kDa as marked, proving that the cell lines are successfully expressed.

[0080] Pulse target antigen for T2-HLA-A*11:01 cell line and T2-HLA-A*24:02 cell line:

[0081] (1) T2 cell line culture medium is RPMI1640 medium + 10% (volume percentage) FBS + 1x penicillin-streptomycin. The lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) used in the present application is simply synthesized by Merrifield synthesis method (also known as polypeptide solid-phase synthesis method).

[0082] (2) According to the bioinformatics prediction function of NetMHCpan (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ), the affinity of the lncRNA LINC02717 polypeptide is predicted to determine that the lncRNA LINC02717 polypeptide binds to the HLA-A*11:01 subtype.

[0083] (3) The density of T2-HLA-A*11:01 cell line and T2-HLA-A*24:02 cell line is adjusted to 1x10 6 6 / mL using RPMI1640 complete medium, and 100 μL per well is added to a low adsorption U-shaped 96-well plate. 25 μM lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) is added to each well, and the plate is incubated in a 37°C incubator for 12 h.

[0084] (4) Centrifuge at 400g for 5 min, discard the supernatant, and then resuspend the cells with 100 μL of 3% FBS-PBS (i.e. PBS buffer containing 3% fetal bovine serum). Add 1 μL of HLA-I flow antibody (biolegend, 311410) to each tube, and incubate at 4°C for 30 min in the dark. The HLA-I flow antibody can bind to the HLA molecule presenting the polypeptide on the surface of the T2 cells, and the more polypeptide presented on the surface of the cells, the stronger the signal of the HLA-I antibody.

[0085] (5) Centrifuge at 400g for 5 min, discard the supernatant. Then resuspend the cells with 500 μL of 3% FBS-PBS, centrifuge at 400g for 5 min, and discard the supernatant to remove excess antibody.

[0086] (6) Resuspend the cells with 250 μL of 3% FBS-PBS, and perform flow cytometry analysis to detect the fluorescence intensity of the HLA-I antibody. When the lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) has a high affinity for HLA-A*11:01, the HLA-A*11:01 protein presents a large amount of target polypeptide on the cell membrane surface, so the expression amount of HLA-I protein on the surface of T2 cell line increases. The results are as follows Figure 2The results showed that T2-HLA-A*11:01 can present a large amount of lncRNALINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK), and the expression amount of HLA-I protein is significantly higher than that of the control group.

[0087] Example 3, co-culture of tumor antigen and HLA paired peripheral blood lymphocytes

[0088] This example uses peripheral blood-derived peripheral mononuclear cells (Peripheral blood monoculearcell, PBMC) from a kidney cancer patient positive for HLA-A*11:01 molecule identified lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) to detect the immune response of lncRNA LINC02717 polypeptide. At the same time, this example sets up the addition of NY-ESO-1 (157-165) polypeptide (SEQ ID No. 2: SLLMWITQC) as an irrelevant polypeptide group, the irrelevant polypeptide is derived from the tumor antigen in the NY-ESO-1 gene, and is known to have high affinity with HLA-A*02:01, but will not produce affinity with HLA-A*11:01 (SEQ ID No. 3) and HLA-A*24:02 (SEQ ID No. 4).

[0089] 1. On day 0, thaw the patient's PBMC and resuspend the PBMC using complete medium. The complete medium includes: AIM-V medium (item number: 31035025, Eppendorf), 10% (volume percentage) heat-inactivated FBS, 1x pen-strep, 20U / mL IL-2, 10ng / mL IL-7, 10g / mL IL-15. After diluting the cells to 1x10 6 PBMC cells per well in a U-shaped low-adsorption 96-well plate, and incubated at 37°C for 24h. 5

[0090] 2. On day 1, add the corresponding lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) to the cells of step 1 at a final concentration of 25μM per well, and repeat the three-well cells. At the same time, set up a negative control, i.e. add an equal amount of DMSO solution for dissolving the polypeptide, and then incubate uniformly in a 37°C incubator for 3 days.

