WO2023226295A1 - Pd-l1相关疫苗及其应用 - Google Patents

Pd-l1相关疫苗及其应用 Download PDF

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WO2023226295A1
WO2023226295A1 PCT/CN2022/128039 CN2022128039W WO2023226295A1 WO 2023226295 A1 WO2023226295 A1 WO 2023226295A1 CN 2022128039 W CN2022128039 W CN 2022128039W WO 2023226295 A1 WO2023226295 A1 WO 2023226295A1
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cancer
cells
hla
cell
immune
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French (fr)
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李波
李冬丽
张乐
黄英
刘耿
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深圳吉诺因生物科技有限公司
武汉华大吉诺因生物科技有限公司
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Publication of WO2023226295A1 publication Critical patent/WO2023226295A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes

Definitions

  • the present disclosure relates to the field of biomedicine. Specifically, the present disclosure relates to PD-L1 related vaccines and their applications.
  • the immune system contains many types of regulatory immune cells. Their role is to control the intensity of the body's immune response and maintain immune balance. Regulatory immune cells mainly include regulatory T cells (Tregs, Regulatory T cells), M2 macrophages (M2 macrophage), myeloid-derived suppressor cells (MDSCs, myeloid-derived suppressor cells) and different dendritic cells (DCs, dendritic cell) subgroup.
  • Tregs regulatory T cells
  • M2 macrophages M2 macrophage
  • MDSCs myeloid-derived suppressor cells
  • DCs dendritic cell subgroup.
  • the immunosuppressive process involving regulatory immune cells is a mechanism that controls the scale and duration of specific immune responses. The difference between immunosuppression and immune tolerance is that immunomodulation is an active immune activation.
  • regulatory immune cells are considered to have positive significance in the immune environment of healthy people, and can prevent excessive immune responses from damaging their own cells.
  • regulatory immune cells may inhibit the body's immune killing response to malignant cells.
  • studies have shown that many types of cancer cells have adapted to the phenotype of regulatory immune cells and, like regulatory immune cells, can express a variety of immunosuppressive proteins to inhibit the activity of anti-tumor-specific T cells in the tumor microenvironment. .
  • mechanisms to antagonize immunosuppression are also formed in the immune system.
  • some pro-inflammatory T cells can target and recognize regulatory immune cells expressing immunosuppressive proteins, reduce the number of regulatory immune cells, antagonize the immunosuppressive effect of tumors, and assist by secreting pro-inflammatory cytokines at the inflammatory site.
  • regulatory immune cells expressing immunosuppressive proteins
  • reduce the number of regulatory immune cells antagonize the immunosuppressive effect of tumors
  • assist by secreting pro-inflammatory cytokines at the inflammatory site we define this type of cells as Anti-TIS cells.
  • Anti-TIS Tumor ImmunoSuppression
  • HLA is the expression product of the human major histocompatibility complex (MHC) and is a highly polymorphic allogeneic antigen. There will be differences in HLA allele typing among people in different regions. For example, Europe and the United States have higher coverage. The main allele type is HLA-A02:01, and the top two allele types with the highest coverage in China are HLA-A11:01 and HLA-A24:02.
  • MHC human major histocompatibility complex
  • Programmed cell death ligand 1 also known as the surface antigen cluster of differentiation 274 (CD274), is a transmembrane protein in humans that is involved in the self-regulation of the immune system closely related.
  • Programmed cell death receptor (PD-1) binds to PD-L1 on the surface of cancer cells or immune regulatory cells, which can transmit immunosuppressive signals, reduce the expansion and activation of CD8 + T cells, and reduce antigen-specific T cells in lymph nodes. The aggregation of cells leads to immune evasion of cancer cells.
  • the inventors selected PD-L1 protein from many cancer-related genes to screen targets for anti-tumor immune suppression. Furthermore, the full length of the PD-L1 protein, 290 amino acids, was sheared according to 1-step displacement sliding, and a total of 1126 8, 9, 10, and 11mer peptide segments could be obtained.
  • the inventor analyzed all the peptides and found that only a very small number of peptides can bind to HLA-A11:01 or HLA-A24:02 molecules to form HLA complexes, and further combine with T cell receptors in the body to stimulate The body produces an immune response, but most of the remaining peptides do not have HLA affinity and immunogenicity, and have no research value in developing anti-tumor immunosuppressive vaccines. Therefore, the inventors screened numerous epitopes to obtain epitopes with immunological functions. These peptides can be used as vaccines or drugs.
  • the disclosure provides an isolated polypeptide.
  • the isolated polypeptide has SEQ ID NO: 1, 3, 5, 7, 8, 9, 11, 13, 14, 17, 18, 19, 21, 24, 25, 27, 28.
  • the above-mentioned polypeptides have affinity for HLA-A11:01 and/or HLA-A24:02, can be naturally presented to the cell surface and bind to specific immune effector cells, and specifically eliminate stromal cells, immune cells and other negative cells in the tumor microenvironment. Regulatory cells and tumor cells expressing PD-L1 have potential value as targets for immunotherapy and are of great significance in preventing and treating cancer.
  • the present disclosure provides an isolated nucleic acid.
  • the isolated nucleic acid encodes the aforementioned polypeptide or a functional analog thereof.
  • the present disclosure provides an antigen-presenting cell.
  • the antigen-presenting cells are used to present the polypeptides described above.
  • the present disclosure provides an immune cell.
  • the binding target of the immune cell is the aforementioned isolated polypeptide or the aforementioned antigen-presenting cell.
  • the disclosure provides an antibody.
  • the antibody specifically recognizes the aforementioned isolated polypeptide.
  • the present disclosure provides a vaccine.
  • the vaccine includes: the aforementioned isolated polypeptide, isolated nucleic acid, antigen-presenting cells, immune cells and/or antibodies.
  • the present disclosure provides a medicament.
  • the drug includes: the aforementioned isolated polypeptide, isolated nucleic acid, antigen-presenting cells, immune cells and/or antibodies.
  • the present disclosure proposes the use of the aforementioned isolated polypeptides, isolated nucleic acids, antigen-presenting cells, immune cells and/or antibodies in the preparation of vaccines.
  • the vaccine is used to prevent cancer.
  • the present disclosure proposes the use of the aforementioned isolated polypeptides, isolated nucleic acids, antigen-presenting cells, immune cells and/or antibodies in the preparation of medicines.
  • the medicament is used to treat cancer.
  • the present disclosure provides a method of preventing or treating cancer.
  • the method includes administering to a subject the aforementioned isolated polypeptide, the isolated nucleic acid, the antigen-presenting cell, the immune cell, and/or the antibody.
  • Figures 1 to 4 respectively show polypeptide mass spectra according to embodiments of the present disclosure.
  • the upper half of the mass spectrum is eluted, and the lower half is synthesis;
  • Figure 5 shows electron microscopy images of polypeptides and in vitro immunogenicity ELISPOTs detection according to embodiments of the present disclosure
  • Figures 6 and 7 respectively show a schematic diagram of target cell analysis of immune cell-specific killing and presentation of polypeptides according to embodiments of the present disclosure
  • FIGS 8 and 9 respectively show schematic diagrams of analysis of mouse tumor growth controlled by polypeptide vaccines according to embodiments of the present disclosure.
  • the disclosure provides an isolated polypeptide.
  • the isolated polypeptide has SEQ ID NO: 1, 3, 5, 7, 8, 9, 11, 13, 14, 17, 18, 19, 21, 24, 25, 27, 28 , the amino acid sequence shown in any one of 30 to 34 or a functional analog thereof or at least 60% (such as 65%, 70%, 75%, 85%, 91%, 92%, 94%, 96%, 98) %) homologous amino acid sequence.
  • the above-mentioned polypeptides have affinity for HLA-A11:01 and/or HLA-A24:02, can be naturally presented to the cell surface and bind to specific immune effector cells, and specifically eliminate stromal cells, immune cells and other negative cells in the tumor microenvironment. Regulatory cells and tumor cells expressing PD-L1 have potential value as targets for immunotherapy and are of great significance in preventing and treating cancer.
  • the amino acid sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 8, 11, 14, 17, 24, 32, 33, 34 or its functional analogue or its Polypeptides with amino acid sequences that are at least 60% homologous work better.
  • the polypeptide of the amino acid sequence shown in any one of SEQ ID NO: 3, 5, 8, 24, 32, 34 or its functional analog or its amino acid sequence with at least 60% homology is more effective and can be expressed after identification
  • the HLA molecules presented on the cell surface have strong immunogenicity and the ability to stimulate the production of specific T cells with high killing efficiency.
  • the present disclosure provides an isolated nucleic acid.
  • the isolated nucleic acid encodes the aforementioned polypeptide or its complementary sequence.
  • the polypeptide expressed by the nucleic acid proposed in the above embodiments of the present disclosure under appropriate conditions can be used to prevent or treat tumors, thereby exerting an immune killing effect.
  • the polypeptide expressed by the nucleic acid proposed in the embodiments of the present disclosure under appropriate conditions can be used to prevent or treat tumors.
  • the isolated nucleic acid has SEQ ID NO: 35, 37, 39, 41, 42, 43, 45, 47, 48, 51, 52, 53, 55, 58, 59, 61,
  • nucleotide sequence shown in any one of SEQ ID NO: 35, 37, 39, 41, 42, 45, 48, 51, 58, 66, 67, 68 or its function is similar
  • the nucleic acid effect is better if it is a substance or a nucleic acid sequence having at least 60% homology with it.
  • the nucleotide sequence shown in any one of SEQ ID NO: 37, 39, 42, 58, 66, 68 or its functional analogue or Nucleic acids with at least 60% homology to the nucleic acid sequence are more effective.
  • nucleic acids mentioned in the specification and claims of this disclosure actually include either or both complementary double strands.
  • the gene sequence in this application includes DNA form or RNA form, and disclosing one of them means that the other one is also disclosed.
  • the present disclosure provides an antigen-presenting cell.
  • the antigen-presenting cells are used to present the isolated nucleic acid as described above. Therefore, the antigen-presenting cells of the present disclosure can be used to deliver polypeptides to anti-tumor specific T cells, thereby activating the specific killing function of T cells and effectively used for the prevention or treatment of tumors.
