HLA-A*24: 02 renal cell carcinoma tumor antigen short peptide derived from long-chain non-coding RNA ENSG00000286147

Abstract

The invention discloses an HLA (Human Leukocyte Antigen)-A * 24: 02 renal cell carcinoma tumor antigen short peptide derived from a long-chain non-coding RNA (Ribonucleic Acid) ENSG00000286147. The invention provides a tumor antigen peptide. The amino acid sequence of the tumor antigen peptide is as shown in SEQ ID No. 1. Researches find that a compound formed by the oligopeptide and a corresponding HLA molecule (HLA-A * 24: 02) can be recognized by T cells, and immune response is activated. The invention provides a potential target spot for treatment of renal cell carcinoma, and provides a new target spot screening direction for subsequent tumor antigen treatment of renal carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a HLA-A*24:02 renal cell carcinoma tumor antigen short peptide derived from long-chain non-coding RNA ENSG00000286147. Background Art

[0002] Renal carcinoma is a common malignant tumor. Once advanced renal cancer metastasizes to distant sites, the patient's 5-year survival rate is only 10%-18%. Renal cancer 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 an estimated 400,000 new reported cases and more than 175,000 deaths each year. In 2020, the age-standardized incidence rate (ASR) of renal cancer worldwide was 4.6 per 100,000 people, ranking 16th among all cancer incidences. The ASR incidence rate in men (6.1 per 100,000 people) is higher than that in women (3.2 per 100,000 people), and the incidence rate is relatively higher in more developed countries. At present, the common treatment for renal cell carcinoma is surgical resection. When the tumor is too large or metastatic, immune checkpoint and targeted combination therapy are generally used, but the treatment effect is limited by multiple factors such as the patient's immune microenvironment. Therefore, it is necessary to develop new drugs to treat renal cancer.

[0003] Long non-coding RNA (lncRNA) is a non-coding RNA with a length greater than 200 nucleotides. Traditional theory holds that lncRNA does not have coding ability and mainly plays a role in epigenetics, RNA transcription and post-transcriptional regulation. However, in recent years, more and more evidence has confirmed that lncRNA is not completely without amino acid coding ability. Although lncRNA has no start codon and cannot encode complete macromolecular proteins, lncRNA can encode short amino acid peptides through small open reading frames (sORF). The polypeptides encoded by lncRNA also play an important role in the biological process of tumors. For example, some tumor-specific lncRNA polypeptides can serve as antigens for T cells to recognize tumors and induce tumor immune responses. At present, only about 1% of the genome is marked as protein coding regions, but 75% of the genome can be transcribed and theoretically translated. Through the study of non-coding antigens represented by lncRNA, we can increase the possibility of finding tumor-specific and patient-shared antigens in tumors.

[0004] For tumors, T cells can only play an immune function if they recognize antigens presented on tumor cells. The antigen presentation process of tumors starts in the endoplasmic reticulum in the cytoplasm. Endogenous proteins of tumor cells are degraded in the cytoplasm to form short peptides, and in this process a polypeptide library formed by the decomposition of multiple proteins is produced. Part of these peptides is transferred to the endoplasmic reticulum through a transporter associated with antigen processing (TAP). Once the short peptide enters the endoplasmic reticulum, it will be promoted by internal enzymes to assemble and fold with human leukocyte antigen (HLA) to form a complex, and then finally presented to the cell surface. Those skilled in the art are committed to discovering and determining these polypeptide fragments presented on the surface of target cells. Summary of the invention

[0005] The purpose of the present invention is to provide a HLA-A*24:02 renal cell carcinoma tumor antigen short peptide derived from long non-coding RNA ENSG00000286147.

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

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

[0008] In the second aspect, the present invention claims a multi-epitope peptide formed by connecting multiple epitope peptides.

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

[0010] Furthermore, the multi-epitope peptide is 2 or more epitope peptides, and can be formed by connecting 2-12 epitope peptides.

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

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

[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 described in (A1);

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

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

[0017] (A5) A recombinant cell containing the nucleic acid molecule described in (A1).

