Breast cancer related ctl epitope peptide combination and application thereof in tumor treatment

Abstract

The application belongs to the technical field of biological medicine manufacturing, and provides a breast cancer related CTL antigen epitope peptide combination and application thereof in tumor treatment. The breast cancer related CTL antigen epitope peptide combination is a combination of 20 polypeptides of 7 targets, and the amino acid sequences of the 20 polypeptides are shown as SEQ ID NO. 1-SEQ ID NO. 20. The breast cancer related CTL antigen epitope peptide combination has the following characteristics: high expression and wide spectrum coverage, HLA full spectrum layout (precise HLA adaptation covering >95% of the population), and multi-level immune activation of tumor specificity. The HLA has high affinity, can activate T cells to produce cytokine secretion, and can be used for co-culture preparation of tumor specific T cells after sensitizing DC cells, so that good tumor specific killing effect can be achieved.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical manufacturing technology. Specifically, this application provides a combination of breast cancer-related CTL (Cytotoxic T Lymphocyte) antigen epitope peptides and their application in tumor treatment. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women worldwide. Triple-negative breast cancer (TNBC), which lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounts for approximately 15–20% of all breast cancers, with a higher incidence and poorer prognosis in Chinese women. Immunotherapy, as an emerging treatment strategy, has received widespread attention in recent years. Targeted therapies such as trastuzumab have significantly improved patient prognosis, but drug resistance and immune tolerance issues still limit its efficacy. Tumor antigen peptide vaccines, by activating tumor-specific T cells, provide a new and precise active immunotherapy strategy for TNBC. Research on tumor antigen epitopes based on T cell recognition is an important direction in breast cancer immunotherapy, but the selection of breast cancer antigen epitopes, optimization of immunogenicity, and overcoming immune tolerance remain current research challenges. Tumor antigen epitopes can be presented by major histocompatibility complex (MHC) molecules and recognized by T cells, thereby stimulating specific anti-tumor immune responses. Presentation of tumor-associated antigens to T lymphocytes using dendritic cells has been shown to induce antigen-specific T cell responses in vivo, and in some cases, objective clinical responses. Bioinformatics prediction combined with experimental validation can efficiently identify immunogenic and clinically relevant tumor antigen epitopes. This approach not only improves the efficiency of epitope screening but also provides new possibilities for personalized cancer immunotherapy.

[0003] A 7-target, 20-peptide combination centered on MAM-A, WT-1, Survivin, NY-ESO-1, TP53, Galectin-3, and HER2 is an innovative DC (dendritic cell) vaccine candidate designed based on HLA epidemiological data of the Chinese population, TNBC biological characteristics, and existing clinical evidence. This combination balances tumor-specific targets, broad HLA coverage, multi-target anti-escape capabilities, and immunogenicity.

[0004] MAM-A is a breast tissue-specific secretory protein, encoding the gene SCGB2A2. It is highly expressed in >80% of breast cancers, while its expression is restricted in normal tissues, making it an ideal specific immune target for breast cancer. Clinical studies have demonstrated that MAM-A DNA vaccines can induce multifunctional CD4+. + and CD8 +T-cell responses, in synergy with neoadjuvant endocrine therapy, can enhance local immune infiltration in breast cancer. Bioinformatics studies of multi-epitope vaccines based on MAM-A, MAGE-A, and Gal-3 have also validated the important role of MAM-A in multi-target combination strategies.

[0005] WT-1 is listed as a top-priority tumor antigen by the National Cancer Institute (NCI), with a protein expression rate exceeding 70% in TNBC, and is independently associated with poor prognosis. Clinical trials have confirmed the safety and antitumor immunogenicity of the WT-1 peptide vaccine.

[0006] Survivin belongs to the inhibitor of apoptosis (IAP) protein family, exhibiting an expression rate as high as 80–90% in TNBC, while being virtually absent in normally differentiated tissues, demonstrating high tumor specificity. Survivin not only regulates tumor cell anti-apoptosis but also participates in mitotic regulation, making it a dual target with both functional intervention and immune targeting significance. Studies in the mouse 4T1 TNBC model have confirmed that Survivin peptide microparticle vaccines can significantly inhibit tumor growth. The classic MHC-I epitope LMLGEFLKL has been validated in multiple clinical trials and is one of the globally recognized potent immunogenic tumor peptides.

