OBSCN new antigen epitope peptide and its application in preparing drugs for preventing and treating tumors

Abstract

The present invention belongs to the field of biomedicine technology, and specifically discloses an OBSCN new antigen epitope peptide and its application in the preparation of a drug for preventing and / or treating tumors. The amino acid sequence of the OBSCN new antigen epitope peptide provided by the present invention is shown in SEQ ID NO: 1-4, and the epitope peptide can effectively stimulate and induce the production of a mutant epitope peptide-specific CD8 with tumor killing function. + T lymphocytes specifically kill tumor cells that positively express new antigens. In addition, the patient coverage rate of the antigen epitopes contained in the epitope peptide is high, and it has good therapeutic potential and application prospects to make it into drugs (such as vaccines, cell preparations, etc.) for preventing and / or treating tumors. The drug provided by the present invention contains the OBSCN new antigen epitope peptide or its encoding nucleic acid, or a T cell antigen receptor or chimeric antigen receptor or its encoding nucleic acid that specifically recognizes the OBSCN new antigen epitope, and has a high clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an OBSCN new antigen epitope peptide and an application thereof in preparing a drug for preventing and / or treating tumors. Background Art

[0002] Tumor immunotherapy is a new generation of tumor treatment methods that has developed rapidly after traditional treatment methods such as surgery, chemotherapy, and radiotherapy, and has great clinical application prospects. Tumor immunotherapy mainly regulates the body's immune system to activate or restart the body's anti-tumor function, thereby achieving the purpose of killing tumors.

[0003] Studies have shown that the clinical efficacy of tumor immunotherapy depends largely on the presence of tumor antigen-specific CD8 + T lymphocytes, and tumor antigens play an important role in them. The ideal tumor antigen is a tumor-specific antigen that is expressed only in tumor cells and not in normal cells. Neoantigens are a type of tumor-specific antigen produced by tumor-specific mutations. Because they are not expressed in normal cells, they have high immunogenicity and can activate CD4 + T lymphocytes and CD8 + T lymphocytes produce a strong immune response and are considered one of the most ideal targets for tumor immunotherapy. Epitopes in new antigens that can specifically activate T lymphocytes are also called new epitopes, which have shown unique advantages in tumor immunotherapy.

[0004] Studies have found that OBSCN gene mutations often occur in various cancers, especially breast cancer. OBSCN plays an important role in cancer susceptibility and tumor progression, and its functional mutations may affect the Wnt signaling pathway through β-catenin. It is worth noting that among more than 13,000 candidate genes, OBSCN and TP53 are co-mutated in breast cancer and colorectal cancer. There have been many studies on TP53, but the mechanism of OBSCN in tumorigenesis is still unclear. In normal breast epithelial cells, OBSCN may have the effect of inhibiting tumor formation and metastasis.

[0005] Chinese patent CN116970058B discloses a new antigen polypeptide based on MHC-II class molecules targeting the R249S mutation on the tumor driver gene TP53, whose amino acid sequence is MGGMNRSPILTIITL or SSCMGGMNRSPILTI. The former can activate T cells with HLA typing HLA-DRB1*07:01, and the latter can activate T cells with HLA typing HLA-DQA1*02:01 / DQB1*03:01, and enhance the killing ability of T cells against tumor cells with TP53R249S mutation, and can be used to prepare drugs for preventing and / or treating tumors. In addition, Chinese patent CN116948004B discloses a new antigen polypeptide targeting the H36P mutation on the tumor driver gene CTNNB1, whose amino acid sequence is QSYLDSGIPSGATTT or PSGATTTAPSLSGKG, both of which can activate T cells with HLA typing HLA-DQA1*02:01 / DQB1*03:01, and enhance the killing ability of T cells against tumor cells with CTNNB1 H36P mutation, filling the gap of personalized antigen peptides in the treatment of tumor patients with both HLA-DQA1*02:01 / DQB1*03:01 genotype and CTNNB1-H36P somatic mutation.

