Hla-a*11:01 short peptides derived from long non-coding rna slc12a5-as1 and use thereof

Abstract

The application discloses an HLA-A*11:01 renal cell carcinoma tumor antigen short peptide derived from a long-chain non-coding RNA SLC12A5-AS1 and application thereof. The application provides a tumor antigen peptide, and an amino acid sequence of the tumor antigen peptide is shown as SEQ ID No. 1. It is found by the application that a 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 invention relates to the field of biotechnology, and in particular to an HLA-A*11:01 renal cell carcinoma tumor antigen short peptide derived from long-chain non-coding RNA SLC12A5-AS1 and applications thereof. Background Art

[0002] In the past five years, there have been a total of 1.2 million newly reported cases of kidney cancer worldwide, and the global incidence is increasing. In 2024, it is estimated that there will be more than 400,000 newly reported cases and more than 175,000 deaths, of which approximately 25% of newly reported patients will have metastatic disease at the time of diagnosis. In addition, 30% of kidney cancer patients develop distant metastases after radical nephrectomy, and the 5-year survival rate of these patients is only 10-18%. Currently, the common treatment for renal cell carcinoma is surgical resection. When the tumor is too large or metastatic, immune checkpoint and targeted combination therapy is usually used, but the treatment effect is limited by multiple factors such as the patient's immune microenvironment. Therefore, it is very necessary to develop new drugs to treat kidney cancer.

[0003] Long non-coding RNA (lncRNA) is a type of non-coding RNA consisting of more than 200 bases. Traditional theories posit that lncRNAs lack coding capacity and primarily play a role in epigenetic regulation, RNA transcription, and post-transcriptional regulation. However, recent evidence has confirmed that lncRNAs are not completely devoid of amino acid coding capacity. While lncRNAs lack start codons and cannot encode complete macromolecular proteins, they can encode short amino acid peptides through small open reading frames (sORFs). Peptides encoded by lncRNAs also play a crucial role in tumor biology. For example, certain tumor-specific lncRNA peptides can serve as tumor antigens for T cell recognition and induce immune responses. Currently, only approximately 1% of the genome is marked as protein-coding, yet 75% of the genome is transcribed and, theoretically, translated. Research on non-coding antigens, represented by lncRNAs, can enhance the potential for identifying tumor-specific and patient-shared antigens in tumors.

[0004] For tumors, T cells can only exert their 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 finally presented on 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 present invention aims to provide an HLA-A*11:01 renal cell carcinoma tumor antigen short peptide derived from long-chain non-coding RNA SLC12A5-AS1 and its application.

[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 SLC12A5-AS1.

[0008] In the second aspect, the present invention claims protection for a polyepitope 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 treating renal cell carcinoma.

[0012] In a third aspect, the present invention claims protection for a biomaterial 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 or the multi-epitope peptide described in the second aspect;

[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. Furthermore, 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 and the HLA-A*11:01 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 comprise 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*11:01-positive cells in vitro to obtain antigen-presenting cells capable of presenting the tumor antigen peptide on the cell surface. Loading the tumor antigen peptide described in the first aspect or the multi-epitope peptide described in the second aspect onto HLA-A*11:01-positive cells may be accomplished by co-incubating the tumor antigen peptide or the multi-epitope peptide with the HLA-A*11:01-positive cells.

[0022] In one embodiment of the present invention, the tumor antigen peptide described in the first aspect above is co-incubated with T2-HLA-A*11:01 cells (cultured in a cell culture incubator at 37° C. for 12 hours).

[0023] 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*11:01 molecule), or the level of the cell presenting the complex (the complex formed by the tumor antigen peptide and the HLA-A*11:01 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, to further induce the presentation of the peptide.

[0024] In a sixth aspect, the present invention claims protection for antigen-presenting cells prepared using the method described in the fifth aspect above.

[0025] In a seventh aspect, the present invention claims an effector T cell inducer.

