WO2018045509A1 - 多肽及其应用 - Google Patents

多肽及其应用 Download PDF

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Publication number
WO2018045509A1
WO2018045509A1 PCT/CN2016/098328 CN2016098328W WO2018045509A1 WO 2018045509 A1 WO2018045509 A1 WO 2018045509A1 CN 2016098328 W CN2016098328 W CN 2016098328W WO 2018045509 A1 WO2018045509 A1 WO 2018045509A1
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Prior art keywords
polypeptide
cancer
tumor
cell
seq
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PCT/CN2016/098328
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English (en)
French (fr)
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罗顺涛
李波
侯勇
林秀妹
安婷
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武汉华大吉诺因生物科技有限公司
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Priority to PCT/CN2016/098328 priority Critical patent/WO2018045509A1/zh
Priority to CN201680088999.7A priority patent/CN109803978B/zh
Publication of WO2018045509A1 publication Critical patent/WO2018045509A1/zh
Priority to US16/295,653 priority patent/US11213563B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—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
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/86—Lung

Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to polypeptides and uses thereof, and more particularly to polypeptides and their use in the preparation of kits, medicaments, vaccines, and polypeptides for preventing or treating a subject
  • the use of a disease associated with a mutation in the COL21A1 gene relates to a nucleic acid, a nucleic acid construct, an expression vector, a host cell, a pharmaceutical composition, an antigen presenting cell, an immune effector cell, a vaccine, an antibody, and to a therapeutic method, a diagnostic method, and a diagnostic system.
  • Targeted therapies mainly include monoclonal antibodies (sometimes classified as passive immunotherapy) and small molecule targeted drugs, while immunotherapy mainly includes cytokine therapy, immunoassay monoclonal antibody, adoptive cell reinfusion and tumor vaccine.
  • Immunotherapy enhances the tumor microenvironment's anti-tumor immunity by regulating the immune system of the motor, thereby controlling and killing tumor cells. Therefore, it has the advantages of high efficiency, specificity, and good tolerance, and has broad prospects in cancer treatment.
  • Tumor immunotherapy vaccines mainly include tumor cell vaccine, dendritic cell (DC cell) vaccine, protein & peptide vaccine, nucleic acid vaccine, genetic engineering vaccine and anti-idiotypic tumor vaccine.
  • the main mechanism by which these vaccines can kill tumors is by causing patients to target tumor-specific antigen immune responses, including antigen-antibody reactions and cytotoxic T lymphocyte (CTL)-specific killing, among which CTL-specific killing is in tumor immune response. It has played a big role.
  • a tumor-specific polypeptide is a tumor-specific antigen that primarily causes CTL-specific killing, and includes tumor-mutated polypeptides as well as tumor-specific highly expressed polypeptides.
  • the polypeptide with tumor mutation is a specific target of tumor immunotherapy because it is only present in the tumor tissue of patients, and has the characteristics of good safety and small side effects.
  • Immunotherapy targeting tumor mutant polypeptides, with polypeptide-specific DC-CTL, and tumor-infiltrating lymphocytes (TIL) The method of loss and so on is representative and has a good therapeutic effect.
  • Tumor-specific polypeptides are recognized by CTL or TIL cells and require the antigen presentation function of the human leukocyte antigen HLA.
  • Human leukocyte antigens are mainly divided into two subtypes I and II.
  • Type I HLA is mainly divided into three subtypes A, B and C, each of which is based on its sequence, A, B and C.
  • Subtypes can be divided into multiple subtypes.
  • HLA-A0201 is one of the HLA-A subtypes, accounting for 13% of the Chinese population and has a high proportion. The binding of different polypeptides to the HLA-A0201 subtype is different.
  • the HLA subtype determines that only a portion of the mutant polypeptide can bind to its HLA and is presented to CTL or TIL cells by its HLA.
  • the inventors found that the mutation of the COL21A1 gene (the wild type COL21A1 gene encodes the a chain of XXI-type collagen, which plays a role in maintaining the integrity of the extracellular matrix) results in the amino acid encoded by the 45th amino acid by serine. (Ser, S) is mutated to leucine (Leu, L), and the COL21A1 gene after mutation can be expressed at a high level in tumor tissues, and the polypeptide encoded by the mutant gene is expressed at a high level in tumor tissues. Moreover, the inventors have experimentally verified that the mutant polypeptide sequence has high affinity with HLA-A0201.
  • the present invention proposes an isolated polypeptide.
  • the polypeptide is selected from the group consisting of: (1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; or (2) having at least 70%, at least 75%, at least 80 compared to (1) a polypeptide having %, at least 85%, at least 90%, at least 95%, at least 99% identity; or (3) a polypeptide having one or more amino acid substitutions, deletions, and/or additions compared to (1).
  • the substitution, deletion and/or addition of the at least one or more amino acids is a substitution of amino acid position 2 and/or amino acid 9 of the amino acid sequence set forth in SEQ ID NO: 1.
  • the substitution, deletion and/or addition of the at least one or more amino acids is such that the amino acid substitution at position 2 of the amino acid sequence set forth in SEQ ID NO: 1 is L or M, and/or the amino acid substitution at position 9 Is L.
  • the polypeptide has the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. Wherein (2) the polypeptide or (3) the polypeptide has the same function as the polypeptide of (1).
  • FILDGLYSV SEQ ID NO: 1.
  • FILDGLYSL SEQ ID NO: 2.
  • FMLDGLYSV (SEQ ID NO: 3).
  • FMLDGLYSL (SEQ ID NO: 5).
  • the polypeptide has a high affinity for HLA-A0201 and has the ability to activate specific T cell immunity.
  • the invention provides the use of an agent for detecting the above polypeptide in the preparation of a kit
  • the kit is for diagnosing a tumor, optionally, the tumor simultaneously expresses HLA-A0201 and the polypeptide, optionally, the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophagus Cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumors.
  • the above polypeptide is specifically expressed in tumor tissues, and the inventors have further verified by experiments that a kit prepared by using the reagent for detecting the above polypeptide can be effectively used for diagnosing a tumor;
  • the inventors have surprisingly found that the above polypeptide has high affinity with HLA-A0201, and can be presented to CTL or TIL cells by the presenting cells expressing HLA-A0201 to activate specific T cell immunity, when the tumor simultaneously expresses HLA-A0201 And the above polypeptide, the safety and effectiveness of the kit is significantly improved; at the same time, the inventors found that lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin Cancer, prostate cancer, cervical cancer, leukemia or brain tumor tissue specifically express the above polypeptide, and when the tumor is the above tumor, the validity and sensitivity of the kit diagnosis can be further improved.
  • the invention provides the use of the above polypeptide for the preparation of a medicament for the prophylaxis or treatment of a tumor, optionally, wherein the tumor simultaneously expresses HLA-A0201 and the polypeptide, optionally
  • the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.
  • the inventors have found that the above polypeptide is specifically highly expressed in tumor tissues, and the inventors have further verified by experiments that the drug prepared by the above polypeptide can be effectively used for preventing or treating a tumor;
  • the safety and efficacy of treatment or prevention are significantly improved;
  • the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreas
  • the effectiveness and sensitivity of treatment or prevention can be further improved in cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumors.
  • the invention provides an isolated nucleic acid.
  • the nucleic acid is a nucleic acid encoding the above polypeptide or a complement thereof.
  • the nucleic acid is capable of specifically encoding the above polypeptide.
  • the polypeptide has a high affinity with HLA-A0201 and has the ability to activate specific T cell immunity.
  • the nucleic acid proposed in the examples of the present invention is in a suitable condition.
  • the polypeptide expressed below can be used for the prevention or treatment of tumors, especially when the tumor simultaneously expresses HLA-A0201 and the above polypeptide, and the treatment or prevention is safer and more effective.
  • the invention proposes a nucleic acid construct.
  • the nucleic acid construct comprises a coding sequence which is a nucleic acid as described above, and an optional control sequence operably linked to the coding sequence.
  • the control sequence is one or more control sequences that direct expression of the polypeptide in a host.
  • the nucleic acid construct of the present invention can efficiently express the above polypeptide in a suitable host cell after being ligated to an expression vector under suitable conditions, and can be effectively used for tumors, especially simultaneously expressing HLA-A0201 and the above. Specific treatment or prevention of a tumor of a polypeptide.
  • the invention proposes an expression vector.
  • the vector comprises the nucleic acid construct described above.
  • the expression vector proposed in the embodiments of the present invention can efficiently express the above polypeptide in an expression host under suitable conditions, and the expression vector can be effectively used for tumors, particularly tumors simultaneously expressing HLA-A0201 and the above polypeptides. Sexual treatment or prevention.
  • the invention proposes a host cell.
  • the cell The nucleic acid construct or expression vector described above is optionally obtained by transfection or transformation of the nucleic acid construct or expression vector.
  • the host cell can efficiently express the above polypeptide under suitable conditions, and the host cell can be effectively used for specific treatment or prevention of tumors, particularly tumors simultaneously expressing HLA-A0201 and the above polypeptides. .
  • the invention proposes a pharmaceutical composition.
  • the pharmaceutical composition comprises: a polypeptide as described above; and a pharmaceutically acceptable adjuvant.
  • the inventors have found through extensive experiments that a pharmaceutical composition comprising the aforementioned polypeptide and a pharmaceutically acceptable adjuvant can significantly stimulate the proliferation and secretion of CTL or TIL, and can significantly kill tumor cells presenting the above polypeptide antigen, with significant treatment or Prevention of tumors, especially the efficacy of tumors that specifically express the above polypeptide antigens.
  • the invention provides the use of a polypeptide as described above for the preparation of a vaccine for the prevention or treatment of a tumor, optionally, wherein the tumor simultaneously expresses HLA-A0201 and the polypeptide
  • the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.
  • the inventors have found that the above polypeptide is specifically highly expressed in tumor tissues, and the inventors have further verified by experiments that the vaccine prepared by the above polypeptide can be effectively used for preventing or treating tumors, and the safety thereof is further improved. High, side effects are smaller; when the tumor simultaneously expresses HLA-A0201 and the above polypeptide, the safety and efficacy of treatment or prevention is significantly improved; when the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, The effectiveness and sensitivity of treatment or prevention can be further improved in gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.
  • the invention provides an antigen presenting cell.
  • the antigen presenting cell can present a polypeptide as described above.
  • the antigen presenting cell which presents the polypeptide described above is effective to cause an immune response of the patient to the tumor-specific antigen-the above polypeptide, thereby activating the CTL-specific killing function, and the antigen presenting cell proposed by the embodiment of the present invention It has remarkable efficacy in treating tumors expressing HLA-A0201 and the above polypeptides, and the therapeutic effect thereof is remarkable and the safety is high.
  • the invention provides an immune effector cell.
  • the immune effector cell can recognize the aforementioned polypeptide or recognize an antigen presenting cell that presents the aforementioned polypeptide on the surface of the cell.
  • the immune effector cells specifically kill tumor cells that co-express HLA-A0201 and the above polypeptide.
  • the invention proposes a vaccine.
  • the vaccine comprises a nucleic acid as described above, or a nucleic acid construct as described above, or an expression vector as described above, or a host cell as described above, or an antigen presenting cell as described above , or the immune effector cells described above.
  • the nucleic acid or nucleic acid construct or expression vector of the present invention expresses the aforementioned polypeptide under suitable conditions, and the nucleic acid or nucleic acid construct or expression vector of the present invention can be used for the treatment or prevention of expression.
  • the tumor of the polypeptide, the antigen presenting cells proposed in the embodiments of the present invention have significant efficacy in treating tumors expressing HLA-A0201 and the polypeptide, and the immune effector cells proposed in the embodiments of the present invention have significant specific killer presentation Antigen - the role of the target cell of the polypeptide.
  • the vaccine proposed in the examples of the present invention has a remarkable effect of treating or preventing a tumor expressing HLA-A0201 and the polypeptide, which is safer and has fewer side effects.
  • the invention provides an antibody.
  • the antibody specifically recognizes a polypeptide as described above.
  • the antibody proposed in the embodiments of the present invention can specifically bind to the polypeptide, and can specifically recognize tumor cells which specifically express the polypeptide, and the antibody proposed in the embodiment of the invention plays a huge role in tumor diagnosis, treatment or prevention. effect.
  • the invention provides a method of treatment.
  • the method of treatment comprises: administering to the patient a therapeutically effective amount of a polypeptide as described above, a nucleic acid as described above, a nucleic acid construct as described above, an expression vector as described above, as described above Host cell, a pharmaceutical composition as described above, an antigen presenting cell as described above, an immune effector cell as described above, a vaccine as described above or an antibody as described above.
  • the therapeutic method proposed in the examples of the present invention comprises administering any effective amount of the aforementioned polypeptide or the like to effectively treat or prevent a tumor expressing HLA-A0201 and the polypeptide.
  • the invention provides the use of a polypeptide as described above for the prevention or treatment of a disease associated with a mutation in the COL21A1 gene in a subject.
  • the polypeptide of the embodiment of the present invention is useful for preventing or treating a disease associated with a mutation of the COL21A1 gene in a subject.
  • the invention proposes a diagnostic method.
  • the diagnostic method comprises: detecting whether a biological sample derived from a patient carries a polypeptide as described above; determining whether the patient has a tumor based on whether the biological sample carries the polypeptide, optionally The tumor simultaneously expresses HLA-A0201 and the polypeptide, and optionally, the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer , prostate cancer, cervical cancer, leukemia or brain tumors.
  • the inventors have found that the polypeptide is specifically highly expressed in tumor tissues, whereas the polypeptide is absent from normal tissues.
  • the diagnostic method proposed by the embodiments of the present invention can effectively diagnose a tumor patient that specifically expresses the polypeptide.
  • the inventors found that lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor specific high expression
  • the polypeptide, and further, the method according to the embodiment of the present invention further improves the diagnostic accuracy of the above tumor.
