Use of a neo-antigen esr1-derived ctl epitope peptide or coding nucleic acid in the preparation of a medicament

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses application of a new antigen ESR1-derived CTL epitope peptide or coding nucleic acid thereof in preparation of a medicine for treating tumors. The application identifies and obtains an HLA-A2-restricted CTL epitope peptide derived from the new antigen ESR1 through analysis of a COSMIC database, epitope prediction and in-vivo and in-vitro immune activity experiments. The mutant epitope peptide is derived from a high-frequency mutation of ESR1, can effectively stimulate and induce production of a new epitope-specific cytotoxic T lymphocyte, specifically distinguishes between a wild type and a mutant sequence, kills tumor cells expressing the mutant epitope, and has a good anti-tumor effect. The medicine for treating tumors prepared therefrom can contain the new antigen ESR1-derived CTL epitope peptide or the coding nucleic acid thereof, or contain a T cell receptor, a chimeric antigen receptor or the coding nucleic acid thereof that specifically recognize the mutant epitope, and has good treatment potential and a clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a CTL epitope peptide or its encoded nucleic acid derived from a neoantigen ESR1 shared / hotspot mutation in the preparation of a drug for treating tumors. Background Technology

[0002] Breast cancer is one of the leading causes of cancer death among women worldwide, and its incidence and mortality rates continue to rise annually. Estrogen receptor (ER)-positive breast cancer accounts for approximately 70% of all breast cancer cases. For ER+ breast cancer patients, endocrine therapy (anti-estrogen therapy) is the primary clinical treatment. However, endocrine therapy resistance is a major problem faced by ER+ breast cancer patients, ultimately leading to recurrence and metastasis.

[0003] Studies have shown that acquired somatic mutations in ESR1 are a key driver of endocrine therapy resistance in ER+ metastatic breast cancer patients, leading to persistent transcriptional activity and reduced sensitivity to endocrine therapy. These mutations are highly detrimental to metastatic breast cancer (MBC) and are present in up to 36% of patients with metastatic breast cancer.

[0004] ESR1 is the gene that encodes ER. ESR1 gene abnormalities typically include gene amplification, rearrangement, and mutation. Studies have shown that missense mutations in ESR1 are often associated with the progression of drug-resistant and metastatic breast cancer, and most mutations are concentrated in the ligand-binding domain (LBD) of ESR1.

[0005] In recent years, tumor immunotherapy has been considered one of the most promising treatment methods due to its ability to induce and activate tumor-specific immune cells in patients to recognize and kill tumors and establish immune memory. The clinical efficacy of tumor immunotherapy depends on tumor antigen-specific T lymphocytes in patients, and tumor antigens play a crucial role in this process. Ideally, tumor antigens are tumor-specific antigens expressed only in tumor cells and not in normal cells. Neoantigens are a class of tumor-specific antigens generated by specific mutations in tumor cells. Because these mutated antigens are acquired and not expressed in normal tissues, they possess strong immunogenicity and tumor specificity, and are considered one of the most ideal tumor antigens currently available. Epitopes among neoantigens that can specifically activate T lymphocytes, also known as neoepitaphs, have shown unique advantages in tumor immunotherapy.

[0006] However, the vast majority of neoantigens identified so far are patient-specific, resulting in low population coverage and severely limiting the clinical application of neoepitope-based tumor immunotherapy. Therefore, shared neoantigens with broad population coverage will be key to future clinical applications.

[0007] With the development of next-generation sequencing and bioinformatics technologies, mutation information from a large number of cancer patients has been included in online databases such as COSMIC and TIMER. Furthermore, prediction software combining different algorithms will facilitate the screening of neoantigens specific to shared / hotspot mutation sites originating from ESR1 and the prediction of mutation-specific epitope peptides. However, the accuracy of epitope peptide prediction is currently low, requiring validation through activity assays to be practically meaningful. In addition, the HLA-A2 subtype accounts for approximately 50% of the Chinese population. Therefore, identifying HLA-A2-restricted shared neoepitaxe peptides has broad population coverage and can provide new candidates and strategies for neoantigen-based tumor immunotherapy. Summary of the Invention

[0008] To overcome the problem of low population coverage for neoantigens, this invention analyzed the COSMIC online database and found that the ESR1 mutation rate in endocrine therapy-resistant samples was 76.06%. Analysis of ESR1 point mutations revealed that ESR1 mutations were mainly concentrated at four sites: 303, 380, 537, and 538, with some tumor samples exhibiting two or more mutation sites simultaneously. These four ESR1 mutation sites can cover more than 80% of drug-resistant mutation samples, demonstrating broad population coverage and making them ideal targets for immunotherapy.

[0009] The neoantigen epitope peptide provided by this invention is derived from a high-frequency mutation of ESR1. It can effectively stimulate and induce the production of neoepitaxytoplasmic-specific cytotoxic T lymphocyte clones, specifically distinguishing between wild-type and mutant sequences, and killing tumor cells expressing the mutant epitope, exhibiting good anti-tumor effects. Furthermore, this neoantigen epitope peptide has high patient coverage and good therapeutic potential and application prospects. Therefore, the main technical problem solved by this invention is to provide the application of a CTL epitope peptide derived from the neoantigen ESR1 or its encoded nucleic acid in the preparation of drugs for treating tumors.