[0007] NY-ESO-1 (CTAG1B) is the most immunogenic cancer testis antigen (CTA) discovered to date, with a protein expression rate of approximately 15–30% in TNBC, demonstrating extremely high potential for clinical benefit. The classic MHC-I epitope SLLMWITQC has achieved CD8+ expression in multiple clinical studies of TCR-T cell therapy, DC vaccines, and protein vaccines. + T-cell response confirmed.

[0008] TP53 mutation is the most common somatic mutation in TNBC, with an incidence rate exceeding 80%, far higher than other breast cancer subtypes (approximately 30–40%). The tumor-specific neoantigens generated by TP53 mutations possess natural immunogenicity and tumor specificity. Hotspot mutant peptides such as R175H, R248W, R273H, and G245S can circumvent central immune tolerance and activate genuine tumor-specific T cell responses.

[0009] Galectin-3 is a β-galactosyl-binding lectin that is highly expressed in TNBC tumor cells and the tumor microenvironment (TME), mediating tumor immune escape, promoting TAM (tumor-associated macrophage) M2 polarization, and tumor metastasis. The G98S mutant of Galectin-3 produces tumor-specific neoantigen peptides in its N-terminal region (aa 1-26), exhibiting a good targeting safety window.

[0010] The HER2-low subtype accounts for approximately 50–55% of TNBC. In HER2-low patients, low expression of the HER2 protein leads to the presentation of HER2-derived antigenic peptides by dendritic cells (DCs), inducing HER2-specific CD8⁺ T cells. Short MHC-I peptides derived from the hypervariable region of the HER2 kinase domain can serve as an important supplementary target for TNBC combination therapies, providing an additional immune activation pathway for HER2-low subgroup patients.

[0011] Single-target vaccines face a major challenge of tumor antigen loss: once the expression of the target antigen is downregulated, the single-specific CTL loses its effector target. Summary of the Invention

[0012] To address the above issues, this invention achieves multi-target synergy through the following three levels: Tumor-specific stratification: MAM-A provides breast tissue specificity, while the TP53 mutant peptide provides true tumor neoantigen specificity, both jointly anchoring the core immune targeting of TNBC tumor cells; High expression and broad-spectrum coverage: Survivin (80–90%), WT-1 (>70%), and Gal-3 (>70%) are all highly expressed targets in TNBC, ensuring that most patients' tumor cells simultaneously express more than three combined targets, forming multiple killing mechanisms; HLA full-spectrum layout: 20 peptides from 7 targets are optimized for A*11:01, A*02:01, A*24:02, and B*40:01 / B*46:01, with HLA-I class coverage exceeding 95% and HLA-II class (DRB1*09:01 / 07:01) coverage exceeding 80%, achieving precise HLA adaptation covering >95% of Chinese TNBC patients.

[0013] On the one hand, this application provides a combination of breast cancer-related CTL antigen epitope peptides, which is a combination of 20 polypeptides targeting 7 sites, and the amino acid sequences of the 20 polypeptides are shown in SEQ ID NO.1-SEQ ID NO.20.

[0014] On the other hand, this application provides a combination of mRNAs (Messenger Ribonucleic Acid) encoding the above-mentioned combination of breast cancer-related CTL antigen epitope peptides.

[0015] On the other hand, this application provides the use of the above-mentioned combination of breast cancer-related CTL antigen epitope peptides or mRNAs in the preparation of drugs for treating breast cancer.

[0016] Furthermore, the drug is a peptide vaccine, a peptide-DC vaccine, an mRNA vaccine, a saRNA (Self-Amplifying RNA Vaccine), an mRNA-DC vaccine, a DNA vaccine, a DNA-DC vaccine, or a tumor-specific DC-T cell drug.