[0018] Wherein, the expression cassette refers to a DNA capable of expressing the tumor antigen peptide described in the first aspect above or the multi-epitope peptide described in the second aspect above in a host cell, and the DNA may include not only a promoter for initiating transcription of the relevant coding gene, but also a termination sequence for terminating transcription. Further, the expression cassette may also include an enhancer sequence. The recombinant vector may be a recombinant plasmid carrying the expression cassette. The recombinant bacteria (such as prokaryotic cells such as Escherichia coli or yeast) and the recombinant cells (such as animal cell lines or human cell lines) may carry the recombinant vector.

[0019] In a fourth aspect, the present invention claims protection for a complex formed by the tumor antigen peptide described in the first aspect above and the HLA-A*24:02 molecule.

[0020] In a fifth aspect, the present invention claims a method for preparing antigen presenting cells.

[0021] The method for preparing antigen-presenting cells claimed in the present invention may include the following steps: loading the tumor antigen peptide described in the first aspect or the multi-epitope peptide described in the second aspect onto HLA-A*24:02 molecule-positive cells in vitro to obtain antigen-presenting cells capable of presenting the tumor antigen peptide on the cell surface.

[0022] Among them, loading the tumor antigen peptide described in the first aspect or the multi-epitope peptide described in the second aspect onto HLA-A*24:02 molecule-positive cells can be completed by co-incubating the tumor antigen peptide or the multi-epitope peptide with the HLA-A*24:02 molecule-positive cells.

[0023] In one embodiment of the present invention, the tumor antigen peptide described in the first aspect above is co-incubated with T2-HLA-A*24:02 cells (cultured in a cell culture incubator at 37° C. for 12 hours).

[0024] Wherein, 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 may not naturally present the complex of the present invention (i.e., the complex formed by the tumor antigen peptide and the HLA-A*24:02 molecule), or the level of the cell presenting the complex (the complex formed by the tumor antigen peptide and the HLA-A*24:02 molecule) is higher than that in the natural state. Such cells can be obtained by pulse treatment with the tumor antigen peptide or the multi-epitope peptide of the present invention. Pulse treatment involves incubating cells with the tumor antigen peptide or the multi-epitope peptide for several hours, preferably, the concentration of the peptide used is 25 μM, and further inducing the presentation of the peptide.

[0025] In a sixth aspect, the present invention claims protection for antigen presenting cells prepared by the method described in the fifth aspect above.

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

[0027] The effector T cell inducer claimed in the present invention comprises the tumor antigen peptide described in the first aspect above or the multi-epitope peptide described in the first aspect above or the antigen presenting cell described in the sixth aspect above.

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

[0029] The method for preparing effector T cells claimed in the present invention may include the following steps (B1) or (B2):

[0030] (B1) stimulating HLA-A*24:02-positive PBMCs in vitro with the tumor antigen peptide described in the first aspect or the multi-epitope peptide described in the second aspect, thereby obtaining effector T cells from the stimulated cells;

[0031] (B2) Stimulating T cells in vitro with the antigen presenting cells described in the sixth aspect to obtain effector T cells.

[0032] In (B1), since PBMCs contain both 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 multi-epitope peptide is co-incubated with PBMCs positive for HLA-A*24:02 molecules, the HLA-A*24:02 molecules on the antigen presenting cells bind to the tumor antigen peptides and form a ternary complex with the TCR on the T cells, thereby activating the T cells to obtain effector T cells.

[0033] In (B2), since the HLA-A*24:02 molecule on the antigen presenting cell binds to the tumor antigen peptide, when it is co-cultured with the T cell, the HLA-A*24:02 molecule, the tumor antigen peptide and the TCR on the T cell form a ternary complex, thereby activating the T cell to obtain effector T cells.

[0034] Furthermore, the method further comprises the step of adding IL-2, IL-7 and IL-5 to the stimulation system.

[0035] In one embodiment of the present invention, the effector T cells are activated CD8 + T cells.

[0036] In a ninth aspect, the present invention claims protection for effector T cells prepared using the method described in the eighth aspect above.

[0037] In a tenth aspect, the present invention claims a drug.