[0010] In addition, the present invention also provides a drug for treating tumors.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0012] The use of a CTL epitope peptide derived from a neoantigen ESR1 or its encoded nucleic acid in the preparation of a medicament for treating tumors, wherein the amino acid sequence of the CTL epitope peptide derived from the neoantigen ESR1 is shown in any one or more of SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, 14.

[0013] This invention, through analysis of the COSMIC database and epitope prediction, obtained 14 antigenic peptides associated with the neoantigen ESR1 and hotspot mutations, whose amino acid sequences are shown below:

[0014] ESR1-K303R: R NSLALSLTADQMV (SEQ ID NO: 1);

[0015] ESR1-K303R: Arg -Asn-Ser-Leu-Ala-Leu-Ser-Leu-Thr-Ala-Asp-Gln-Met-Val.

[0016] ESR1-E380Q:LL Q CAWLEI (SEQ ID NO: 2);

[0017] ESR1-E380Q: Leu-Leu- Gln -Cys-Ala-Trp-Leu-Glu-Ile.

[0018] ESR1-E380Q(10aa): HLL Q CAWLEI (SEQ ID NO: 3);

[0019] ESR1-E380Q(10aa):His-Leu-Leu- Gln -Cys-Ala-Trp-Leu-Glu-Ile.

[0020] ESR1-Y537S: VPL S DLLLEM (SEQ ID NO: 4);

[0021] ESR1-Y537S: Val-Pro-Leu- Ser -Asp-Leu-Leu-Leu-Glu-Met.

[0022] ESR1-Y537S-D538G(1): VPL SG LLLEM (SEQ ID NO: 5);

[0023] ESR1-Y537S-D538G(1): Val-Pro-Leu- Ser - Gly -Leu-Leu-Leu-Glu-Met.

[0024] ESR1-Y537S-D538G(2): NVVPL SGLLL(SEQ ID NO:6);

[0025] ESR1-Y537S-D538G(2):Asn-Val-Val-Pro-Leu- Ser - Gly -Leu-Leu-Leu。

[0026] ESR1-Y537N:VPL N DLLLEM(SEQ ID NO:7);

[0027] ESR1-Y537N:Val-Pro-Leu- Asn -Asp-Leu-Leu-Leu-Glu-Met。

[0028] ESR1-Y537N-D538G(1):NVVPL NG LLL(SEQ ID NO:8);

[0029] ESR1-Y537N-D538G(1):Asn-Val-Val-Pro-Leu- Asn - Gly -Leu-Leu-Leu。

[0030] ESR1-Y537N-D538G(2):VPL NG LLLEM(SEQ ID NO:9);

[0031] ESR1-Y537N-D538G(2):Val-Pro-Leu- Asn - Gly -Leu-Leu-Leu-Glu-Met。

[0032] ESR1-Y537C:VPL C DLLLEM(SEQ ID NO:10);

[0033] ESR1-Y537C:Val-Pro-Leu- Cys -Asp-Leu-Leu-Leu-Glu-Met。

[0034] ESR1-Y537C-D538G(1):VPL CG LLLEM(SEQ ID NO:11);

[0035] ESR1-Y537C-D538G(1):Val-Pro-Leu- Cys - Gly-Leu-Leu-Leu-Glu-Met.

[0036] ESR1-Y537C-D538G(2): NVVPL CG LLL(SEQ ID NO: 12);

[0037] ESR1-Y537C-D538G(2):Asn-Val-Val-Pro-Leu- Cys - Gly -Leu-Leu-Leu.

[0038] ESR1-D538G(10aa): VPLY G LLLEM (SEQ ID NO: 13);

[0039] ESR1-D538G(10aa): Val-Pro-Leu-Tyr- Gly -Leu-Leu-Leu-Glu-Met.

[0040] ESR1-D538G: LY G LLLEML (SEQ ID NO: 14);

[0041] ESR1-D538G: Leu-Tyr- Gly -Leu-Leu-Leu-Glu-Met-Leu.

[0042] Among them, except for the mutant epitope peptides shown in SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, and 14, the other mutant peptides are not specific or have low immunogenicity.

[0043] Specifically, the nucleic acid encoding the CTL epitope peptide derived from the neoantigen ESR1 includes DNA or RNA. It can be modified according to codon preferences without altering the encoded amino acid sequence.

[0044] In a preferred embodiment of the present invention, the drug includes therapeutic tumor vaccines, cell preparations, etc.

[0045] Specifically, the drug is a therapeutic tumor vaccine or cell preparation that can induce the body to produce neoantigen-specific T cell clones. The therapeutic tumor vaccine can be a nucleic acid vaccine, containing a nucleic acid sequence encoding a CTL epitope peptide derived from the neoantigen ESR1, such as a DNA vaccine or an RNA vaccine; or, the therapeutic tumor vaccine can be a peptide vaccine, a recombinant protein vaccine, a synthetic long peptide vaccine, a peptide pool vaccine, or a vaccine containing an antigen conjugate, etc., wherein the peptide vaccine, recombinant protein vaccine, synthetic long peptide vaccine, etc., all contain the amino acid sequence of a CTL epitope peptide derived from the neoantigen ESR1. The cell preparation contains antigen-presenting cells (such as dendritic cells), which contain the amino acid sequence of a CTL epitope peptide derived from the neoantigen ESR1.