[0006] However, the vast majority of neoantigens identified so far are specific to tumor patients, with low population coverage, which seriously restricts the clinical application of neo-epitope-based tumor immunotherapy. HLA-A2 subtype accounts for about 50% of the Chinese population, so the identification of HLA-A2-restricted neoantigen epitopes has a wide population coverage and can provide new candidates and strategies for neoantigen-based tumor immunotherapy. Summary of the invention

[0007] The main technical problem solved by the present invention is to provide a new OBSCN antigen epitope, and the use of a polypeptide containing the new antigen epitope or its encoding nucleic acid in the preparation of a drug for preventing and / or treating tumors.

[0008] At the same time, the present invention also provides a medicine for preventing and / or treating tumors.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] OBSCN neoantigen epitope peptide, wherein the neoantigen epitope peptide comprises an OBSCNV327L mutation or an A1348V mutation.

[0011] As a preferred embodiment of the present invention, the new antigen epitope peptide comprises an amino acid sequence as shown in SEQ ID NO: 1 or 2.

[0012] OBSCNV327L(5aa):LV[L]VR(SEQ ID NO:1);

[0013] OBSCNA1348V(5aa): SEPK[V] (SEQ ID NO: 2).

[0014] As a preferred embodiment of the present invention, the amino acid sequence of the new antigen epitope peptide is shown in SEQ ID NO: 3 or 4:

[0015] OBSCNV327L(10aa):VLV[L]VREPAV(SEQ ID NO:3);

[0016] OBSCNA1348V(10aa): FHLDVSEPK[V] (SEQ ID NO: 4).

[0017] The nucleic acid encoding the new OBSCN antigen epitope peptide is DNA or RNA.

[0018] As a preferred embodiment of the present invention, the nucleic acid sequence can be modified according to the codon preference without changing the encoded amino acid sequence.

[0019] The application of the OBSCN neoantigen epitope peptide or its encoding nucleic acid is specifically: application in the preparation of drugs for preventing and / or treating tumors.

[0020] As a preferred embodiment of the present invention, the medicine includes but is not limited to vaccines and cell preparations.

[0021] Specifically, the drug is a vaccine that can induce the body to produce specific cytotoxic T cell clones for tumors with OBSCN V327L mutation or A1348V mutation. The vaccine can be a nucleic acid vaccine, which contains a nucleic acid sequence encoding an OBSCN new antigen epitope peptide, such as a DNA vaccine or an RNA vaccine. Alternatively, the vaccine is a recombinant protein vaccine, a synthetic peptide vaccine, a mixed peptide pool vaccine, etc., which contains an OBSCN new antigen epitope peptide sequence. Alternatively, the vaccine is a cell vaccine, which contains antigen presenting cells (such as dendritic cells), and the antigen presenting cells contain the OBSCN new antigen epitope peptide sequence.

[0022] Specifically, the drug is a cell preparation that can prevent and / or treat tumors that express positive OBSCNV327L mutation or A1348V mutation. The cell preparation comprises modified cells, including but not limited to T cells, NK cells, CIK cells, and tumor infiltrating lymphocytes that contain T cell antigen receptors (TCR) or chimeric antigen receptors (CAR) or nucleic acids encoding them, and the T cell antigen receptors or chimeric antigen receptors can specifically recognize the new antigen epitopes of OBSCN.

[0023] As a preferred embodiment of the present invention, the OBSCN new antigen epitope peptide can be prepared by chemical synthesis, including but not limited to solid phase synthesis, liquid phase synthesis, solid-liquid phase synthesis. Synthesis strategies include C-terminal synthesis, N-terminal synthesis, segmented synthesis, etc., and conventional modifications can be performed at the C-terminal or N-terminal to include a modification group.

[0024] As a preferred embodiment of the present invention, the tumor is a tumor that positively expresses the OBSCN neoantigen, including a malignant tumor.

[0025] Specifically, the tumor includes but is not limited to endometrial cancer, hepatocellular carcinoma, colon adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, lung adenocarcinoma, esophageal cancer, breast cancer, low-grade brain glioma, renal papillary cell carcinoma, intranodal parisha myofibroblastoma, neuromuscular aberrant tumor, desmoid tumor, juvenile angiofibroma, enamel-type craniopharyngiocarcinoma, hepatoblastoma, pancreatic ductal carcinoma, biliary tract cancer, cerebral hemangioma, skin Merkel cell carcinoma, skin squamous cell carcinoma, melanoma in situ, and skin basal cell carcinoma.