[0038] The active ingredients of the drug claimed in the present invention include the tumor antigen peptide described in the first aspect above, or the multi-epitope peptide described in the second aspect above, or the biological material 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.

[0039] In an eleventh aspect, the present invention claims a detection reagent for detecting the effector T cells described in the ninth aspect above.

[0040] The detection reagent for detecting the effector T cells claimed in the present invention includes the complex described in the fourth aspect above.

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

[0042] (C1) Use of HLA-A*24:02 molecule-positive cells in the preparation of a product for detecting the tumor antigen peptide described in the first aspect above.

[0043] (C2) Use of the tumor antigen peptide described in the first aspect above in the preparation of a product for detecting cells positive for the HLA-A*24:02 molecule.

[0044] In one embodiment of the present invention, the HLA-A*24:02 molecule-positive cells are T2 cells expressing HLA-A*24:02 molecules.

[0045] (C3) Use of the effector T cells described in the ninth aspect of the preceding text to kill target cells in vitro; the target cells are cells (HLA-A*24:02 molecule positive) presenting the tumor antigen peptides described in the first aspect of the preceding text on their surface.

[0046] (C4) Use of the tumor antigen peptide described in the first aspect above, or the multi-epitope 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 medicament for killing target cells; the target cells are cells (HLA-A*24:02 molecule positive) whose surface presents the tumor antigen peptide described in the first aspect above.

[0047] The target cells may be all tumor cells that are HLA-A*24:02 positive, such as renal cancer cells.

[0048] In one embodiment of the present invention, the target cells are T2 cells expressing HLA-A*24:02 molecules stimulated by the tumor antigen peptide described in the first aspect above.

[0049] (C5) Use of the tumor antigen peptide described in the first aspect above, or the multi-epitope 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) Use of the complex described in the fourth aspect above in detecting the effector T cells described in the ninth aspect above.

[0051] The present invention relates to a newly discovered antigenic short peptide (REWGPIFNIL) derived from a long non-coding RNA in renal cell carcinoma tissue. The complex formed by the short peptide and the corresponding HLA molecule (HLA-A*24:02) can be recognized by T cells and activate immune response. The present invention provides a potential target for the treatment of renal cell carcinoma and provides a new target screening direction for subsequent tumor antigen treatment of renal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The representative mass spectra of the short peptides of the present invention are shown in Figure 2 .

[0053] Figure 2The present invention lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) and the corresponding HLA molecule (HLA-A*24:02) and other non-corresponding HLA molecules (HLA-A*02:01) are presented in T2 cells. A is the identification of T2-HLA-A*24:02 cell line and T2-HLA-A*11:01 cell line (protein immunoblotting to detect the expression of HLA-A*11:01 protein / HLA-A*24:02 protein). B is the present invention lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) and the corresponding HLA molecule (HLA-A*24:02) and the irrelevant HLA molecule (HLA-A*11:01) T2 cell presentation diagram. In the figure, the T2-HLA-A*24:02 control group is the T2-HLA-A*24:02 cell line that was not stimulated by any peptide. The mean fluorescence intensity of HLA-I represents the efficiency of T2 cells presenting peptides. The higher the value, the stronger the affinity of the peptide to the corresponding HLA. In the figure, *** indicates P < 0.001, and the difference is extremely significant.

[0054] Figure 3 This is the CD137 activation detection after co-culture of the lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) of the present invention with the corresponding HLA-A*24:02 patient PBMC. In the figure, *** indicates P < 0.001, and the difference is extremely significant; ** indicates P < 0.01, and the difference is extremely significant.

[0055] Figure 4 The IFNγ ELISPOT secretion was detected after the lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) of the present invention was co-cultured with PBMC of the corresponding HLA-A*24:02 patient. In the figure, *** indicates P<0.001, and the difference is extremely significant.