[0017] Furthermore, the drug is a tumor-specific DC-T cell drug. In the application, the above-mentioned combination of breast cancer-related CTL antigen epitope peptides is used to sensitize DC cells, and the sensitized DC cells are co-cultured with T cells to obtain tumor-specific T cells.

[0018] Further, dendritic cells (DCs) are sensitized using the above-mentioned breast cancer-associated CTL antigen epitope peptide combination at a concentration of 3-8 μg / mL. Preferably, DCs are sensitized using the above-mentioned breast cancer-associated CTL antigen epitope peptide combination at a concentration of 5 μg / mL.

[0019] Furthermore, sensitized DC cells and T cells were co-cultured at a ratio of 1:8-12 to obtain tumor-specific T cells; the culture medium used for co-culture contained 800-1200 U / mL rhIL-2 (Recombinant Human Interleukin-2); the co-culture time was 18-30 hours.

[0020] Preferably, sensitized DC cells and T cells are co-cultured at a ratio of 1:10 to obtain tumor-specific T cells; the culture medium used for co-culture contains 1000 U / mL rhIL-2; and the co-culture time is 24 hours.

[0021] Furthermore, the drug is an injectable drug.

[0022] Furthermore, the drug is a drug that can be injected locally or intravenously.

[0023] On the other hand, this application provides tumor-specific T cells, the preparation process of which includes sensitizing DC cells with the above-mentioned breast cancer-related CTL antigen epitope peptide, and co-culturing the sensitized DC cells with T cells to obtain tumor-specific T cells.

[0024] Furthermore, dendritic cells were sensitized using the above-mentioned combination of breast cancer-associated CTL antigen epitope peptides at a concentration of 3-8 μg / mL.

[0025] Furthermore, sensitized DC cells and T cells are co-cultured at a ratio of 1:8-12 to obtain tumor-specific T cells; the culture medium used for co-culture contains 800-1200 U / mL rhIL-2.

[0026] Preferably, sensitized DC cells and T cells are co-cultured at a ratio of 1:10 to obtain tumor-specific T cells; the culture medium used for co-culture contains 1000 U / mL rhIL-2.

[0027] On the other hand, this application provides a medicament for treating breast cancer, the medicament comprising the aforementioned tumor-specific T cells.

[0028] Excipients used in drugs containing cells include, but are not limited to, solvents, antioxidants, osmotic pressure regulators, pH regulators, suspending agents, and other excipients known in the art.

[0029] The DC cells and T cells used in this application may be derived from individual cancer patients or from other sources. For example, these cells can be isolated or induced from cell populations such as PBMCs (e.g., using the methods described in the examples).

[0030] The beneficial effects of this invention include: The breast cancer tumor-specific antigen peptide combination in this invention is formulated based on the epidemiological characteristics and HLA typing of the Chinese population. This combination balances target tumor specificity, broad HLA coverage, multi-target anti-escape ability, and immunogenicity strength. Tumor-specific stratification: MAM-A provides breast tissue specificity, while TP53 mutant peptide provides true tumor neoantigen specificity. Together, they anchor the core immune targeting of TNBC tumor cells. High expression and broad spectrum coverage: Survivin (80–90%), WT-1 (>70%), and Gal-3 (>70%) are all highly expressed targets in TNBC, ensuring that the tumor cells of most patients express more than three combined targets at the same time, forming a multiple killing basis; HLA full-spectrum layout: 20 peptides from 7 targets are optimized for A*11:01, A*02:01, A*24:02 and B*40:01 / B*46:01, with HLA-I class coverage of over 95% and HLA-II class (DRB1*09:01 / 07:01) coverage of over 80%, achieving precise HLA adaptation for more than 95% of Chinese TNBC patients.

[0031] Verification has shown that the antigenic polypeptide combination in this invention has high HLA affinity and can activate T cells to produce cytokine secretion. This indicates that the breast cancer-specific antigenic epitope polypeptide has the potential to be formulated into polypeptide drugs.