[0026] A drug for preventing and / or treating tumors, comprising one or more of the following:

[0027] (1) OBSCN new antigen epitope peptide sequence;

[0028] (2) Nucleic acid sequences encoding new OBSCN antigen epitope peptides;

[0029] (3) A T cell antigen receptor or chimeric antigen receptor that specifically recognizes the neoantigen epitope of OBSCN or a nucleic acid encoding the same.

[0030] As a preferred embodiment of the present invention, the drug is a biological product, including a preventive or therapeutic biological product, such as a vaccine, a cell preparation, and the like.

[0031] Specifically, the drug is a vaccine that can induce the body to produce specific cytotoxic T cell clones for tumors with OBSCN V327L mutation or A1348V mutation. The vaccine includes but is not limited to nucleic acid vaccines (such as DNA, RNA vaccines, which contain nucleic acid sequences encoding OBSCN new antigen epitope peptides), recombinant protein vaccines, synthetic peptide vaccines, mixed peptide pool vaccines (which contain OBSCN new antigen epitope peptide sequences), and cell vaccines (which contain antigen presenting cells, which contain OBSCN new antigen epitope peptide sequences).

[0032] Specifically, the drug is a cell preparation that can prevent and / or treat tumors that positively express OBSCNV327L mutation or A1348V mutation. The cell preparation comprises modified cells, including but not limited to T cells, NK cells, CIK cells, and tumor infiltrating lymphocytes that contain T cell antigen receptors or chimeric antigen receptors or nucleic acids encoding them, wherein the T cell antigen receptors or chimeric antigen receptors can specifically recognize the new antigen epitopes of OBSCN.

[0033] As a preferred embodiment of the present invention, when the drug is a vaccine, it may also include an immunomodulator or adjuvant, including but not limited to poly-ICLC, 1018ISS, Amplivax, MF59, AS03, AS04, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, cyclic dinucleotides (such as STING), dSLIM, GM-CSF, IL-2, IC30, IC31, Montanide ISA TM (such as Montanide ISA51).

[0034] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection, spray, and dispersion.

[0035] As a preferred embodiment of the present invention, the administration route of the drug is a pharmaceutically acceptable route, including but not limited to intradermal, subcutaneous, intramuscular, intravenous, and intranasal or oral inhalation.

[0036] As a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier or excipient, including but not limited to a solvent, a solubilizer, a preservative, a stabilizer, a wetting agent, an emulsifier, a salt for adjusting osmotic pressure, and a buffer.

[0037] As a preferred embodiment of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage.

[0038] Beneficial effects of the present invention:

[0039] The OBSCN new antigen epitope peptide provided by the present invention (amino acid sequence as shown in SEQ ID NO: 1-4) can effectively stimulate and induce the production of specific cytotoxic T lymphocytes to kill tumor cells that positively express the new antigen. Due to the high patient coverage of the antigen epitope, it has good therapeutic potential and application prospects when made into drugs (such as vaccines, cell preparations, etc.) for preventing and / or treating tumors.

[0040] The drug for preventing and / or treating tumors provided by the present invention comprises an OBSCN neoantigen epitope peptide or its encoding nucleic acid, or a T cell antigen receptor or a chimeric antigen receptor that specifically recognizes an OBSCN neoantigen epitope or its encoding nucleic acid, which can induce the production of a mutant epitope peptide-specific CD8 T cell receptor with tumor killing function in vitro and in vivo. + T lymphocytes have high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The experimental example shows the secretion of IFN-γ by T lymphocytes induced by mutant epitope peptides in healthy volunteers.

[0042] Figure 2 CD8 T lymphocytes secreting IFN-γ induced by mutant epitope peptides in healthy volunteers + The condition of T lymphocytes.

[0043] Figure 3 CD8 granzyme B secreting T lymphocytes induced by mutant epitope peptides in healthy volunteers + The condition of T lymphocytes.

[0044] Figure 4 CD8 secretion of IFN-γ in T lymphocytes induced by mutant epitope peptides in breast cancer patients + The condition of T lymphocytes.

[0045] Figure 5 CD8 secretion of granzyme B in T lymphocytes induced by mutant epitope peptides in breast cancer patients + The condition of T lymphocytes.