  • HLA-A0201 has a high proportion in the Chinese population, and HLA-A0201 has a strong affinity with the polypeptide, and the polypeptide stimulates a series of immune responses by binding to the cell surface HLA-A0201. . Therefore, the diagnostic method proposed in the examples of the present invention diagnoses a tumor patient who simultaneously expresses HLA-A0201 and the polypeptide with a higher probability.
  • the invention provides a diagnostic system.
  • the diagnostic system includes: a polypeptide detecting device, configured to detect whether a biological sample derived from a patient carries the polypeptide described above; a diagnostic result determining device, the diagnostic result determining device and The polypeptide detecting device is operative to determine whether the patient has a tumor based on whether the biological sample carries the polypeptide, optionally, the tumor simultaneously expresses HLA-A0201 and the polypeptide, optionally,
  • the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.
  • the inventors have found that the polypeptide is specifically highly expressed in tumor tissues, whereas the polypeptide is absent from normal tissues.
  • the diagnostic system proposed by the embodiments of the present invention can be used to effectively determine a tumor patient that specifically expresses the polypeptide.
  • the inventors found that lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor specific high expression Polypeptide, as mentioned in the examples of the present invention
  • the diagnostic system has further improved the diagnostic accuracy of the above tumors.
  • HLA-A0201 has a high proportion in the Chinese population, and HLA-A0201 has a strong affinity with the polypeptide, and the polypeptide stimulates a series of immune responses by binding to the cell surface HLA-A0201. .
  • the diagnostic system proposed by the embodiments of the present invention diagnoses a tumor patient who simultaneously expresses HLA-A0201 and the polypeptide with a higher probability.
  • FIG. 1 is a schematic structural view of a diagnostic system according to an embodiment of the present invention.
  • FIG. 2 is a graph showing the results of flow cytometry detection of affinity of T2 cells loaded with polypeptide and HLA-A0201 according to an embodiment of the present invention
  • FIG. 3 is a graph showing the results of an ELISPOTs method for verifying a polypeptide-activated CD8 + T cell immune response according to an embodiment of the present invention
  • FIG. 4 is a graph showing the results of specific killing of activated CD8 + T cells to target cells loaded with a polypeptide according to an embodiment of the present invention
  • Figure 5 is a graph showing the results of immunotherapy of a polypeptide according to an embodiment of the present invention.
  • A shows an adjuvant, adjuvant + wild type (FILDGSYSV (SEQ ID NO: 7) polypeptide group, FILDGLYSV (SEQ ID NO: 1) polypeptide or a variable form polypeptide thereof (SEQ ID NO: 2 to 6)
  • FILDGSYSV SEQ ID NO: 7
  • FILDGLYSV SEQ ID NO: 1
  • FILDGLYSV SEQ ID NO: 2 to 6
  • a sequence shows the effect of inhibiting tumor growth after treatment with each group + adjuvant
  • FILDGSYSV FILDGSYSV
  • FILDGLYSV SEQ ID NO: 1 polypeptide or its variable form polypeptide (SEQ ID NO: 2 to 6)
  • the sequence shows the results of the survival rate of the mice after each group + adjuvant treatment;
  • Figure 6 shows a graph of the results of immunotherapy of a polypeptide according to an embodiment of the present invention
  • A shows a DC-loaded wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide, a DC-loaded FILDGLYSV (SEQ ID NO: 1) mutant polypeptide or a variant thereof (SEQ ID NO: 2-6) a sequence of peptides that inhibit tumor growth after treatment,
  • Figure 7 shows the results of the immunotherapy of peptides
  • A shows a nucleic acid sequence encoding a wild type or mutant polypeptide, or a nucleic acid sequence encoding a nucleic acid sequence of a mutant polypeptide variable form polypeptide, which is used for immunotherapy after transfecting DC, and inhibiting tumor growth effect,
  • B shows a graph of the survival rate of mice using a nucleic acid sequence encoding a wild-type or mutant polypeptide, or a nucleic acid sequence encoding a nucleic acid sequence of a mutant polypeptide variable form polypeptide, for transfection of DCs;
  • Figure 8 shows the results of the immunotherapy of peptides
  • A shows a graph showing inhibition of tumor growth after DC-loaded wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide + CTL, DC-loaded mutant polypeptide or its variable form + CTL treatment,
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first” and “second” may include one or more of the features either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
  • the invention proposes an isolated polypeptide.
  • the polypeptide is selected from the group consisting of: (1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; or (2) having at least 70%, at least 75%, at least 80% compared to (1) a polypeptide having at least 85%, at least 90%, at least 95%, at least 99% identity; or (3) a polypeptide having one or more amino acid substitutions, deletions, and/or additions compared to (1).
  • the polypeptide proposed in the examples of the present invention is derived from a tumor mutant polypeptide, is absent in a human in which the mutation does not occur, and is only present in the tumor tissue of the patient who has the mutation, and the normal tissue does not contain the mutation. Since it is only found in the patient's tumor tissue, and there is no normal tissue, its specificity is higher, and the specificity of the immune response caused is also higher.
  • the substitution, deletion and/or addition of the at least one or more amino acids described above is a substitution of amino acid position 2 and/or amino acid 9 of the amino acid sequence set forth in SEQ ID NO: 1.
  • substitution of amino acid sequence 2 and/or amino acid 9 of the amino acid sequence of SEQ ID NO: 1 does not alter the specificity between the amino acid sequence and T cells.
  • the substitution, deletion and/or addition of the at least one or more amino acids described above is such that the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 is substituted with L or M, and / Or the amino acid at position 9 is substituted with L.
  • the above polypeptide has the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • FILDGLYSV (SEQ ID NO: 1), FILDGLYSL (SEQ ID NO: 2), FMLDGLYSV (SEQ ID NO: 3), FLLDGLYSV (SEQ ID NO: 4), FMLDGLYSL (SEQ ID NO: 5) FLLDGLYSL (SEQ ID NO: 6) has high affinity with HLA-A0201 and both have the ability to activate specific T cell immunity.
  • variable forms of the polypeptide are FILDGLYSL (SEQ ID NO: 2), FMLDGLYSV (SEQ ID NO: 3), FLLDGLYSV (SEQ ID NO: 4), FMLDGLYSL (SEQ ID NO: 5), FLLDGLYSL (SEQ ID NO) :6), the second or ninth position of the polypeptide FILDGLYSV (SEQ ID NO: 1) is changed, the amino acid substitution at position 2 is L or M, and/or the amino acid substitution at position 9 is L, and this substitution Enhanced more The binding of the peptide to HLA-A0201 without altering its specificity with T cells.
  • both the SEQ ID NO: 2-6 polypeptide and the FILDGLYSV (SEQ ID NO: 1) polypeptide have the ability to activate specific T cell immunity.
  • the present invention provides, in one aspect, the use of an agent for detecting the above polypeptide in the preparation of a kit, the use of the above polypeptide in the preparation of a medicament, and the use of the above polypeptide in the preparation of a vaccine for use in a kit, a medicament or a vaccine. Diagnose, prevent or treat tumors.
  • the tumor simultaneously expresses HLA-A0201 and the polypeptide.
  • the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.
  • the above polypeptide is specifically expressed in tumor tissues, and the inventors have further verified by experiments that the kit for preparing the above polypeptide or the above polypeptide preparation medicine or vaccine can be effectively used. In the diagnosis of tumors, it is safer and has fewer side effects. At the same time, the inventors have surprisingly found that the above polypeptides have high affinity with HLA-A0201 and can be presented to CTL or TIL cells by presenting cells expressing HLA-A0201.
  • kits, drug or vaccine diagnosis or treatment are significantly improved; at the same time, the inventors found that lung cancer, melanoma, Breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor tissue specifically express the above polypeptide, and when the tumor is the above tumor, The effectiveness of the kit, drug or vaccine diagnosis and treatment can be further improved.
  • the invention provides the use of a polypeptide as described above for preventing or treating a disease associated with a mutation in the COL21A1 gene in a subject.
  • the inventors have found through extensive screening experiments that the mutation of the COL21A1 gene causes the amino acid at the 45th position encoded by it to be mutated from serine (Ser, S) to leucine (Leu, L).
  • the polypeptide of the embodiment of the present invention has the same antigenic property as the COL21A1 gene mutation-encoding polypeptide, and the specific immune response caused by the polypeptide has obvious effect on the specific recognition and killing effect of the COL21A1 gene mutant cell, and further
  • the polypeptide of the embodiment of the invention can be used for the prevention or treatment of a disease associated with mutation of the OL21A1 gene.
  • the inventors have found through experiments that the polypeptide is effective for preventing or treating a disease associated with mutation of the OL21A1 gene.
  • the invention provides an isolated nucleic acid.
  • the nucleic acid is a nucleic acid encoding the above polypeptide or a complement thereof.
  • the nucleic acid is capable of specifically encoding the above polypeptide.
  • the polypeptide has a high affinity with HLA-A0201 and has the ability to activate specific T cell immunity.
  • the nucleic acid proposed in the examples of the present invention is in a suitable condition.
  • the polypeptide expressed below can be used for the prevention or treatment of tumors, especially when the tumor simultaneously expresses HLA-A0201 and the above polypeptide, and the treatment or prevention is safer and more effective.
  • the invention provides a nucleic acid construct.
  • the nucleic acid construct comprises a coding sequence which is a nucleic acid as described above, and an optional control sequence operably linked to the coding sequence.
  • the control sequence is one that directs expression of the polypeptide in the host or Multiple control sequences.
  • the control sequences include, but are not limited to, U6, H1, CMV, EF-1, LTR or RSV promoters.
  • the nucleic acid construct of the present invention can efficiently express the above polypeptide in a suitable host cell after being ligated to an expression vector under suitable conditions, and can be effectively used for tumors, especially simultaneously expressing HLA-A0201 and the above. Specific treatment or prevention of a tumor of a polypeptide.
  • the invention provides an expression vector.
  • the vector comprises the nucleic acid construct described above.
  • the type of the expression vector is not particularly limited as long as the nucleic acid construct described above can be efficiently expressed in a recipient cell, and the expression vector includes, but is not limited to, a retrovirus vector, a lentiviral vector, and/or a gland. Virus-associated viral vector.
  • the expression vector proposed in the embodiments of the present invention can efficiently express the above polypeptide in an expression host under suitable conditions, and the expression vector can be effectively used for tumors, particularly tumors simultaneously expressing HLA-A0201 and the above polypeptides. Sexual treatment or prevention.
  • the invention provides a host cell.
  • the cell carries the nucleic acid construct or expression vector described above, optionally obtained by transfection or transformation of the nucleic acid construct or expression vector. Transformation or transfection can be carried out by means of electroporation, viral transfection or transformation of competent cells. The manner in which transfection or transformation is employed is determined by the nature of the host cell and the nature of the nucleic acid construct or expression vector to be transduced, as long as the high expression of the aforementioned polypeptide can be achieved in the host cell and the host cell A good cell state does not have a large effect.
  • the host cell can efficiently express the above polypeptide under suitable conditions, and the host cell can be effectively used for specific treatment or prevention of tumors, particularly tumors simultaneously expressing HLA-A0201 and the above polypeptides. .
  • suitable conditions refer to conditions suitable for expression of the polypeptide described herein. It will be readily understood by those skilled in the art that conditions suitable for expression of the polypeptide include, but are not limited to, suitable transformation or transfection formats, suitable transformation or transformation conditions, healthy host cell status, appropriate host cell density, and suitable cell culture environment. Suitable cell culture time. The “suitable conditions” are not particularly limited, and those skilled in the art can optimize the conditions for expression of the polypeptide according to the specific environment of the laboratory.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises: a polypeptide as described above; and a pharmaceutically acceptable adjuvant.
  • the inventors have found through extensive experiments that a pharmaceutical composition comprising the aforementioned polypeptide and a pharmaceutically acceptable adjuvant can significantly stimulate the proliferation and secretion of CTL or TIL, and can significantly kill tumor cells presenting the above polypeptide antigen, with significant treatment or Prevention of tumors, especially the efficacy of tumors that specifically express the above polypeptide antigens.
  • the invention provides an antigen presenting cell.
  • the antigen presenting cell can present a polypeptide as described above.
  • the antigen presenting cell which presents the polypeptide described above is effective to cause an immune response of the patient against the tumor-specific antigen-the above polypeptide, thereby activating the CTL-specific killing function, and the antigen presenting according to the embodiment of the present invention
  • the cells have remarkable efficacy in treating tumors expressing HLA-A0201 and the above polypeptides, and the therapeutic effect thereof is remarkable and the safety is high.
  • the antigen presenting cell is obtained by at least one of the following: contacting a cell having antigen presenting ability with the polypeptide; or the nucleic acid described above, or the nucleic acid construct described above Or the expression vector described above is introduced into the antigen-presenting cell.
  • the inventors discovered through experiments that the antigen The presenting cell can effectively present the polypeptide described above by any one or more of the above methods, and exposing the polypeptide described above to the surface of the presenting cell, and presenting the antigen presenting cell of the aforementioned polypeptide can effectively cause the patient to tumor Specific antigen - The immune response of the above polypeptide, which in turn activates the CTL-specific killing function.
  • the antigen presenting cell is a dendritic cell.
  • Dendritic cells have strong antigen endocytosis and processing ability to present antigen on the surface of cells.
  • the inventors chose dendritic cells as antigen presenting cells, and antigen presenting cells initiate, regulate and maintain an immune response against the polypeptide in the body.
  • the invention provides an immune effector cell.
  • the immune effector cell can recognize the polypeptide described above or recognize an antigen presenting cell that presents the aforementioned polypeptide on the cell surface.
  • the immune effector cells are obtained by contacting the antigen presenting cells described above with cells having immunogenic effect.
  • the antigen presenting cell activates an immunogenic cell, activates the antigen, the aforementioned polypeptide, and activates
  • the cells having an immunogenic effect produce a large amount of immune effector cells, and the immune effector cells have a function of specifically killing the target cells which present the antigen-the polypeptide.
  • the cells having immunogenic effect ability are T lymphocytes, and the inventors have found that CD8 + T cells are preferred, and CD8 + T cells are more capable of undergoing antigen presenting cell activation, and CD8 + T obtained is obtained.