  • the antigen-presenting cells are dendritic cells, B cells or mononuclear phagocytes, preferably dendritic cells.
  • Dendritic cells have extremely strong ability to internalize and process antigens and can present antigens on the cell surface.
  • the inventors selected dendritic cells as antigen-presenting cells.
  • Antigen-presenting cells initiate, regulate and maintain a stronger immune response against the polypeptide in the body.
  • the antigen-presenting cells express HLA-A11:01 and/or HLA-A24:02.
  • the above-mentioned polypeptides have affinity for HLA-A11:01 and/or HLA-A24:02, can be naturally presented and combined with specific immune effector cells, and specifically eliminate stromal cells, immune cells and other negatively regulated cells in the tumor microenvironment.
  • the present disclosure provides an immune cell.
  • the binding target of the immune cell is the aforementioned isolated polypeptide or the aforementioned antigen-presenting cell. As a result, the immune killing function can be exerted.
  • the immune cells are obtained by contacting the aforementioned antigen-presenting cells with cells with immune effector capabilities, or sorting and culturing them from tumor tissues (such as tumor-infiltrating T cells, Tumor-infiltrating lymphocytes (TILs), or the T cell receptor (TCR) gene that can recognize the previously isolated polypeptide is transferred into T cells (such as TCR-T).
  • tumor tissues such as tumor-infiltrating T cells, Tumor-infiltrating lymphocytes (TILs), or the T cell receptor (TCR) gene that can recognize the previously isolated polypeptide is transferred into T cells (such as TCR-T).
  • TILs Tumor-infiltrating lymphocytes
  • TCR T cell receptor
  • the cells with immune effector ability are CD8 + T cells.
  • CD8 + T cells to accept the activation effect of antigen-presenting cells is stronger, and the specific killing effect of the obtained CD8 + T cells on the target cells presenting the antigen-described isolated peptide is stronger.
  • the disclosure provides an antibody.
  • the antibody specifically recognizes the isolated polypeptide described above.
  • tumor antigens can be specifically recognized.
  • the antibody can specifically recognize an antigen and specifically bind to tumor cells or cells infected by viruses (such as HBV, HCV, HIV, EBV, etc.), thereby causing tumor cells or cells infected by viruses to Infected cells are engulfed by phagocytes to achieve specific elimination of tumor cells or virus-infected cells.
  • viruses such as HBV, HCV, HIV, EBV, etc.
  • the present disclosure provides a vaccine.
  • the vaccine includes: the aforementioned isolated polypeptide, isolated nucleic acid, antigen-presenting cells, immune cells and/or antibodies. Therefore, when the vaccine according to the embodiment of the present disclosure enters the body, it can specifically activate specific T cells (such as CTL, TIL) to achieve specific elimination of negatively regulated cells and expressions such as stromal cells and immune cells in the tumor microenvironment.
  • T cells such as CTL, TIL
  • PD-L1 tumor cells have good safety and are of great significance in preventing cancer.
  • the vaccine further includes an adjuvant.
  • an adjuvant This disclosure does not strictly limit the type of adjuvant. Conventional adjuvants in the field can be selected, and can be selected flexibly according to actual needs.
  • the present disclosure provides a medicament.
  • the drug includes: the aforementioned isolated polypeptide, isolated nucleic acid, antigen-presenting cells, immune cells and/or antibodies. Therefore, when drugs according to embodiments of the present disclosure enter the body, they can specifically activate specific T cells (such as CTL, TIL) to achieve specific elimination of negatively regulated cells such as stromal cells and immune cells in the tumor microenvironment, as well as expression PD-L1 tumor cells have good safety and are of great significance in the treatment of cancer.
  • specific T cells such as CTL, TIL
  • the medicament further includes pharmaceutically acceptable excipients.
  • the present disclosure proposes the use of the aforementioned isolated polypeptides, isolated nucleic acids, antigen-presenting cells, immune cells and/or antibodies in the preparation of vaccines.
  • the vaccine is used to prevent cancer.
  • the vaccine can specifically activate specific T cells (such as CTL, TIL) to specifically eliminate negatively regulated cells such as stromal cells and immune cells in the tumor microenvironment, as well as tumor cells expressing PD-L1. Cancer is of great significance.
  • the present disclosure proposes a method for preventing cancer, comprising: administering the aforementioned isolated polypeptides, isolated nucleic acids, antigen-presenting cells, immune cells and/or antibodies to a subject.
  • the subject of the vaccine expresses HLA-A11:01 and/or HLA-A24:02.
  • the above-mentioned polypeptides have affinity for HLA-A11:01 and/or HLA-A24:02, can be naturally presented to the cell surface and bind to specific immune effector cells, and specifically eliminate stromal cells, Negatively regulated cells such as immune cells and tumor cells expressing PD-L1 are highly safe and of great significance in preventing cancer.
  • the present disclosure proposes the use of the aforementioned isolated polypeptides, isolated nucleic acids, antigen-presenting cells, immune cells and/or antibodies in the preparation of medicines.
  • the medicament is used to treat cancer.
  • drugs enter the body they can specifically activate specific T cells (such as CTL, TIL) to specifically eliminate negatively regulated cells such as stromal cells and immune cells in the tumor microenvironment, as well as tumor cells expressing PD-L1.
  • T cells such as CTL, TIL
  • the cancer is breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia, thyroid cancer , lymphoma, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, gallbladder cancer, oral cancer, laryngeal cancer, bone cancer, testicular cancer or brain cancer.
  • the subject of the drug expresses HLA-A11:01 and/or HLA-A24:02.
  • the above-mentioned polypeptides have affinity for HLA-A11:01 and/or HLA-A24:02, can be naturally presented to the cell surface and bind to specific immune effector cells, and specifically eliminate stromal cells, Negatively regulated cells such as immune cells and tumor cells expressing PD-L1 are highly safe and of great significance in the treatment of cancer.
  • the present disclosure provides a method of preventing or treating cancer.
  • the method includes administering to a subject the aforementioned isolated polypeptide, the isolated nucleic acid, the antigen-presenting cell, the immune cell, and/or the antibody.
  • the isolated peptides, isolated nucleic acids, antigen-presenting cells, immune cells, and antibodies enter the body and can specifically activate specific T cells (such as CTL, TIL) to specifically remove the matrix in the tumor microenvironment.
  • T cells such as CTL, TIL
  • Negatively regulated cells such as cells, immune cells, and tumor cells expressing PD-L1 are of great significance in the treatment of cancer.
  • the subject of the vaccine expresses HLA-A11:01 and/or HLA-A24:02.
  • the above-mentioned polypeptides have affinity for HLA-A11:01 and/or HLA-A24:02, can be naturally presented to the cell surface and bind to specific immune effector cells, and specifically eliminate stromal cells, Negatively regulated cells such as immune cells and tumor cells expressing PD-L1 are highly safe and of great significance in the treatment of cancer.
  • the cancer is breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia, thyroid cancer , lymphoma, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, gallbladder cancer, oral cancer, laryngeal cancer, bone cancer, testicular cancer or brain cancer.
  • this disclosure uses a bioinformatics analysis process to cut the full length of the PDL1 protein into a polypeptide sequence of 8-11mer amino acids.
  • the length of the protein encoded by the PDL1 gene It has 290 amino acids, a molecular weight of 33275 Daltons, and 1126 cleaved polypeptide sequences.
  • the presentation ability prediction and affinity prediction were performed on these 1126 polypeptides.
  • the presentation ability score value is expressed as a numerical value from 0 to 1. The higher the score value, the stronger the presentation ability. The score value greater than 0.1 indicates that the polypeptide has the presentation ability.
  • the affinity score is expressed by the IC50 score.
  • An IC50 less than 500 indicates that the polypeptide has affinity, and an IC50 less than 50 indicates that the polypeptide has high affinity.
  • the filtering condition is to meet one of the following two conditions: (1) submission score value > 0.5; (2) submission score value > 0.1 and IC50 ⁇ 500.
  • the polypeptide sequences that meet the conditions (Table 2 and Table 3) are used for the next step of T2 affinity verification.
  • T2 cells Take 2 ⁇ 10 5 T2 cells, spread them in 500 ⁇ L of IMDM serum-free medium containing human ⁇ 2 microglobulin (final concentration, 3 ⁇ g/ml), resuspend them in a 24-well plate, and add the polypeptides in Table 1 (final concentration: 100 ⁇ M ), incubate overnight in an incubator (37°C, 5% CO 2 ). Two duplicate wells per group; T2 cells without peptide added were used as background control. Centrifuge the cells at 200g for 5 minutes to collect the cells. After the cells were washed twice with PBS, the cells were directly incubated with FITC monoclonal antibody against HLA-A11:01/HLA-A24:02 and maintained at 4°C for 30 minutes.
  • MFI>1.5 indicates that the peptide has high affinity for HLA-A11:01 or HLA-A24:02 molecules
  • 1.0 ⁇ FI ⁇ 1.5 indicates that the peptide has moderate affinity for HLA-A11:01 or HLA-A24:02 molecules.
  • Affinity, and 0.5 ⁇ FI ⁇ 1.0 indicates that the peptide is a HLA-A11:01 or HLA-A24:02 molecule with low affinity.
  • the results are shown in the table below.
  • the 12 peptides with high affinity verified by T2 affinity were selected for subsequent mass spectrometry presentation verification, in vitro stimulation and expansion of CD8 + T cells, and ELISPOTs method verification.
  • Example 3 Mass spectrometry experiments verify that polypeptides are presented by HLA molecules on the surface of tumor cells
  • This disclosure uses a combination of co-immunoprecipitation and mass spectrometry to enrich polypeptide-MHC complexes on the cell surface, and identify whether the MHC molecules on the surface of tumor cells present the polypeptide.
  • the specific method is as follows:
  • Isolation and purification of MHC-I restricted T cell epitope peptides use pan-MHC-I A/B/C antibody (clone number: w6/32) and sepharose CL-4B beads coupled to surface protein A molecules Bind for 1 hour at 4°C. Use NanoDrop to detect the residual antibody content of the supernatant. The antibody binding rate is >90% as qualified. Prepare pan-MHC-I A/B/C combined sepharose and set aside at 4°C.