[0046] Figure 6 The figure shows the killing of target cells by T lymphocytes induced by mutant epitope peptides in healthy volunteers.

[0047] The significance analysis symbols in the figures are as follows: * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0048] In order to more clearly illustrate the technical solution to be protected by the present invention, the drawings in the embodiments or experimental examples are briefly introduced above. It should be understood that the above drawings should not be regarded as any limitation on the protection scope of the present invention. For ordinary technicians in this field, other related drawings can also be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0049] The technical solution of the present invention will be clearly and completely explained below in conjunction with specific embodiments and experimental examples.

[0050] Those skilled in the art should understand that the following embodiments and experimental examples are only used to illustrate the technical solutions and technical effects of the present invention and should not be regarded as any limitation on the protection scope of the present invention. Based on the following embodiments, other technical solutions obtained by those skilled in the art without creative work, such as technical solutions obtained after modification, deformation or simple replacement, all belong to the protection scope of the present invention.

[0051] Unless otherwise specified, the raw materials, reagents, and equipment used in the examples or experimental examples are all commercially available products.

[0052] Example 1

[0053] This embodiment provides a new OBSCN antigen epitope peptide, comprising the OBSCN V327L mutation, and its amino acid sequence is as follows:

[0054] OBSCNV327L(10aa): VLV[L]VREPAV (SEQ ID NO: 3).

[0055] This embodiment also provides a codon-optimized nucleic acid encoding the above-mentioned new OBSCN antigen epitope peptide.

[0056] Example 2

[0057] This embodiment provides an OBSCN neoantigen epitope peptide, comprising an OBSCNA1348V mutation, and its amino acid sequence is shown below:

[0058] OBSCNA1348V(10aa): FHLDVSEPK[V] (SEQ ID NO: 4).

[0059] This embodiment also provides a codon-optimized nucleic acid encoding the above-mentioned new OBSCN antigen epitope peptide.

[0060] Example 3

[0061] This example provides an application of an OBSCN neoantigen epitope peptide or its encoding nucleic acid (same as in Example 1) in the preparation of a drug for preventing and / or treating tumors.

[0062] This embodiment also provides a drug for preventing tumors, which is an mRNA vaccine, which contains an effective amount of mRNA delivered by lipid nanoparticles (LNP) (same as Example 1, and codon optimized), and appropriate amounts of adjuvants and auxiliary materials.

[0063] This embodiment also provides a drug for preventing tumors, which is a synthetic polypeptide vaccine, comprising a pharmaceutically effective amount of a new OBSCN antigen epitope peptide (as shown in SEQ ID NO: 3) prepared by Fmoc solid phase synthesis, and an appropriate amount of adjuvants and auxiliary materials.

[0064] Example 4

[0065] This example provides an application of an OBSCN neoantigen epitope peptide or its encoding nucleic acid (same as in Example 2) in the preparation of a drug for preventing and / or treating tumors.

[0066] This embodiment also provides a drug for preventing tumors, which is an mRNA vaccine, which contains an effective amount of mRNA delivered by lipid nanoparticles (LNP) (same as Example 2, and codon optimized), and appropriate amounts of adjuvants and auxiliary materials.

[0067] This embodiment also provides a drug for preventing tumors, which is a mixed peptide pool vaccine, which contains a pharmaceutically effective amount of a new OBSCN antigen epitope peptide (as shown in SEQ ID NO: 3 and 4) prepared by Fmoc solid phase synthesis, and an appropriate amount of adjuvants and auxiliary materials.

[0068] Example 5

[0069] This embodiment provides a drug for treating tumors, which is a cell preparation, comprising a pharmaceutically effective amount of CAR-T cells that can specifically recognize the OBSCN neoantigen epitope (the same as the antigen epitope contained in the OBSCN neoantigen epitope peptide in Example 2), and an appropriate amount of adjuvants and auxiliary materials.