[0046] In a preferred embodiment of the present invention, when the drug is a therapeutic tumor vaccine, it may further include an immunomodulator or adjuvant.

[0047] Specifically, the immunomodulator or adjuvant is selected from poly-ICLC, 1018ISS, Amplivax, aluminum salts, PLGA microparticles, virions or other virus-like particles, β-glucan, SRL172, 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 One or more of the following: (such as Montanide ISA51).

[0048] In a preferred embodiment of the present invention, when the drug is a cell preparation, it may further include an affinity peptide that targets antigen-presenting cells (such as DC cells).

[0049] In the second scenario, the drug is a cell preparation that can treat tumors expressing the neoantigen ESR1 positively. The cell preparation comprises modified cells, such as T cells, NK cells, NK-T cells, and CD4+ cells transfected with nucleic acids encoding T cell receptors (TCRs) or chimeric antigen receptors (CARs). + T cells, CD8 + T cells, tumor-infiltrating lymphocytes (including tumor-infiltrating T lymphocytes), or T cells modified with T cell receptors or chimeric antigen receptors, NK cells, NK-T cells, CD4+ + T cells, CD8 + T cells, tumor-infiltrating lymphocytes, etc. The T cell receptor or chimeric antigen receptor can specifically recognize CTL epitope peptides derived from the neoantigen ESR1. This cell preparation can also be prepared using the aforementioned vaccine.

[0050] In a preferred embodiment of the present invention, the CTL epitope peptide derived from the neoantigen ESR1 can be prepared by chemical synthesis. The chemical synthesis methods include solid-phase synthesis, liquid-phase synthesis, and solid-liquid-phase synthesis. Synthetic strategies include C-terminal synthesis, N-terminal synthesis, and segmented synthesis, with conventional modifications performed at the C-terminus or N-terminus during synthesis to include modifying groups.

[0051] In a preferred embodiment of the present invention, the tumor is a tumor that positively expresses the neoantigen ESR1, including malignant tumors such as cancer.

[0052] Specifically, the tumors include breast cancer, endometrial cancer, hepatocellular carcinoma, thyroid cancer, cervical cancer, prostate cancer, etc.

[0053] A medicament for treating tumors, the medicament comprising a CTL epitope peptide derived from the neoantigen ESR1 or its encoded nucleic acid, or comprising a T cell receptor, chimeric antigen receptor or its encoded nucleic acid that specifically recognizes the CTL epitope peptide derived from the neoantigen ESR1, wherein the amino acid sequence of the CTL epitope peptide derived from the neoantigen ESR1 is shown as any one or more of SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, 14.

[0054] Specifically, the nucleic acid encoding the CTL epitope peptide derived from the neoantigen ESR1 includes DNA or RNA. It can be modified according to codon preferences without altering the encoded amino acid sequence.

[0055] In a preferred embodiment of the present invention, the drug is a therapeutic tumor vaccine, cell preparation, etc.

[0056] In the first scenario, the drug is a therapeutic tumor vaccine or cell preparation that can induce the body to produce neoantigen-specific T cell clones. As mentioned above, the therapeutic tumor vaccine includes nucleic acid vaccines (such as DNA vaccines, RNA vaccines), peptide vaccines, recombinant protein vaccines, synthetic long peptide vaccines, mixed peptide pool vaccines, vaccines containing antigen conjugates, etc. The cell preparation contains antigen-presenting cells (such as dendritic cells), and the antigen-presenting cells contain CTL epitope peptides derived from the neoantigen ESR1.

[0057] In a preferred embodiment of the present invention, when the drug is a therapeutic tumor vaccine, it may further include an immunomodulator or adjuvant.

[0058] Specifically, the immunomodulator or adjuvant is selected from poly-ICLC, 1018ISS, Amplivax, aluminum salts, PLGA microparticles, virions or other virus-like particles, β-glucan, SRL172, 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 One or more of the following: (such as Montanide ISA51).

[0059] In a preferred embodiment of the present invention, when the drug is a cell preparation, it may further include an affinity peptide that targets antigen-presenting cells (such as DC cells).

[0060] In the second scenario, the drug is a cell preparation that can treat tumors expressing the neoantigen ESR1 positively. As described above, the cell preparation comprises modified cells, such as modified T cells, NK cells, NK-T cells, and CD4+ cells. + T cells, CD8 + T cells, tumor-infiltrating lymphocytes, etc. The modified cells are cells transfected with nucleic acids encoding T cell receptors or chimeric antigen receptors, or cells modified with T cell receptors or chimeric antigen receptors, wherein the T cell receptors or chimeric antigen receptors specifically recognize CTL epitope peptides derived from the neoantigen ESR1. This cell preparation can also be prepared using the aforementioned vaccine.