[0056] Figure 5 This is a killing test of the lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) of the present invention after co-culture with PBMC of the corresponding HLA-A*24:02 patient and co-culture with T2 cells presenting the polypeptide. In the figure, ** indicates P < 0.01, and the difference is extremely significant; *** indicates P < 0.001, and the difference is extremely significant. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

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

[0059] Example 1: Human long non-coding RNA polypeptide search

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

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

[0062] Table 1. Sample storage solution (100 mL)

[0063]

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

[0065] (2) Before the surgical tissue is processed in the laboratory, it is rinsed at least five times with PBS containing penicillin and streptomycin, each time for 1-2 minutes. The sample is then cut into 100 mm pieces with a scalpel. 3 Small Block

[0066] (3) The renal cancer and adjacent tissues were divided into 1.5 mL centrifuge tubes and chopped in the tubes. 500 μL of lysis buffer 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) Centrifuge the lysed sample at 4°C, 12000 g for 30 min and collect the supernatant. Then filter out impurities in the supernatant using a 0.45 μm filter. Place the filtered supernatant in a clean 15 mL centrifuge tube for later use.

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

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

[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 samples were frozen at -80°C for later use.

[0072] (9) Place the sample in a 3KD ultrafiltration tube and centrifuge at 12,000 rpm for 30 min at 4°C to remove large proteins in the eluate and retain the peptide solution.

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

[0074] The mass spectrometer used was a Dionex Ultimate3000-Thermo QE Plus system: the mass spectrometer used the ddms2 analysis method. The liquid chromatography used: pre-column: (Thermo) Acclaim 100, 100μm×2cm, nanoViper, C18, 5μm, 100A, 164564, analytical column: (Thermo) Acclaim 100, 75μm×15cm, nanoViper, C18, 3μm, 100A, 164568. The mobile phase A of the mass spectrometer was 98% water, 2% acetonitrile, 0.1% formic acid, and the mobile phase B was 98% acetonitrile, 2% water, 0.1% formic acid, and the mobile phase gradient was 74 minutes. The mobile phase B increased from 5% to 50% in 74 minutes. The total running time was 90 minutes.

[0075] (11) Mass spectrometry analysis results. Using the search software ProteinPilot and Peaks, we searched the Uniprot database of human proteins. The database predicted that the long non-coding RNA ENSG00000286147 would encode a small peptide sequence (SEQ ID No. 1: REWGPIFNIL) in renal cancer tissues. However, this sequence was not found in adjacent tissues. The mass spectrometry results are shown in Figure 2. Figure 1 shown.

[0076] Example 2: T2-HLA-A*24:02 cell line pulsed with target antigen

[0077] T2 cells are a type of human lymphocyte hybridoma cell that lacks antigen peptide transport proteins, which prevents endogenous antigens from being localized to the endoplasmic reticulum and Golgi apparatus, and therefore cannot be degraded into antigen peptides. Therefore, by expressing the target HLA protein in T2 cells, an antigen presenting cell line corresponding to HLA can be obtained. By co-culturing the target polypeptide with the cell line, the polypeptide can bind to the surface of T2 cells. T2 cells are often used to study the antigen presentation process and the mutual recognition between T cells and HLA molecules.

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

[0079] The specific construction methods of the T2-HLA-A*24:02 cell line and the T2-HLA-A*11:01 cell line used in this example are as follows: First, 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) were obtained from the National Center for Biotechnology Information (NCBI) of the United States (https: / / www.ncbi.nlm.nih.gov / ), and the full-length sequences were synthesized by Qingke Biotechnology and cloned into the eukaryotic cell expression vector pcDNA3.1-2A-GFP (see SEQ ID No. 5 for the full sequence of the vector) between the HindIII and NotI restriction sites to obtain pcDNA3.1-HLA-A*11:01-GFP plasmid and pcDNA3.1-HLA-A*24:02-GFP plasmid. Then, the pcDNA3.1-HLA-A*11:01-GFP plasmid and the pcDNA3.1-HLA-A*24:02-GFP plasmid were transfected 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 the expression of HLA-A*11:01 protein in the T2-WT cell line (wild-type T2 cell line), 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 were detected by western blotting. The results are shown in FIG. Figure 2 As shown in A. 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 indicated, proving that the cell line is successfully expressed.

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

[0081] (1) The culture medium of T2 cell line is RPMI1640 culture medium + 10% (volume percentage) FBS + 1× penicillin-streptomycin. The lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) used in the present invention is simply synthesized by Merrifield synthesis method (also known as polypeptide solid phase synthesis method).