[0032] The mRNA and DNA sequences corresponding to the polypeptide sequences in this invention can be designed into mRNA and DNA tumor vaccines with the same efficacy. Therefore, they can be used for different forms of tumor vaccines, such as polypeptide vaccines, polypeptide-DC vaccines, mRNA vaccines, saRNA vaccines, mRNA-DC vaccines, DNA vaccines, and DNA-DC vaccines.

[0033] Verification has shown that the breast cancer-specific polypeptide combination of this invention, when sensitized dendritic cells (DCs) and co-cultured with expanded T cells, can yield CD8+ cells that simultaneously exhibit tumor-specific killing activity. + The purity of T cells and CD4+ T cells can reach over 90%. This indicates that DC vaccine presentation can effectively mobilize T cells and break immune tolerance in cancer patients. Tumor-specific T cells obtained through this method can also be directly used to treat cancer patients.

[0034] Pharmacodynamic results showed that T cells co-cultured and expanded with breast cancer-specific dendritic cells had a good killing effect on breast cancer cells and a good inhibitory effect on tumor growth in animal models of breast cancer. 1) ELISPOT assay showed that the levels of cytokine γ-IFN secreted by T cells co-cultured with breast cancer-specific DC cells were significantly higher than those secreted by conventionally cultured CTLs.

[0035] 2) T cells co-cultured and expanded with breast cancer-specific DC cells have specific killing ability: they have a significant killing effect on breast cancer tumor cells, which is significantly higher than that on non-breast cancer cells, and also significantly higher than the killing rate of breast cancer tumor cells by ordinary cultured CTLs.

[0036] 3) Pharmacodynamic experiments on microtumor organoids showed that they had a good inhibitory effect on tumor growth. Attached Figure Description

[0037] Figure 1 The cell morphology after DC induction in Example 3 is shown.

[0038] Figure 2 This demonstrates the tumor cell killing effect of T lymphocytes sensitized with tumor-specific antigens. Detailed Implementation

[0039] Example 1: Design of breast cancer-specific antigen peptide combinatorial design Epitope prediction: Seven targets were selected, primarily MAM-A, WT-1, Survivin, NY-ESO-1, TP53, Galectin-3, and HER2. Based on the protein sequences published on the official website https: / / www.ncbi.nlm.nih.gov / , the functional domains and HLA allele anchoring of each target protein were analyzed. The HLA class I peptide binding prediction program from the Bioinformatics and Molecular Analysis Division of the National Institutes of Health (NIH) and NetMHCpan were used to predict MHC types I and II. Peptide combinations were also developed considering the HLA genotyping characteristics of the Chinese population.

[0040] Taking the target MAM-A (mammary globin A) as an example, the design process for the polypeptide sequence is as follows: Protein sequence source: The full sequence of the mature MAM-A peptide (NCBI NP_002402.1, 75 aa after removing the signal peptide): MASRAEAQQEMVQKFQTIEEILNQNLASYSEFNRQQNQAVFLQNLMQNPPQQNMPQNMIPPPPMQNMPQNMIPP (SEQ ID NO.21). Note: When mature secreted proteins are processed in tumor cells, they can produce MHC-I presented peptides after degradation by the proteasome, which is the source of CTL epitopes.

[0041] Candidate peptide sliding window scanning: A 9-mer sliding window scan was performed on the full-length 93aa protein (including the signal region). Key regions: Core region aa20–60: rich in hydrophobic residues, with concentrated proteasome cleavage sites. C-terminal region aa70–95: a region rich in known T cell epitopes.

[0042] Using the full-length MAM-A protein sequence (93 aa, NCBI NP_002402.1) as input, a sliding window method was used to sequentially extract all possible 9-mer and 10-mer peptides, generating a total of 87 9-mer peptides (position 1→87, step size 1) and 86 10-mer peptides (position 1→86, step size 1), for a total of 173 original candidate peptides, which served as the initial peptide library.