  • the specific killer of the cells presents the antigen - the target cells of the polypeptide are more potent.
  • the invention provides a vaccine.
  • the vaccine comprises a nucleic acid, a nucleic acid construct, an expression vector, a host cell, an antigen presenting cell as described above, or an immune effector cell as described above.
  • the nucleic acid, the nucleic acid construct, the expression vector, and the host cell of the embodiments of the present invention can be used for specific killing of a tumor highly expressing the polypeptide, and the antigen presenting cells proposed by the embodiments of the present invention have remarkable treatment.
  • the efficacy of the tumor expressing HLA-A0201 and the polypeptide, in addition, the immune effector cells proposed in the examples of the present invention have a significant specific killing effect on the antigen-presenting target cells of the polypeptide.
  • the vaccine according to the embodiment of the present invention comprises the nucleic acid, the nucleic acid construct, the expression vector, the host cell, the antigen presenting cell or the immune effector cell as described above, which has significant treatment or prevention of a tumor expressing HLA-A0201 and the polypeptide. Its role is to be safer and have fewer side effects.
  • the invention provides an antibody.
  • the antibody specifically recognizes a polypeptide as described above.
  • the antibody proposed in the embodiments of the present invention can specifically bind to the polypeptide, and can specifically recognize tumor cells which specifically express the polypeptide, and the antibody proposed in the embodiment of the invention plays a huge role in tumor diagnosis, treatment or prevention. effect.
  • the antibody can be obtained by collecting serum of an animal immunized with the polypeptide described above; and purifying the antibody of interest from the serum.
  • the polypeptide proposed in the embodiment of the present invention has higher specificity and higher specificity of the immune response because it is only found in the tumor tissue of the patient, and there is no normal tissue, and other tumor polypeptide vaccines. Compared with the advantages of being safer, having fewer side effects, and rarely causing a serious immune reaction, and because of its simple structure and easy artificial synthesis, it can be used as a vaccine, a pharmaceutical composition, etc., to cause an immune response against a tumor.
  • FILDGLYSV SEQ ID NO: 1
  • the polypeptide or a variable form thereof can be used as a target or a vaccine for tumor biological therapy directed against simultaneous expression of HLA-A0201 and the mutant polypeptide, and has an immune response.
  • the polypeptide + adjuvant, the polypeptide-supported antigen presenting cell vaccine, or the polypeptide-specific DC-CTL, DC-CIK vaccine, etc. can specifically kill tumor cells, prevent and treat cancer, including lung cancer expressing the polypeptide sequence, Melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia, brain tumor and other cancer types.
  • the present invention proposes a method of treatment.
  • the method of treatment comprises: administering to the patient a therapeutically effective amount of a polypeptide as described above, a nucleic acid as described above, a nucleic acid construct as described above, an expression vector as described above, as described above Host cell, a pharmaceutical composition as described above, an antigen presenting cell as described above, an immune effector cell as described above, a vaccine as described above or an antibody as described above.
  • the therapeutic method proposed in the examples of the present invention comprises administering any effective amount of the aforementioned polypeptide or the like to effectively treat or prevent a tumor expressing HLA-A0201 and the polypeptide.
  • administering refers to introducing a predetermined amount of a substance into a patient in some suitable manner.
  • the polypeptide, nucleic acid, nucleic acid construct, expression vector, host cell, pharmaceutical composition, antigen presenting cell, immune effector cell, vaccine or antibody in the embodiments of the present invention can be administered by any common route as long as it can reach the expected organization.
  • Various modes of administration are contemplated, including peritoneal, venous, muscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the invention is not limited to these exemplary modes of administration.
  • the active ingredient of the orally administered composition should be coated or formulated to prevent its degradation in the stomach.
  • the compositions of the invention may be administered in an injectable preparation.
  • the pharmaceutical compositions of the invention may be administered using a particular device that delivers the active ingredient to the target cells.
  • the frequency and dose of administration of the polypeptide, nucleic acid, nucleic acid construct, expression vector, host cell, pharmaceutical composition, antigen presenting cell, vaccine or antibody in the embodiments of the present invention can be determined by a plurality of related factors, including The type of disease being treated, the route of administration, the age of the patient, the sex, the severity of the body weight and the disease, and the type of drug as the active ingredient.
  • the daily dose may be divided into 1 dose, 2 doses or multiple doses in a suitable form for administration once, twice or more times throughout the time period, as long as a therapeutically effective amount is achieved. .
  • treatment refers to an amount sufficient to significantly ameliorate certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • treatment is used to mean obtaining the desired pharmacological and/or physiological effect.
  • treatment encompasses the administration of a polypeptide, nucleic acid, nucleic acid construct, expression vector, host cell, pharmaceutical composition, antigen presenting cell, immune effector cell, vaccine or antibody in an embodiment of the invention to a subject for treatment, including but not It is limited to those individuals in need of administration as described herein.
  • the present invention proposes a diagnostic method.
  • the diagnostic method comprises: detecting whether the biological sample from which the patient is derived carries the polypeptide as described above; determining whether the patient has a tumor based on whether the biological sample carries the polypeptide. Since the polypeptide proposed in the embodiment of the present invention is only found in cancer tissues, the free polypeptide present in the serum can be detected by mass spectrometry, and the polypeptide is used as a tumor marker in the diagnosis of cancer to determine whether the patient has a disease. cancer. The inventors have found that the polypeptide is specifically highly expressed in tumor tissues, the present invention The diagnostic method proposed in the examples can effectively diagnose tumor patients that specifically express the polypeptide.
  • lung cancer melanoma
  • breast cancer nasopharyngeal cancer
  • liver cancer gastric cancer
  • esophageal cancer colorectal cancer
  • pancreatic cancer skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor specific high expression
  • the method according to the embodiment of the present invention further improves the diagnostic accuracy of the above tumor.
  • the inventors have found that HLA-A0201 has a high proportion in the Chinese population, and thus, the diagnostic method proposed in the examples of the present invention diagnoses a tumor patient who simultaneously expresses HLA-A0201 and the polypeptide with a higher probability.
  • the diagnostic system includes: a polypeptide detecting device 100; a diagnostic result determining device 200.
  • the polypeptide detecting device 100 is configured to detect whether the biological sample derived from the patient carries the polypeptide described above, and the diagnostic result determining device 200 is connected to the polypeptide detecting device 100 for determining whether the biological sample carries the polypeptide. Whether the patient has a tumor.
  • the free peptide present in the serum of the patient may be detected by a mass spectrometer, and then the presence or absence of the free polypeptide in the serum of the patient is determined by a mass spectrometry data analysis device to determine whether the patient has a tumor.
  • a mass spectrometry data analysis device to determine whether the patient has a tumor.
  • the diagnostic system proposed by the embodiment of the present invention further improves the diagnostic accuracy of the above tumor.
  • HLA-A0201 has a high proportion in the Chinese population, and HLA-A0201 has a strong affinity with the polypeptide, and the polypeptide stimulates a series of immune responses by binding to the cell surface HLA-A0201. .
  • the diagnostic system proposed by the embodiments of the present invention diagnoses a tumor patient who simultaneously expresses HLA-A0201 and the polypeptide with a higher probability.
  • the polypeptide according to the embodiment of the present invention and the use thereof, the nucleic acid encoding the polypeptide, the nucleic acid construct, the expression vector, the host cell, the pharmaceutical composition, the antigen presenting cell, the immune effector cell, the vaccine, the antibody, the treatment The method and system for diagnosing cancer are discovered and completed by the inventor of the present application after painstaking creative labor and optimization work.
  • the affinity prediction of the polypeptides was separately performed using the self-developed "tumor DNA and RNA sequencing-based mutant polypeptide binding ability prediction software" (software copyright number: 2016SR002835).
  • the predicted results are expressed as IC 50 scores, an IC 50 of less than 500 nM indicates that the polypeptide has an affinity, and an IC 50 of less than 50 nM indicates that the polypeptide has high affinity.
  • the inventors predicted the affinity of the wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide, the mutant polypeptide and the variable form polypeptide thereof, and finally screened the mutant polypeptide and its variable form polypeptide with an IC 50 score of less than 500 nM, and the mutant polypeptide and the same alternative forms of the polypeptide IC 50 is less than the score of wild type (FILDGSYSV (SEQ ID NO: 7 )) polypeptide.
  • the predicted results of the affinity of the polypeptide are shown in Table 1. Based on the results, the next step T2 affinity verification was performed.
  • variable polypeptide mutant form of the polypeptide of IC 50 were lower than 50 nM, and alternative forms described mutant polypeptide polypeptide predicted, are high affinity polypeptides.
  • Polypeptide and its mutant form of the polypeptide of the variable is less than the wild type IC 50 (FILDGSYSV (SEQ ID NO: 7 )) polypeptide. Therefore, it is predicted that the affinity of the mutant polypeptide and its variable form polypeptide is higher than that of the wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide.
  • polypeptides involved in the examples of the invention were synthesized according to standard solid phase synthesis methods and purified by reverse phase HPLC. The purity (>90%) and identity of the polypeptide were determined by HPLC and mass spectrometry, respectively.
  • T2 cells are HLA-A2-positive T and B lymphocyte hybridoma cells, which can express HLA-A0201 on the cell surface, but cannot be transported due to defects in the essential antigen polypeptide transporter (TAP) in the endogenous antigen presentation pathway. Endogenous antigen. T2 cells were purchased from ATCC (number: CRL-1992).
  • T2 cells were purchased from ATCC (number: CRL-1992).
  • T2 cells 2 ⁇ 10 5 T2 cells were taken and resuspended in a 24-well plate with 500 ⁇ l of IMDM serum-free medium containing human ⁇ 2 microglobulin (final concentration, 3 ⁇ g/ml), and synthetic wild-type FILDGSYSV (SEQ ID NO: 7) Polypeptide, FILDGLYSV (SEQ ID NO: 1) polypeptide or its five variable form polypeptides (final concentration 100 ⁇ M) were cultured overnight in an incubator (37 ° C, 5% CO 2 ). Two replicate wells per group; T2 cells without polypeptide added were used as background controls, and CMV polypeptide (NLVPMVATV (SEQ ID NO: 8)) was added as a positive control.
  • the cells were collected by centrifugation of 200 g of cells for 5 minutes. After the cells were washed twice with PBS, the cells were directly incubated with anti-HLA-A*02:01 FITC monoclonal antibody and maintained at 4 ° C for 30 minutes.
  • flow cytometry BD FACSJazz TM software to detect and analyze their mean fluorescence intensity.
  • FI>1.5 indicates that the peptide has high affinity for HLA-A*0201 molecule
  • 1.0 ⁇ FI ⁇ 1.5 indicates that the peptide has moderate affinity for HLA-A*02:01 molecule
  • 0.5 ⁇ FI ⁇ 1.0 indicates that the peptide is HLA-A*0201 molecule has low affinity.
  • PBMC Peripheral blood mononuclear cells
  • Ficoll lymphocyte separation solution was used to separate peripheral blood mononuclear cells (PBMC).
  • PBMCs were used to attach mononuclear cells, and CD8 magnetic beads were used to screen PBMCs.
  • GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) were used to induce adherent monocytes to be immature DCs, followed by IFN-gamma (100 U/ml) and CD40L (100 U/ml).
  • FILDGLYSV SEQ ID NO: 1
  • FILDGLYSV SEQ ID NO: 1
  • IL-21 was added.
  • IL-2 and IL-7 were added, and then IL-addition was performed on the 5th and 7th days.
  • 2 and IL-7 co-cultured cells were counted on day 10, and subsequent ELISPOTs and LDH assays. The result of the counting is shown in Table 3:
  • Example 3 The cells co-cultured in Example 3 were separately cultured and detected with T2 cells loaded with the over-mutated polypeptide FILDGLYSV (SEQ ID NO: 1) and wild-type (FILDGSYSV (SEQ ID NO: 7) polypeptide into human IFN-gamma ELISPOTs plates. . The spots produced by the ELISPOT experiment were finally counted.
  • FILDGLYSV SEQ ID NO: 1
  • FILDGSYSV wild-type polypeptide
  • the requirement for immunogenicity of the mutant polypeptide is as follows: number of spots (mutant polypeptide) / number of spots (wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide) > 2, ie, the number of spots caused by the mutant polypeptide exceeds that of the wild type (FILDGSYSV ( SEQ ID NO: 7)) More than twice the number of polypeptide spots is a requirement for the polypeptide to be immunogenic.
  • CD8 + T cells can specifically recognize the complex of HLA-A0201 and polypeptide, the difference in polypeptide sequence, and the T cell population of the complex of recognition polypeptide and HLA-A0201 are also different. Since T2 cells express HLA-A0201, CD8 + T cells can specifically recognize T2 cells loaded with the mutant polypeptide FILDGLYSV (SEQ ID NO: 1), but cannot recognize wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide. Loaded T2 cells. After specifically recognizing the complex of HLA-A0201 and the polypeptide, the polypeptide-specific CD8 + T cells can reactivate and secrete IFN-gamma interferon.
  • the IFN-gamma interferon secreted by CD8 + T cells can be captured by antibodies on the ELISPOTs plate.
  • the antibody that finally recognizes IFN-gamma can catalyze the coloration of the substrate through the enzyme coupled to the antibody, eventually producing spots. .
  • the number of spots represents the number of cells that are activated to secrete IFN-gamma interferon.
  • Example 5 LDH release assay demonstrates CD8 + T cell killing activity
  • Example 3 The cells co-cultured in Example 3 were co-cultured with T2 cells carrying the over-mutated polypeptide FILDGLYSV (SEQ ID NO: 1), or the wild-type (FILDGSYSV (SEQ ID NO: 7)) polypeptide, or the unloaded polypeptide, and set in the experiment.