[0026] 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.

[0027] In an eighth aspect, the present invention claims a method for preparing effector T cells.

[0028] The method for preparing effector T cells claimed in the present invention may include the following steps (B1) or (B2):

[0029] (B1) stimulating HLA-A*11:01-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;

[0030] (B2) Stimulating T cells in vitro with the antigen-presenting cells described in the sixth aspect to obtain effector T cells.

[0031] 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 HLA-A*11:01 molecule-positive PBMCs, 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 to obtain effector T cells.

[0032] In (B2), since the HLA-A*11:01 molecules on the antigen-presenting cells used bind to the tumor antigen peptide, when they are co-cultured with T cells, the HLA-A*11:01 molecules, the tumor antigen peptide and the TCR on the T cells form a ternary complex, thereby activating the T cells to obtain effector T cells.

[0033] Furthermore, the method further comprises the step of adding IL-2, IL-7 and IL-5 to the stimulation system.

[0034] In one embodiment of the present invention, the effector T cells are activated CD8 + T cells.

[0035] In a ninth aspect, the present invention claims protection for effector T cells prepared using the method described in the eighth aspect above.

[0036] In a tenth aspect, the present invention claims protection for a drug.

[0037] 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 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.

[0038] In the eleventh aspect, the present invention claims a detection reagent for detecting the effector T cells described in the ninth aspect above.

[0039] The detection reagent for detecting the effector T cells claimed in the present invention includes the complex described in the fourth aspect above.

[0040] In a twelfth aspect, the present invention claims protection for any of the following applications:

[0041] (C1) Use of HLA-A*11:01 molecule-positive cells in the preparation of a product for detecting the tumor antigen peptide described in the first aspect above.

[0042] (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*11:01 molecule.

[0043] In one embodiment of the present invention, the HLA-A*11:01 molecule-positive cells are T2 cells expressing HLA-A*11:01 molecules.

[0044] (C3) Use of the effector T cells described in the ninth aspect above in killing target cells in vitro; the target cells are cells (HLA-A*11:01 molecule positive) whose surface presents the tumor antigen peptides described in the first aspect above.

[0045] (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 cell is a cell (HLA-A*11:01 molecule positive) presenting the tumor antigen peptide described in the first aspect above on its surface.

[0046] The target cells may be all HLA-A*11:01 positive tumor cells, such as renal cancer cells.

[0047] In one embodiment of the present invention, the target cells are T2 cells expressing HLA-A*11:01 molecules that are stimulated by the tumor antigen peptide described in the first aspect above.

[0048] (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.

[0049] (C6) Use of the complex described in the fourth aspect above in detecting the effector T cells described in the ninth aspect above.

[0050] The present invention relates to a newly discovered antigenic peptide (RVLVPGLRR) derived from a long noncoding RNA in renal cell carcinoma tissue. The complex formed by this peptide and the corresponding HLA molecule (HLA-A*11:01) can be recognized by T cells, activating an immune response. This invention provides a potential target for the treatment of renal cell carcinoma and offers a new target screening direction for subsequent tumor antigen therapy for renal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The following is a representative mass spectrum of the short peptide of the present invention identified by mass spectrometry.

[0052] Figure 2Figure 1 is a T2 cell presentation diagram of the lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) 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) of the present application. 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 diagram of the lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) and the corresponding HLA molecule (HLA-A*11:01) and irrelevant HLA molecule (HLA-A*24:02) of the present application. 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 T2 cells to polypeptide, and the higher the value, the stronger the affinity of the polypeptide to the corresponding HLA. In the figure, ** indicates P<0.01, extremely significant difference.

[0053] Figure 3 Figure 2 is the detection of CD137 activation after co-culturing the lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) and the corresponding HLA-A*11:01 patient PBMC of the present application. In the figure, *** indicates P<0.001, extremely significant difference.