  • Mass spectrometry identification of MHC-I restricted T cell epitope peptides The concentrated MHC-I restricted epitope peptide solution was analyzed by a Q Exactive mass spectrometer (Thermo Fisher Scientific) connected to nanoflow HPLC (Thermo Fisher Scientific) online. Use ReproSil-Pur C18-AQ 1.9um packing to manually fill a 15cm long, 75um inner diameter separation column for separation, use a linear gradient of 2-30% buffer B (80% ACN/0.5% acetic acid) to elute the peptide, and set the flow rate 250nl/min, elution time 90min. The secondary mass spectrometry uses HCD for fragmentation, and the data-dependent "Top 20" method is selected for data acquisition.
  • the acquisition resolution of the MS spectrum is 70,000, 200m/z, and the target value is 3E6 ions; the ions with the top 10 ion strengths are usually separated and accumulated using a maximum injection time of 120ms until the value of the automatic gain controller displays 1E5.
  • the peptide matching option is set to "disable” and the MS/MS resolution is set to 17,500 (200m/z).
  • Mass spectrometry data analysis of MHC-I restricted T cell epitope peptides uses MaxQuant (version1.3.10.15) to compare mass spectrometry profiles with the human full protein library (Uniprot, 86,749 proteins), tumor-associated antigens, tumors A list of spectra generated from a data set of 247 common contaminants (keratin, bovine serum albumin, and proteases) for specific mutated peptides. Variable modification detection settings: N-terminal acetylation and methionine oxidation.
  • the second peptide identification setting enable; specific enzyme digestion setting: unspecific; peptide identification FDR (false discovery rate) is set to 1%, protein identification FDR is not set; the sequence matching length limit is set to 8-15aa, and the maximum peptide mass is set to 1500Da, maximum charge state set to 3.
  • the initial allowable mass deviation of the lead ion was set to 6 ppm, and the maximum fragment mass deviation was set to 20 ppm.
  • the "match between runs" setting is on.
  • the identification result output is saved in the "peptide.txt" file. After removing the peptides matching the reverse library and contamination library, the rest are the identification results of MHC-I restricted epitopes.
  • Example 4 Polypeptide stimulates and expands CD8 + T cells in vitro
  • PBMC cells from HLA-A11:01 subtype-positive healthy volunteers 2 ⁇ 10 6 PBMC cells, were used to isolate monocytes using the adhesion method (adhesion for 3 hours), and CD8 + T cells were isolated using CD8 magnetic beads.
  • Polypeptide sequences with high affinity induce adherent cells into peptide-specific mature DC cells.
  • Mature DC cells loaded with polypeptides were irradiated and co-cultured with CD8 + T cells from the same volunteer, and IL-21 was added. After 3 days, IL-2 and IL-7 were supplemented, and then on days 5 and 7. IL-2 and IL-7 were added once, and on the 10th day, the co-cultured cells were collected for counting and subsequent ELISPOTs and LDH detection. The same operation is performed for HLA-A24:02 subtype. The counting results are as follows, which shows that the polypeptides in Table 5 and Table 6 can stimulate and expand CD8 + T cells in vitro.
  • Example 5 ELISPOTs method to verify that polypeptide activates CD8 + T cell immune response
  • the T cells cultured in Example 4 and T2 loaded with the experimental polypeptide or irrelevant polypeptide were added to the human IFN-gamma interferon ELISPOTs plate for culture. After 20 hours, the ELISPOTs test was performed (see the kit instructions). Spots generated by the ELISPOT experiment were counted.
  • the requirements for immunogenicity of experimental polypeptides are as follows: number of spots (polypeptide)/number of spots (unrelated polypeptide) ⁇ 2, that is, the number of spots caused by the experimental polypeptide exceeds twice or more the number of spots caused by the irrelevant polypeptide.
  • T2 cells can present HLA-A11:01/HLA-A24:02 antigen peptides to T cells.
  • CD8 + T cells specifically recognize the complex of HLA-A11:01/HLA-A24:02 and the polypeptide, the T cells can be activated again and secrete IFN-gamma interferon.
  • the IFN-gamma interferon secreted by activated CD8 + T cells can be captured by the antibodies on the ELISPOTs plate.
  • the antibody that recognizes IFN-gamma can degrade the substrate through the enzyme coupled to the antibody and develop the color, eventually producing spots.
  • the number of spots represents the number of cells activated to secrete IFN-gamma interferon.
  • the ELISPOTs results are shown in Figure 5 and Table 7.
  • the number of spots in the experimental group with a total of 7 polypeptides exceeded the number of spots in the unrelated group by more than 2 times, indicating strong immunogenicity.
  • Example 6 LDH release experiment demonstrates the specific killing activity of CD8 + T cell polypeptides
  • Example 4 The cells cultured in Example 4 were co-cultured with T2 cells loaded with experimental polypeptides or irrelevant polypeptides or unloaded polypeptides. In the experiment, maximum release holes, volume correction holes, culture medium control holes, spontaneous release holes, and different effective targets were set. Ratio (number ratio of T cells to T2 cells) was used as a control. Three duplicate wells were set up in each group. After 4 hours, 50 ⁇ l of the co-cultured cell supernatant was taken out and added to 50 ⁇ l of LDH substrate mixture to allow the cell supernatant to catalyze LDH substrate reaction, finally read the 490nm wavelength and 680nm reference wavelength, and calculate the target cell killing activity of T2 according to the following formula according to the control well.
  • killing efficiency (%) (experimental well - spontaneous release of effector cells - spontaneous release of target cells + culture well) / (maximum release of target cells - volume correction well - spontaneous release of target cells + culture well) ⁇ 100%.
  • the DNA sequence corresponding to the polypeptide was synthesized, and the corresponding lentiviral vector pHBLV-Puro was constructed respectively.
  • the lentiviral plasmid was co-transfected with pSPAX2 and pMD2G helper plasmids into 293T cells, and the polypeptide lentivirus was packaged.
  • the human melanoma cell line SKMEL5 was purchased from ATCC (No. HTB-70), and its HLA subtype is HLA-A11:01 positive.
  • the human colorectal cancer cell line HCT8 was purchased from ATCC (No.: CCL-244), and its HLA subtype is HLA-A24:02.
  • Cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and streptomycin. Culture in a 37°C, 5% CO2 incubator.
  • the packaged lentivirus was transfected into the SKMEL5 and HCT8 cell lines respectively, and Puromycin antibiotic (puromycin) was used to continuously screen the surviving SKMEL5 and HCT8 cell lines, and finally establish the SKMEL5 and HCT8 cell lines expressing the polypeptide.
  • Puromycin antibiotic puromycin
  • PBMC peripheral blood mononuclear cells
  • NOD SCID mice were excluded from immune leakage. Each mouse was intraperitoneally injected with 2 ⁇ 10 7 /0.5ml of PBMC, and human immune reconstitution was performed on NOD SCID mice. Mice with successful immune reconstitution after 4 weeks were selected to prepare the human melanoma cell line and human colorectal cancer cell line models.
  • the established human melanoma cell lines and human colorectal cancer cell lines were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and streptomycin. Culture in a 37°C, 5% CO 2 incubator. Tumor cells were collected, centrifuged at 200 g/min, and washed three times with sterile physiological saline. Make appropriate dilution, add 40 ⁇ l of cell suspension to 10 ⁇ l of 0.4% tryphenol blue staining and count under microscope to make a tumor cell suspension with a concentration of 1 ⁇ 10 8 cells/ml. Select NOD/ after immune reconstitution. SCID mice, each mouse was inoculated subcutaneously with 100 ml of tumor cell suspension.
  • the vaccination site will be observed daily for infection and whether the tumor will naturally subside after growth. After 7 days, tumors about the size of rice grains could be palpated in the subcutaneous tumors of mice, indicating that the SKMEL5 or HCT8 subcutaneous tumor model NOD/SCID mouse model was successfully constructed.
  • the SKMEL5 subcutaneous tumor model NOD/SCID mice with immune reconstitution for 4 weeks were randomly divided into 6 groups: adjuvant group, adjuvant + irrelevant peptide group, adjuvant + NAFTVTVPK peptide group, adjuvant + GVALTFIFR peptide group, adjuvant + GIQDTNSKK
  • the initial immunization dose for unrelated peptide groups and peptide combinations is 100ml/animal.
  • HCT8 subcutaneous tumor model NOD/SCID mice with immune reconstitution for 4 weeks were randomly divided into 6 groups: adjuvant group, adjuvant + irrelevant peptide group, adjuvant + IFMTYWHLL peptide group, adjuvant + TYWHLLNAF peptide group, adjuvant + PYNKINQRI Polypeptide group and adjuvant + polypeptide (polypeptide sequence is SEQ ID NO: 3, 5 and 24) combination group, each group has 8 animals.
  • the initial immunization dose for unrelated peptide groups and peptide combinations is 100ml/animal.