  • FILDGLYSV SEQ ID NO: 1
  • FILDGSYSV wild-type polypeptide
  • the formula for calculating the killing activity is:
  • Killing efficiency (experimental well-effect cell spontaneous release - target cell spontaneous release + medium well) / (target cell maximum release - volume correction hole - target cell spontaneous release + medium well) ⁇ 100%
  • lactate dehydrogenase is one of the enzymes contained in the cytoplasm of living cells, and under normal conditions, it cannot penetrate the cell membrane.
  • LDH lactate dehydrogenase
  • the released LDH converts oxidized coenzyme I (NAD+) into reduced coenzyme I (NADH) during the catalysis of lactic acid to produce pyruvate, which is then passed through the hydrogen donor-phenazine dimethyl sulfate (PMS).
  • the original iodine nitroazolidine blue (INT) or the nitrochlorotetrazolium blue (T) forms a colored formazan compound with a high absorption peak at a wavelength of 490 nm or 570 nm, using the read OD value,
  • the effector cell activity can be known by calculation.
  • T cells specifically recognize and kill target cells loaded with experimental peptides
  • the mutant polypeptide and its variable form polypeptide-activated T cells are capable of killing the mutant polypeptide FILDGLYSV (SEQ ID NO: 1) at a target ratio of 1:1 or 1:10.
  • T2 cells, but not T2 cells carrying the wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide which further validates the experimental group polypeptide (the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 and its variable form polypeptide)
  • the activated T cells specifically kill the target cells carrying the mutant polypeptide FILDGLYSV (SEQ ID NO: 1).
  • Example 6 Establishment of a subcutaneous xenograft model of H2087-FILDGLYSV (SEQ ID NO: 1) or its variable form polypeptide
  • the DNA sequence of the synthetic FILDGLYSV (SEQ ID NO: 1) polypeptide is shown in SEQ ID NO: 9, TTCATCCTGGACGGCCTGTACAGCGTG (SEQ ID NO: 9),
  • variable form FILDGLYSL SEQ ID NO: 2 polypeptide is set forth in SEQ ID NO: 10, TTCATCCTGGACGGCCTGTACAGCCTG (SEQ ID NO: 10),
  • variable form FMLDGLYSV SEQ ID NO: 3
  • SEQ ID NO: 11 DNA sequence of the variable form FMLDGLYSV (SEQ ID NO: 3) polypeptide is set forth in SEQ ID NO: 11, TTCATGCTGGACGGCCTGTACAGCGTG (SEQ ID NO: 11),
  • polypeptide DNA sequence of its variable form FLLDGLYSV (SEQ ID NO: 4) is set forth in SEQ ID NO: 12, TTCCTGCTGGACGGCCTGTACAGCGTG (SEQ ID NO: 12),
  • variable form FMLDGLYSL SEQ ID NO: 5
  • DNA sequence of the variable form FMLDGLYSL SEQ ID NO: 13
  • TTCATGCTGGACGGCCTGTACAGCCTG SEQ ID NO: 13
  • variable form FLLDGLYSL SEQ ID NO: 6
  • SEQ ID NO: 14 TTCCTGCTGGACGGCCTGTACAGCCTG
  • the DNA sequence corresponding to the wild type FILDGSYSV (SEQ ID NO: 7) polypeptide is shown in SEQ ID NO: 15, TTCATCTTAGATGGCTCTTATAGTGTT (SEQ ID NO: 15).
  • the lentiviral vector pHBLV-Puro expressing the polypeptide of the wild type polypeptide FILDGSYSV (SEQ ID NO: 7), FILDGLYSV (SEQ ID NO: 1) and its variable form polypeptide, respectively, was constructed. They are named pHBLV-FILDGSYSV-Puro, pHBLV-FILDGLYSV-Puro, pHBLV-FILDGLYSL-Puro, pHBLV-FMLDGLYSV-Puro, pHBLV-FLLDGLYSV-Puro, pHBLV-FMLDGLYSL-Puro, pHBLV-FLLDGLYSL-Puro.
  • lentiviral plasmids were co-transfected into 293T cells with pSPAX2 and pMD2G helper plasmids, respectively, and the lentivirus expressing the wild-type polypeptide, FILDGLYSV (SEQ ID NO: 1) polypeptide and its variable form polypeptide was packaged.
  • the human non-small cell line lung adenocarcinoma cell line NCI-H2087 was purchased from ATCC (number: CRL-5922) and its HLA subtype was HLA-A*0201 positive.
  • the cells were cultured in DMEM medium containing 10% fetal calf serum, 100 U/mL penicillin and streptomycin. Incubate in a 37 ° C, 5% CO 2 incubator.
  • the packaged FILDGLYSV (SEQ ID NO: 1) lentivirus was transfected into the H2087 cell line, and the surviving H2087 cell line was continuously screened with the Puromycin antibiotic (puromycin) to finally establish the FILDGLYSV (SEQ ID NO: 1) polypeptide.
  • H2087 cell line Named H2087-FILDGLYSV (SEQ ID NO: 1) cell line.
  • PBMC peripheral blood mononuclear cells
  • NOD SCID mice Thirty-six NOD SCID mice were excluded from the immune leak, and each of the NOD SCID mice was subjected to human immune reconstitution by intraperitoneal injection of PBMC 2 ⁇ 10 7 /0.5 ml. Further, mice selected 4 weeks later were prepared to inoculate a human lung cancer cell line model.
  • H2087-FILDGLYSV The established human non-small cell line lung adenocarcinoma cell line H2087-FILDGLYSV (SEQ ID NO: 1) was cultured in DMEM medium containing 10% fetal calf serum, 100 U/mL penicillin and streptomycin. Incubate in a 37 ° C, 5% CO 2 incubator. H2087-FILDGLYSV (SEQ ID NO: 1) tumor cells were collected, centrifuged at 3000 rpm, and the tumor cells were washed 3 times with sterile physiological saline.
  • H2087-FILDGLYSV SEQ ID NO: 1 subcutaneous tumor model NOD/SCID mice immunized for 4 weeks were subjected to polypeptide + complete Freund's adjuvant vaccine, or polypeptide + DC vaccine, or lentivirus-infected DC cell vaccine, As well as DC-CTL vaccine treatment, the tumor volume and mouse survival rate were recorded every 2 days.
  • the H2087-FILDGLYSV (SEQ ID NO: 1) subcutaneous tumor model NOD/SCID mice immunized for 4 weeks were randomly divided into 8 groups: wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide group, adjuvant group, adjuvant Agent ten FILDGLYSV (SEQ ID NO: 1) or five variable form polypeptide groups of 6 each.
  • the first immunization dose of wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide, FILDGLYSV (SEQ ID NO: 1) polypeptide or 5 variably form polypeptides was 100 ⁇ g/head.
  • the polypeptide was resuspended in PBS, mixed with 150 ⁇ l of Freund's complete adjuvant, adjusted to 300 ⁇ l/cell with PBS, and injected subcutaneously at the back. After 2 weeks, the same dose was used for booster immunization (the first use of complete Freund's adjuvant, followed by incomplete Freund's adjuvant), and a total of 4 immunizations.
  • the general characteristics of the mice were observed daily, including mental state, activity, response, diet, body weight, and tumor growth. The longest diameter (long) and shortest diameter (width) of the tumor were measured with a vernier caliper every 2 days.
  • the calculation formula of tumor volume is: 1/2 ⁇ length ⁇ width 2 ;
  • the results are shown in Figure 5.
  • PBMC peripheral blood mononuclear cells
  • PBL peripheral blood lymphocytes
  • GM-CSF 1000 U/ml
  • IL-4 1000 U/ml
  • IFN-gamma 100 U/ml
  • CD40L 10 ng/ml
  • Wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide, FILDGLYSV (SEQ ID NO: 1) polypeptide and variable form polypeptide thereof (concentration: 10 ⁇ g/ml) were added to induce adherent cells to be mature DC cells, and mature DCs were harvested. Wash 3 times with physiological saline. The DC after loading the polypeptide was adjusted to (4.0 ⁇ 0.5) ⁇ 10 7 /ml with physiological saline for subsequent experiments.
  • mice were randomized into 7 groups: DC-loaded wild-type (FILDGSYSV (SEQ ID NO: 7)) polypeptide group, DC-loaded FILDGLYSV (SEQ ID NO: 1) polypeptide, and DC-loaded 5 variably polypeptides Group, 6 in each group.
  • the inner side of the thigh of the mouse was intradermally injected, 0.1 ml per side, and once a week.
  • PBMC peripheral blood mononuclear cells
  • PBMC cells were harvested, incubated at 37 ° C for 2 h, washed away unattached cells, recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), recombination Human white medium-4 (rhIL-4) cultured DC cells.
  • rhGM-CSF granulocyte-macrophage colony-stimulating factor
  • rhIL-4 recombination Human white medium-4
  • the medium was changed and the cell density was adjusted to 1 ⁇ 10 6 cells/ml; and the appropriate amount of the wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide, which was constructed and expressed in Example 6, was added, A lentiviral fluid of FILDGLYSV (SEQ ID NO: 1) and its variable form polypeptide (shown in any of SEQ ID NOS: 2-6).
  • FILDGSYSV wild type polypeptide
  • a lentiviral fluid of FILDGLYSV SEQ ID NO: 1
  • its variable form polypeptide shown in any of SEQ ID NOS: 2-6.
  • the virus culture medium was removed, and a culture medium containing 50 ng/ml rhIL-4, 100 ng/ml rh GM-CSF, 100 U/ml IFN- ⁇ and 100 U/ml CD40L was added and placed in a 37 ° C 5% CO 2 incubator. Cultivate.
  • mice were randomized into 7 groups: wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide-DC group, FILDGLYSV (SEQ ID NO: 1) polypeptide-DC group, and its five variability forms (SEQ ID NO : 2 to 6 of any sequence) polypeptide-DC group, 6 in each group.
  • wild type FILDGSYSV (SEQ ID NO: 7)
  • FILDGLYSV SEQ ID NO: 1 polypeptide-DC group
  • its five variability forms SEQ ID NO : 2 to 6 of any sequence
  • Figure 7 shows the results of lentiviral-infected DCs expressing FILDGLYSV (SEQ ID NO: 1) or its variable form polypeptide (shown in any of SEQ ID NOs: 2-6) gene package relative to the wild-type polypeptide control group.
  • the vaccine has significant tumor suppressive effects and can significantly prolong the survival of mice.
  • Example 8 The PBL collected in Example 8 was subjected to magnetic bead sorting to obtain CD8 + T and DC carrying the wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide, and the FILDGLYSV (SEQ ID NO: 1) polypeptide or its five variable forms were loaded.
  • the second week was again loaded with FILDGLYSV (SEQ ID NO: 1).
  • the DC of the polypeptide or its five variable form polypeptides (shown by any of SEQ ID NOS: 2 to 6)
  • DC, DC loaded with a wild type (FILDGSYSV (SEQ ID NO: 7)) polypeptide is subjected to a second round of stimulation.
  • Three rounds of co-stimulation were performed, and the medium was appropriately added during the culture.
  • CTL Cell-cytotoxic T lymphocytes
  • the cells were resuspended in physiological saline, resuspended in a volume of 0.2 ml, and returned via the tail vein.
  • the number of cells per mouse in each tumor model was about 1 ⁇ 10 8 cells.
  • the mice were observed for vital signs, and the size of the tumor was measured with a vernier caliper every 2 days. The result is shown in Figure 8.
  • Figure 8 shows that the DC-CTL vaccine activated by FILDGLYSV (SEQ ID NO: 1) or its variable form polypeptide (shown in any of SEQ ID NOs: 2 to 6) is apparent relative to the wild-type polypeptide control group.
  • the polypeptide of the present invention can be effectively applied to the preparation of a kit, a drug or a vaccine, and the specificity of the immune reaction caused by the drug or vaccine is also higher, and it is safer and has fewer side effects than other tumor polypeptide vaccines. It has the advantages of less serious immune reaction, and because of its simple structure and easy artificial synthesis, it can be used as a vaccine, a pharmaceutical composition, etc., to cause an immune response against a tumor.