[0043] Initial screening of candidate peptides (combined with anchoring rules): First round of screening: Structure and source filtering (173 → 36). The following filtering rules were applied to the 173 original peptides to eliminate peptides that were structurally unsuitable for MHC-I presentation: ① Remove unanchored peptides in the signal peptide region (aa1–22): The MAM-A signal peptide (aa1–22) is cleaved in the endoplasmic reticulum and does not produce effective CTL epitopes; 16 peptides in this region with polar residues (A / Q / E) at both P2 / P ends are eliminated.

[0044] ②Removal of Pro / Gly enriched peptides (Pro+Gly ≥ 3): Polyproline peptides cannot bend to fit the MHC-I binding groove due to their rigid structure, and polyglycine regions lack side chain contact points; peptides containing ≥ 3 P or G were eliminated, resulting in the elimination of 41 peptides.

[0045] ③Removal of charged extreme peptides (net charge > +3 or < -3): High net charge peptides have extremely low affinity for the hydrophobic binding groove of MHC-I; elimination of peptides with Arg / Lys accumulation or Asp / Glu accumulation, 28 peptides were eliminated.

[0046] ④ Remove completely duplicate / highly overlapping peptides: Peptides that differ from adjacent windows by 1 amino acid and have the same core anchor position (P2 / P end) are retained as the best representative; redundancy is removed, and 52 peptides are discarded.

[0047] Second round of screening: HLA anchoring rule filtering (36 to 17). Based on the binding motif of the target HLA allele, each of the 36 peptides is checked to see if it matches the preferred anchoring residue of the corresponding HLA. After performing anchoring rule checks: strict double anchor matching: 11 rules; single anchor condition retained: 6 rules (P2 strict matching, P last position is a tolerant residue); eliminated (no effective anchoring): 19 rules.

[0048] The third round of screening: comprehensive evaluation (17 to 8), the 17 candidate peptides were comprehensively scored from the following 4 dimensions, including anchoring rule compliance, proteasome cleavage probability, sequence hydrophobicity, and self-protein homology, and the top 8 in comprehensive score were retained.

[0049] The final 8 candidate peptides entered the NetMHCpan prediction process: 1) IC 50 ≤100 nM (SB standard, suitable for DC load); 2) %Rank≤0.5 (NetMHCpan SB threshold); 3) Coverage of different HLA alleles (maximizing population coverage); 4) No MHC-II interfering epitopes (avoiding non-specific activation).

[0050] The final selected peptides are shown in Table 1.

[0051] Table 1. Final selection of peptides MAM-A-P01 MAM-A KKVNLELLEK (SEQ ID NO.1) A*02:01 10mer aa 35-44 MAM-A-P02 MAM-A EQLAKQHISR (SEQ ID NO.2) A*11:01 10mer aa 70-79 MAM-A-P03 MAM-A KFHQKFQSEL (SEQ ID NO.3) A*24:02 10mer aa 46-55 Using the same technical approach, peptide sequences targeting 7 sites, including WT-1, were designed to form 20 peptide combinations containing 7 targets, as shown in Table 2.

[0052] Table 2. Target sites, epitope sequences, and HLA typing in antigen-peptide combinations. .

[0053] Example 2: Verification of affinity of mammary globin-specific antigen peptides Peptide synthesis: Based on the mammary globin (MAM) antigen epitope from Example 1, peptide synthesis was performed using a solid-phase synthesis method. Solid-phase synthesis involves immobilizing the first amino acid on a solid support, then adding the next amino acid one by one, and reacting. The synthesized peptides were purified and analyzed using methods including high-performance liquid chromatography (HPLC) and mass spectrometry (MS).

[0054] Affinity analysis: Using the HLA tetramer method: 1) HLA tetramers of the antigenic peptide were prepared using the MBL tetramer kit, as shown in Table 3.

[0055] Table 3 HLA tetramers of antigenic peptides TE-M01 HLA-A\*02:01 / KKVNLELLEK (SEQ ID NO.1) A\*02:01 MAM-A aa 35-44 PE TE-M02 HLA-A\*11:01 / EQLAKQHISR (SEQ ID NO.2) A\*11:01 MAM-A aa 70-79 PE TE-M03 HLA-A\*24:02 / KFHQKFQSEL (SEQ ID NO.3) A\*24:02 MAM-A aa 46-55 PE TE-MLP HLA-DRB1\*09:01 / HFQKFQSELQELQA (SEQ ID NO.17) DRB1\*09:01 MAM-A aa 45-59 APC 2) Peripheral blood mononuclear cells (PBMCs), at a concentration of 2-5 × 10⁻⁵. 7 The cells were resuspended in FACS Buffer (PBS + 2% fetal bovine serum) at a density of 0.5 mL to prepare a cell suspension.