[0061] In a preferred embodiment of the present invention, the drug can be administered via local administration.

[0062] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmacologically acceptable dosage form, including but not limited to tablets, granules, capsules, powders, pills, sprays, powders for injection, and injection solutions.

[0063] In a preferred embodiment of the present invention, to prepare the drug into a specific dosage form, the drug further comprises pharmaceutically acceptable excipients, including but not limited to coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH adjusters, flavoring agents, etc. The excipient components can be rationally selected based on pharmaceutical knowledge.

[0064] In a preferred embodiment of the present invention, the dosage of the drug is a therapeutically acceptable dosage.

[0065] The beneficial effects of this invention are:

[0066] This invention, through analysis of the COSMIC database, epitope prediction, and in vitro and in vivo immunomodulatory activity experiments, identified eight HLA-A2-restricted CTL epitope peptides derived from the neoantigen ESR1, with amino acid sequences shown in SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, and 14. These mutant epitope peptides originate from high-frequency mutations in ESR1 and can effectively stimulate and induce the production of neotope-specific cytotoxic T lymphocytes, specifically distinguishing between wild-type and mutant sequences, and specifically killing tumor cells expressing the mutant epitope, exhibiting good anti-tumor effects. These mutant epitope peptides have high patient coverage and are ideal targets for immunotherapy. Their preparation into anti-tumor drugs, such as tumor vaccines and cell preparations, has good therapeutic potential and broad application prospects.

[0067] The tumor treatment drug provided by this invention comprises a CTL epitope peptide derived from the neoantigen ESR1 or its encoded nucleic acid, or comprises a T-cell receptor, chimeric antigen receptor, or its encoded nucleic acid that specifically recognizes the mutated epitope peptide. This drug can induce the production of CD8 specific to the mutated peptide with tumor-killing function in vitro and in vivo. + T lymphocytes, which can accurately distinguish between wild-type and mutant types, have high clinical application value. Attached Figure Description

[0068] Figure 1 Figure 1. Intracellular factor staining results of T2A2 cells carrying mutant / wild-type epitope peptides as target cells, based on 14 mutant epitope peptides derived from the neoantigen ESR1.

[0069] Figure 2 Figure 1 shows the experimental results of the killing effect of specific CTLs induced in vitro by eight mutant epitope peptides derived from the neoantigen ESR1 on T2A2 cells loaded with mutant / wild-type epitope peptides.

[0070] Figure 3 Figure 1 shows the experimental results of the killing effect of specific CTLs induced in vitro by eight mutant epitope peptides derived from the neoantigen ESR1 on tumor cell line (MDA-MB-231-WT / MUT).

[0071] Figure 4 Eight mutant epitope peptides derived from the neoantigen ESR1 were used in a peptide pool in conjunction with CpG-ODN1826 to immunize HLA-A2.1 / K. b Figure 1. Results of intracellular factor staining and killing effects experiments of specific CTLs obtained from transgenic mice as target cells of T2A2 cells loaded with mutant / wild-type epitope peptide pools.

[0072] Figure 5Eight mutant epitope peptides derived from the neoantigen ESR1 were used in a peptide pool in conjunction with CpG-ODN1826 to immunize HLA-A2.1 / K. b Figure 1. Experimental results of the killing effect of specific CTLs obtained from transgenic mice on T2A2 cells loaded with a single mutant / wild-type epitope peptide as target cells;

[0073] Figure 6 Eight mutant epitope peptides derived from the neoantigen ESR1 were used in a peptide pool in conjunction with CpG-ODN1826 to immunize HLA-A2.1 / K. b Figure 1 shows the results of intracellular factor staining experiments using specific CTLs obtained from transgenic mice as target cells of the tumor cell line (MDA-MB-231-WT / MUT).

[0074] Figure 7 Figure 1 shows the in vivo antitumor effect of CTLs induced by eight mutant epitope peptides derived from the neoantigen ESR1 and adopted back into tumor-bearing NOD / SCID mice.

[0075] In the above figures, the significance indicators are as follows: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0076] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings obtained in the experimental examples have been briefly described above. It should be understood that the above drawings only show some experimental examples of the present invention and should not be considered as any limitation on the scope of protection of the claims. For those skilled in the art, other related drawings can be obtained based on these drawings without any creative effort. Detailed Implementation

[0077] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and experimental examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other implementation schemes obtained by those skilled in the art without creative effort, such as implementation schemes obtained by modification, variation or simple substitution, should fall within the scope of protection of the present invention.

[0078] Unless otherwise specified, the experimental methods used in the following examples and experimental cases are conventional methods; the raw materials, reagents, instruments, etc. used are commercially available unless otherwise specified; the terms and abbreviations used have their conventional meanings in the art, such as PBS for phosphate buffer.

[0079] Example 1

[0080] This embodiment provides a drug for treating tumors (i.e., tumors positively expressing CTL epitope peptides derived from the neoantigen ESR1). The drug is a synthetic polypeptide vaccine containing a pharmaceutically effective amount of CTL epitope peptides derived from the neoantigen ESR1 prepared by Fmoc solid-phase synthesis, and appropriate amounts of adjuvants, etc. The amino acid sequence of the CTL epitope peptides derived from the neoantigen ESR1 is shown in any one of SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, 14.