[0082] (2) Based on the bioinformatics prediction function of NetMHCpan (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ), the affinity of lncRNA ENSG00000286147 peptide was predicted, and it was determined that lncRNA ENSG00000286147 peptide binds to HLA-A*24:02 subtype.

[0083] (3) Use RPMI1640 complete medium to adjust the density of T2-HLA-A*24:02 and T2-HLA-A*11:01 cell lines to 1×10 6 / mL, added to a low-adsorption U-shaped 96-well plate, 100 μL per well. Add 25 μM tumor antigen (SEQ ID No. 1: REWGPIFNIL) to each well and place in an incubator at 37°C for 12 hours.

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

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

[0086] (6) Add 250 μL 3% FBS-PBS to resuspend the cells, perform flow cytometry analysis, and detect the fluorescence intensity of HLA-I antibody. When the lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) has a high affinity with HLA-A*24:02, the HLA-A*24:02 protein presents a large amount of target polypeptide on the cell membrane surface, so the expression of HLA-I protein on the surface of the T2 cell line increases. The results are shown in Figure 2. Figure 2As shown in B. The results showed that T2-HLA-A*24:02 could present a large amount of ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL), and the expression level of HLA-I protein was significantly higher than that of the control group.

[0087] Example 3: Co-culture of tumor antigens and HLA-paired peripheral blood lymphocytes

[0088] In this example, peripheral blood mononuclear cells (PBMCs) from peripheral blood of patients with renal cancer who were positive for HLA-A*24:02 molecules and identified lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) were used to detect the immune response of lncRNA ENSG00000286147 polypeptide. (157-165) The polypeptide (SEQ ID No.2: SLLMWITQC) is an irrelevant polypeptide group, which 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 not with HLA-A*11:01 (SEQ ID No.3) and HLA-A*24:02 (SEQ ID No.4).

[0089] 1. On day 0, take the frozen PBMC of the patient and resuscitate them in a 37℃ water bath, and resuspend the PBMC in complete culture medium. The complete culture medium includes: AIM-V culture medium (Cat. No.: 31035025, Invitrogen), 10% (volume percentage) heat-inactivated FBS, 1× penicillin-streptomycin, 20U / mL IL-2, 10ng / mL IL-7, 10g / mL IL-15. Dilute the cells to 1×10 6 After that, 1×10 5 PBMC cells were plated in U-shaped low-adhesion 96-well plates and cultured at 37°C for 24 h.

[0090] 2. Day 1: Add the patient tumor lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) corresponding to PBMC to the cells in step 1 at a final concentration of 25 μM per well, and repeat for three wells. At the same time, set up a negative control, that is, add an equal amount of DMSO solution dissolved in the polypeptide, and then culture uniformly in a 37°C incubator for 3 days.

[0091] 3. Day 4: Perform half-change of medium in all wells one by one, replace each well with new complete medium (same formula as step 1) and a final concentration of 25 μM polypeptide, mix well by pipetting, and continue to culture in a 37°C incubator for 3 days.

[0092] 4. Day 7: Perform half-change of medium in all wells one by one, replace each well with new complete medium (same formula as step 1) and a final concentration of 25 μM polypeptide, mix well by pipetting, and continue to culture in a 37°C incubator for 3 days.

[0093] 5. Day 9: Replace all cells in all wells with complete culture medium without cytokines (remove IL-2, IL-7 and IL-15 based on the complete culture medium formula in step 1), culture in a 37°C incubator for 12 hours, and then proceed with subsequent operations.

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

[0095] CD137 is a T cell marker expressed by T cells after they recognize cells carrying antigens. CD137 is a member of the tumor necrosis factor receptor superfamily and can be expressed in antigen-activated T cells. It has a co-stimulatory function, can upregulate survival-related genes, and enhance cell division.

[0096] (1) Collect the peripheral blood lymphocytes cultured above and stimulated by multiple rounds of antigens, and adjust the number of cells in each sample to 1×10 5 Centrifuge at 400 g for 5 min.