[0056] 3) Add 25 μl of cell suspension to a microplate or flow cytometer.

[0057] 4) Prepare the tetramer (1:50~1:100) + Anti-CD8 / FITC antibody prepared in 2×Staining Cocktail: FACS Buffer + 1). Take 25 μl and add it to the microplate or flow cytometer tube in step 3), and mix it with the cell suspension.

[0058] 5) Incubate in an ice bath away from light for 60 minutes.

[0059] 6) Add 150 μl of FACS Buffer to each well of the microplate or 2-3 ml of FACS Buffer to the flow cytometer tube, mix well, centrifuge at 1200 rpm for 5 min, and carefully remove the supernatant.

[0060] 7) Repeat step 6) twice.

[0061] 8) Resuspend the cells in 200 μl of fixative (PBS + 1% paraformaldehyde (PFA)) and perform sample analysis using flow cytometry.

[0062] Affinity analysis results: The results of the MAM-A peptide tetramer affinity assay are shown in Table 4.

[0063] Table 4 MAM-A-P01 (KKVNLELLEK) (SEQ ID NO.1) A\*02:01 ~45 0.14 ±0.03 Positive MAM-A-P02 (EQLAKQHISR) (SEQ ID NO.2) A\*11:01 ~120 0.11 ±0.02 Positive MAM-A-P03 (KFHQKFQSEL) (SEQ ID NO.3) A\*24:02 ~65 0.07 ±0.01 Positive MAM-A-LP (HFQKFQSELQELQA) (SEQ ID NO.17) DRB1\*09:01 ~67 0.09 ±0.01 Positive .

[0064] Immunogenicity analysis of epitope peptides: Peripheral blood mononuclear cells (PBMCs) were co-cultured with 5 μg / ml antigen peptide and 30 μg / ml rhIL-2, respectively. After three stimulations with the antigen peptide, activated T cells were collected, and ELISPOT was used to detect the high concentration of cytokine γ-IFN secreted around the activated T cells.

[0065] The immunogenicity results are shown in the γ-IFN column of Table 5.

[0066] Table 5 Immunogenicity analysis of epitope peptides MAM-A-P01 (KKVNLELLEK) (SEQ ID NO.1) A\*02:01 ~45 112 ±18 Positive MAM-A-P02 (EQLAKQHISR) (SEQ ID NO.2) A\*11:01 ~120 96 ±16 Positive MAM-A-P03 (KFHQKFQSEL) (SEQ ID NO.3) A\*24:02 ~65 68 ±12 Positive MAM-A-LP (HFQKFQSELQELQA) (SEQ ID NO.17) DRB1\*09:01 ~67 82 ±14 Positive .

[0067] Other peptides: Similarly, for each peptide, solid-phase synthesis was performed to prepare HLA tetramers of the antigenic peptide (as shown in Table 6). Peptide tetramer affinity testing and epitope peptide immunogenicity analysis were performed, and the results were all positive.

[0068] Table 6 Tetramer affinity detection of other peptides .

[0069] Example 3: Tumor-specific antigen peptide combination loaded with dendritic cells (1) Take 100 mL of fresh peripheral blood from a healthy volunteer (HLA-A 02:01) (dispensed into 5 mL anticoagulant tubes) and separate mononuclear cells using countercurrent centrifugation. Count the cells using a hemocytometer and adjust the cell density to 1×10⁻⁶ cells / mL with 1640 cell culture medium. 6 Incubate at 37℃ in a 5% CO2 incubator with cells / mL.