[0081] Example 2

[0082] This embodiment provides a drug for treating tumors (i.e., tumors with positive expression of mutant epitope peptides). The drug is an mRNA vaccine, which contains a pharmaceutically effective amount of mRNA delivered by lipid nanoparticles (LNPs), and appropriate amounts of adjuvants, delivery systems, etc. The mRNA is codon-optimized and can encode a CTL epitope peptide derived from the neoantigen ESR1. The amino acid sequence of the CTL epitope peptide derived from the neoantigen ESR1 is shown in any one of SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, 14.

[0083] Example 3

[0084] This embodiment provides a drug for treating tumors (i.e., tumors with positive expression of mutant epitope peptides). The drug is a cell preparation containing a drug-effective amount of modified T cells and an appropriate amount of adjuvant, etc. The modified T cells are inserted with T cell receptors that can specifically recognize CTL epitope peptides derived from the neoantigen ESR1. The amino acid sequence of the CTL epitope peptide derived from the neoantigen ESR1 is shown in any one of SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, 14. The T cell receptors are obtained through screening.

[0085] Example 4

[0086] This embodiment provides the application of a CTL epitope peptide derived from the neoantigen ESR1 in the preparation of a drug for treating tumors. The application includes: mixing a pharmaceutically effective amount of the CTL epitope peptide derived from the neoantigen ESR1, prepared by Fmoc solid-phase synthesis, with an appropriate amount of adjuvants in a certain order to prepare a mixed peptide pool vaccine for treating tumors (i.e., tumors with positive expression of mutant epitope peptides); the amino acid sequence of the CTL epitope peptide derived from the neoantigen ESR1 is shown in SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, 14.

[0087] Experimental Example

[0088] I. Mutation Analysis of Neoantigen ESR1

[0089] First, hotspot mutation sites and mutated amino acids of ESR1 were analyzed using the COSMIC database, and the mutation frequency of ESR1 in different tumor subtypes was further analyzed. Finally, HLA-A2-restricted CTL epitope peptides derived from the neoantigen ESR1 were predicted using a recognized epitope prediction website. Information on the neoantigen ESR1 and antigenic peptides associated with hotspot mutations used in the following experimental examples is shown in Table 1, and the epitope prediction results are shown in Table 2.

[0090] Table 1 Neoantigen ESR1 and antigenic peptides associated with hotspot mutations

[0091]

[0092] The above-mentioned mutant epitope peptides were prepared by Fmoc solid-phase synthesis, and mass spectrometry analysis confirmed that their molecular weights met the theoretical values.

[0093] II. In vitro induction of neoantigen ESR1 and hotspot mutation-related antigenic peptide-specific T lymphocytes

[0094] The in vitro induction method for neoantigen ESR1 and hotspot mutation-related antigen peptide-specific T lymphocytes includes the following steps:

[0095] (1) Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by density gradient centrifugation, and the cells were resuspended in RPMI-1640 medium to adjust the cell density to 1×10⁻⁶. 6 Cells / mL, 5 mL / well, were seeded into 6-well plates and cultured at 37°C in a 5% CO2 incubator for 4 hours.

[0096] (2) After culturing for 4 hours, aspirate the non-adherent cells from the well plate, centrifuge, and then spray with 5×10⁻⁶ cells. 6 Cells were frozen at a concentration of cells / mL for later use. Adherent cells were induced to differentiate into dendritic cells (DCs) using RPMI-1640 complete medium containing 1000 U / mL GM-CSF and 500 U / mL rhIL-4. On day 5, LPS (100 ng / mL) was added to induce DC maturation.

[0097] (3) Four hours after loading mature DCs with mutant epitope peptides, the mature DCs loaded with mutant epitope peptides were co-cultured with T lymphocytes at a DCs:T lymphocytes ratio of 1:10. Stimulation was performed once a week for a total of three cycles, with half the medium changed every two days. Cytokines IL-2 (final concentration: 100 U / mL) and IL-7 (final concentration: 10 ng / mL) were supplemented simultaneously. After three rounds of stimulation, the induced cytotoxic T lymphocytes were collected. Using T2A2 cells loaded with wild-type or mutant epitope peptides and tumor cell lines expressing wild-type or mutant neoantigens (MDA-MB-231-WT / MDA-MB-231-MUT) as target cells, intracellular factor staining and LDH lactate dehydrogenase cytotoxicity assays were performed. The results are as follows: Figure 1-3 As shown.

[0098] III. In vivo induction of neoantigen ESR1 and hotspot mutation-related antigenic peptide-specific T lymphocytes

[0099] The in vivo induction method for neoantigen ESR1 and hotspot mutation-related antigen peptide-specific T lymphocytes includes the following procedures:

[0100] (1) HLA-A2.1 / K in 6-8 week old infants b Transgenic mice were randomly assigned to two experimental groups: the CpG group (CpG-ODN 1826 + saline solution for dissolving peptides) and the CpG+Peptide pool group (containing 8 mutant epitope peptides SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, 14 and CpG-ODN 1826).