[0070] Experimental Example 1

[0071] 1. Screening and synthesis of new antigen epitope peptides

[0072] (1) Download data from The Cancer Genome Atlas (TCGA) cohort

[0073] The mutational landscape of breast cancer was reviewed using somatic missense mutation data from The Cancer Genome Atlas (TCGA). At the same time, RNA sequencing data revealed the infiltration of immune cells in the tumor microenvironment. The comprehensive dataset, including mutation profiles, gene expression data, and related clinical information (all obtained from the TCGA data portal https: / / portal.gdc.com, as of October 3, 2021), covers data from 1,101 breast cancer patients and 113 paired adjacent normal breast tissue samples. The mutation data were downloaded and visualized using the maftools package in R software.

[0074] (2) Tumor-specific somatic mutations shared by breast cancer patients

[0075] The most common non-synonymous mutations were selected as the options in the “Genes with mutation sites” field, and the frequency of each mutation was calculated by the following formula: mutation frequency = number of samples with missense mutations / total number of samples.

[0076] (3) Prediction and synthesis of candidate antigen epitope peptides

[0077] Using different algorithms, 4 programs were used to predict potential antigenic epitopes: IEDB, NetMHC 4.0, NetCTL 1.2, and SYFPEITHI, with the goal of identifying peptides of 8-11 amino acids that have binding potential to the high-frequency human HLA-A*02 subtype, specifically A*0201, A*0203, A*0206, and A*6802. The prevalence of HLA-A2 monomers in the Chinese population is 37.7%, and HLA-A2-restricted CD8 + The CTL neoepitope was synthesized using standard laboratory techniques, purified and confirmed by electrospray mass spectrometry, and then dissolved in phosphate buffer at pH 7.2 to a final concentration of 10 mg / mL.

[0078] 2. Screening results of new antigen epitope peptides

[0079] (1) Gene mutation sites with higher frequency in breast cancer samples

[0080] Somatic missense mutations are considered to be the main origin of neoantigens (or neoepitopes) in cancer cells. Therefore, missense mutations that occur frequently in breast cancer were comprehensively calculated and analyzed and displayed using a 21-mer peptide containing a mutant amino acid at position 11.

[0081] The results showed that the frequently mutated sites were consistent with the hotspot mutations commonly reported in the existing literature. The identified missense mutations with a frequency estimated to be greater than 1% were added to the list of missense mutations, as shown in Table 1.

[0082] Table 1 High-frequency mutation sites and their mutation frequencies in breast cancer samples

[0083]

[0084]

[0085] (2) Screening results of potential new epitopes binding to HLA-A2 molecules

[0086] 8-10 amino acid peptides targeting high-frequency human HLA-A class I gene products were designed, with the target subtype being HLA-A*02 (A*0201, A*0203, A*6802, and A*0206), using four different algorithm programs for prediction, including IEDB, NetMHC4.0, NetCTL 1.2, and SYFPEITHI. The design process needs to consider factors such as proteasomal C-terminal cleavage, transport efficiency of the transporter associated with antigen processing (TAP), and the half-dissociation time of the peptide-HLA class I molecule. The results obtained in the epitope prediction analysis were ranked, with NetMHC 4.0 (IC50 < 500 nM) as the main tool, and supported by other programs, as shown in Table 2.

[0087] Table 2 Prediction scores of new epitopes at high-frequency mutation sites in breast cancer

[0088]

[0089] Experimental Example 2

[0090] 1. Binding affinity of the neoepitope / HLA-A2 molecule complex

[0091] The predicted neo-epitopes were synthesized by standard laboratory techniques and assessed for their binding affinity to HLA-A2 transgenic T2 (T2A2) cells.

[0092] The binding strength test protocol is as follows:

[0093] (1) The prepared candidate peptide was dissolved in PBS (pH 7.2) to a concentration of 1 mg / mL and stored at -80°C until use.

[0094] (2) Obtain T2 cells in good growth condition, centrifuge at 4°C, 2000 rpm for 10 min, wash twice with serum-free IMDM medium, and then adjust the density to 2×10 6 / mL, spread in 24-well plates, 1mL / well.

[0095] (3) Thaw the dissolved peptide in a 4°C refrigerator. After plating, add human β2-microglobulin (β2-M) (concentration 0.5 μg / mL) first, and then add the dissolved antigen peptide (50 μg / mL).

[0096] Design different groups: experimental group, negative control group (PBS), positive control group (COX-2 321-329 ) and background group (T2A2 cells) were incubated in a 37°C cell culture incubator for 18 h.