[0091] 3. On day 4, all well cells were individually half-replaced, each well was replaced with new complete medium (formula synchronized with step 1) and polypeptide at a final concentration of 25μM, and then mixed uniformly by blowing and continued to be incubated uniformly in a 37°C incubator for 3 days.

[0092] ​4. Day 7: Half medium change for all wells, replace with fresh complete medium (recipe in step 1) and 25 μΜ polypeptide per well, mix well and continue incubation for 3 days.

[0093] 5. Day 9: Change medium for all wells to complete medium without cytokines (remove IL-2, IL-7 and IL-15 from the recipe in step 1), incubate for 12 h at 37 °C, and continue with the following steps.

[0094] 6. Flow cytometry to detect activation of peripheral blood lymphocyte tumor-reactive T cells (effector T cells):

[0095] CD137 is a T cell marker expressed after T cells recognize antigen-bearing cells. CD137 is a member of the tumor necrosis factor receptor superfamily, expressed in antigen-activated T cells, has a costimulatory function, can up-regulate survival-related genes, and enhance cell division.

[0096] (1) Collect the peripheral blood lymphocytes cultured above, which have been stimulated with antigen for multiple rounds, adjust the number of cells in each sample to 1 x 10 5 at 400 g for 5 min.

[0097] (2) Discard the supernatant, add 400 μΐ^of 3% FBS-PBS to each sample, resuspend the cells, wash away the excess medium, and centrifuge at 400 g for 5 min.

[0098] (3) Discard the supernatant, add 100 μΐ^of 3% FBS-PBS to each sample, and resuspend the cells.

[0099] (4) Add 1 μΐ^of CD3, CD8 and CD137 flow cytometry antibodies to each sample, respectively, and incubate at 4 °C in the dark for 30 min.

[0100] (5) After incubation, centrifuge at 400 g for 5 min, discard the supernatant, and repeat the washing with 200 μΐ^of 3% FBS-PBS once at 400 g for 5 min.

[0101] (6) Resuspend the cells with 200 μΐ^of 3% FBS-PBS, and perform flow cytometry detection using a BD LSRFortessa.

[0102] The results are shown in Figure 3 . The results show the percentage of CD3 + CD8 + double-positive cells that are CD137 +The ratio of cells. The results show that compared with the irrelevant polypeptide group and the DMSO control group, the CD3 + CD8 + T cells were significantly activated.

[0103] Example 4, Enzyme-linked immunospot assay for detecting IFN-γ cytokine secretion of activated T cells in PBMC

[0104] Enzyme-linked immunospot (ELISpot) can detect the IFN-γ secretion of single activated T cells. IFN-γ ELISPOT is a reliable method for evaluating T cell immune response to tumor antigens, which has high sensitivity and can detect the secretion of IFN-γ of single cells to evaluate the activation level of T cells. IFN-γ, a cytokine secreted by immune active cells, plays a major role in inducing antiviral immunity, mainly secreted by CD8 cells that recognize antigens. The level of IFN-γ secretion represents the activation level of T cells after recognition by antigens.

[0105] The ELIspot kit is a product of Self-Dakai Biotechnology Co., Ltd. The plates in the kit are pre-embedded with antibodies before leaving the factory.

[0106] The cells (containing activated T cells) after co-culturing the tumor antigens with HLA matched peripheral blood lymphocytes in Example 3 were taken out 1 / 2 for the related experiments of this example.

[0107] (1) Add 200 μL of serum-free medium to each experimental well with a syringe, activate the pre-coated plate, and stand at room temperature for 10 min before removing.

[0108] (2) Add 1×10 5 cells per well to the experimental well, and add 10 μL of PMA stimulator to the positive control well. Then cover the plate, and place it in a 37°C, 5% CO2 incubator for 24 h.

[0109] (3) On the second day, pour out the cells and medium in the well. Add pre-cooled deionized water and place it in a 4°C refrigerator for 10 min to lyse the cells.