[0101] (2) The mutant epitope peptide was administered at a dose of 100 μg / mouse, and CpG-ODN 1826 was administered at a dose of 30 μg / mouse. Immunization was carried out by subcutaneous injection at multiple points at the base of the tail. Mice were immunized three times, on days 0, 7, and 14.

[0102] (3) Mice were sacrificed on day 19, and their spleens and lymph nodes were obtained, ground and digested. The cell suspension was then filtered through a sterile filter into sterile centrifuge tubes and collected by centrifugation.

[0103] (4) Discard the supernatant and add 5 mL of erythrocyte lysis buffer. After lysis, wash with PBS and collect cells by centrifugation. After centrifugation, discard the supernatant and resuspend the cells in GT-T551 medium, adjusting the cell concentration to 5 × 10⁻⁶ cells / mL. 6 Cells per milliliter were seeded into 6-well plates and cultured in a 37°C, 5% CO2 incubator.

[0104] (5) On day 1, add the mutant epitope peptide (peptide pool, 2.5 μg / mL) and mIL-2 (50 U / mL). Replace half the medium and add mIL-2 every other day. Collect induced peptide-specific T lymphocytes on day 5. Using T2A2 cells loaded with wild-type or mutant epitope peptides and tumor cell lines expressing wild-type or mutant neoantigens (MDA-MB-231-WT / MDA-MB-231-MUT) as target cells, intracellular factor staining and LDH lactate dehydrogenase cytotoxicity assays were performed. The results are as follows: Figure 4-6 As shown.

[0105] IV. Antitumor Activity Experiment

[0106] The experimental methods for antitumor activity include the following procedures:

[0107] (1) Select 6-8 week old NOD / SCID mice and place MDA-MB-231-MUT tumor cell suspension (5×10⁻⁶ mcg) in the mice. 6 (1 cell / mouse) was injected subcutaneously into the right back of mice. The tumor was allowed to grow to 80-100 mm in size. 3 The mice were randomly divided into 3 groups;

[0108] (2) NOD / SCID mice were adopted via tail vein injection three times, once a week, with each infusion consisting of 200 μL. The PBS group received PBS buffer, and the T cell group received 5 × 10⁶ μL of PBS buffer. 6 One uninduced T cell; 5 × 10 6 T cells induced by the epitope peptide pool. Mice were sacrificed 5 days after the third adoptive infusion. Mouse weight was recorded every 3 days during the adoptive infusion period. The length, width, and height of the mouse tumors were measured using digital calipers, and the tumor volume V = 1 / 2 × a (length) × b (width) × c (height) was calculated. Results are as follows... Figure 7 As shown.

[0109] V. Testing Methods

[0110] 1. Intracellular factor staining experiment

[0111] Intracellular factor staining assays include the following procedures:

[0112] (1) Target cell preparation: Collect T2 cells by centrifugation, and load them with mutant or wild-type epitope peptides at a final concentration of 80 μg / mL. Incubate at 37°C and 5% CO2 for 4 hours. For tumor cell lines, trypsin digestion and resuspending followed by washing with serum-free medium. After centrifugation and washing, adjust the cell density to 2 × 10⁶ cells / mL. 5 per milliliter.

[0113] (2) Preparation of mutant epitope peptide-specific T lymphocytes: Collect the induced T lymphocytes, wash them with serum-free medium and resuspend them, and adjust the cell density to 2×10⁶ cells / year. 6 per milliliter.

[0114] (3) Peptide-specific T lymphocytes and target cells were seeded in 96-well U-shaped plates at a ratio of 10:1 and incubated at 37°C in a 5% CO2 cell culture incubator for 4 hours. After co-incubation for 4 hours, the inhibitor BFA was added to each well and the cells were cultured for another hour.

[0115] (4) Collect cells from each well into a 1.5 mL centrifuge tube, centrifuge and wash, then add 50 μL of diluted cell surface antibodies (CD3, CD8, and FasL), vortex to mix, and incubate at 4 °C in the dark for 30 min. Wash twice with PBS after incubation. Set up a blank control group, a CD3 monopositive group, a CD8 monopositive group, and an isotype control group.

[0116] (5) Add 200 μL of fixative to each tube of cells and vortex to mix. Incubate at room temperature in the dark for 30 min. After incubation, add 800 μL of diluted cell permeabilizing agent and then centrifuge to collect the cells.

[0117] (6) Add 50 μL of intracellular factor antibody (IFN-γ, except for blank group, single positive group and isotype control group) diluted with membrane permeabilizing agent, vortex mix well and incubate at 4°C in the dark for 30 min. After incubation, wash twice with PBS.

[0118] (7) Discard the supernatant, resuspend the cells in 200 μL PBS, and analyze by flow cytometry.

[0119] 2. LDH lactate dehydrogenase cytotoxicity assay

[0120] The LDH lactate dehydrogenase cytotoxicity assay includes the following procedures:

[0121] (1) Target cell preparation: Collect T2 cells by centrifugation, and load them with mutant or wild-type epitope peptides at a final concentration of 80 μg / mL. Incubate at 37°C and 5% CO2 for 4 hours. For tumor cell lines, wash with serum-free medium after trypsin digestion and resuspension. After centrifugation and washing of the target cells, adjust the cell density to 1×10⁶ cells / mL. 5 per milliliter.