[0054] Figure 4 Figure 3 is the detection of IFNγ ELISPOT secretion after co-culturing the lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) and the corresponding HLA-A*11:01 patient PBMC of the present application. In the figure, *** indicates P<0.001, extremely significant difference.

[0055] Figure 5 Figure 4 is the killing detection after co-culturing the lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) and the corresponding HLA-A*11:01 patient PBMC and the polypeptide-presenting T2 cells of the present application. In the figure, *** indicates P<0.001, extremely significant difference; ** indicates P<0.01, extremely significant difference. DETAILED DESCRIPTION

[0056] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0057] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0058] Example 1: Human long non-coding RNA polypeptide search

[0059] (1) After resection, surgical tissue (including tumor tissue and adjacent tissue at the lesion site of renal cell carcinoma patients) should be quickly preserved in sample preservation fluid, transported to the laboratory via cold chain within 2 hours, and weighed and recorded.

[0060] The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

[0061] Table 1. Sample storage solution (100 mL)

[0062]

[0063]

[0064] After the sample preservation solution is prepared, aliquot it into 15 mL centrifuge tubes, 5 mL per tube. The aliquots can be stored at 4°C for 1 month.

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

[0066] (3) The renal cancer and adjacent tissues were divided into 1.5 mL centrifuge tubes and minced in the tubes. 500 μL of lysis buffer was added to each centrifuge tube, and the tissues were ground in a cryogenic tissue grinder for 2 min, followed by tissue lysis.

[0067] (4) Centrifuge the lysed sample at 12,000 g for 30 min at 4°C and collect the supernatant. Filter the supernatant using a 0.45 μm filter to remove impurities. Place the filtered supernatant in a clean 15 mL centrifuge tube for later use.

[0068] (5) Anti-HLA-I antibody (Proteintech, 15240-1-AP) and protein-A beads (Invitrogen, 101041) were added to the tissue lysate for immunoprecipitation experiments 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 SLC12A5-AS1 in renal cancer tissues encoded a small peptide sequence (SEQ ID No. 1: RVLVPGLRR). 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 human lymphocyte hybridoma cells that lack antigen peptide transporters, preventing endogenous antigens from being localized to the endoplasmic reticulum and Golgi apparatus and, therefore, from being degraded into antigenic peptides. Therefore, by expressing target HLA proteins in T2 cells, an antigen-presenting cell line corresponding to the HLA can be obtained. By co-culturing the target peptide with the cell line, the peptide can bind to the T2 cell surface. 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 a product of the American Type Culture Collection (ATCC), with the serial number CRL-1992.

[0079] The specific construction methods of the T2-HLA-A*11:01 cell line and the T2-HLA-A*24:02 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) (https: / / www.ncbi.nlm.nih.gov / ). 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 vector sequence) between the HindIII and NotI restriction sites to obtain the pcDNA3.1-HLA-A*11:01-GFP plasmid and pcDNA3.1-HLA-A*24:02-GFP plasmid, respectively. 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. The expression of HLA-A*11:01 protein in the T2-WT cell line (wild-type T2 cell line) and 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 middle A. HLA-A*11:01 and HLA-A*24:02 proteins are highly expressed in T2 cells, with the protein size being the indicated 40 kDa, demonstrating successful expression in the cell line.

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

[0081] (1) The culture medium for the T2 cell line consisted of RPMI1640 medium + 10% (volume percentage) FBS + 1× penicillin-streptomycin. The lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) used in the present invention was synthesized using the Merrifield synthesis method (also known as solid-phase peptide synthesis).

[0082] (2) Based on the bioinformatics prediction function of NetMHCpan (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ), the affinity of lncRNA SLC12A5-AS1 peptide was predicted, and it was determined that lncRNA SLC12A5-AS1 peptide binds to HLA-A*11:01 subtype.