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

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

[0099] (4) Add 1 μL of CD3, CD8, and CD137 flow cytometry antibodies to each sample 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, wash once with 200 μL 3% FBS-PBS, and centrifuge at 400 g for 5 min.

[0101] (6) Resuspend the cells in 200 μL 3% FBS-PBS and perform flow cytometry detection using BD LSRFortessa.

[0102] The results are as follows Figure 3 As shown, the results show that CD3 + CD8 + CD137 in double positive cells +The results showed that compared with the irrelevant peptide group and the DMSO control group, after the lncRNA ENSG00000286147 peptide (SEQ ID No. 1: REWGPIFNIL) stimulated peripheral blood mononuclear cells from HLA-A*24:02-positive renal cancer patients, CD3 + CD8 + T cells were significantly activated.

[0103] Example 4: Detection of IFN-γ cytokine secretion by activated T cells in PBMC by ELISA

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

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

[0106] Half of the cells (containing activated T cells) obtained after co-culture of tumor antigens and HLA-paired peripheral blood lymphocytes in Example 3 were taken out for use in the related experiments of this example.

[0107] (1) Use a dispenser to add 200 μL of serum-free culture medium to each experimental well to activate the pre-coated plate. Let it stand at room temperature for 10 min and then remove it.

[0108] (2) The cells in static culture were cultured at a rate of 1×10 5 Add 10 μL of PMA stimulant to each well of the experiment well, and add 10 μL of PMA stimulant to each well of the positive control experiment well. Then cover the plate and place it in a 37°C, 5% carbon dioxide incubator for 24 hours.

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

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

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

[0112] (6) Remove the liquid and add 200 μL of 1× Washing buffer (provided in the kit) to each well. Let it stand for 1 min and remove the liquid. Repeat 6 times.

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

[0114] (8) Remove the liquid and add 200 μL of 1× Washing buffer (provided in the kit) to each well. Let it stand for 1 min and then remove the liquid. Repeat this process 6 times. Finally, make sure that all the liquid in the wells is removed.

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

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

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

[0118] The results are as follows Figure 4 Compared with the DMSO control group and the irrelevant polypeptide stimulation group, the results showed that the antigen polypeptide lncRNA ENSG00000286147 (SEQ ID No. 1: REWGPIFNIL) can significantly activate T cells in the patient's peripheral blood 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 cell 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 cell apoptosis, the membrane phosphatidylserine flips from the inner side of the lipid membrane to the outer side. Annexin V, as a phospholipid binding protein, has a high affinity for phosphatidylserine. It binds to the cell 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 cell apoptosis.

[0121] (1) PBMC cells of volunteers (normal persons or patients) with positive HLA-A*24:02 molecules were obtained. After stimulating PBMCs with lncRNA ENSG00000286147 polypeptide (SEQ ID No. 1: REWGPIFNIL) according to the scheme in Example 3, all cells were collected as effector cells (mainly T cells activated by the lncRNA ENSG00000286147 polypeptide of the present invention). The control group was cultured for 10 days without antigen stimulation (unactivated T cell group). Since there was no specific antigen to activate T cells in the unactivated T cell group, the T cells were still in a resting state and could be used as a negative control of T cells.

[0122] (2) Pulsing T2-HLA-A*24:02 cells (see Example 2) with polypeptides according to the scheme in Example 2, so that the surface of T2-HLA-A*24:02 cells presents lncRNA ENSG00000286147 polypeptide (SEQ ID No.1: REWGPIFNIL), and collecting cells as target cells. Since the surface of T2-HLA-A*24:02 cells after polypeptide pulse can present lncRNA ENSG00000286147 polypeptide (SEQ ID No.1: REWGPIFNIL), and T2-HLA-A*24:02 cells are essentially tumor cells, T2 cells can be used as antigen presenting cells in antigen experiments, and can also be used as target cells for T cells to recognize antigens.

[0123] (3) The effector cells and target cells were co-cultured at a ratio of 10:1, and the co-cultured cells were collected at 0h, 24h, and 48h to detect the viability of T2-HLA-A*24:02 cells. A control group (T2-HLA-A*24:02 group) without the addition of effector cells was also set up.