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Abstract

一种多肽，编码所述多肽的核酸，包含上述核酸的核酸构建体、表达载体、宿主细胞，在细胞表面呈递上述多肽的抗原呈递细胞及其免疫效应细胞，包含上述多肽的药物组合物，包含上述核酸、核酸构建体、表达载体、宿主细胞、抗原呈递细胞、免疫效应细胞的疫苗，识别上述多肽的抗体。施用上述多肽、核酸、药物组合物、疫苗、抗体的治疗方法。检测上述多肽的诊断方法和诊断装置。上述多肽在用于制备疫苗、诊断肿瘤试剂盒或药物组合物中的用途，以及上述多肽、核酸作为检查靶标在肿瘤诊断中的用途。

Description

多肽及其应用
优先权信息
无。
技术领域
本发明涉及生物医药领域，具体而言，本发明涉及多肽及其应用，更具体地，本发明涉及多肽及其在制备试剂盒、药物、疫苗中的用途，涉及多肽在预防或治疗受试者中与COL21A1基因突变相关的疾病的用途，涉及核酸、核酸构建体、表达载体、宿主细胞、药物组合物、抗原呈递细胞、免疫效应细胞、疫苗、抗体，涉及治疗方法、诊断方法和诊断系统。
背景技术
癌症，由于细胞内基因突变导致细胞增殖失控的一种疾病。目前已成为人类健康的重大威胁，是导致人类死亡的主要原因之一。世界卫生组织(WHO)在发表的《全球癌症报告2014》中指出，2012年全球癌症患者和死亡病例都在迅速增加，而新增癌症病例有近一半出现在亚洲，其中大部分在中国，中国新增癌症病例高居第一位。《2012年中国肿瘤登记年报》数据显示，中国每年新增癌症病例约350万，约有250万人因此死亡。因此，寻找高效特异的癌症治疗方法具有重大的临床价值。
传统的肿瘤治疗方法主要包括手术、放疗和化疗，但这几种方法都具有较大的局限性，比如由于癌细胞的近端入侵或远端转移，手术切除后的肿瘤转移复发率较高，而放疗和化疗对于机体自身的正常细胞尤其是造血系统和免疫系统会造成严重的损害，因此对于已发生肿瘤转移的患者也很难达到较好的远期疗效。随着肿瘤分子机制的深入研究和生物技术的进一步发展，靶向药物治疗和免疫治疗在肿瘤的综合治疗中发挥着愈来愈大的作用。靶向疗法主要包括单克隆抗体(有时归为被动免疫疗法)和小分子靶向药物，而免疫疗法主要包括细胞因子疗法、免疫检验点单抗、过继细胞回输和肿瘤疫苗等。免疫疗法通过调动机体的免疫系统，增强肿瘤微环境抗肿瘤免疫力，从而控制和杀伤肿瘤细胞，因此有效率高，特异性强，耐受性好的优点，在肿瘤治疗中具有广阔的前景。
肿瘤免疫治疗疫苗主要包括肿瘤细胞疫苗、树突状细胞(DC细胞)疫苗、蛋白&多肽疫苗、核酸疫苗、基因工程疫苗和抗独特型肿瘤疫苗。这些疫苗能够杀伤肿瘤的主要机制即是通过引起患者针对于肿瘤特异性抗原免疫反应，包括抗原抗体反应和细胞毒性T淋巴细胞(CTL)特异性杀伤等，其中CTL特异性杀伤在肿瘤免疫反应中起了很大的作用。肿瘤特异性多肽是一种肿瘤特异性抗原，主要引起CTL特异性杀伤，它包括肿瘤突变的多肽以及肿瘤特异性高表达多肽。其中肿瘤突变的多肽由于其只存在于患者肿瘤组织，是肿瘤免疫治疗的一个特异性靶点，具有安全性好，及副作用小等特点。靶向肿瘤突变多肽的免疫治疗，以多肽特异性DC-CTL，以及肿瘤浸润淋巴细胞(TIL)过继回 输等方法为代表，具有良好的治疗效果。
肿瘤特异性多肽能够被CTL或TIL细胞识别，需要人类白细胞抗原HLA的抗原呈递功能。人白细胞抗原主要分为I和II两种亚型，I型HLA又主要分为A，B，C三种亚型，其中每种亚型，又根据其序列的不同，A，B，C三种亚型又可以分为多种亚型，HLA-A0201是HLA-A亚型中的一种，在中国人群中占比13％，具有较高的比例。不同的多肽与HLA-A0201亚型的结合力是不同的。在特定HLA亚型的肿瘤患者体内，HLA亚型决定了只能有部分突变多肽能与其HLA具有结合能力，并被其HLA递呈给CTL或TIL细胞。
然而，肿瘤免疫治疗仍有待进一步深入研究和开发。
发明内容
本发明是基于发明人对下列事实和问题的发现而提出的：
发明人经过大量的筛选实验，发现COL21A1基因(野生型COL21A1基因编码XXI型胶原的a链，起到维持胞外基质的完整性的作用)的突变导致其编码的第45位点的氨基酸由丝氨酸(Ser,S)突变为亮氨酸(Leu，L)，突变后的COL21A1基因能够在肿瘤组织中高水平表达，肿瘤组织内高水平表达该突变基因编码的多肽。并且，发明人通过实验验证了该突变多肽序列与HLA-A0201具有高亲和力。
基于上述的研究发现，在本发明的第一方面，本发明提出了一种分离的多肽。根据本发明的实施例，所述多肽选自：(1)具有SEQ ID NO：1所示氨基酸序列的多肽；或(2)与(1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的多肽；或(3)与(1)相比具有一个或者多个氨基酸的取代、缺失和/或添加的多肽。任选地，所述至少一个或多个氨基酸的取代、缺失和/或添加为如SEQ ID NO：1所述氨基酸序列的第2位和/或第9位氨基酸的取代。任选地，所述至少一个或多个氨基酸的取代、缺失和/或添加为如SEQ ID NO:1所示氨基酸序列的第2位氨基酸取代为L或M，和/或第9位氨基酸取代为L。任选地，所述多肽具有如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的氨基酸序列。其中，(2)所述多肽或(3)所述多肽具有和(1)所述多肽相同的功能。
FILDGLYSV(SEQ ID NO:1)。
FILDGLYSL(SEQ ID NO:2)。
FMLDGLYSV(SEQ ID NO:3)。
FLLDGLYSV(SEQ ID NO:4)。
FMLDGLYSL(SEQ ID NO:5)。
FLLDGLYSL(SEQ ID NO:6)。
根据本发明的实施例，所述多肽具有与HLA-A0201高亲和能力，具有激活特异性T细胞免疫的能力。
在本发明的第二方面，本发明提出了检测上述多肽的试剂在制备试剂盒中的用途，所 述试剂盒用于诊断肿瘤，任选地，所述肿瘤同时表达HLA-A0201和所述多肽，任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。基于发明人的实验研究发现，上述多肽在肿瘤组织中特异性高表达，进而发明人通过进一步的实验验证并提出，使用检测上述多肽的试剂制备的试剂盒可有效地用于诊断肿瘤；同时，发明人惊奇地发现，上述多肽与HLA-A0201有高亲和力，进而可被表达HLA-A0201的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，当所述肿瘤同时表达HLA-A0201和上述多肽时，该试剂盒诊断的安全性和有效性显著提高；同时，发明人发现肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤组织特异性高表达上述多肽，进而当肿瘤为上述肿瘤时，该试剂盒诊断的有效性和灵敏性可进一步提高。
在本发明的第三方面，本发明提出了上述多肽在制备药物中的用途，所述药物用于预防或治疗肿瘤，任选地，所述肿瘤同时表达HLA-A0201和所述多肽，任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。如前所述，发明人发现，上述多肽在肿瘤组织中特异性高表达，进而发明人通过进一步的实验验证并提出，上述多肽所制备的药物可有效用于预防或治疗肿瘤；当所述肿瘤同时表达HLA-A0201和上述多肽时，其治疗或预防的安全性和有效性显著提高；当肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤时，其治疗或预防的有效性和灵敏性可进一步提高。
在本发明的第四方面，本发明提出了一种分离的核酸。根据本发明的实施例，所述核酸为编码上述多肽的核酸或其互补序列。所述核酸能够特异性编码上述多肽，如前所述，上述多肽具有与HLA-A0201高亲和能力，具有激活特异性T细胞免疫的能力，进而，本发明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，尤其是肿瘤同时表达HLA-A0201和上述多肽时，其治疗或预防的安全性和有效性更高。
在本发明的第五方面，本发明提出了一种核酸构建体。根据本发明的实施例，所述核酸构建体包含编码序列，所述编码序列为上述的核酸，以及可选的控制序列，所述控制序列与所述编码序列可操作地连接。其中，所述控制序列为可指导多肽在宿主中表达的一个或多个控制序列。本发明实施例所提出的核酸构建体可在适合条件下，与表达载体连接后，在适合的宿主细胞中高效表达上述多肽，进而可有效用于对肿瘤，特别是同时表达HLA-A0201和上述多肽的肿瘤的特异性治疗或预防。
在本发明的第六方面，本发明提出了一种表达载体。根据本发明的实施例，所述载体包含上述的核酸构建体。本发明实施例所述提出的表达载体可在适合条件下，在表达宿主中高效表达上述多肽，所述表达载体可有效用于对肿瘤，特别是同时表达HLA-A0201和上述多肽的肿瘤的特异性治疗或预防。
在本发明的第七方面，本发明提出了一种宿主细胞。根据本发明的实施例，所述细胞 携带上述的核酸构建体或表达载体，可选地，是通过转染或者转化所述核酸构建体或表达载体获得的。根据本发明的实施例，所述宿主细胞在合适条件下可高效表达上述多肽，所述宿主细胞可有效用于对肿瘤，特别是同时表达HLA-A0201和上述多肽的肿瘤的特异性治疗或预防。
在本发明的第八方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：前面所述的多肽；以及药学上可接收的佐剂。发明人通过大量的实验发现，包括前面所述多肽和药学上可接受佐剂的药物组合物可显著刺激CTL或TIL的增值和分泌，可显著杀伤呈递上述多肽抗原的肿瘤细胞，具有显著治疗或预防肿瘤，尤其是特异性高表达上述多肽抗原的肿瘤的功效。
在本发明的第九方面，本发明提出了前面所述的多肽在制备疫苗中的用途，所述疫苗用于预防或治疗肿瘤，任选地，所述肿瘤同时表达HLA-A0201和所述多肽，任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。如前所述，发明人发现，上述多肽在肿瘤组织中特异性高表达，进而发明人通过进一步的实验验证并提出，上述多肽所制备的疫苗可有效用于预防或治疗肿瘤，其安全性更高，副作用更小；当所述肿瘤同时表达HLA-A0201和上述多肽时，其治疗或预防的安全性和有效性显著提高；当肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤时，其治疗或预防的有效性和灵敏性可进一步提高。
在本发明的第十方面，本发明提出了一种抗原呈递细胞。根据本发明的实施例，所述抗原呈递细胞可呈递前面所述的多肽。根据本发明的实施例，呈递前面所述多肽的抗原呈递细胞可有效引起患者针对肿瘤特异性抗原-上述多肽的免疫反应，进而激活CTL特异性杀伤功能，本发明实施例所提出的抗原呈递细胞具有显著的治疗表达HLA-A0201和上述多肽的肿瘤的功效，其治疗的效果显著，安全性高。
在本发明的第十一方面，本发明提出了一种免疫效应细胞。根据本发明的实施例，所述免疫效应细胞可识别前面所述多肽或者识别在细胞表面呈递前面所述多肽的抗原呈递细胞。根据本发明的实施例，所述免疫效应细胞可特异性杀伤共表达HLA-A0201和上述多肽的肿瘤细胞。
在本发明的第十二方面，本发明提出了一种疫苗。根据本发明的实施例，所述疫苗包含前面所述的核酸，或前面所述的核酸构建体，或前面所述的表达载体，或前面所述的宿主细胞，或前面所述的抗原呈递细胞，或前面所述的免疫效应细胞。如前所述，本发明实施例的核酸或核酸构建体或表达载体在合适的条件下表达前面所述的多肽，本发明实施例的核酸或核酸构建体或表达载体可用于治疗或预防表达前面所述多肽的肿瘤，本发明实施例所提出的抗原呈递细胞具有显著的治疗表达HLA-A0201和所述多肽的肿瘤的功效，本发明实施例所提出的免疫效应细胞具有显著的特异性杀伤呈递抗原-所述多肽的靶细胞的作用。本发明实施例所提出的疫苗具有显著的治疗或预防表达HLA-A0201和所述多肽的肿瘤的作用，其安全性更高、副作用更小。
在本发明的第十三方面，本发明提出了一种抗体。根据本发明的实施例，所述抗体特异性识别前面所述的多肽。本发明实施例所提出的抗体可特异结合所述多肽，进而可特异性识别特异性高表达所述多肽的肿瘤细胞，本发明实施例所提出的抗体在肿瘤诊断、治疗或预防中发挥巨大的作用。
在本发明的第十四方面，本发明提出了一种治疗方法。根据本发明的实施例，所述治疗方法包括：对患者给予治疗有效量的前面所述的多肽、前面所述的核酸、前面所述的核酸构建体、前面所述的表达载体、前面所述的宿主细胞、前面所述的药物组合物、前面所述的抗原呈递细胞、前面所述的免疫效应细胞、前面所述的疫苗或者前面所述的抗体。如前所述，本发明实施例所提出的治疗方法，包括给予任一种有效量的前面所述的多肽等，均可有效治疗或预防表达HLA-A0201和所述多肽的肿瘤。
在本发明的第十五方面，本发明提出了前面所述的多肽用于预防或治疗受试者中与COL21A1基因突变相关的疾病的用途。本发明实施例的多肽用于预防或治疗受试者中与COL21A1基因突变相关的疾病，效果显著。
在本发明的第十六方面，本发明提出了一种诊断方法。根据本发明的实施例，所述诊断方法包括：检测患者来源的生物样品是否携带前面所述的多肽；基于所述生物样品是否携带所述多肽，确定所述患者是否患有肿瘤，任选地，所述肿瘤同时表达HLA-A0201和所述多肽，任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。发明人发现，所述多肽在肿瘤组织中特异性高表达，而正常组织中不存在所述多肽。本发明实施例所提出的诊断方法可有效诊断出特异性高表达所述多肽的肿瘤患者。其中，发明人发现，肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤特异性高表达所述多肽，进而，本发明实施例所提出的方法对上述肿瘤的诊断准确性进一步提高。同时，发明人发现，HLA-A0201在中国人群中具有较高的比例，HLA-A0201与所述多肽有着较强的亲和力，所述多肽通过与细胞表面HLA-A0201结合进而激发一系列的免疫反应。因此本发明实施例所提出的诊断方法诊断出同时表达HLA-A0201和所述多肽的肿瘤患者的几率更高。