[0097] (4) After incubation for 18 h, the cells were collected in a 1.5 mL EP tube, centrifuged at 4°C and 2000 rpm for 10 min, and washed twice with pre-cooled PBA buffer (containing 2% calf serum and an appropriate amount of NaN3).

[0098] (5) After discarding the supernatant, incubate the HLA-A2 antibody at 4°C in the dark for 30 min, and then blow and wash to mix.

[0099] (6) After incubation, add 1 mL PBA buffer to wash, centrifuge at 4°C and 3000 rpm for 10 min, discard the supernatant, add 200 μL pre-cooled PBA buffer, and perform detection by flow cytometry.

[0100] (7) The calculation formula of fluorescence index FI is as follows: FI = (MFI of experimental group - MFI of background group) / MFI of background group. The FI value represents the binding ability of antigen epitope peptide to HLA-A2 molecule. The experimental results are shown in Table 3.

[0101] Table 3 Binding affinity of the screened epitope peptides to HLA-A2

[0102]

[0103] FI a =(MFI of the givenpeptide-MFI of the PBS control groupwithoutpeptides) / MFI of the PBS control

[0104] groupwithoutpeptides.

[0105] As can be seen from Table 3, the two MUT neo-epitopes from the OBSCNV327L and OBSCNA1348V mutation sites showed higher binding affinity (FI>1). The remaining neo-epitope peptides showed weaker binding affinity (FI<0.5, data not shown). Therefore, the OBSCNV327L and OBSCNA1348V mutation epitope peptides were selected for the next step of immunological activity verification.

[0106] 2. Induction of human peripheral blood mononuclear cells (PBMCs)

[0107] The experimental plan is as follows:

[0108] (1) HLA-A2 typing was performed on the peripheral blood of healthy people or breast cancer patients. 40 mL of peripheral blood was drawn from each of the identified HLA-A2 volunteers.

[0109] (2) The extracted peripheral blood was mixed and diluted with PBS (pH 7.2) at a ratio of 1:1. Take a 50-mL centrifuge tube, add 15 mL of separation solution, and then add 30 mL of diluted peripheral blood. Centrifuge at 2000 rpm for 30 min. The centrifuge tube can be divided into four layers from top to bottom: plasma layer, lymphocyte layer, separation solution layer, and red blood cell layer.

[0110] (3) Prepare another 40 mL centrifuge tube and add 30 mL of PBS (pH 7.2) to each tube. Carefully pipette the mononuclear cells from the "white film layer" into the centrifuge tube and centrifuge and wash twice: 2000 rpm, 20 min.

[0111] (4) Inducing CTL: HLA-A2 has been isolated by the above-mentioned density gradient centrifugation method + Peripheral blood mononuclear cells from healthy donors or colorectal cancer patients were resuspended in GT-T551 medium and the cell density was adjusted to 1-1.5×10 6 cells / mL, and plated in a 6-well plate at 5 mL / well, and placed in a 37°C, 5% CO2 cell culture incubator for 4 h. Then, non-adherent cells (peripheral blood lymphocytes) in the supernatant of the well plate were slowly aspirated, collected and frozen in liquid nitrogen at a freezing density of 1×10 7 cells / mL for subsequent incubation with mature autologous DCs.

[0112] The remaining wells were supplemented with IMDM medium containing fetal bovine serum, and 1000U / mL GM-CSF and 1000U / mL IL-4 cytokines were added to induce the adherent cells in the 6-well plates to differentiate into dendritic cells. Thereafter, half of the medium was replaced and the corresponding cytokines were supplemented every 2 days. On the 5th day, 100ng / mL LPS was added to induce DC maturation.

[0113] (5) Add 50 μg / mL of epitope peptide to the induced mature dendritic cells and incubate for 24 h. The DCs pulsed with epitope peptides and autologous peripheral blood lymphocytes (PBL) previously frozen in liquid nitrogen are cultured in a 6-well plate at a ratio of DC:PBL=1:10. The cells are cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 100 U / mL rhIL-2 and 10 ng / mL rhIL-7 are added. The medium is replaced by half every 2 days to supplement the corresponding cytokines. One week later, the autologous DCs are pulsed with epitope peptides again to restimulate the PBLs. After three rounds of stimulation, the induced cytotoxic T lymphocytes are collected for the next functional analysis.