[0070] (2) DC induction experiment: Add cell growth factors 100 ng / mL rhGM-CF and 50 ng / mL rhIL-4. Change half the medium every other day, and observe cell growth and morphological changes daily (results are shown in the figure). Figure 1 (As shown).

[0071] (3) Transfection of DC cells with antigen peptide combination: On the 6th day of culture, 5 μg / ml of the antigen peptide combination in Example 1 (0.25 μg / ml for each peptide) was added to sensitize the growing DC cells.

[0072] (4) Detection of DC transfection efficiency: w6 / 32 antibody staining was used to detect the expression of transfected DC protein, and the flow cytometry signal showed a significant increase.

[0073] (5) Add cell growth factors rhTNF-α, IL-1β, and PG-E2 to allow sensitized DCs to grow into further mature DCs. Change half the medium every other day.

[0074] (6) DC cell counting and maturity detection 1) Flow cytometry detection of DC phenotype: Cells were digested with EDTA-free trypsin at 1500 rpm for 5 min, and the cells were collected and washed once with phosphate-buffered saline (PBS). 1 × 10⁻⁶ cells were collected from each cell. 4 Cells were collected in flow cytometry tubes and centrifuged at 1500 rpm for 5 min, then the supernatant was discarded. Cells were resuspended in 100 μL of PBS, and fluorescently labeled antibodies (1 μL each for HLA-DR, CCR7, CD86, and CD80) were added. The cells were stained at room temperature in the dark for 30 min. After washing and centrifuging again, each tube was resuspended in PBS. Analyzed using a flow cytometer.

[0075] 2) Detection of cytokines in supernatant: Antigen peptides stimulated DC cells to secrete more γ-IFN and IL-12p70, promoting maturation (results are shown in Table 7).

[0076] Table 7 Results of DC cell number, viability, and phenotypic detection .

[0077] Example 4: In vitro killing experiment of tumor-specific T cells Cell isolation and purification: 100 mL of fresh peripheral blood was collected from healthy volunteers, and PBMCs were isolated using Ficoll. Cell counting: Cells were counted using a hemocytometer, and the cell density was adjusted to 1 × 10⁻⁶ cells / mL with 1640 cell culture medium. 6 Incubate at 37℃ in a 5% CO2 incubator with cells / mL.

[0078] PBMCs were induced and cultured with 500 U / mL rhIL-2, 0.5 ug / mL anti-CD3, and 0.5 ug / mL anti-CD28. After two days, the culture medium was changed to contain only 500 U / mL rhIL-2 and cultured for 7 days to differentiate into T cells.

[0079] Activation and expansion of tumor-specific T cells: The cultured T cells were co-cultured with the mature DC cells from Example 3 using a 24-well culture plate (DC: 1×10⁶). 5 T cells / mL: 1×10 6 (T cells / mL), and 1000 U / mL rhIL-2 was added. After co-culturing for 24 h, supernatants from each group were collected. T cell proliferation was detected using the CCK8 kit; cytokines such as TNF-α, IL-12, and γ-IFN were detected using the ELISA method.

[0080] Flow cytometry detection of cell phenotype: Collect the cultured cells, take 1×10 4 Cells were collected in flow cytometry tubes and centrifuged at 1500 rpm for 5 min, then the supernatant was discarded. Cells were resuspended in 100 μL of PBS, and fluorescently labeled antibodies (CD4, CD8, CD16, CD28, CD56, CD25, CD45RO, CD19, CD127, etc.) were added. Staining was performed at room temperature in the dark for 30 min. After washing and centrifugation again, each tube was resuspended in PBS. Analyzed using a flow cytometer, and the data were analyzed.

[0081] The results are shown in Table 8: CD3 + T cells account for more than 95%, and also contain CD3, which has the strongest tumor-killing activity. + CD8 + Cells (CTLs) and CD3 + CD4 + T cells suggest that DC vaccine presentation can effectively mobilize T cells and break immune tolerance in cancer patients.

[0082] Table 8 Results of T cell count, viability, cytokines, and phenotype detection .