[0083] (3) Use RPMI1640 complete medium to adjust the density of T2-HLA-A*11:01 cell line and T2-HLA-A*24:02 cell line to 1×10 6 100 μL / well of the low-adsorption U-shaped 96-well plate was added to each well. 25 μM tumor antigen (SEQ ID No. 1: RVLVPGLRR) was added to each well and cultured 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 of 3% FBS-PBS (PBS buffer containing 3% fetal bovine serum) to resuspend the cells. Add 1 μL of HLA-I flow cytometry antibody (biolegend, 311410) to each tube and incubate at 4°C in the dark for 30 min. HLA-I flow cytometry antibody can bind to HLA molecules presenting peptides on the surface of T2 cells. The more peptides presented on the cell surface, the stronger the HLA-I antibody signal.

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

[0086] (6) Add 250 μL of 3% FBS-PBS to resuspend the cells, perform flow cytometry analysis, and detect the fluorescence intensity of HLA-I antibodies. When the lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) has a high affinity with 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 of HLA-I protein on the surface of the T2 cell line increases. The results are shown in Figure 2. Figure 2 As shown in B. The results showed that T2-HLA-A*11:01 could present a large amount of lncRNASLC12A5-AS1 polypeptide (SEQ ID No.1: RVLVPGLRR), 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 with HLA-matched peripheral blood lymphocytes

[0088] In this example, the immune response to lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) was detected using peripheral blood mononuclear cells (PBMCs) from patients with renal cancer who were HLA-A*11:01 positive. (157-165) The polypeptide (SEQ ID No. 2: SLLMWITQC) is an unrelated polypeptide group. The unrelated polypeptide is derived from a 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, cryopreserved PBMCs were thawed in a 37°C water bath and resuspended in complete culture medium. The complete culture medium includes: AIM-V medium (Cat. No. 31035025, Invitrogen), 10% (volume percentage) heat-inactivated FBS, 1× penicillin-streptomycin, 20 U / mL IL-2, 10 ng / mL IL-7, 10 g / mL IL-15. The cells were diluted 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] Day 1: Add the PBMC-specific lncRNA SLC12A5-AS1 peptide (SEQ ID No. 1: RVLVPGLRR) and an unrelated peptide to the cells prepared in step 1 at a final concentration of 25 μM per well in triplicate. A negative control was also established by adding an equal amount of DMSO solution containing the peptide. The cells were then cultured uniformly at 37°C 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 (formula is the same as step 1) and a final concentration of 25 μM polypeptide, mix thoroughly by pipetting, and continue to culture uniformly 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 (formula is the same as step 1) and a final concentration of 25 μM polypeptide, mix thoroughly 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 (excluding 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 to subsequent operations.

[0094] 6. Flow cytometry detection of 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 antigen-bearing cells. A member of the tumor necrosis factor receptor superfamily, CD137 is expressed in antigen-activated T cells and has a co-stimulatory function, upregulating survival-related genes and enhancing 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 of 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, and 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 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 SLC12A5-AS1 peptide (SEQ ID No.1: RVLVPGLRR) stimulated the peripheral blood mononuclear cells of HLA-A*11:01 molecule-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 PBMCs by ELISpot

[0104] The enzyme-linked immunospot (ELISpot) assay can detect IFN-γ secretion by single activated T cells. IFN-γ ELISPOT is a reliable method for evaluating T cell immune responses to tumor antigens. It is highly sensitive and can detect IFN-γ secretion by single cells, thereby assessing T cell activation levels. IFN-γ, a cytokine secreted by immune-competent cells, plays a major role in inducing antiviral immunity and is primarily secreted by CD8 cells that recognize antigens. The level of IFN-γ secretion represents the level of T cell activation following antigen recognition.

[0105] The ELIspot kit is a product of Dakota 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) Add 200 μL of serum-free culture medium to each experimental well using a dispenser to activate the pre-coated plate. Let it stand at room temperature for 10 minutes and then remove it.