[0110] (4) Remove the liquid, add 200 μL of 1×Washing buffer working solution (provided in the kit) to each well, stand for 1 min, and remove the liquid, repeat 6 times.

[0111] (5) Add 100 μL of 1x Biotinylated Antibody working solution (provided in the kit) to each well, and incubate at 37°C for 1 hour.

[0112] (6) Remove the liquid, add 200 μL of 1x Washing buffer working solution (provided in the kit) to each well, and stand for 1 min, then remove the liquid, repeat 6 times.

[0113] (7) Add 100 μL of 1x Streptavidin-HRP working solution (provided in the kit) to each well, and incubate at 37°C for 1 hour.

[0114] (8) Remove the liquid, add 200 μL of 1x Washing buffer working solution (provided in the kit) to each well, and stand for 1 min, then remove the liquid, repeat 6 times, and finally ensure that the liquid in the well is removed.

[0115] (9) Prepare AEC developing solution according to the AEC Dilution: AEC Solution I (20x): AEC Solution II (20x): AEC Solution III (200x) (provided in the kit) at a volume ratio of 180:20:20:1. Add 100 μL of the prepared developing solution to each well, and stand at room temperature in the dark for 30 min.

[0116] (10) Pour out the liquid in the well, remove the base, and rinse the experimental well and the back repeatedly with deionized water to terminate the color development. Place the rinsed plate in a fume hood to dry.

[0117] (11) Use Mabtech IRIS instrument to read the ELIspot plate, and count the number of IFN-γ spots produced by activated T cells in each experimental well.

[0118] The results are shown in Table 1. Figure 4 Compared with the DMSO control group and the irrelevant polypeptide stimulation group, the results show that the antigen polypeptide lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) can significantly activate T cells in the peripheral blood of patients to produce an immune response.

[0119] Example 5, Detection of the immune killing effect of tumor antigens on tumor cells

[0120] Annexin V is a reagent for detecting apoptosis. In normal cells, phosphatidylserine is only distributed on the inner side of the lipid bilayer of the cell membrane. In the early stage of apoptosis, the membrane phosphatidylserine is flipped from the inner side to the outer side of the lipid membrane. Annexin V, as a phospholipid binding protein, has a high affinity for phosphatidylserine. It binds to the membrane of early apoptotic cells through the phosphatidylserine exposed on the outer side of the cell. Therefore, Annexin V is a sensitive indicator for detecting early apoptosis of cells.

[0121] (1) Take PBMC cells of HLA-A*11:01 molecule positive volunteers (normal people or kidney cancer patients) and stimulate the PBMC according to the scheme in Example 3 using the lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK). Collect all cells as effector cells (mainly T cells activated by the lncRNA LINC02717 polypeptide of the application), and the control group is cultured for 10 days without antigen stimulation (unactivated T cell group). The unactivated T cell group does not activate T cells with specific antigens, so the T cells are still in a resting state and can be used as a negative control for T cells.

[0122] (2) Pulse the T2-HLA-A*11:01 cells (see Example 2) according to the final scheme in Example 2 to present the lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) on the surface of the T2-HLA-A*11:01 cells, and collect the cells as target cells. Since the T2-HLA-A*11:01 cells after polypeptide pulsing can present the lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) on the surface, and the T2-HLA-A*11:01 cells are essentially tumor cells, the T2 cells can act as both antigen-presenting cells and target cells for T cell recognition of antigens in the antigen experiment.

[0123] (3) Co-culture the effector cells and target cells at a ratio of 10:1, and at 0h, 24h, 48h, take the co-cultured cells to detect the viability of T2-HLA-A*11:01 cells. The experiment also sets up a control group without adding effector cells (T2-HLA-A*11:01 group).

[0124] (4) After the co-culture is completed, the cell culture medium is aspirated into a 1.5 mL centrifuge tube, and the cell suspension is centrifuged at 1000 rpm for 5 min in a pre-cooled centrifuge at 4°C, and the supernatant is discarded.