[0114] 3. ELISPOT method to detect the ability of neo-epitope-specific CTL to secrete IFN-γ in vitro

[0115] The experimental plan is as follows:

[0116] (1) Activation of pre-coated plates: Take out the required ELISPOT strips, add 200 μL of serum-free IMDM medium to each well for blocking, let stand for 10 min, and then discard the medium.

[0117] (2) Cell plating: The above-induced CTLs were used as effector cells, and the peptide-loaded T2A2 were used as stimulator cells. The cell concentration was adjusted to 2×10 6 / mL, set the amount added to each well:

[0118] Experimental wells: add 50 μL effector cells and 50 μL stimulator cells to each well;

[0119] Negative control wells: add 100 μL of serum-free IMDM medium to each well;

[0120] Positive control wells: add 50 μL of effector cells and 10 μL of PHA to each well, and then add 40 μL of serum-free IMDM medium.

[0121] After plating, place the plate in an incubator and incubate at 37°C, 5% CO2 for 18 hours.

[0122] (3) Cell lysis: After incubation, pour off the medium in the wells, add 200 μL of sterile deionized water to each well, and lyse the cells at 4°C for 10 min. Pour off the liquid in the wells, add 200 μL of 1× Washing Buffer for washing, and wash six times, each time for 60 s. After washing, pat dry on absorbent paper.

[0123] (4) Add detection antibody and incubate: Add 100 μL of biotin-labeled antibody and incubate at 37°C for 1 h. After incubation, pour out the liquid in the wells and add 200 μL of 1× Washing Buffer for washing. Wash 6 times, each time for 60 s. After washing, pat dry on absorbent paper.

[0124] (5) Add enzyme-linked avidin and incubate: Add 100 μL of enzyme-linked avidin and incubate at 37°C for 1 h. After incubation, pour out the liquid in the wells and add 200 μL of 1× Washing Buffer to each well for washing. Wash 6 times, each time for 60 s. After washing, pat dry on absorbent paper.

[0125] (6) Color development: Add 100 μL of freshly prepared AEC color development solution and incubate at room temperature (25°C) in the dark for 30 min to develop the color. After color development is complete, pour out all the liquid in the wells and open the base of the plate. Wash with deionized water to stop color development. Place in a ventilated place and let dry at room temperature. Then use an ELISPOT image analyzer to count the number of spots in each well of the plate. The results are compared as shown in the figure below. Figure 1 shown.

[0126] from Figure 1 It can be seen that T lymphocytes induced by mutant epitope peptides in healthy volunteers can secrete more IFN-γ.

[0127] 4. Intracellular factor staining experiment

[0128] (1) Target cell preparation: Collect T2 cells and adjust the density to 2×10 6 / mL, spread into 24-well plates, add β2-M at 3μg / mL, add epitope peptide at 50μg / mL, and incubate at 37℃, 5% CO2 for 4h.

[0129] (2) Co-incubation of target cells with induced T cells: 500 μL of peptide-loaded T2 or transfected tumor cells and CTL were added to 1.5 mL EP tubes and co-incubated for 4 h at 37°C and 5% CO2. A control group, PBS group, CD3 single-positive group, CD8 single-positive group, isotype control group, positive control PHA group and experimental group were set up. After co-incubation, the blocker BFA was added to inhibit the secretion of intracellular factors by CTLs.

[0130] (3) Incubate with CD3 and CD8 antibodies, mix well and incubate at 4°C in the dark for 30 min, add 200 μL of fixation buffer to resuspend the cells, incubate at room temperature in the dark for 30 min, then add 200 μL of 1× membrane permeabilization agent, centrifuge at 1500 rpm for 3 min, and repeat the operation once.

[0131] (4) Incubation with intracellular factor antibodies: Add 100 μL of 1× membrane permeabilization agent (resuspend cells) and appropriate amounts of PE Mouse Anti-Human IFN-γ, granzyme B, and Perforin antibodies, incubate at 4°C in the dark for 30 min. After the antibody incubation, add FACS buffer and centrifuge at 1500 rpm for 3 min to wash once. Then add FACS buffer to resuspend cells and detect on a flow cytometer. The results are shown in Figure 2. Figure 2-5 shown.