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials, or features are included in at least one embodiment or example of the present disclosure. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

Abstract

提供了PD-L1相关疫苗及其应用，所述PD-L1相关疫苗包括分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体，所述分离的多肽具有如SEQ ID NO：1、3、5、7、8、9、11、13、14、17、18、19、21、24、25、27、28、30-34任一项所示的氨基酸序列或为其功能类似物或与其具有至少60%的同源性。

Description

PD-L1相关疫苗及其应用 技术领域
本公开涉及生物医药领域。具体地，本公开涉及PD-L1相关疫苗及其应用。
背景技术
免疫系统中包含许多类型的调节性免疫细胞，它们的作用是控制机体免疫反应的强度，维持免疫平衡。调节性免疫细胞主要包括调节性T细胞(Tregs，Regulatory T cells)、M2巨噬细胞(M2 macrophage)、骨髓来源抑制细胞(MDSCs，myeloid-derived suppressor cells)和不同的树突状细胞(DCs，dendritic cell)亚群。调节性免疫细胞参与的免疫抑制过程，是控制特定免疫反应规模和时长的一种机制。免疫抑制与免疫耐受的不同之处在于免疫调节是一种主动的免疫激活。已有研究表明，调节性免疫细胞能够表达多种免疫抑制相关蛋白，如IDO、PD-L1、TDO、Arginase、CCL22等，减弱免疫应答效果，其机理有以下三种：(1)分解氨基酸代谢酶，减少免疫细胞生长和功能所需氨基酸，如色氨酸和精氨酸；(2)诱导靶细胞和T细胞上调免疫检查点相关分子表达，如PD-1、PD-L1、PD-L2；(3)分泌趋化因子，招募更多调节性免疫细胞亚群到炎症区域。
调节性免疫细胞在健康人的免疫环境中被认为是有正向意义的，能够避免过强的免疫反应损伤自身细胞。然而，在肿瘤免疫环境中，调节性免疫细胞可能会抑制机体对恶性细胞的免疫杀伤反应。而且已有研究表明，许多类型的癌细胞已经适应了调节性免疫细胞的表型，和调节性免疫细胞一样能够表达多种免疫抑制蛋白，抑制抗肿瘤特异性T细胞在肿瘤微环境中的活性。为了平衡调节性免疫细胞引起的免疫抑制，免疫系统中同时也形成了拮抗免疫抑制的机制。在肿瘤免疫微环境中，部分促炎T细胞能够靶向识别表达免疫抑制蛋白的调节性免疫细胞，减少调节性免疫细胞数目，拮抗肿瘤免疫抑制作用，并通过在炎症部位分泌促炎细胞因子辅助增强适应性免疫应答，我们将这类细胞定义为Anti-TIS细胞。
Anti-TIS(Tumor ImmunoSuppression)技术即是基于Anti-TIS细胞的作用机理所提出的一种创新型的免疫治疗方案，旨在激活人体抗肿瘤免疫抑制的细胞，靶向调节性免疫细胞和肿瘤细胞，在可接受的毒性范围内，打破免疫抑制，尽可能多地激活免疫反应，产生抗肿瘤治疗效果。
发明内容
需要说明的是，本公开是基于发明人的下列发现而完成的：
HLA是人类的主要组织相容性复合体(MHC)的表达产物，是具有高度多态性的同种异体抗原，不同地域人群的HLA等位分型会存在差异，例如欧美地区覆盖度较高的等位分型主要是HLA-A02:01，中国覆盖度最高的前两种等位分型是HLA-A11:01和HLA-A24:02。
细胞程序性死亡配体1(Programmed cell death 1ligand 1，PD-L1)也称为表面抗原分化簇274(cluster of differentiation 274，CD274)是人类体内的一种跨膜蛋白，与免疫系统的自我调节密切相关。细胞程序性死亡受体(PD-1)与癌细胞或免疫调节细胞表面的PD-L1结合，可以传导免疫抑制的信号，减低CD8 +T细胞的扩增和活化，减少淋巴结中抗原特异性T细胞的聚集，从而导致癌细胞的免疫逃逸。
有鉴于此，基于上述机制，发明人从众多癌症相关基因中选取PD-L1蛋白来筛选抗肿瘤免疫抑制的靶标。进一步地，PD-L1蛋白全长290个氨基酸，按照1步长位移滑动剪切，共计可得到8、9、10、11mer肽段1126条。发明人对所有的肽段进行分析，发现仅有极少数的肽段能够结合HLA-A11:01或HLA-A24:02分子形成HLA复合物，并与机体中的T细胞受体进一步结合，刺激机体产生免疫反应，而其余绝大多数肽段不具备HLA亲和力和免疫原性，不存在开发抗肿瘤免疫抑制疫苗的研究价值。因而，发明人从众多表位中筛选得到有免疫学功能的表位。这些多肽可以作为疫苗或药物，能够以免疫抑制蛋白PDL1表位作为靶点，提高肿瘤微环境中的Anti-TIS细胞活性、数目，特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞、表达PDL1的肿瘤细胞。另外，由于Anti-TIS细胞在体内自然存在，机体必然存在一种确保免疫稳态以控制它们的机制，因此触发自身免疫相关不良事件的风险极低。目前已开展的试验表明，接种了上述多肽的小鼠没有发生不良反应，而临床研究数据也证明了这种方法的安全性。由此，在预防和治疗癌症方面具有重要意义。
在本公开的一个方面，本公开提出了一种分离的多肽。根据本公开的实施例，所述分离的多肽具有如SEQ ID NO：1、3、5、7、8、9、11、13、14、17、18、19、21、24、25、27、28、30～34任一项所示的氨基酸序列或其功能类似物或与其具有至少60％同源性的氨基酸序列。上述多肽对HLA-A11:01和/或HLA-A24:02具有亲和力，能够自然呈递到细胞表面并与特定的免疫效应细胞结合，并特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，具备成为免疫治疗靶点的潜在价值，在预防和治疗癌症方面具有重要意义。
在本公开的另一方面，本公开提出了一种分离的核酸。根据本公开的实施例，所述分离的核酸编码前述所述多肽或其功能类似物。
在本公开的又一方面，本公开提出了一种抗原递呈细胞。根据本公开的实施例，所述 抗原递呈细胞用于递呈前面所述多肽。
在本公开的又一方面，本公开提出了一种免疫细胞。根据本公开的实施例，所述免疫细胞的结合靶标为前述分离的多肽或者前述抗原呈递细胞。
在本公开的又一方面，本公开提出了一种抗体。根据本公开的实施例，所述抗体特异性识别前述分离的多肽。
在本公开的又一方面，本公开提出了一种疫苗。根据本公开的实施例，所述疫苗包括：前述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体。
在本公开的又一方面，本公开提出了一种药物。根据本公开的实施例，所述药物包括：前述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体。
在本公开的又一方面，本公开提出了前述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体在制备疫苗中的用途。根据本公开的实施例，所述疫苗用于预防癌症。
在本公开的又一方面，本公开提出了前述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体在制备药物中的用途。根据本公开的实施例，所述药物用于治疗癌症。
在本公开的又一方面，本公开提出了一种预防或治疗癌症的方法。根据本公开的实施例，所述方法包括：向受试者施加前面所述分离的多肽、所述分离的核酸、所述抗原递呈细胞、所述免疫细胞和/或所述抗体。
本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1～图4分别显示了根据本公开实施例的多肽质谱图，质谱图的上半部分是eluted，下半部分是synthesis；
图5显示了根据本公开实施例的多肽与体外免疫原性ELISPOTs检测的电镜图；
图6和图7分别显示了根据本公开实施例的免疫细胞特异性杀伤呈递多肽的靶细胞分析示意图；
图8和图9分别显示了根据本公开实施例的多肽疫苗控制小鼠肿瘤生长分析示意图。
具体实施方式
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开， 而不能理解为对本公开的限制。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
在本公开的一个方面，本公开提出了一种分离的多肽。根据本公开的实施例，该分离的多肽具有如SEQ ID NO：1、3、5、7、8、9、11、13、14、17、18、19、21、24、25、27、28、30～34任一项所示的氨基酸序列或其功能类似物或与其具有至少60％(例如65％、70％、75％、85％、91％、92％、94％、96％、98％)同源性的氨基酸序列。上述多肽对HLA-A11:01和/或HLA-A24:02具有亲和力，能够自然呈递到细胞表面并与特定的免疫效应细胞结合，并特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，具备成为免疫治疗靶点的潜在价值，在预防和治疗癌症方面具有重要意义。
具体地，在上述多肽中，SEQ ID NO：1、3、5、7、8、11、14、17、24、32、33、34任一项所示的氨基酸序列或其功能类似物或与其具有至少60％同源性的氨基酸序列的多肽效果较佳。SEQ ID NO：3、5、8、24、32、34任一项所示的氨基酸序列或其功能类似物或与其具有至少60％同源性的氨基酸序列的多肽效果更佳，经鉴定能够表达并呈递于细胞表面的HLA分子，具有较强的免疫原性，具有刺激产生高杀伤效率特异性T细胞的能力。
在本公开的另一方面，本公开提出了一种分离的核酸。根据本公开的实施例，所述分离的核酸编码前述多肽或其互补序列。如前所述，上述本公开实施例所提出的核酸在合适条件下表达的多肽能够用于预防或治疗肿瘤，从而发挥免疫杀伤作用。进而，本公开实施例所提出的核酸在合适条件下表达的多肽能够用于预防或治疗肿瘤。
根据本公开的实施例，所述分离的核酸具有如SEQ ID NO：35、37、39、41、42、43、45、47、48、51、52、53、55、58、59、61、62、64～68任一项所示的核苷酸序列或其功能类似物或与其具有至少60％(例如65％、70％、75％、85％、91％、92％、94％、96％、98％)同源性的核酸序列。具体的，上述分离的核酸中，SEQ ID NO：35、37、39、41、42、45、48、51、58、66、67、68任一项所示的核苷酸序列或其功能类似物或与其具有至少60％同源性的核酸序列的核酸效果较佳，SEQ ID NO：37、39、42、58、66、68任一项 所示的核苷酸序列或其功能类似物或与其具有至少60％同源性的核酸序列的核酸效果更佳。
表1多肽和核酸序列