[0122] (2) Preparation of mutant epitope peptide-specific T lymphocytes: Collect the induced T lymphocytes, wash them with serum-free medium and resuspend them, and adjust the cell density to 5 × 10⁶ cells / year. 6 per milliliter.

[0123] (3) Peptide-specific T lymphocytes and target cells were seeded into 96-well U-shaped plates at effector-to-target ratios (E:T) of 12.5:1, 25:1, and 50:1, respectively. The number of target cells was 5000 per well, and the final volume per well was 100 μL. The cells were incubated at 37°C in a 5% CO2 cell culture incubator for 4 hours.

[0124] Setting up experimental groups:

[0125] Target cell spontaneous release group: 50 μL target cells + 50 μL serum-free culture medium;

[0126] Maximum target cell release group: 50 μL target cells + 50 μL serum-free culture medium + 10 μL lysis buffer;

[0127] Background control group: 100 μL serum-free culture medium;

[0128] Volume correction group: 100 μL serum-free IMDM + 10 μL lysis buffer;

[0129] Experimental group: 50 μL target cells + 50 μL T lymphocytes with different effector-to-target ratios.

[0130] (4) 45 min before the end of incubation, add 10 μL of lysis buffer to each of the target cell maximum release group and the volume correction group. Transfer 50 μL of supernatant to another flat-bottomed 96-well plate, add 50 μL of diluted substrate mixture to each well, and incubate at room temperature in the dark for 30 min. Add 50 μL of stop solution to each well, remove air bubbles, and detect at 490 nm wavelength within 1 h.

[0131] VI. Experimental Results and Analysis

[0132] Based on Table 1, analysis of the COSMIC online database revealed that the ESR1 mutation rate in endocrine therapy-resistant samples was 76.06%. Analysis of ESR1 point mutations showed that mutations were mainly concentrated at four sites: 303, 380, 537, and 538, with some tumor samples exhibiting two or more mutation sites simultaneously. These four ESR1 mutation sites cover over 80% of drug-resistant mutation samples, demonstrating broad population coverage and making them ideal targets for immunotherapy.

[0133] Table 2. Prediction results of HLA-A2 restricted mutant epitopes derived from the neoantigen ESR1.

[0134]

[0135] As shown in Table 2, based on the four hotspot mutation sites K303R, E380Q, Y537S / N / C and D538G of ESR1, the prediction of HLA-A2 restriction epitopes from ESR1 was carried out using Net MHC 4.0 Server and the IEDB online website. Combining the prediction results of the two websites, 14 mutant epitope peptides were obtained.

[0136] Figure 1 Figure 1 shows the intracellular factor staining results of T2A2 cells carrying the 14 mutant epitope peptides derived from the neoantigen ESR1, with the mutant / wild-type epitope peptides as the target cells. Figure 2 shows the CD8+ staining results obtained from healthy donor-derived PBMCs induced with the mutant epitope peptides, using T2A2 cells carrying the mutant / wild-type epitope peptides as the target cells. + T lymphocytes are effector cells. Figure 1 It can be seen that the eight mutant epitope peptides SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, and 14 can induce the production of CD8 specific to the mutant epitope peptides in vitro. + T lymphocytes, and the CD8 produced + T lymphocytes were able to significantly distinguish between mutant and wild-type epitope peptides. For the E380Q site, although SEQ ID NO:2 showed better predicted affinity compared to SEQ ID NO:3, intracellular factor staining results indicated that CTLs induced by SEQ ID NO:2 contained IFN-γ... + CD8 + T and FasL + CD8 + The proportion of T cells was low, and T2A2 cells carrying mutant / wild-type epitope peptides could not be well distinguished. SEQ ID NO: 9 showed better predictive affinity than SEQ ID NO: 8, but intracellular factor staining results showed that CTLs induced by SEQ ID NO: 9 contained IFN-γ... + CD8 + T and FasL + CD8 + The proportion of T cells is low, and T2A2 cells carrying mutant / wild-type epitope peptides cannot be well distinguished. Therefore, most mutant epitope peptides obtained solely through epitope peptide prediction methods cannot differentiate between wild-type and mutant types. For the development of vaccines related to mutant epitope peptides, efficacy verification through activity experiments is essential for practical application.

[0137] Figure 2Figure 1 shows the cytotoxic effects of specific CTLs induced in vitro by eight mutant epitope peptides derived from the neoantigen ESR1, targeting T2A2 cells carrying the mutant / wild-type epitope peptides. The figure also shows the CD8+ cytotoxicity of healthy donor-derived PBMCs induced with the mutant epitope peptides, using T2A2 cells carrying either the mutant or wild-type epitope peptides as target cells. + T lymphocytes are effector cells; the efficiency of effector cell killing of target cells was detected by a lactate dehydrogenase cytotoxicity assay. Figure 2 It can be seen that, compared with the T2A2 target cell group loaded with wild-type epitope peptides, the eight mutant epitope peptides SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, and 14 induced HLA-A2 mutations in healthy donors. + CTLs obtained from PBMCs can specifically kill T2A2 cells loaded with mutant epitope peptides, and the killing efficiency increases with the increase of effector-target ratio, indicating that these 8 mutant epitope peptides have good immunogenicity.