[0132] from Figure 2 It can be seen that in healthy volunteers, the mutant epitope peptide-induced T lymphocytes had more IFN-γ-secreting CD8 + T lymphocytes.

[0133] from Figure 3 It can be seen that in healthy volunteers, the T lymphocytes induced by the mutant epitope peptides have more CD8 secreting granzyme B + T lymphocytes.

[0134] from Figure 4 It can be seen that in breast cancer patients, the T lymphocytes induced by the mutant epitope peptides have more IFN-γ-secreting CD8 + T lymphocytes.

[0135] from Figure 5 It can be seen that in breast cancer patients, the T lymphocytes induced by the mutant epitope peptide have more CD8 secreting granzyme B + T lymphocytes.

[0136] 5. Cytotoxic activity assay to detect the killing of target cells by in vitro induced CTLs

[0137] (1) Preparation of target cells and effector cells

[0138] The cells without or with peptide T2A2 were used as target cells and the cell density was adjusted to 1×10 5 / mL, 5000 / 50μL were added to each well, and different numbers and volumes of effector cells were added to form different effector-target ratios in the experimental groups, which were 12.5:1, 25:1, and 50:1, respectively. Serum-free IMDM medium was supplemented to a total volume of 100μL, and 3 replicates were set up for each group. At the same time, the following control groups were set up: target cell spontaneous release group, target cell maximum release group, effector cell spontaneous release group, background control group and total volume correction control group. Each control group also had 3 replicates, and the total volume of each well was 100μL.

[0139] (2) LDH detection

[0140] The 96-well cell culture plate was placed in a 37°C, 5% CO2 incubator for 4 hours. 45 minutes before the end, 10 μL of 10× lysis buffer was added to the target cell maximum release well and volume correction well. After the culture was completed, the culture plate was centrifuged at 1000 rpm for 10 minutes, taken out, and 50 μL of supernatant was gently pipetted into the corresponding well of another 96-well plate. After all the transfers were completed, 50 μL of lactate dehydrogenase substrate mixture was quickly added to each well, and the plates were incubated at room temperature and away from light for 30 minutes. After the incubation was completed, 50 μL of stop solution was added to each well, and the absorption OD value was detected at 490 nm on a microplate reader. The killing rate was calculated as follows: killing rate = (experimental group release - effector cell spontaneous release - target cell spontaneous release) / (target cell maximum release - target cell spontaneous release) × 100%. The results are shown in Figure 2. Figure 6 shown.

[0141] from Figure 6 It can be seen that the T lymphocytes induced by the mutant epitope peptides in healthy volunteers have better ability to kill target cells.

[0142] The above experimental results show that the OBSCN new antigen epitope peptide (amino acid sequence as shown in SEQ ID NO: 1-4) provided by the present invention can effectively stimulate and induce the production of specific cytotoxic T lymphocytes to kill tumor cells that positively express the new antigen. Due to the high coverage rate of patients with this antigen epitope, it has good therapeutic potential and application prospects to make it into a drug (such as a vaccine, cell preparation, etc.) for the prevention and / or treatment of tumors.

[0143] Although the technical solution of the present invention has been described in detail above with general descriptions, specific implementation methods and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the protection scope of the present invention. Simple deformation, modification or improvement based on the technical concept of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. OBSCN new antigen epitope peptide, characterized by: The amino acid sequence of the new antigen epitope peptide is shown in SEQ ID NO:

4.

2. A nucleic acid encoding the OBSCN neoantigen epitope peptide according to claim 1.

3. Use of the OBSCN neoantigen epitope peptide according to claim 1, or the nucleic acid encoding the OBSCN neoantigen epitope peptide according to claim 2 in the preparation of a drug for preventing and / or treating tumors; The tumor is breast cancer.

4. A drug for preventing and / or treating tumors, characterized in that: The drug comprises the OBSCN new antigen epitope peptide as claimed in claim 1.

5. The drug according to claim 4, characterized in that: The drug is a vaccine that can induce the body to produce specific cytotoxic T cell clones targeting tumors with OBSCNA1348V mutation.

6. The drug according to claim 4 or 5, characterized in that: The dosage form of the medicine is powder injection or injection solution.

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