基因 氨基酸位点 SEQ ID 多肽序列 SEQ ID 核酸序列
PDL1 6-15 NO:1 VFIFMTYWHL NO:35 GTCTTTATATTCATGACCTACTGGCATTTG
PDL1 6-16 NO:2 VFIFMTYWHLL NO:36 GTCTTTATATTCATGACCTACTGGCATTTGCTG
PDL1 8-16 NO:3 IFMTYWHLL NO:37 ATATTCATGACCTACTGGCATTTGCTG
PDL1 8-17 NO:4 IFMTYWHLLN NO:38 ATATTCATGACCTACTGGCATTTGCTGAAC
PDL1 11-19 NO:5 TYWHLLNAF NO:39 ACCTACTGGCATTTGCTGAACGCATTT
PDL1 11-21 NO:6 TYWHLLNAFTV NO:40 ACCTACTGGCATTTGCTGAACGCATTTACTGTC
PDL1 15-25 NO:7 LLNAFTVTVPK NO:41 TTGCTGAACGCATTTACTGTCACGGTTCCCAAG
PDL1 17-25 NO:8 NAFTVTVPK NO:42 AACGCATTTACTGTCACGGTTCCCAAG
PDL1 32-41 NO:9 YGSNMTIECK NO:43 TATGGTAGCAATATGACAATTGAATGCAAA
PDL1 33-41 NO:10 GSNMTIECK NO:44 GGTAGCAATATGACAATTGAATGCAAA
PDL1 36-46 NO:11 MTIECKFPVEK NO:45 ATGACAATTGAATGCAAATTCCCAGTAGAAAAA
PDL1 37-46 NO:12 TIECKFPVEK NO:46 ACAATTGAATGCAAATTCCCAGTAGAAAAA
PDL1 79-89 NO:13 SSYRQRARLLK NO:47 AGTAGCTACAGACAGAGGGCCCGGCTGTTGAAG
PDL1 97-105 NO:14 AALQITDVK NO:48 GCTGCACTTCAGATCACAGATGTGAAA
PDL1 105-113 NO:15 KLQDAGVYR NO:49 AAATTGCAGGATGCAGGGGTGTACCGC
PDL1 110-118 NO:16 GVYRCMISY NO:50 GGGGTGTACCGCTGCATGATCAGCTAT
PDL1 116-124 NO:17 ISYGGADYK NO:51 ATCAGCTATGGTGGTGCCGACTACAAG
PDL1 116-125 NO:18 ISYGGADYKR NO:52 ATCAGCTATGGTGGTGCCGACTACAAGCGA
PDL1 117-126 NO:19 SYGGADYKRI NO:53 AGCTATGGTGGTGCCGACTACAAGCGAATT
PDL1 120-129 NO:20 GADYKRITVK NO:54 GGTGCCGACTACAAGCGAATTACTGTGAAA
PDL1 126-136 NO:21 ITVKVNAPYNK NO:55 ATTACTGTGAAAGTCAATGCCCCATACAACAAA
PDL1 127-136 NO:22 TVKVNAPYNK NO:56 ACTGTGAAAGTCAATGCCCCATACAACAAA
PDL1 129-136 NO:23 KVNAPYNK NO:57 AAAGTCAATGCCCCATACAACAAA
PDL1 133-141 NO:24 PYNKINQRI NO:58 CCATACAACAAAATCAACCAAAGAATT
PDL1 153-162 NO:25 LTCQAEGYPK NO:59 CTGACATGTCAGGCTGAGGGCTACCCCAAG
PDL1 159-167 NO:26 GYPKAEVIW NO:60 GGCTACCCCAAGGCCGAAGTCATCTGG
PDL1 168-178 NO:27 TSSDHQVLSGK NO:61 ACAAGCAGTGACCATCAAGTCCTGAGTGGTAAG
PDL1 169-178 NO:28 SSDHQVLSGK NO:62 AGCAGTGACCATCAAGTCCTGAGTGGTAAG
PDL1 181-189 NO:29 TTNSKREEK NO:63 ACCACCAATTCCAAGAGAGAGGAGAAG
PDL1 189-198 NO:30 KLFNVTSTLR NO:64 AAGCTTTTCAATGTGACCAGCACACTGAGA
PDL1 202-212 NO:31 TTNEIFYCTFR NO:65 ACAACTAATGAGATTTTCTACTGCACTTTTAGG
PDL1 252-260 NO:32 GVALTFIFR NO:66 GGTGTAGCACTGACATTCATCTTCCGT
PDL1 255-263 NO:33 LTFIFRLRK NO:67 CTGACATTCATCTTCCGTTTAAGAAAA
PDL1 273-281 NO:34 GIQDTNSKK NO:68 GGCATCCAAGATACAAACTCAAAGAAG
需要说明的是，对于本公开说明书和权利要求书中所提及的核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本 申请中的基因序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。
在本公开的又一方面，本公开提出了一种抗原递呈细胞。根据本公开的实施例，所述抗原递呈细胞用于呈递前面所述分离的核酸。由此，利用本公开的抗原递呈细胞可以将多肽递送至抗肿瘤特异性T细胞上，从而激活T细胞特异性杀伤功能，有效地用于肿瘤的预防或治疗。
根据本公开的实施例，所述抗原递呈细胞为树突状细胞、B细胞或单核-吞噬细胞，优选树突细胞。树突细胞具有极强的抗原内吞和加工处理能力，可将抗原呈递在细胞的表面。发明人选择树突细胞作为抗原呈递细胞，抗原呈递细胞在机体内启动、调节和维持针对所述多肽的免疫反应更加强烈。
根据本公开的实施例，所述抗原递呈细胞表达HLA-A11:01和/或HLA-A24:02。上述多肽对HLA-A11:01和/或HLA-A24:02具有亲和力，能够自然呈递并与特定的免疫效应细胞结合，并特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，具备成为免疫治疗靶点的潜在价值，在预防和治疗癌症方面具有重要意义。
在本公开的又一方面，本公开提出了一种免疫细胞。根据本公开的实施例，所述免疫细胞的结合靶标为前面所述的分离的多肽或者前面所述抗原呈递细胞。由此，可以发挥免疫杀伤功能。
根据本公开的实施例，所述免疫细胞是通过下列方式获得的：将前面所述的抗原呈递细胞与具有免疫效应能力的细胞接触，或从肿瘤组织中分选培养(如肿瘤浸润T细胞，Tumor-infiltrating lymphocytes，TILs)，或将能识别前面所述分离的多肽的T细胞受体(T cell recepter，TCR)基因转入T细胞(如TCR-T)。所具体地，所述具有免疫效应能力的细胞为CD8 +T细胞。通过呈递前面所述的分离的肽的抗原呈递细胞与具有免疫效应能力的细胞接触，抗原呈递细胞可活化具有免疫效应能力的未激活细胞，递呈抗原-前面所述的多肽，进而激活具有免疫效应能力的细胞，大量产生免疫效应细胞，该免疫效应细胞具有特异性杀伤呈递抗原-所述多肽的靶细胞的作用。CD8 +T细胞接受抗原呈递细胞激活作用的能力更强，获得的CD8 +T细胞的特异性杀伤呈递抗原-所述分离的肽的靶细胞的作用更强。
在本公开的又一方面，本公开提出了一种抗体。根据本公开的实施例，所述抗体特异性识别前面所述的分离的多肽。由此，可特异性识别肿瘤抗原。根据本公开的实施例，公开发现，所述抗体可特异性识别抗原，与肿瘤细胞或被病毒(如HBV、HCV、HIV、EBV等)感染的细胞特异性结合，进而使得肿瘤细胞或被病毒感染的细胞被吞噬细胞吞噬，实现对肿瘤细胞或被病毒感染的细胞的特异性清除。
在本公开的又一方面，本公开提出了一种疫苗。根据本公开的实施例，所述疫苗包括：前面所述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体。由此，根据本公 开实施例的疫苗进入机体内，可以特异性激活特异性T细胞(如CTL、TIL)，达到特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，安全性好，在预防癌症方面具有重要意义。
根据本公开的实施例，所述疫苗进一步包括佐剂。本公开对于佐剂的类型不做严格限定，可以选择本领域常规佐剂，具体可以根据实际需要灵活选择。
在本公开的又一方面，本公开提出了一种药物。根据本公开的实施例，所述药物包括：前面所述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体。由此，根据本公开实施例的药物进入机体内，可以特异性激活特异性T细胞(如CTL、TIL)，达到特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，安全性好，在治疗癌症方面具有重要意义。
根据本公开的实施例，所述药物进一步包括药学上可接受的辅料。
在本公开的又一方面，本公开提出了前述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体在制备疫苗中的用途。根据本公开的实施例，所述疫苗用于预防癌症。疫苗进入机体内，可以特异性激活特异性T细胞(如CTL、TIL)，达到特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，在预防癌症方面具有重要意义。
相应地，本公开提出一种预防癌症的方法，包括：向受试者施用前述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体。
根据本公开的实施例，所述疫苗的受试者表达HLA-A11:01和/或HLA-A24:02。如前所述，上述多肽对HLA-A11:01和/或HLA-A24:02具有亲和力，能够自然呈递到细胞表面并与特定的免疫效应细胞结合，并特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，安全性高，在预防癌症方面具有重要意义。
在本公开的又一方面，本公开提出了前述分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞和/或抗体在制备药物中的用途。根据本公开的实施例，所述药物用于治疗癌症。药物进入机体内，可以特异性激活特异性T细胞(如CTL、TIL)，达到特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，在治疗癌症方面具有重要意义。