[0125] (5) Add 300 μL of Annexin V-FITC binding solution in the kit to resuspend, and ensure the solution volume is consistent. Then add Annexin V-EGFP staining solution in the kit to the centrifuge tube, and incubate at room temperature for 20 min in the dark.

[0126] (6) Flow cytometry analysis is performed to detect the proportion of Annexin V in T2 cells, and the proportion of tumor cells (T2-HLA-A*11:01 cells) negative for Annexin V, i.e., the survival rate of tumor cells, is calculated.

[0127] The results are shown in Table 1. Figure 5 The results show that the activated T cells after stimulation of PBMC with lncRNA LINC02717 polypeptide (SEQ ID No. 1: AVILPQPPK) and HLA-A*11:01 molecules can recognize T2-HLA-A*11:01 cells presenting LINC02717 polypeptide and effectively kill T2-HLA-A*11:01 cells.

[0128] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a specific example, it should be understood that the application can be further improved. In general, according to the principle of the application, the present application is intended to include any changes, uses or improvements of the application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

Claims

1. A tumor antigen peptide, the amino acid sequence of which is shown in SEQ ID No.

1.

2. A biomaterial related to the tumor antigen peptide according to claim 1: (A1) a nucleic acid molecule encoding the tumor antigen peptide according to claim 1; (A2) an expression cassette containing the nucleic acid molecule described in (A1); (A3) a recombinant vector containing the nucleic acid molecule described in (A1); (A4) a recombinant bacterium containing the nucleic acid molecule described in (A1); (A5) A recombinant cell containing the nucleic acid molecule described in (A1).

3. A complex formed by the tumor antigen peptide according to claim 1 and an HLA-A*11:01 molecule.

4. A non-disease diagnostic and therapeutic method for preparing antigen-presenting cells, comprising the following steps: loading the tumor antigen peptide of claim 1 onto HLA-A*11:01 molecule-positive cells in vitro to obtain antigen-presenting cells capable of presenting the tumor antigen peptide on the cell surface.

5. Antigen-presenting cells prepared by the method of claim 4. An effector T cell inducer comprising the tumor antigen peptide according to claim 1 or the antigen-presenting cell according to claim 5.

7. A non-disease diagnostic and therapeutic method for preparing effector T cells, comprising the following steps (B1) or (B2): (B1) stimulating HLA-A*11:01 molecule-positive PBMCs in vitro with the tumor antigen peptide according to claim 1, and then obtaining effector T cells from the stimulated cells; (B2) Stimulating T cells in vitro with the antigen-presenting cells according to claim 5 to obtain effector T cells.

8. Effector T cells prepared by the method of claim 7.

9. A medicament, the active ingredient of which comprises the tumor antigen peptide according to claim 1, the biomaterial according to claim 2, the complex according to claim 3, the antigen-presenting cell according to claim 5, or the effector T cell according to claim 8.

10. A detection reagent for detecting the effector T cells according to claim 8, comprising the complex according to claim 3.

11. Any of the following applications: (C1) Non-disease diagnostic and therapeutic use of the effector T cells of claim 8 in killing target cells in vitro; the target cells are cells presenting the tumor antigen peptide of claim 1 on their surface; (C2) Use of the tumor antigen peptide of claim 1, the biomaterial of claim 2, the complex of claim 3, the antigen-presenting cell of claim 5, or the effector T cell of claim 8 in the preparation of a medicament for killing target cells; the target cells are cells presenting the tumor antigen peptide of claim 1 on their surface; the target cells are renal cancer cells; (C3) Use of the tumor antigen peptide according to claim 1, the biomaterial according to claim 2, the complex according to claim 3, the antigen-presenting cell according to claim 5, or the effector T cell according to claim 8 in the preparation of a medicament for treating renal cancer; (C4) Non-disease diagnostic and therapeutic use of the complex of claim 3 in detecting the effector T cells of claim 8.

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