在本发明的第十七方面，本发明提出了一种诊断系统。根据本发明的实施例，所述诊断系统包括：多肽检测装置，所述多肽检测装置用于检测患者来源的生物样品是否携带前面所述的多肽；诊断结果确定装置，所述诊断结果确定装置与所述多肽检测装置相连，用于基于所述生物样品是否携带所述多肽，确定所述患者是否患有肿瘤，任选地，所述肿瘤同时表达HLA-A0201和所述多肽，任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。发明人发现，所述多肽在肿瘤组织中特异性高表达，而正常组织中不存在所述多肽。本发明实施例所提出的诊断系统可用于有效确定特异性高表达所述多肽的肿瘤患者。其中，发明人发现，肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤特异性高表达所述多肽，本发明实施例所提 出的诊断系统对上述肿瘤的诊断准确性进一步提高。同时，发明人发现，HLA-A0201在中国人群中具有较高的比例，HLA-A0201与所述多肽有着较强的亲和力，所述多肽通过与细胞表面HLA-A0201结合进而激发一系列的免疫反应。因而，本发明实施例所提出的诊断系统诊断出同时表达HLA-A0201和所述多肽的肿瘤患者的几率更高。
本发明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，或通过本发明的实践了解到。
附图说明
本发明的上述和/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是根据本发明实施例的诊断系统的结构示意图；
图2是根据本发明实施例的负载多肽的T2细胞与HLA-A0201亲和力的流式细胞仪检测结果图；
图3是根据本发明实施例的ELISPOTs方法验证多肽激活CD8+T细胞免疫反应的结果图；
图4是根据本发明实施例的激活的CD8+T细胞对负载多肽的靶细胞的特异性杀伤的结果图；
图5是根据本发明实施例的多肽免疫治疗的结果图，
其中，A显示了进行佐剂、佐剂+野生型(FILDGSYSV(SEQ ID NO:7)多肽组、FILDGLYSV(SEQ ID NO:1)多肽或其可变形式多肽(SEQ ID NO:2～6任一序列所示)各组+佐剂治疗后抑制肿瘤生长效果图，
B显示了进行佐剂、佐剂+野生型(FILDGSYSV(SEQ ID NO:7))多肽组、FILDGLYSV(SEQ ID NO:1)多肽或其可变形式多肽(SEQ ID NO:2～6任一序列所示)各组+佐剂治疗后小鼠生存率的结果图；
图6显示了根据本发明实施例的多肽免疫治疗的结果图，
其中，A显示了进行DC-负载野生型(FILDGSYSV(SEQ ID NO:7))多肽、DC-负载FILDGLYSV(SEQ ID NO:1)突变多肽或其可变形式(SEQ ID NO:2～6任一序列所示)多肽治疗后，抑制肿瘤生长效果图，
B显示了进行DC-负载野生型(FILDGSYSV(SEQ ID NO:7))多肽、DC-负载FILDGLYSV(SEQ ID NO:1)突变多肽或其可变形式(SEQ ID NO:2～6任一序列所示)多肽治疗后，小鼠生存率的结果图；
图7显示了多肽免疫治疗的结果图，
其中，A显示了携带编码野生型或突变多肽的核酸序列，或编码突变多肽可变形式多肽的核酸序列的慢病毒载体转染DC后用于免疫治疗，抑制肿瘤生长效果图，
B显示了携带编码野生型或突变多肽的核酸序列，或编码突变多肽可变形式多肽的核酸序列的慢病毒载体转染DC后用于免疫治疗，小鼠生存率的结果图；以及
图8显示了多肽免疫治疗的结果图，
其中，A显示了进行DC-负载野生型(FILDGSYSV(SEQ ID NO:7))多肽+CTL、DC负载突变多肽或其可变形式+CTL治疗后，抑制肿瘤生长效果图，
B显示了进行DC-负载野生型(FILDGSYSV(SEQ ID NO:7))多肽+CTL、DC负载突变多肽或其可变形式+CTL治疗后，小鼠生存率的结果图。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
多肽
在本发明的第一方面，本发明提出了一种分离的多肽。根据本发明的实施例，该多肽选自：(1)具有SEQ ID NO：1所示氨基酸序列的多肽；或(2)与(1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的多肽；或(3)与(1)相比具有一个或者多个氨基酸的取代、缺失和/或添加的多肽。本发明实施例所提出的多肽来源于肿瘤突变多肽，在未发生该突变的人体内不存在，且只存在于发生该突变的患者的肿瘤组织，正常组织不包含该突变。由于其只发现存在于患者肿瘤组织，而不存在正常组织，因此其特异性更高，引起的免疫反应的特异性也更高。
具体地，根据本发明的实施例，上述至少一个或多个氨基酸的取代、缺失和/或添加为如SEQ ID NO：1所述氨基酸序列的第2位和/或第9位氨基酸的取代。发明人发现，SEQ ID NO：1所述氨基酸序列的第2位和/或第9位氨基酸的取代不改变氨基酸序列与T细胞之间的特异性。
更具体地，根据本发明的实施例，上述至少一个或多个氨基酸的取代、缺失和/或添加为如SEQ ID NO:1所示氨基酸序列的第2位氨基酸取代为L或M，和/或第9位氨基酸取代为L。例如，上述多肽具有如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的氨基酸序列。根据本发明的实施例，FILDGLYSV(SEQ ID NO:1)、FILDGLYSL(SEQ ID NO:2)，FMLDGLYSV(SEQ ID NO:3)，FLLDGLYSV(SEQ ID NO:4)，FMLDGLYSL(SEQ ID NO:5)，FLLDGLYSL(SEQ ID NO:6)均与HLA-A0201具有高亲和能力，均具有激活特异性T细胞免疫的能力。发明人发现，多肽的可变形式FILDGLYSL(SEQ ID NO:2)，FMLDGLYSV(SEQ ID NO:3)，FLLDGLYSV(SEQ ID NO:4)，FMLDGLYSL(SEQ ID NO:5)，FLLDGLYSL(SEQ ID NO:6)，改变了多肽FILDGLYSV(SEQ ID NO:1)的第2位或第9位位点，第2位氨基酸取代为L或M，和/或第9位氨基酸取代为L，这种取代增强了多 肽与HLA-A0201的结合力，而不改变其与T细胞之间的特异性。因此，SEQ ID NO:2～6多肽与FILDGLYSV(SEQ ID NO:1)多肽均具有激活特异性T细胞免疫的能力。
用途
在应用方面，本发明一方面提出了检测上述多肽的试剂在制备试剂盒中的用途，上述多肽在制备药物中的用途以及上述多肽在制备疫苗中的用途，该试剂盒、药物或疫苗用于诊断、预防或治疗肿瘤。任选地，所述肿瘤同时表达HLA-A0201和所述多肽。任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。基于发明人的实验研究发现，上述多肽在肿瘤组织中特异性高表达，进而发明人通过进一步的实验验证并提出，检测上述多肽的试剂所制备的试剂盒或上述多肽制备药物或疫苗可有效用于诊断肿瘤，其安全性更高，副作用更小；同时，发明人惊奇地发现，上述多肽与HLA-A0201有高亲和力，进而可被表达HLA-A0201的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，当所述肿瘤同时表达HLA-A0201和上述多肽时，该试剂盒、药物或疫苗诊断或治疗的安全性和有效性显著提高；同时，发明人发现肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤组织特异性高表达上述多肽，进而当肿瘤为上述肿瘤时，该试剂盒、药物或疫苗诊断、治疗的有效性可进一步提高。
另一方面，本发明提出了上述多肽在预防或治疗受试者中与COL21A1基因突变相关的疾病的用途。发明人经过大量的筛选实验，发现COL21A1基因的突变导致其编码的第45位点的氨基酸由丝氨酸(Ser,S)突变为亮氨酸(Leu，L)。本发明实施例所述的多肽具有与COL21A1基因突变编码多肽相同的抗原特性，所述多肽所引起的特异性免疫反应，其效应细胞对于COL21A1基因突变细胞的特异性识别和杀伤效果显著，进而本发明实施例的多肽可用于OL21A1基因突变相关疾病的预防或治疗。且发明人通过实验发现，所述多肽用于预防或治疗OL21A1基因突变相关疾病的效果显著。
治疗组合物
一方面，本发明提出了一种分离的核酸。根据本发明的实施例，所述核酸为编码上述多肽的核酸或其互补序列。所述核酸能够特异性编码上述多肽，如前所述，上述多肽具有与HLA-A0201高亲和能力，具有激活特异性T细胞免疫的能力，进而，本发明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，尤其是肿瘤同时表达HLA-A0201和上述多肽时，其治疗或预防的安全性和有效性更高。
需要说明的是，对于本发明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。
相应地，另一方面，本发明提出了一种核酸构建体。根据本发明的实施例，所述核酸构建体包含编码序列，所述编码序列为上述的核酸，以及可选的控制序列，所述控制序列与所述编码序列可操作地连接。其中，所述控制序列为可指导多肽在宿主中表达的一个或 多个控制序列。根据本发明的实施例，控制序列包括但不限于U6，H1，CMV，EF-1，LTR或RSV启动子。本发明实施例所提出的核酸构建体可在适合条件下，与表达载体连接后，在适宜的宿主细胞中高效表达上述多肽，进而可有效用于对肿瘤，特别是同时表达HLA-A0201和上述多肽的肿瘤的特异性治疗或预防。
相应地，另一方面，本发明提出了一种表达载体。根据本发明的实施例，所述载体包含上述的核酸构建体。所述表达载体的类型不受特别限制，只要能够实现前面所述的核酸构建体在受体细胞中高效表达即可，表达载体包括但不限于反转录病毒载体、慢病毒载体和/或腺病毒相关病毒载体。本发明实施例所述提出的表达载体可在适合条件下，在表达宿主中高效表达上述多肽，所述表达载体可有效用于对肿瘤，特别是同时表达HLA-A0201和上述多肽的肿瘤的特异性治疗或预防。
相应地，另一方面，本发明提出了一种宿主细胞。根据本发明的实施例，所述细胞携带上述的核酸构建体或表达载体，可选地，是通过转染或者转化所述核酸构建体或表达载体获得的。转化或转染可采用电转、病毒转染或感受态细胞转化的方式进行。采用何种转染或转化的方式是根据宿主细胞的性质以及待转核酸构建体或表达载体的性质所决定的，只要能够在所述宿主细胞中实现前面所述多肽的高效表达并对宿主细胞的良好的细胞状态不产生较大影响即可。根据本发明的实施例，所述宿主细胞在合适条件下可高效表达上述多肽，所述宿主细胞可有效用于对肿瘤，特别是同时表达HLA-A0201和上述多肽的肿瘤的特异性治疗或预防。
需要说明的是，本申请说明书中所述的“适合条件”，是指适合本申请所述多肽表达的条件。本领域技术人员容易理解的是，适合多肽表达的条件包括但不限于合适的转化或转染方式、合适的转化或转条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的所述多肽表达的条件。
再一方面，本发明提出了一种药物组合物。根据本发明的实施例，该药物组合物包括：前面所述的多肽；以及药学上可接收的佐剂。发明人通过大量的实验发现，包括前面所述多肽和药学上可接受佐剂的药物组合物可显著刺激CTL或TIL的增值和分泌，可显著杀伤呈递上述多肽抗原的肿瘤细胞，具有显著治疗或预防肿瘤，尤其是特异性高表达上述多肽抗原的肿瘤的功效。
另一方面，本发明提出了一种抗原呈递细胞。根据本发明的实施例，该抗原呈递细胞可呈递前面所述的多肽。根据本发明的实施例，呈递前面所述的多肽的抗原呈递细胞可有效引起患者针对肿瘤特异性抗原-上述多肽的免疫反应，进而激活CTL特异性杀伤功能，本发明实施例所提出的抗原呈递细胞具有显著的治疗表达HLA-A0201和上述多肽的肿瘤的功效，其治疗的效果显著，安全性高。
根据本发明的具体示例，所述抗原呈递细胞通过下列至少之一的方式获得：将具有抗原呈递能力的细胞与所述多肽接触；或将前面所述的核酸、或者前面所述的核酸构建体、或者前面所述的表达载体导入所述具有抗原呈递能力的细胞。发明人通过实验发现，抗原 呈递细胞通过上述方式任何一种或几种，可有效呈递前面所述的多肽，将前面所述的多肽暴露在呈递细胞的表面，呈递了前面所述多肽的抗原呈递细胞可有效引起患者对肿瘤特异性抗原-上述多肽的免疫反应，进而激活CTL特异性杀伤功能。
根据本发明的具体实施例，所述抗原呈递细胞为树突细胞。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。
再一方面，本发明提出了一种免疫效应细胞。根据本发明的实施例，所述免疫效应细胞可识别前面所述的多肽或识别在细胞表面呈递前面所述多肽的抗原呈递细胞。根据本发明的实施例，该免疫效应细胞是通过前面所述的抗原呈递细胞与具有免疫效应能力的细胞进行接触获得的。发明人发现，通过呈递前面所述的多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。根据本发明的再一具体示例，具有免疫效应能力的细胞为T淋巴细胞，发明人发现，优选CD8+T细胞，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-所述多肽的靶细胞的作用更强。
再一方面，本发明提出了一种疫苗。根据本发明的实施例，该疫苗包含前面所述的核酸、核酸构建体、表达载体、宿主细胞、前面所述的抗原呈递细胞，或前面所述的免疫效应细胞。如前所述，本发明实施例的核酸、核酸构建体、表达载体、宿主细胞能够用于高表达所述多肽的肿瘤的特异性杀伤，本发明实施例所提出的抗原呈递细胞具有显著的治疗表达HLA-A0201和所述多肽的肿瘤的功效，另外，本发明实施例所提出的免疫效应细胞具有显著的特异性杀伤呈递抗原-所述多肽的靶细胞的作用。本发明实施例所提出的疫苗包含前面所述的核酸、核酸构建体、表达载体、宿主细胞、抗原呈递细胞或免疫效应细胞，其具有显著的治疗或预防表达HLA-A0201和所述多肽的肿瘤的作用，其安全性更高、副作用更小。
再一方面，本发明提出了一种抗体。根据本发明的实施例，该抗体特异性识别前面所述的多肽。本发明实施例所提出的抗体可特异结合所述多肽，进而可特异性识别特异性高表达所述多肽的肿瘤细胞，本发明实施例所提出的抗体在肿瘤诊断、治疗或预防中发挥巨大的作用。另外，根据本发明的实施例，所述抗体可通过如下方式获得：采集使用前面所述的多肽进行免疫接种的动物的血清；以及从所述血清中纯化出目的抗体。利用根据本发明实施例的制备抗体的方法，可方便、快捷、有效地制备出具有特异性识别所述多肽的抗体，所制备的抗体可有效用于肿瘤的诊断、治疗和预防。
综上，本发明实施例所提出的多肽由于其只发现存在于患者肿瘤组织，而不存在正常组织，因此其特异性更高，引起的免疫反应的特异性也更高，与其他肿瘤多肽疫苗相比，具有更为安全，副作用小，很少引起严重的免疫反应的优点，又因其结构简单、易于人工合成，可作为疫苗、药物组合物等，引起针对肿瘤的免疫反应。FILDGLYSV(SEQ ID NO:1) 该多肽或其可变形式可以被作为靶点或者疫苗应用于针对于同时表达HLA-A0201和该突变多肽的肿瘤生物治疗，具有引起机体免疫反应。可以采用多肽+佐剂，或多肽负载的抗原呈递细胞疫苗，或多肽特异性DC-CTL,DC-CIK疫苗等方式，特异性杀伤肿瘤细胞，预防以及治疗癌症，包括表达该多肽序列的肺癌，黑色素瘤，乳腺癌，鼻咽癌，肝癌，胃癌，食道癌，结直肠癌，胰腺癌，皮肤癌，前列腺癌，宫颈癌，白血病，脑肿瘤等癌症类型。