根据本公开的实施例，所述癌症为乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、黑色素瘤、皮肤癌、前列腺癌、宫颈癌、白血病、甲状腺癌、淋巴瘤、膀胱癌、肾癌、子宫体癌、卵巢癌、胆囊癌、口腔癌、喉癌、骨癌、睾丸癌或脑癌。
根据本公开的实施例，所述药物的受试者表达HLA-A11:01和/或HLA-A24:02。如前所述，上述多肽对HLA-A11:01和/或HLA-A24:02具有亲和力，能够自然呈递到细胞表面并 与特定的免疫效应细胞结合，并特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，安全性高，在治疗癌症方面具有重要意义。
在本公开的又一方面，本公开提出了一种预防或治疗癌症的方法。根据本公开的实施例，所述方法包括：向受试者施加前面所述分离的多肽、所述分离的核酸、所述抗原递呈细胞、所述免疫细胞和/或所述抗体。如前所述的分离的多肽、分离的核酸、抗原递呈细胞、免疫细胞、抗体进入机体内，可以特异性激活特异性T细胞(如CTL、TIL)，达到特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，在治疗癌症方面具有重要意义。
根据本公开的实施例，所述疫苗的受试者表达HLA-A11:01和/或HLA-A24:02。如前所述，上述多肽对HLA-A11:01和/或HLA-A24:02具有亲和力，能够自然呈递到细胞表面并与特定的免疫效应细胞结合，并特异性清除肿瘤微环境中基质细胞、免疫细胞等负向调节的细胞以及表达PD-L1的肿瘤细胞，安全性高，在治疗癌症方面具有重要意义。
根据本公开的实施例，所述癌症为乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、黑色素瘤、皮肤癌、前列腺癌、宫颈癌、白血病、甲状腺癌、淋巴瘤、膀胱癌、肾癌、子宫体癌、卵巢癌、胆囊癌、口腔癌、喉癌、骨癌、睾丸癌或脑癌。
下面将结合实施例对本公开的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1多肽的免疫原性预测
本公开根据选定的HLA-A11:01和HLA-A24:02等位基因分型，利用生物信息分析流程将PDL1蛋白全长切割成8-11mer氨基酸的多肽序列，PDL1基因编码的蛋白的长度为290个氨基酸，分子量大小为33275道尔顿，切割的多肽序列1126条。对这1126条多肽分别进行呈递能力预测和亲和力预测。呈递能力打分值用0-1的数值表示，打分值越高表明呈递能力越强，打分值大于0.1则表示该多肽具有呈递能力。亲和力打分值用IC50分值表示，IC50小于500表示该多肽具有亲和力，IC50小于50表示该多肽具有高亲和力。筛选条件是满足以下两个条件之一：(1)呈递打分值>0.5；(2)呈递打分值>0.1且IC50<500。满足条件的多肽序列(表2和表3)进行下一步T2亲和力验证。
表2多肽序列与HLA-A11:01等位的呈递能力和亲和力预测得分
SEQ ID 基因 氨基酸位点 多肽序列 HLA分型 呈递打分值 亲和力打分值
NO:7 PDL1 15-25 LLNAFTVTVPK HLA-A11:01 0.2182 89.9590
NO:8 PDL1 17-25 NAFTVTVPK HLA-A11:01 0.7874 15.0000
NO:9 PDL1 32-41 YGSNMTIECK HLA-A11:01 0.1470 375.2523
NO:10 PDL1 33-41 GSNMTIECK HLA-A11:01 0.3712 34.6272
NO:11 PDL1 36-46 MTIECKFPVEK HLA-A11:01 0.5907 6.7074
NO:12 PDL1 37-46 TIECKFPVEK HLA-A11:01 0.1814 31.6022
NO:13 PDL1 79-89 SSYRQRARLLK HLA-A11:01 0.4621 38.1700
NO:14 PDL1 97-105 AALQITDVK HLA-A11:01 0.5077 90.5887
NO:15 PDL1 105-113 KLQDAGVYR HLA-A11:01 0.2106 73.2814
NO:16 PDL1 110-118 GVYRCMISY HLA-A11:01 0.2449 13.5977
NO:17 PDL1 116-124 ISYGGADYK HLA-A11:01 0.2952 8.1088
NO:18 PDL1 116-125 ISYGGADYKR HLA-A11:01 0.1865 71.6428
NO:20 PDL1 120-129 GADYKRITVK HLA-A11:01 0.1239 58.2933
NO:21 PDL1 126-136 ITVKVNAPYNK HLA-A11:01 0.3871 85.5169
NO:22 PDL1 127-136 TVKVNAPYNK HLA-A11:01 0.3067 23.8387
NO:23 PDL1 129-136 KVNAPYNK HLA-A11:01 0.1047 246.4766
NO:25 PDL1 153-162 LTCQAEGYPK HLA-A11:01 0.3360 15.1653
NO:27 PDL1 168-178 TSSDHQVLSGK HLA-A11:01 0.3165 37.5170
NO:28 PDL1 169-178 SSDHQVLSGK HLA-A11:01 0.4771 56.6550
NO:29 PDL1 181-189 TTNSKREEK HLA-A11:01 0.3763 36.1821
NO:30 PDL1 189-198 KLFNVTSTLR HLA-A11:01 0.2815 25.7653
NO:31 PDL1 202-212 TTNEIFYCTFR HLA-A11:01 0.1389 122.2114
NO:32 PDL1 252-260 GVALTFIFR HLA-A11:01 0.6331 18.2811
NO:33 PDL1 255-263 LTFIFRLRK HLA-A11:01 0.2900 6.6192
NO:34 PDL1 273-281 GIQDTNSKK HLA-A11:01 0.5256 24.7917
表3多肽序列与HLA-A24:02等位的呈递能力和亲和力预测得分
SEQ ID 基因 氨基酸位点 多肽序列 HLA分型 呈递打分值 亲和力打分值
NO:1 PDL1 6-15 VFIFMTYWHL HLA-A24:02 0.3786 11.7606
NO:2 PDL1 6-16 VFIFMTYWHLL HLA-A24:02 0.1079 22.6598
NO:3 PDL1 8-16 IFMTYWHLL HLA-A24:02 0.8124 8.3178
NO:4 PDL1 8-17 IFMTYWHLLN HLA-A24:02 0.1026 69.4832
NO:5 PDL1 11-19 TYWHLLNAF HLA-A24:02 0.9231 7.5832
NO:6 PDL1 11-21 TYWHLLNAFTV HLA-A24:02 0.1202 28.6888
NO:19 PDL1 117-126 SYGGADYKRI HLA-A24:02 0.2008 155.6688
NO:24 PDL1 133-141 PYNKINQRI HLA-A24:02 0.8545 17.0088
NO:26 PDL1 159-167 GYPKAEVIW HLA-A24:02 0.1978 74.1338
实施例2多肽T2亲和力验证
取2×10 5个T2细胞铺用500μL含有人类β 2微球蛋白(最终浓度，3μg/ml)的IMDM无血清培养基重悬到24孔板里，加入表1中的多肽(最终浓度100μM)，在培养箱(37℃,5％CO 2)，培养过夜。每个组2个复孔；没有加多肽的T2细胞被用作背景对照。将细胞200g离心5分钟收集细胞。细胞用PBS洗涤两次后，将细胞直接用抗HLA-A11:01/HLA-A24:02的FITC单克隆抗体孵育，4℃维持30分钟。然后用流式细胞仪进行分析。荧光指数(FI)用下列公式计算：FI＝[平均荧光强度(MFI)样品-MFI background]/MFI background，其中 MFI background代表不含肽的值。FI>1.5表明该肽具有用于HLA-A11:01或HLA-A24:02分子的高亲和性，1.0<FI<1.5表明该肽具有对HLA-A11:01或HLA-A24:02分子中等亲和力，以及0.5<FI<1.0表明该肽为HLA-A11:01或HLA-A24:02分子低亲和力。
结果如下表所示，选择T2亲和力验证具有高亲和力的12条多肽进行后续的质谱呈递验证、体外刺激扩增CD8 +T细胞和ELISPOTs方法验证。
表4多肽序列与HLA等位的亲和力的检测结果
样本 SEQ ID 加入多肽浓度 平均荧光强度 FI 结论
VFIFMTYWHL NO:1 100μM 690 3.57 高亲和力
VFIFMTYWHLL NO:2 100μM 11 -0.93 无亲和力
IFMTYWHLL NO:3 100μM 970 5.42 高亲和力
IFMTYWHLLN NO:4 100μM 103 -0.32 无亲和力
TYWHLLNAF NO:5 100μM 1000 5.62 高亲和力
TYWHLLNAFTV NO:6 100μM 131 -0.13 无亲和力
LLNAFTVTVPK NO:7 100μM 531 2.51 高亲和力
NAFTVTVPK NO:8 100μM 540 2.57 高亲和力
YGSNMTIECK NO:9 100μM 236 0.56 低亲和力
GSNMTIECK NO:10 100μM 102 -0.32 无亲和力
MTIECKFPVEK NO:11 100μM 540 2.57 高亲和力
TIECKFPVEK NO:12 100μM 226 0.50 无亲和力
SSYRQRARLLK NO:13 100μM 299 0.98 低亲和力
AALQITDVK NO:14 100μM 738 3.89 高亲和力
KLQDAGVYR NO:15 100μM 3 -0.98 无亲和力
GVYRCMISY NO:16 100μM 31 -0.80 无亲和力
ISYGGADYK NO:17 100μM 574 2.80 高亲和力
ISYGGADYKR NO:18 100μM 348 1.31 中等亲和力
SYGGADYKRI NO:19 100μM 341 1.26 中等亲和力
GADYKRITVK NO:20 100μM 138 -0.09 无亲和力
ITVKVNAPYNK NO:21 100μM 376 1.49 中等亲和力
TVKVNAPYNK NO:22 100μM 39 -0.74 无亲和力
KVNAPYNK NO:23 100μM 90 -0.41 无亲和力
PYNKINQRI NO:24 100μM 990 5.55 高亲和力