[0138] Figure 3 The figure shows the experimental results of the killing effect of specific CTLs induced in vitro by eight mutant epitope peptides derived from the neoantigen ESR1 on tumor cell line (MDA-MB-231-WT / MUT). Figure 3 It can be seen that the CD8 generated in vitro by the eight epitope peptides SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, and 14 + T lymphocytes can specifically kill the tumor cell line MDA-MB-231-MUT that overexpresses a mutant neoantigen, and the CD8+ produced... + T lymphocytes can significantly distinguish between mutant and wild-type epitopes on the surface of tumor cells.

[0139] Figure 4-6 Eight CTL mutant epitopes derived from the neoantigen ESR1 were used in a peptide pool in combination with CpG-ODN 1826 to immunize HLA-A2.1 / K. b Figure 1 shows the results of intracellular factor staining and killing experiments on specific CTLs obtained from transgenic mice, targeting T2A2 cells with a pool of mutant / wild-type epitope peptides, T2A2 cells with a single mutant / wild-type epitope peptide, and the tumor cell line (MDA-MB-231-WT / MUT). Figure 4-6 It can be seen that, compared with the CpG-only immunization group, the CpG + peptide pool immunization group had higher levels of CD8+ in the spleen and lymph nodes. + T lymphocytes can specifically kill MDA-MB-231-MUT tumor cells and T2A2 cells carrying mutant epitope peptides, and have a higher proportion of IFN-γ. + CD8+ T cells and FasL + CD8 + T cells. This indicates that the mutant epitope peptide also has good immunogenicity in vivo, and the CTLs induced in vivo by the epitope peptide pool can specifically recognize and kill neoantigen-positive tumor cells.

[0140] Figure 7 The figure shows the in vivo antitumor activity of CTLs induced by eight mutant epitope peptides derived from the neoantigen ESR1 and adopted into tumor-bearing NOD / SCID mice. A xenograft model of MDA-MB-231-MUT tumor cells was established in NOD / SCID mice. CTLs induced in vivo by the mutant peptide pool were adopted into tumor-bearing NOD / SCID mice to investigate the in vivo antitumor activity of mutant peptide-induced T lymphocytes. Figure 7 It can be seen that, compared with the PBS group and the uninduced T cell group, the CTLs induced by the mutant peptide pool can significantly inhibit tumor growth.

[0141] The experimental results above demonstrate that this invention, through analysis of the COSMIC database, epitope prediction, and in vitro and in vivo immunomodulatory activity experiments, identified eight HLA-A2-restricted CTL epitope peptides derived from the neoantigen ESR1. Their amino acid sequences are shown in SEQ ID NO: 1, 3, 6, 8, 11, 12, 13, and 14. These mutant epitope peptides originate from high-frequency mutations in ESR1 and can effectively stimulate and induce the production of novel epitope-specific cytotoxic T lymphocytes. They specifically distinguish between wild-type and mutant sequences and specifically kill tumor cells expressing the mutant epitope, exhibiting good anti-tumor effects.

[0142] The mutant epitope peptide identified in this invention has high patient coverage. When prepared into a drug for treating tumors (such as a tumor vaccine, cell preparation, or a drug containing a T-cell receptor, chimeric antigen receptor, or its encoding nucleic acid that specifically recognizes the mutant epitope peptide), it can induce the production of mutant peptide-specific CD8+ with tumor-killing function in vitro and in vivo. + T lymphocytes, which can accurately distinguish between wild-type and mutant types, have good therapeutic potential and broad application prospects.

[0143] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments, 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 scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. The use of a CTL epitope peptide derived from the neoantigen ESR1 or its encoded nucleic acid in the preparation of a drug for treating tumors, characterized in that: The CTL epitope peptides derived from the neoantigen ESR1 include polypeptides with the following amino acid sequences: as shown in SEQ ID NO: 1, 3, 6, 8, 11, 12, 13 and 14; The tumor is a breast cancer that positively expresses a CTL epitope peptide derived from the neoantigen ESR1.

2. The application according to claim 1, characterized in that: The drug is a therapeutic tumor vaccine or cell preparation that can induce the body to produce neoantigen-specific T cell clones.

3. The application according to claim 2, characterized in that: The therapeutic tumor vaccine is a nucleic acid vaccine, which contains a nucleic acid sequence encoding a CTL epitope peptide derived from the neoantigen ESR1; Alternatively, the therapeutic tumor vaccine may be a peptide vaccine containing the amino acid sequence of a CTL epitope peptide derived from the neoantigen ESR1.

4. The application according to claim 2, characterized in that: The cell preparation comprises antigen-presenting cells containing the amino acid sequence of a CTL epitope peptide derived from the neoantigen ESR1.

5. The application according to claim 1, characterized in that: The drug is a cell preparation that can treat tumors that express the neoantigen ESR1 positively.

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