[0108] (2) The cells in static culture were divided into 1×10 5Add 10 μL PMA stimulator to the experimental wells, and add 10 μL PMA stimulator to the positive control wells. Then cover the plate, and place it in a 37°C, 5% CO2 incubator for 24 hours.

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

[0110] (4) Discard the liquid, and add 200 μL of 1x Washing buffer working solution (provided in the kit) to each well. After standing for 1 minute, discard the liquid, and repeat the operation 6 times.

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

[0112] (6) Discard the liquid, and add 200 μL of 1x Washing buffer working solution (provided in the kit) to each well. After standing for 1 minute, discard the liquid, and repeat the operation 6 times.

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

[0114] (8) Discard the liquid, and add 200 μL of 1x Washing buffer working solution (provided in the kit) to each well. After standing for 1 minute, discard the liquid, and repeat the operation 6 times, and finally ensure that the liquid in the wells is completely discarded.

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

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

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

[0118] The results are as follows Figure 4Compared with the DMSO control group and the irrelevant peptide stimulation group, the results showed that lncRNA SLC12A5-AS1 peptide (SEQ ID No. 1: RVLVPGLRR) 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 cell membrane lipid bilayer. In the early stages of apoptosis, the phosphatidylserine in the membrane flips from the inner side to the outer side. Annexin V, a phospholipid-binding protein, has a high affinity for phosphatidylserine. It binds to the cell membrane of cells in the early stages of apoptosis through the exposed phosphatidylserine on the outer side of the cell. Therefore, Annexin V is a sensitive indicator for detecting early apoptosis.

[0121] (1) PBMC cells were obtained from volunteers with positive HLA-A*11:01 molecules. After stimulating the PBMCs with the lncRNA SLC12A5-AS1 polypeptide (SEQ ID No. 1: RVLVPGLRR) according to the protocol in Example 3, all cells were collected and used as effector cells (mainly T cells activated by the lncRNA SLC12A5-AS1 polypeptide of the present invention). The control group was cultured for 10 days without antigen stimulation (unactivated T cell group). Since the unactivated T cell group did not have specific antigens to activate T cells, the T cells were still in a resting state and could be used as a negative control for T cells.

[0122] (2) T2-HLA-A*11:01 cells (see Example 2) were pulsed with peptides according to the protocol in Example 2, so that the lncRNA SLC12A5-AS1 peptide (SEQ ID No. 1: RVLVPGLRR) was presented on the surface of the T2-HLA-A*11:01 cells, and the cells were collected as target cells. Since the surface of T2-HLA-A*11:01 cells after peptide pulses can present the lncRNA SLC12A5-AS1 peptide (SEQ ID No. 1: RVLVPGLRR), and T2-HLA-A*11:01 cells are essentially tumor cells, T2 cells can be used as both antigen-presenting cells and target cells for T cells to recognize antigens in antigen experiments.

[0123] (3) Effector cells and target cells were co-cultured at a ratio of 10:1. At 0 h, 24 h, and 48 h, the viability of T2-HLA-A*11:01 cells was detected. A control group (T2-HLA-A*11:01 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, and 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 conjugate solution in the kit to resuspend the cells, ensuring a consistent volume. 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 min.

[0126] (6) Flow cytometry was used to detect the proportion of Annexin V in T2 cells and to calculate the proportion of Annexin V-negative tumor cells, i.e., the viability of tumor cells (T2-HLA-A*11:01 cells).

[0127] The results are as follows Figure 5 The results show that T cells activated by stimulating HLA-A*11:01-positive PBMCs with lncRNA SLC12A5-AS1 polypeptide (SEQ ID No.1: RVLVPGLRR) can recognize T2-HLA-A*11:01 cells presenting lncRNA SLC12A5-AS1 polypeptide (SEQ ID No.1: RVLVPGLRR) and effectively kill T2-HLA-A*11:01 cells.

[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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using 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 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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