LTCQAEGYPK NO:25 100μM 304 1.01 中等亲和力
GYPKAEVIW NO:26 100μM 29 -0.81 无亲和力
TSSDHQVLSGK NO:27 100μM 237 0.57 低亲和力
SSDHQVLSGK NO:28 100μM 344 1.28 中等亲和力
TTNSKREEK NO:29 100μM 226 0.50 无亲和力
KLFNVTSTLR NO:30 100μM 294 0.95 低亲和力
TTNEIFYCTFR NO:31 100μM 373 1.47 中等亲和力
GVALTFIFR NO:32 100μM 745 3.93 高亲和力
LTFIFRLRK NO:33 100μM 681 3.51 高亲和力
GIQDTNSKK NO:34 100μM 852 4.64 高亲和力
Blank   0μM 151 0.00 无亲和力
CMV   100μM 672 3.45 高亲和力
实施例3质谱实验验证多肽在肿瘤细胞表面被HLA分子呈递
本公开通过免疫共沉淀-质谱联合的方式，富集细胞表面的多肽-MHC复合体，对肿瘤细胞表面的MHC分子是否呈递了所述多肽进行了鉴定。具体方法如下：
1)MHC-I限制性T细胞表位肽的分离与纯化：使用pan-MHC-I A/B/C抗体(克隆号：w6/32)与表面偶联protein A分子的sepharose CL-4B beads在4℃结合1小时，使用NanoDrop检测上清残余的抗体含量，抗体结合率>90％视为合格，制备pan-MHC-I A/B/C结合sepharose，4℃备用。SKMEL5及HCT8细胞样本中分别加入40ml RIPA裂解液，4℃孵育1小时，12000rpm离心30min，上清加入sepharose CL-4B beads进行预杂交，4℃孵育1小时；离心去除beads，上清加入pan-MHC-A/B/C结合sepharose CL-4B beads，4℃孵育过夜(16-18小时)。使用4℃预冷的PBS洗涤beads，重复三次；使用超纯水洗涤beads；离心去除洗涤液，使用0.1N acetic acid洗脱beads表面的抗体-MHC-I蛋白复合物，抗体-MHC-I蛋白复合在酸性条件下解离，进一步，使用3kDa超滤管或C18固相萃取柱(25mg，waters)对洗脱产物中的蛋白和多肽进行分离纯化，使用冷冻真空离心机对纯化产物进行浓缩，浓缩产物-20℃保存至质谱上机。
2)MHC-I限制性T细胞表位肽的质谱鉴定：浓缩的MHC-I限制性表位肽溶液通过在线连接nanoflow HPLC(Thermo Fisher Scientific)的Q Exactive质谱仪(Thermo Fisher Scientific)进行分析，采用ReproSil-Pur C18-AQ 1.9um填料手工填装长15cm，内径75um的分离柱进行分离，使用线性梯度2-30％的buffer B(80％ACN/0.5％acetic acid)洗脱多肽，流速设置250nl/min，洗脱时间90min。二级质谱采用HCD进行碎片化，数据采集选择数据依赖的“Top 20”方法。MS图谱的采集分辨率为70,000，200m/z，目标值为3E6离子；离子强度排行前10的离子通常采用最大注射时间为120ms进行分离和累积直至自动增益控制器的数值显示为1E5。多肽匹配选项设置“disable”，MS/MS分辨率设置17,500(200m/z)。
3)MHC-I限制性T细胞表位肽的质谱数据分析：数据分析采用MaxQuant(version1.3.10.15)比对质谱图谱和人全蛋白库(Uniprot，86,749个蛋白)、肿瘤相关抗原、肿瘤特异性突变肽段以及一个包含247个常见污染物(角蛋白、牛血清蛋白和蛋白酶)的数据集生成的图谱列表。可变修饰检测设置：N端乙酰化和甲硫氨酸氧化。第二位多肽鉴定设置：enable；特异性酶切设置：unspecific；多肽鉴定FDR(false discovery rate)设置1％，蛋白鉴定FDR不设置；序列匹配长度限制设置为8-15aa，最大多肽质量设置为1500Da，最大电荷状态设置为3。前导离子的初始允许质量偏差设置为6ppm，最大碎片质量偏差设置为20ppm。“match between runs”设置开启。鉴定结果输出保存在“peptide.txt”文件中，去 除匹配到反库和污染库中的多肽，其余为MHC-I限制性表位的鉴定结果。
结果显示，表4的12条高亲和力多肽中，有11条可以在本实验中鉴定表达并呈递于细胞表面的HLA分子。相应多肽的质谱谱图见图1～4。
实施例4多肽体外刺激扩增CD8 +T细胞
取HLA-A11:01亚型阳性健康志愿者的PBMC细胞，2×10 6个PBMC细胞，用贴壁法分离单核细胞(贴3h)，以及CD8磁珠的方法分离得到CD8 +的T细胞。采用GM-CSF(1000U/ml)，IL-4(1000U/ml)，诱导贴壁单核细胞为未成熟DC，再用IFN-gamma(100U/ml)，LPS(10ng/ml)，以及表4具有高亲和力的多肽序列诱导贴壁细胞为多肽特异性成熟DC细胞。将负载过多肽的成熟DC细胞辐照，并与同一志愿者的CD8 +T细胞共培养，并加入IL-21,3天后，补加IL-2和IL-7，以后于第5，7天补加一次IL-2和IL-7，第10天取共培养的细胞进行计数，和后续的ELISPOTs以及LDH检测。HLA-A24:02亚型也是进行如上操作。计数结果如下，由此表明，表5和表6中的多肽均可在体外刺激扩增CD8 +T细胞。
表5 HLA-A11:01分型多肽培养后计数结果
多肽序列 HLA分型 培养前细胞总数 培养后细胞总数
LLNAFTVTVPK HLA-A11:01 2.0×10^6 7.87×10^6
NAFTVTVPK HLA-A11:01 2.0×10^6 9.05×10^6
MTIECKFPVEK HLA-A11:01 2.0×10^6 8.28×10^6
AALQITDVK HLA-A11:01 2.0×10^6 8.32×10^6
ISYGGADYK HLA-A11:01 2.0×10^6 8.40×10^6
GVALTFIFR HLA-A11:01 2.0×10^6 8.30×10^6
LTFIFRLRK HLA-A11:01 2.0×10^6 9.87×10^6
GIQDTNSKK HLA-A11:01 2.0×10^6 9.82×10^6
表6 HLA-A24:02分型多肽培养后计数结果
多肽序列 HLA分型 培养前细胞总数 培养后细胞总数
VFIFMTYWHL HLA-A24:02 2.0×10^6 7.51×10^6
IFMTYWHLL HLA-A24:02 2.0×10^6 9.95×10^6
TYWHLLNAF HLA-A24:02 2.0×10^6 8.29×10^6
PYNKINQRI HLA-A24:02 2.0×10^6 9.87×10^6
实施例5 ELISPOTs方法验证多肽激活CD8 +T细胞免疫反应
将实施例4中培养的T细胞与负载过实验多肽或无关多肽(ALADGVQKV)的T2分别加入到人IFN-gamma干扰素ELISPOTs板中进行培养，20小时后，依据ELISPOTs检测(见试剂盒说明书)对ELISPOT实验产生的斑点进行计数。实验多肽具有免疫原性的要求如下：斑点数(多肽)/斑点数(无关多肽)≥2，即实验多肽引起的斑点数超过无关多肽斑点数目的两倍及以上。
ELISPOTs检测方法原理：由于T2细胞表达HLA-A11:01/HLA-A24:02，因此，T2细胞可以向T细胞提呈HLA-A11:01/HLA-A24:02抗原肽。CD8 +T细胞特异识别HLA-A11:01/HLA-A24:02和多肽的复合物之后，T细胞能再次激活并分泌IFN-gamma干扰素。而CD8 +T细胞被激活分泌的IFN-gamma干扰素可以被ELISPOTs板子上的抗体所捕获，识别IFN-gamma的抗体可以通过偶联在抗体上的酶，降解底物显色，最终产生斑点。斑点的数目代表了被激活分泌IFN-gamma干扰素的细胞数目。ELISPOTs结果见图5和表7。累计有7条多肽的实验组斑点数超过无关组斑点数的2倍及以上，具有较强的免疫原性。
表7多肽刺激特异性CD8 +T细胞分泌IFN-gamma干扰素
SEQ ID 多肽序列 多肽斑点数 无关多肽斑点数 倍数(实验/无关)
NO:1 VFIFMTYWHL 18±2.72 22±1.41 0.82
NO:3 IFMTYWHLL 52±3.79 22±1.41 2.36
NO:5 TYWHLLNAF 49±0.18 22±1.41 2.23
NO:7 LLNAFTVTVPK 14±0.61 22±1.41 0.64
NO:8 NAFTVTVPK 65±4.32 22±1.41 2.95
NO:11 MTIECKFPVEK 36±3.37 22±1.41 1.64
NO:14 AALQITDVK 47±1.51 22±1.41 2.14
NO:17 ISYGGADYK 15±3.90 22±1.41 0.68
NO:24 PYNKINQRI 48±3.90 22±1.41 2.18
NO:32 GVALTFIFR 64±4.77 22±1.41 2.91
NO:33 LTFIFRLRK 38±3.86 22±1.41 1.73
NO:34 GIQDTNSKK 60±3.94 22±1.41 2.73
实施例6 LDH释放实验证明CD8 +T细胞多肽特异性杀伤活性
将实施例4中培养的细胞与负载过实验多肽或无关多肽或未负载多肽的T2细胞进行共培养，实验中设置最大释放孔、体积校正孔、培养基对照孔、自发释放孔、不同效靶比(T细胞与T2细胞的数目比)作为对照，每组设置3个复孔，4h后，取出共培养的细胞上清 50μl，并加入到50μl LDH底物混合液中，使细胞上清催化LDH底物反应，最终读取490nm波长和680nm参考波长，并根据对照孔，按照下列公式计算靶细胞杀伤T2的杀伤活性。
杀伤活性计算公式为：杀伤效率(％)＝(实验孔-效应细胞自发释放-靶细胞自发释放+培养基孔)/(靶细胞最大释放-体积校正孔-靶细胞自发释放+培养基孔)×100％。
结果见表8、图6和图7，前述具备免疫原性的7条多肽，其相对应的7组特异性T细胞在E:T＝20:1组的杀伤效率显著高于无关肽组及不负载肽组，且杀伤效率均超过50％，表明这7条多肽具有刺激产生高杀伤效率特异性T细胞的能力。
表8 T细胞特异性识别并杀伤呈递实验多肽的靶细胞
  E:T＝1:1 E:T＝10:1 E:T＝20:1
T2 -2.62％ 6.28％ 14.99％
T2+Irrelevant 2.03％ 8.25％ 16.56％
T2+VFIFMTYWHL 4.06％ 8.32％ 12.84％
T2 0.79％ 4.21％ 10.66％
T2+Irrelevant 2.61％ 7.40％ 18.51％
T2+IFMTYWHLL 6.73％ 34.30％ 77.67％
T2 -2.49％ 4.89％ 10.70％
T2+Irrelevant 0.27％ 12.50％ 15.22％