治疗方法
更进一步，本发明提出了一种治疗方法。根据本发明的实施例，该治疗方法包括：对患者给予治疗有效量的前面所述的多肽、前面所述的核酸、前面所述的核酸构建体、前面所述的表达载体、前面所述的宿主细胞、前面所述的药物组合物、前面所述的抗原呈递细胞、前面所述的免疫效应细胞、前面所述的疫苗或者前面所述的抗体。如前所述，本发明实施例所提出的治疗方法，包括给予任一种有效量的前面所述的多肽等，均可有效治疗或预防表达HLA-A0201和所述多肽的肿瘤。
在本文中所使用的术语“给予”指将预定量的物质通过某种适合的方式引入病人。本发明实施例中的多肽、核酸、核酸构建体、表达载体、宿主细胞、药物组合物、抗原呈递细胞、免疫效应细胞、疫苗或抗体可以通过任何常见的途径给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但是本发明不限于这些已举例的给药方式。然而，由于口服给药时，口服给药的组合物的活性成分应该被包被或被配制以防止其在胃部被降解。优选地，本发明的组合物可以注射制剂给药。此外，本发明的药物组合物可以使用将活性成分传送到靶细胞的特定器械来给药。
本发明实施例中的多肽、核酸、核酸构建体、表达载体、宿主细胞、药物组合物、抗原呈递细胞、疫苗或抗体的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些实施例，日剂量可分为适宜形式的1剂、2剂或多剂，以在整个时间段内以1次、2次或多次给药，只要达到治疗有效量即可。
术语“治疗有效量”是指足以显著改善某些与疾病或病症相关的症状的量，也即为给定病症和给药方案提供治疗效果的量。术语“治疗”用于指获得期望的药理学和/或生理学效果。本文使用的“治疗”涵盖将发明实施例中的多肽、核酸、核酸构建体、表达载体、宿主细胞、药物组合物、抗原呈递细胞、免疫效应细胞、疫苗或抗体给予个体以治疗，包括但不限于将含本文所述的给予有需要的个体。
诊断方法
另外，本发明提出了一种诊断方法。根据本发明的实施例，该诊断方法包括：检测患者来源的生物样品是否携带前面所述的多肽；基于所述生物样品是否携带所述多肽，确定所述患者是否患有肿瘤。由于本发明实施例所提出的多肽仅发现于癌组织中，进而可以通过质谱的方式检测到血清中存在的该游离多肽，所述多肽作为肿瘤标志物应用于癌症的诊断，确定患者是否患有癌症。发明人发现，所述多肽在肿瘤组织中特异性高表达，本发明 实施例所提出的诊断方法可有效诊断出特异性高表达所述多肽的肿瘤患者。
其中，发明人发现，肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤特异性高表达所述肿瘤，进而，本发明实施例所提出的方法对上述肿瘤的诊断准确性进一步提高。
同时，发明人发现，HLA-A0201在中国人群中具有较高的比例，从而，本发明实施例所提出的诊断方法诊断出同时表达HLA-A0201和所述多肽的肿瘤患者的几率更高。
诊断系统
最后，本发明提出了一种诊断系统。根据本发明的实施例，参考图1，该诊断系统包括：多肽检测装置100；诊断结果确定装置200。其中，多肽检测装置100用于检测患者来源的生物样品是否携带前面所述的多肽，诊断结果确定装置200与所述多肽检测装置100相连，用于基于生物样品是否携带所述多肽，确定所述患者是否患有肿瘤。根据本发明具体实施例，可以采用质谱仪检测患者血清中是否存在的该游离多肽，进而通过质谱数据分析装置确定患者血清中是否存在该游离多肽，确定所述患者是否患有肿瘤。发明人发现，所述多肽在肿瘤组织中特异性高表达，本发明实施例所提出的诊断系统可用于有效确定特异性高表达所述多肽的肿瘤患者。
另外，发明人发现，肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤特异性高表达所述肿瘤，本发明实施例所提出的诊断系统对上述肿瘤的诊断准确性进一步提高。
同时，发明人发现，HLA-A0201在中国人群中具有较高的比例，HLA-A0201与所述多肽有着较强的亲和力，所述多肽通过与细胞表面HLA-A0201结合进而激发一系列的免疫反应。因而，本发明实施例所提出的诊断系统诊断出同时表达HLA-A0201和所述多肽的肿瘤患者的几率更高。
需要说明的是，根据本发明实施例的多肽及其用途、编码所述多肽的核酸、核酸构建体、表达载体、宿主细胞、药物组合物、抗原呈递细胞、免疫效应细胞、疫苗、抗体、治疗和诊断癌症的方法和系统是本申请的发明人经过艰苦的创造性劳动和优化工作才发现和完成的。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。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(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)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，例如可以采购自Illumina公司。
实施例1多肽的亲和力预测
根据选定的HLA等位基因分型，利用自主开发的“基于肿瘤DNA和RNA测序的突变多肽结合能力预测软件”(软件著作权号：2016SR002835)对多肽分别进行亲和力预测。预测结果用IC50分值表示，IC50小于500nM表示该多肽有亲和力，IC50小于50nM表示该 多肽具有高亲和力。发明人对野生型(FILDGSYSV(SEQ ID NO：7))多肽、突变多肽及其可变形式多肽进行亲和力预测，最后筛选突变多肽及其可变形式多肽IC50得分小于500nM，并且突变多肽及其可变形式多肽的IC50得分小于野生型(FILDGSYSV(SEQ ID NO：7))多肽。多肽的亲和力预测结果见表1。根据该结果，进行下一步T2亲和力验证。
表1多肽与HLA-A0201的亲和力预测结果
Figure PCTCN2016098328-appb-000001
经计算机软件预测，突变多肽及其可变形式多肽的IC50均低于50nM，说明突变多肽及其可变形式多肽经预测，均为高亲和力的多肽。且突变多肽及其可变形式多肽的IC50小于野生型(FILDGSYSV(SEQ ID NO：7))多肽。因此，经预测可知突变多肽及其可变形式多肽的亲和力高于野生型(FILDGSYSV(SEQ ID NO：7))多肽。
实施例2多肽T2亲和力验证
(一)、多肽的合成与纯化
依照标准固相合成方法合成本发明实施例中所涉及的各种类型多肽，并通过反相HPLC进行纯化。多肽的纯度(>90％)和身份分别通过HPLC和质谱测定。
(二)、亲和力验证
T2细胞是HLA-A2阳性的T、B淋巴细胞杂交瘤细胞，其细胞表面可表达HLA-A0201,但因其内源性抗原呈递途径中必需的抗原多肽转运蛋白(TAP)缺陷，故不能转运内源性抗原。T2细胞购自ATCC(编号：CRL-1992)。
取2×105个T2细胞，用500μl含有人类β2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，加入合成的野生型FILDGSYSV(SEQ ID NO：7)多肽、FILDGLYSV(SEQ ID NO：1)多肽或其5种可变形式多肽(最终浓度100μM)，在培养 箱(37℃,5％CO2)，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，加入CMV多肽(NLVPMVATV(SEQ ID NO：8))作为阳性对照。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-A*02:01的FITC单克隆抗体孵育，4℃维持30分钟。然后用流式细胞仪(BD FACSJazzTM)及其软件检测并分析其平均荧光强度。荧光指数(FI)用下列公式计算：FI＝[平均荧光强度(MFI)样品-MFIbackground]/MFIbackground，其中MFIbackground代表不含肽的值。FI>1.5表明该肽对HLA-A*0201分子具有高亲和性，1.0<FI<1.5表明该肽对HLA-A*02:01分子具有中等亲和力，以及0.5<FI<1.0表明该肽为HLA-A*0201分子低亲和力。多肽的亲和力检测结果见表2和图2。
表2：多肽与HLA-A0201亲和力的检测结果
Figure PCTCN2016098328-appb-000002
经实验预测，背景对照的FI为0，CMV的阳性多肽FI为1.88，两个均正常。而野生型(FILDGSYSV(SEQ ID NO：7))多肽和突变多肽及其5种可变形式多肽的FI都大于1.5，进一步证明野生型(FILDGSYSV(SEQ ID NO：7))多肽、突变多肽及其可变形式多肽对HLA-A0201分子都是具有高亲和力的。
实施例3多肽体外刺激CD8+T细胞
取HLA-A0201亚型阳性的健康志愿者的外周血100ml，采用Ficoll淋巴细胞分离液，分离外周血单个核细胞(PBMC)，将PBMC用贴壁法获取单核细胞，并用CD8磁珠筛选PBMC细胞中的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，再加入IFN-gamma(100U/ml)，CD40L(100U/ml)，最后分 别加入多肽(FILDGLYSV(SEQ ID NO:1))及其5种可变形式多肽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细胞与志愿者的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。计数结果表3所示：
表3：培养后计数结果
Figure PCTCN2016098328-appb-000003
培养10天后，细胞有明显增殖，总的细胞数目扩增倍数在4-7倍之间。
实施例4ELISPOTs方法验证多肽激活CD8+T细胞免疫反应
实施例3中共培养的细胞分别与负载过突变多肽FILDGLYSV(SEQ ID NO：1)、野生型(FILDGSYSV(SEQ ID NO：7))多肽的T2细胞加入到人γ干扰素ELISPOTs板中培养与检测。最终对ELISPOT实验产生的斑点进行计数。突变多肽具有免疫原性的要求如下：斑点数(突变多肽)/斑点数(野生型(FILDGSYSV(SEQ ID NO：7))多肽)>2，即突变多肽引起的斑点数超过野生型(FILDGSYSV(SEQ ID NO：7))多肽斑点数目的两倍以上是多肽具有免疫原性的要求。
结果见表4和图3。
其中，ELISPOTs检测方法原理是：CD8+T细胞能够特异性识别HLA-A0201与多肽的复合物，多肽序列的不同，识别多肽与HLA-A0201的复合物的T细胞的群体也不同。由于T2细胞表达HLA-A0201，因此，CD8+T细胞能够特异性识别负载了突变多肽FILDGLYSV(SEQ ID NO：1)的T2细胞，而不能识别野生型(FILDGSYSV(SEQ ID NO：7))多肽负载的T2细胞。在特异识别HLA-A0201和多肽的复合物之后，多肽特异性CD8+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，最终识别IFN-gamma的抗体可以通过偶联在抗体上的酶，催化底物显色，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。
表4：多肽刺激特异性CD8+T细胞分泌IFN-gamma干扰素结果
Figure PCTCN2016098328-appb-000004
实施例5LDH释放实验证明CD8+T细胞杀伤活性
实施例3中共培养的细胞与负载过突变多肽FILDGLYSV(SEQ ID NO：1)、或野生型(FILDGSYSV(SEQ ID NO：7))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(即效应细胞(T细胞)与靶细胞(T2细胞)的数目比)等对照，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，计算CD8+T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％
其中，LDH释放试验的原理是：乳酸脱氢酶(LDH)是活细胞胞浆内含酶之一，在正常情况下，不能透过细胞膜。当靶细胞受到效应细胞的攻击而损伤时，细胞膜通透性改变，LDH可释放至介质中。释放出来的LDH在催化乳酸生成丙酮酸的过程中，使氧化型辅酶I(NAD+)变成还原型辅酶I(NADH)，后者再通过递氢体-吩嗪二甲酯硫酸盐(PMS)还 原碘硝基氯化氮唑蓝(INT)或硝基氯化四氮唑蓝(T)形成有色的甲臜类化合物，在490nm或570nm波长处有一高吸收峰，利用读取的OD值，经过计算即可得知效应细胞活性。
结果见表5和图4。
表5：T细胞特异性识别并杀伤负载实验多肽的靶细胞
Figure PCTCN2016098328-appb-000005
从上表以及图4中可看出，在效靶比1:1或1:10时，突变多肽及其可变形式多肽激活的T细胞，能够杀伤负载了突变多肽FILDGLYSV(SEQ ID NO：1)的T2细胞，而不能杀伤负载野生型(FILDGSYSV(SEQ ID NO：7))多肽的T2细胞，这进一步验证实验组多肽(SEQ ID NO：1所示氨基酸序列的多肽及其可变形式多肽)激活的T细胞能特异性杀伤负载突变多肽FILDGLYSV(SEQ ID NO：1)的靶细胞。
实施例6H2087-FILDGLYSV(SEQ ID NO：1)或其可变形式多肽的皮下移植瘤模型的建立
(一)构建并包装FILDGLYSV(SEQ ID NO：1)多肽或其可变形式多肽的重组慢病毒
合成FILDGLYSV(SEQ ID NO：1)多肽的DNA序列如SEQ ID NO:9所示，TTCATCCTGGACGGCCTGTACAGCGTG(SEQ ID NO:9)，
及其可变形式FILDGLYSL(SEQ ID NO:2)多肽的DNA序列如SEQ ID NO:10所示，TTCATCCTGGACGGCCTGTACAGCCTG(SEQ ID NO:10)，
及其可变形式FMLDGLYSV(SEQ ID NO:3)多肽的DNA序列如SEQ ID NO:11所示，TTCATGCTGGACGGCCTGTACAGCGTG(SEQ ID NO:11)，
及其可变形式FLLDGLYSV(SEQ ID NO:4)的多肽DNA序列如SEQ ID NO:12所示，TTCCTGCTGGACGGCCTGTACAGCGTG(SEQ ID NO:12)，
及其可变形式FMLDGLYSL(SEQ ID NO:5)多肽的DNA序列如SEQ ID NO:13所示，TTCATGCTGGACGGCCTGTACAGCCTG(SEQ ID NO:13)，