[0124] (4) After the co-culture is completed, the cell culture medium is aspirated into a 1.5 mL centrifuge tube, the cell suspension is centrifuged at 1000 rpm for 5 min in a centrifuge precooled to 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 that the solution volume is consistent. Then add the Annexin V-EGFP staining solution in the kit to the centrifuge tube and incubate at room temperature in the dark for 20 minutes.

[0126] (6) Flow cytometry was used to detect the proportion of Annexin V in T2 cells and to calculate the proportion of tumor cells that were negative for Annexin V, i.e., the viability of tumor cells (T2-HLA-A*24:02 cells).

[0127] The results are as follows Figure 5 The results show that T cells activated by stimulating HLA-A*24:02-positive PBMCs with lncRNA ENSG00000286147 polypeptide (SEQ ID No.1: REWGPIFNIL) can recognize T2-HLA-A*24:02 cells presenting HERV3895 polypeptide and effectively kill T2-HLA-A*24:02.

[0128] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

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

1.

2. A multi-epitope peptide formed by connecting multiple epitope peptides, characterized in that: The multi-epitope peptide comprises the tumor antigen peptide according to claim 1.

3. Biomaterials related to the tumor antigen peptide according to claim 1 or the multi-epitope peptide according to claim 2: (A1) a nucleic acid molecule encoding the tumor antigen peptide according to claim 1 or the multi-epitope peptide according to claim 2; (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).

4. A complex formed by the tumor antigen peptide according to claim 1 and an HLA-A*24:02 molecule.

5. A method for preparing antigen presenting cells, comprising the following steps: loading the tumor antigen peptide according to claim 1 or the multi-epitope peptide according to claim 2 onto HLA-A*24:02 molecule-positive cells in vitro to obtain antigen presenting cells capable of presenting the tumor antigen peptide on the cell surface; or, The antigen presenting cells prepared by the method. 6 . An effector T cell inducer, comprising the tumor antigen peptide according to claim 1 or the multi-epitope peptide according to claim 2 or the antigen presenting cell according to claim 5 .

7. A method for preparing effector T cells, comprising the following steps (B1) or (B2): (B1) stimulating HLA-A*24:02 molecule-positive PBMCs in vitro with the tumor antigen peptide of claim 1 or the multi-epitope peptide of claim 2, and obtaining effector T cells from the stimulated cells; (B2) stimulating T cells in vitro with the antigen presenting cells described in claim 5 to obtain effector T cells; or, The effector T cells prepared by the method.

8. A drug, the active ingredient of which comprises the tumor antigen peptide according to claim 1 or the multi-epitope peptide according to claim 2 or the biomaterial according to claim 3 or the complex according to claim 4 or the antigen presenting cell according to claim 5 or the effector T cell according to claim 7.

9. A detection reagent for detecting the effector T cells according to claim 7, comprising the complex according to claim 4.

10. Any of the following applications: (C1) Use of HLA-A*24:02 molecule-positive cells in the preparation of a product for detecting the tumor antigen peptide according to claim 1; (C2) Use of the tumor antigen peptide according to claim 1 in the preparation of a product for detecting cells positive for HLA-A*24:02 molecules; (C3) Use of the effector T cells of claim 7 in killing target cells in vitro; the target cells are cells presenting the tumor antigen peptides of claim 1 on their surfaces; (C4) Use of the tumor antigen peptide according to claim 1, the multi-epitope peptide according to claim 2, the biomaterial according to claim 3, the complex according to claim 4, the antigen presenting cell according to claim 5, or the effector T cell according to claim 7 in the preparation of a drug for killing target cells; the target cell is a cell presenting the tumor antigen peptide according to claim 1 on its surface; (C5) Use of the tumor antigen peptide according to claim 1, the multi-epitope peptide according to claim 2, the biomaterial according to claim 3, the complex according to claim 4, the antigen presenting cell according to claim 5, or the effector T cell according to claim 7 in the preparation of a drug for treating and / or preventing renal cancer; (C6) Use of the complex described in claim 4 in detecting the effector T cells described in claim 7.

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