[0083] Example 5: In vitro cytological pharmacodynamic assay of breast cancer cell lines The T cells from Example 4 were used. Triple-negative breast cancer cells MDA-MB-231 were cultured using standard 24-well cell plate methods.

[0084] Cellular validation of the tumor-killing effect of T lymphocytes after DC vaccine presentation showed that the killing activities of the prepared T lymphocytes at effector-to-target ratios of 8:1, 4:1, and 2:1 were 93.1%, 87.5%, and 83.2%, respectively, while the corresponding killing activities of unsensitized T lymphocytes at the same effector-to-target ratios were 64.5%, 51.3%, and 39.6%. (e.g.) Figure 2 (As shown).

[0085] 2) Four other TNBC cell lines were selected to cover different triple-negative breast cancer subtypes and HLA types: T lymphocytes presented with DC vaccine also showed good tumor killing effect (the results are shown in Tables 9 and 10).

[0086] Table 9. Characteristics of the other four TNBC cell lines Cell source ATCC ATCC ATCC ATCC TNBC subtype basal sample Human breast duct cancer cells Human breast squamous cell carcinoma mesenchymal sample Transfer characteristics low transfer medium-term transfer low transfer High metastasis (stem cells) HLA-A*02:01 ++++ ++ +++ ++ HLA-A*11:01 + ++ + + HLA-A*24:02 － + － － HLA-B*40:01 + +++ ++ +++ HLA-C + +++ ++ +++ .

[0087] Table 10 Tumor killing effect (%) 2:1 74.5 90.1 76.8 78.5 4:1 82.4 93.6 86.9 90.7 8:1 100 100 91.4 100 .

Claims

1. A combination of breast cancer-associated CTL antigen epitope peptides, characterized in that, The breast cancer-associated CTL antigen epitope peptide combination consists of 20 polypeptides targeting 7 sites, and the amino acid sequences of the 20 polypeptides are shown in SEQ ID NO.1-SEQ ID NO.

20.

2. An mRNA combination encoding the combination of breast cancer-associated CTL antigen epitope peptides according to claim 1.

3. The use of the combination of breast cancer-related CTL antigen epitope peptides according to claim 1 or the combination of mRNAs according to claim 2 in the preparation of a medicament for treating breast cancer.

4. The application according to claim 3, characterized in that, The drug is a peptide vaccine, peptide-DC vaccine, mRNA vaccine, saRNA vaccine, mRNA-DC vaccine, DNA vaccine, DNA-DC vaccine, or tumor-specific DC-T cell drug.

5. The application according to claim 4, characterized in that, The drug is a tumor-specific DC-T cell drug. In this application, the above-mentioned combination of breast cancer-related CTL antigen epitope peptides is used to sensitize DC cells, and the sensitized DC cells are co-cultured with T cells to obtain tumor-specific T cells.

6. The application according to claim 5, characterized in that, The method involves sensitizing DC cells with a combination of breast cancer-associated CTL antigen epitope peptides according to claim 1 at a concentration of 3-8 μg / mL; co-culturing the sensitized DC cells with T cells at a ratio of 1:8-12 to obtain tumor-specific T cells; the culture medium used for co-culturing contains 800-1200 U / mL rhIL-2; and the co-culturing time is 18-30 hours.

7. The application according to any one of claims 3-6, characterized in that, The drug is an injectable drug.

8. Tumor-specific T cells, characterized in that, The process for preparing tumor-specific T cells includes sensitizing DC cells with the combination of breast cancer-associated CTL antigen epitope peptides according to claim 1, and co-culturing the sensitized DC cells with T cells to obtain tumor-specific T cells.

9. The tumor-specific T cells according to claim 8, characterized in that, DC cells were sensitized with a combination of breast cancer-associated CTL antigen epitope peptides according to claim 1 at a concentration of 3-8 μg / mL; the sensitized DC cells were co-cultured with T cells at a ratio of 1:8-12 to obtain tumor-specific T cells; the culture medium used for co-culture contained 800-1200 U / mL rhIL-2.

10. A drug for treating breast cancer, characterized in that, The drug contains tumor-specific T cells as described in claim 9.

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