T2+TYWHLLNAF 8.65％ 42.66％ 74.54％
T2 -1.37％ 8.83％ 14.78％
T2+Irrelevant -0.06％ 5.39％ 15.51％
T2+LLNAFTVTVPK 3.38％ 9.66％ 11.98％
T2 2.06％ 5.28％ 12.47％
T2+Irrelevant 4.66％ 10.02％ 13.33％
T2+NAFTVTVPK 9.48％ 29.29％ 60.41％
T2 -2.09％ 4.20％ 10.40％
T2+Irrelevant -1.68％ 6.21％ 10.68％
T2+MTIECKFPVEK 2.03％ 9.76％ 11.63％
T2 0.06％ 5.93％ 10.57％
T2+Irrelevant 3.59％ 9.18％ 18.69％
T2+AALQITDVK 8.79％ 31.20％ 58.98％
T2 -0.61％ 4.65％ 10.98％
T2+Irrelevant 0.71％ 6.78％ 15.13％
T2+ISYGGADYK 3.17％ 8.69％ 14.22％
T2 -4.90％ 6.22％ 10.47％
T2+Irrelevant 2.45％ 12.48％ 18.35％
T2+PYNKINQRI 9.96％ 39.50％ 57.71％
T2 -0.76％ 6.78％ 9.27％
T2+Irrelevant 1.88％ 8.76％ 15.89％
T2+GVALTFIFR 7.78％ 22.31％ 52.78％
T2 -0.53％ 5.40％ 10.43％
T2+Irrelevant 1.62％ 6.88％ 12.74％
T2+LTFIFRLRK 2.40％ 9.01％ 16.77％
T2 -2.20％ 4.95％ 10.42％
T2+Irrelevant 2.32％ 6.65％ 19.87％
T2+GIQDTNSKK 7.97％ 32.55％ 59.97％
实施例7多肽疫苗的制备及治疗方案
1、构建并包装表达多肽的重组慢病毒
合成多肽对应的DNA序列，并分别构建对应的慢病毒载体pHBLV-Puro。将慢病毒质粒与pSPAX2和pMD2G辅助质粒共同转染293T细胞，并包装出多肽的慢病毒。
2、表达多肽的人源细胞系的建立
人黑色素瘤细胞系SKMEL5购买于ATCC(编号：HTB-70)，其HLA亚型为HLA-A11:01阳性。人结直肠癌细胞系HCT8购买于ATCC(编号:CCL-244)，其HLA亚型为HLA-A24:02。细胞培养于含10％胎牛血清，100U/mL青霉素和链霉素的DMEM培养基中。37℃，5％CO 2的孵箱中培养。将包装好的慢病毒分别转染SKMEL5和HCT8细胞系，并采用Puromycin抗生素(嘌吟霉素)，持续筛选存活的SKMEL5和HCT8细胞系，最终建立表达多肽的SKMEL5和HCT8细胞系。
3、NOD/SCID小鼠人免疫重建
采集健康志愿者抗凝外周血600～900ml。Ficoll分离外周血单个核细胞(peripheral blood mononuclear,PBMC)，收集细胞待用。300只排除免疫渗漏的NOD SCID小鼠，每只腹腔注射PBMC 2×10 7/0.5ml，对NOD SCID小鼠进行人免疫重建。选取4周后免疫重建 成功的小鼠准备接种人黑色素瘤细胞系和人结直肠癌细胞系模型。
4、人黑色素瘤和人结直肠癌肿瘤模型的构建
已建系的人黑色素瘤细胞系和人结直肠癌细胞系，培养于含10％胎牛血清，100U/mL青霉素和链霉素的DMEM培养基中。37℃,5％CO 2的孵箱中培养。收集肿瘤细胞，200g/min离心，用无菌生理盐水洗涤肿瘤细胞3次。做适当稀释，取40微升细胞悬液加入10微升0.4％台酚蓝染色并镜检计数，制成浓度为1×10 8个/ml的肿瘤细胞悬液，选取免疫重建后的NOD/SCID小鼠，每只小鼠皮下接种肿瘤细胞悬液100ml。接种完成后，逐日观察接种部位有无感染，肿瘤生长后有无自然消退。7天后，小鼠皮下瘤可摸到约米粒大小肿瘤，表明SKMEL5或HCT8皮下瘤模型NOD/SCID小鼠模型构建成功。
5、多肽疫苗的制备
(1)选择实施例3和实施例5中等位分型为HLA-A11:01，并且具有双阳性结果(呈递验证阳性且免疫原性验证阳性)的3条多肽序列：NAFTVTVPK(SEQ ID NO:8)、GVALTFIFR(SEQ ID NO:32)和GIQDTNSKK(SEQ ID NO:34)进行多肽疫苗的制备。
将免疫重建4周的SKMEL5皮下瘤模型NOD/SCID小鼠随机分为6组：佐剂组，佐剂+无关多肽组、佐剂+NAFTVTVPK多肽组、佐剂+GVALTFIFR多肽组、佐剂+GIQDTNSKK多肽组和佐剂+多肽(多肽序列为SEQ ID NO:8、32和34)组合组，每组各8只。无关多肽组和多肽组合的首次免疫剂量为100ml/只。上述多肽用PBS重悬后，与150ml/只弗氏完全佐剂混匀后，用PBS调整至300ml/只，于背部皮下双点注射。2周后，使用相同剂量进行加强免疫(第1次使用完全弗氏佐剂，以后均用不完全弗氏佐剂)，共免疫4次。注射结束后观察小鼠生命体征，每3-4天用游标卡尺测量肿瘤纵横大小。肿瘤体积计算为:肿瘤体积＝1/2*长*宽 2。同时，记录小鼠体重变化情况。结果见图8中的A和B。
(2)选择实施例3和实施例5中等位分型为HLA-A24:02，并且具有双阳性结果(呈递验证阳性且免疫原性验证阳性)的3条多肽序列：IFMTYWHLL(SEQ ID NO:3)、TYWHLLNAF(SEQ ID NO:5)和PYNKINQRI(SEQ ID NO:24)进行多肽疫苗的制备。
将免疫重建4周的HCT8皮下瘤模型NOD/SCID小鼠随机分为6组：佐剂组，佐剂+无关多肽组、佐剂+IFMTYWHLL多肽组、佐剂+TYWHLLNAF多肽组、佐剂+PYNKINQRI多肽组和佐剂+多肽(多肽序列为SEQ ID NO:3、5和24)组合组，每组各8只。无关多肽组和多肽组合的首次免疫剂量为100ml/只。上述多肽用PBS重悬后，与150ml/只弗氏完全佐剂混匀后，用PBS调整至300ml/只，于背部皮下双点注射。2周后，使用相同剂量进行加强免疫(第1次使用完全弗氏佐剂，以后均用不完全弗氏佐剂)，共免疫4次。注射结束后观察小鼠生命体征，每3-4天用游标卡尺测量肿瘤纵横大小。肿瘤体积计算为:肿瘤体 积＝1/2*长*宽 2。同时，记录小鼠体重变化情况。结果见图9中的C和D。
结果显示，相对于无关多肽负载的多肽疫苗组和佐剂组，佐剂+核心多肽或多肽组合负载的多肽疫苗组可以明显的减缓小鼠肿瘤的生长，并延长小鼠的生存期。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (20)

  1. 一种分离的多肽，其特征在于，所述分离的多肽具有如SEQ ID NO：1、3、5、7、8、9、11、13、14、17、18、19、21、24、25、27、28、30～34任一项所示的氨基酸序列或其功能类似物或与其具有至少60％同源性的氨基酸序列。
  2. 一种分离的核酸，其特征在于，所述分离的核酸编码权利要求1所述多肽。
  3. 根据权利要求2所述的分离的核酸，其特征在于，所述分离的核酸具有如SEQ ID NO：35、37、39、41、42、43、45、47、48、51、52、53、55、58、59、61、62、64～68任一项所示的核苷酸序列或其功能类似物或与其具有至少60％同源性的核苷酸序列。
  4. 一种抗原递呈细胞，其特征在于，所述抗原递呈细胞用于呈递权利要求1所述分离的多肽。
  5. 根据权利要求4所述抗原递呈细胞，其特征在于，所述抗原递呈细胞为树突状细胞、B细胞或单核-吞噬细胞；
    所述抗原递呈细胞表达HLA-A11:01和/或HLA-A24:02。
  6. 一种免疫细胞，其特征在于，所述免疫细胞的结合靶标为权利要求1所述的分离的多肽或者权利要求4或5所述抗原呈递细胞。
  7. 根据权利要求6所述的免疫细胞，其特征在于，所述免疫细胞是通过下列方式获得的：
    将权利要求4或5所述的抗原呈递细胞与具有免疫效应能力的细胞接触，或从肿瘤组织中分选培养，或将能识别权利要求1所述分离的多肽的T细胞受体基因转入T细胞。
  8. 根据权利要求6或7所述免疫细胞，其特征在于，所述具有免疫效应能力的细胞为CD8 +T细胞。
  9. 一种抗体，其特征在于，所述抗体特异性识别权利要求1所述的分离的多肽。
  10. 一种疫苗，其特征在于，包括：权利要求1所述分离的多肽、权利要求2或3所述分离的核酸、权利要求4或5所述抗原递呈细胞、权利要求6～8任一项所述免疫细胞和/或权利要求9所述抗体。
  11. 根据权利要求10所述疫苗，其特征在于，所述疫苗进一步包括佐剂。
  12. 一种药物，其特征在于，包括：权利要求1所述分离的多肽、权利要求2或3所述分离的核酸、权利要求4或5所述抗原递呈细胞、权利要求6～8任一项所述免疫细胞和/或权利要求9所述抗体。
  13. 根据权利要求12所述药物，其特征在于，所述药物进一步包括药学上可接受的辅 料。
  14. 权利要求1所述分离的多肽、权利要求2或3所述分离的核酸、权利要求4或5所述抗原递呈细胞、权利要求6～8任一项所述免疫细胞和/或权利要求9所述抗体在制备疫苗中的用途，其特征在于，所述疫苗用于预防癌症。
  15. 根据权利要求14所述的用途，其特征在于，所述疫苗的受试者表达HLA-A11:01和/或HLA-A24:02。
  16. 权利要求1所述分离的多肽、权利要求2或3所述分离的核酸、权利要求4或5所述抗原递呈细胞、权利要求6～8任一项所述免疫细胞和/或权利要求9所述抗体在制备药物中的用途，其特征在于，所述药物用于治疗癌症。
  17. 根据权利要求16所述的用途，其特征在于，所述药物的受试者表达HLA-A11:01和/或HLA-A24:02。
  18. 根据权利要求14～17任一项所述用途，其特征在于，所述癌症为乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、黑色素瘤、皮肤癌、前列腺癌、宫颈癌、白血病、甲状腺癌、淋巴瘤、膀胱癌、肾癌、子宫体癌、卵巢癌、胆囊癌、口腔癌、喉癌、骨癌、睾丸癌或脑癌。
  19. 一种预防或治疗癌症的方法，其特征在于，包括：
    向受试者施加权利要求1所述分离的多肽、权利要求2或3所述分离的核酸、权利要求4或5所述抗原递呈细胞、权利要求6～8任一项所述免疫细胞和/或权利要求9所述抗体。
  20. 根据权利要求19所述的方法，其特征在于，所述疫苗的受试者表达HLA-A11:01和/或HLA-A24:02；
    所述癌症为乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、黑色素瘤、皮肤癌、前列腺癌、宫颈癌、白血病、甲状腺癌、淋巴瘤、膀胱癌、肾癌、子宫体癌、卵巢癌、胆囊癌、口腔癌、喉癌、骨癌、睾丸癌或脑癌。
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