及其可变形式FLLDGLYSL(SEQ ID NO:6)多肽的DNA序列如SEQ ID NO:14所示，TTCCTGCTGGACGGCCTGTACAGCCTG(SEQ ID NO:14)，
野生型FILDGSYSV(SEQ ID NO：7)多肽对应的DNA序列如SEQ ID NO:15所示，TTCATCTTAGATGGCTCTTATAGTGTT(SEQ ID NO:15)。
分别构建表达野生型多肽FILDGSYSV(SEQ ID NO：7)、FILDGLYSV(SEQ ID NO：1)的多肽及其可变形式多肽的慢病毒载体pHBLV-Puro。并分别命名为pHBLV-FILDGSYSV-Puro，pHBLV-FILDGLYSV-Puro，pHBLV-FILDGLYSL-Puro，pHBLV-FMLDGLYSV-Puro，pHBLV-FLLDGLYSV-Puro，pHBLV-FMLDGLYSL-Puro，pHBLV-FLLDGLYSL-Puro。再分别将这7个慢病毒质粒与pSPAX2和pMD2G辅助质粒共同转染293T细胞，包装出表达野生型多肽、FILDGLYSV(SEQ ID NO：1)多肽及其可变形式多肽的慢病毒。
(二)表达FILDGLYSV(SEQ ID NO：1)多肽的人源肺癌细胞系的建立
人非小细胞系肺腺癌细胞系NCI-H2087购买于ATCC(编号：CRL-5922),其HLA亚型为HLA-A*0201阳性。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％CO2的孵箱中培养。将包装好的FILDGLYSV(SEQ ID NO：1)慢病毒转染H2087细胞系，并采用Puromycin抗生素(嘌呤霉素)，持续筛选存活的H2087细胞系，最终建立表达FILDGLYSV(SEQ ID NO：1)多肽的H2087细胞系。并命名为 H2087-FILDGLYSV(SEQ ID NO：1)细胞系。
(三)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 2×107/0.5ml，对NOD SCID小鼠进行人免疫重建。进而选取4周后的小鼠准备接种人肺癌细胞系模型。
(四)人非小细胞系肺腺癌肿瘤模型的构建
已建系的人非小细胞系肺腺癌细胞系H2087-FILDGLYSV(SEQ ID NO：1)，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％CO2的孵箱中培养。收集H2087-FILDGLYSV(SEQ ID NO：1)肿瘤细胞，3000转离心，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1×108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100μl。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，用游标卡尺，每2-3天测量肿瘤长径a(长)和短径b(宽)，并计算肿瘤的大小＝a×b×b/2。7天后，小鼠皮下瘤可摸到约米粒大小肿瘤。对免疫重建4周的H2087-FILDGLYSV(SEQ ID NO：1)皮下瘤模型NOD/SCID小鼠分别进行多肽+完全弗氏佐剂疫苗，或多肽+DC疫苗，或慢病毒感染的DC细胞疫苗、以及DC-CTL疫苗治疗，并每2天记录肿瘤的体积和小鼠的生存率。
实施例7多肽疫苗的制备及治疗方案
将免疫重建4周的H2087-FILDGLYSV(SEQ ID NO：1)皮下瘤模型NOD/SCID小鼠随机分为8组：野生型(FILDGSYSV(SEQ ID NO：7))多肽组、佐剂组、佐剂十FILDGLYSV(SEQ ID NO：1)或5种可变性形式多肽组组，每组各6只。野生型(FILDGSYSV(SEQ ID NO：7))多肽、FILDGLYSV(SEQ ID NO：1)多肽或5种可变性形式多肽的首次免疫剂量为100μg/只。上述多肽用PBS重悬后，与150μl/只弗氏完全佐剂混匀后，用PBS调整至300μl/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。每天观察小鼠的一般特征，包括精神状态、活动力、反应、饮食、体重及肿瘤的生长情况等。每2天用游标卡尺测量肿瘤最长径(长)和最短径(宽)。其中，肿瘤体积的计算公式为：1/2×长×宽2；生存期计算公式为：一定时间内生存率＝该时间内存活小鼠/(该时间内存活小鼠+该时间内死亡小鼠) ×100％。结果见图5。
结果显示，相对于单纯佐剂组和野生型(FILDGSYSV(SEQ ID NO：7))多肽组，“FILDGLYSV(SEQ ID NO：1)或其可变形式+弗氏佐剂”组均能够有效的抑制肿瘤的生长，并延长小鼠的生存期。
实施例8DC多肽疫苗的制备及治疗方案
采集健康志愿者抗凝外周血100～150ml。Ficoll分离外周血单个核细胞(peripheral blood mononuclear，PBMC)，收集PBMC细胞，按2～3×106/ml重悬于RPMI 1640培养基中，37℃孵育2h，贴壁细胞即为DC，吸取未贴壁细胞即是外周血淋巴细胞(peripheral blood lymphocyte,PBL)，备用。采用GM-CSF(1000U/ml)，IL-4(1000U/ml)，诱导贴壁单核细胞为未成熟DC，再加入IFN-gamma(100U/ml)，CD40L(10ng/ml)，最后分别加入野生型(FILDGSYSV(SEQ ID NO：7))多肽、FILDGLYSV(SEQ ID NO：1)多肽及其可变形式多肽(浓度为10μg/ml)，诱导贴壁细胞为成熟DC细胞，收获成熟DC，用生理盐水洗涤3次。用生理盐水将负载多肽后的DC调整为(4.0±0.5)×107/ml，用于后续实验。将小鼠随机分为7组:DC-负载野生型(FILDGSYSV(SEQ ID NO：7))多肽组、DC-负载FILDGLYSV(SEQ ID NO：1)多肽、以及DC-负载5种可变性形式多肽组，每组各6只。制备DC-负载野生型(FILDGSYSV(SEQ ID NO：7))多肽，DC-负载FILDGLYSV(SEQ ID NO：1)多肽及其任意一种的可变形式(SEQ ID NO：2～6)的多肽细胞悬液。对小鼠近腹股沟大腿内侧进行皮内注射，每侧注射0.1ml,每周注射1次。剂量为(4.0±0.5)×106细胞/次，共注射2次。注射结束后观察小鼠生命体征，每2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和小鼠生存情况。结果见图6。
结果显示，相对于野生型(FILDGSYSV(SEQ ID NO：7))多肽负载的DC疫苗组，FILDGLYSV(SEQ ID NO：1)或其可变形式(SEQ ID NO：2～6任一项)负载的DC疫苗可以明显的延长小鼠的生存期，以及减缓小鼠肿瘤的生长。
实施例9慢病毒感染的DC细胞疫苗的制备及治疗方案
采集健康志愿者抗凝外周血100～150ml。Ficoll分离外周血单个核细胞(peripheral blood mononuclear，PBMC)，收集PBMC细胞，37℃孵育2h，洗去未贴壁细胞，经重组人粒细胞一巨噬细胞集落刺激因子(rhGM-CSF)、重组人白介-4(rhIL-4)培养DC细胞。培养 至第五天，更换半适量的培养基及调整细胞密度为1×106个/ml；分别加入实施例6中构建表达的含有适量野生型(FILDGSYSV(SEQ ID NO：7))多肽、FILDGLYSV(SEQ ID NO：1)及其可变形式多肽(SEQ ID NO：2～6任一序列所示)的慢病毒液。24h后去除病毒培养液，加入含有50ng/ml rhIL-4、100ng/ml rh GM-CSF，100U/ml的IFN-γ和100U/ml CD40L的培养液，置于37℃5％CO2培养箱中培养。48-72h后荧光显微镜下观察慢病毒感染DC细胞，收集成熟DC细胞。用生理盐水洗3次，并将DC调整为(4.0±0.5)x107个/ml，用于后续实验。将小鼠随机分为7组:野生型(FILDGSYSV(SEQ ID NO：7))多肽-DC组、FILDGLYSV(SEQ ID NO：1)多肽-DC组、及其5种可变性形式(SEQ ID NO：2～6任一序列所示)多肽-DC组，每组各6只。制备DC-负载野生型(FILDGSYSV(SEQ ID NO：7))多肽，DC-负载FILDGLYSV(SEQ ID NO：1)多肽或其五种可变形式多肽(SEQ ID NO：2～6任一序列所示)细胞悬液。对小鼠近腹股沟大腿内侧进行皮内注射，每侧注射0.1ml，每周注射1次。剂量为(4.0±0.5)×106细胞/次，共注射2次。注射结束后观察小鼠生命体征，每2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和小鼠生存情况。结果如图7所示。
图7结果显示，相对于野生型多肽对照组，表达FILDGLYSV(SEQ ID NO：1)或其可变形式多肽(SEQ ID NO：2～6任一序列所示)基因包装的慢病毒感染的DC疫苗，具有明显的肿瘤抑制效果，并且能够显著延长小鼠的生存期。
实施例10多肽特异性DC-CTL疫苗的制备及治疗方案
实施例8收集的PBL经过磁珠分选获得CD8+T与负载野生型(FILDGSYSV(SEQ ID NO：7))多肽的DC，负载FILDGLYSV(SEQ ID NO：1)多肽或其五种可变形式(SEQ ID NO：2～6任一序列所示)多肽的DC共育致敏，细胞比例为DC:CD8+T＝1:4。培养液中加入500IU/ml IL-2和50ng/ml IL-7，37℃5％CO2培养箱共同孵育，培养1周后进行细胞计数；第2周再用负载FILDGLYSV(SEQ ID NO：1)多肽的DC或其五种可变形式多肽(SEQ ID NO：2～6任一序列所示)DC、负载野生型(FILDGSYSV(SEQ ID NO：7))多肽的DC进行第二轮刺激。共刺激三轮，培养期间适当添加培养基。于培养第0,7,14和21天分别计数淋巴细胞数量，计算细胞增殖指数(proliferation index，PI)。其中，PI＝扩增后细胞数/接种细胞数。培养至21天后收获细胞-细胞毒性T淋巴细胞(cytotoxic T lymphocytes，CTL)。将细胞用生理盐水重悬，重悬体积为0.2ml，经尾静脉回输，每只肿瘤模型小鼠回输细胞数约为1x108细胞。注射结束后留心观察小鼠生命体征，每2天用游标卡 尺测量肿瘤纵横大小。结果如图8所示。
图8结果显示，相对于野生型多肽对照组，FILDGLYSV(SEQ ID NO：1)或其可变形式多肽(SEQ ID NO：2～6任一序列所示)激活的DC-CTL疫苗，具有明显的肿瘤抑制效果，并且能够显著延长小鼠的生存期。
工业实用性
本发明的多肽，能够有效地应用于制备试剂盒、药物或疫苗，该药物或疫苗引起的免疫反应的特异性也更高，与其他肿瘤多肽疫苗相比，具有更为安全，副作用小，很少引起严重的免疫反应的优点，又因其结构简单、易于人工合成，可作为疫苗、药物组合物等，引起针对肿瘤的免疫反应。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“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发明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一定指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何的一个或多个实施例或示例中以合适的方式结合。

Claims (20)

  1. 一种分离的多肽，其特征在于，所述多肽选自：
    (1)具有SEQ ID NO：1所示氨基酸序列的多肽；或
    (2)与(1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的多肽；或
    (3)与(1)相比具有一个或者多个氨基酸的取代、缺失和/或添加的多肽；
    任选地，所述至少一个或多个氨基酸的取代、缺失和/或添加为如SEQ ID NO：1所述氨基酸序列的第2位和/或第9位氨基酸的取代，
    任选地，所述至少一个或多个氨基酸的取代、缺失和/或添加为如SEQ ID NO:1所示氨基酸序列的第2位氨基酸取代为L或M，和/或第9位氨基酸取代为L，
    任选地，所述多肽具有如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的氨基酸序列。
  2. 检测权利要求1所述多肽的试剂在制备试剂盒中的用途，所述试剂盒用于诊断肿瘤，
    任选地，所述肿瘤同时表达HLA-A0201和所述多肽，
    任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。
  3. 权利要求1所述的多肽在制备药物中的用途，所述药物用于预防或治疗肿瘤，
    任选地，所述肿瘤同时表达HLA-A0201和所述多肽，
    任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。
  4. 一种分离的核酸，其特征在于，所述核酸为：
    编码权利要求1所述多肽的核酸或其互补序列。
  5. 一种核酸构建体，其特征在于，包含：
    编码序列，所述编码序列为权利要求4所述的核酸，以及
    可选的控制序列，所述控制序列与所述编码序列可操作地连接。
  6. 一种表达载体，其特征在于，所述载体包含权利要求5所述的核酸构建体。
  7. 一种宿主细胞，其特征在于，所述细胞携带权利要求5所述的核酸构建体或权利要求6所述的表达载体，
    可选地，所述宿主细胞是通过转染或者转化所述核酸构建体或表达载体获得。
  8. 一种药物组合物，其特征在于，包括：
    权利要求1所述的多肽；以及
    药学上可接收的佐剂。
  9. 权利要求1所述的多肽在制备疫苗中的用途，所述疫苗用于预防或治疗肿瘤，
    任选地，所述肿瘤同时表达HLA-A0201和所述多肽，
    任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直 肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。
  10. 一种抗原呈递细胞，其特征在于，所述细胞可呈递权利要求1所述的多肽。
  11. 根据权利要求10所述的抗原呈递细胞，其特征在于，所述抗原呈递细胞是通过下列至少之一获得的：
    将具有抗原呈递能力的细胞与所述多肽接触；或
    将权利要求4所述的核酸、或者权利要求5所述的核酸构建体、或者权利要求6所述的表达载体导入所述具有抗原呈递能力的细胞，
    任选地，所述具有抗原呈递能力的细胞为树突细胞。
  12. 一种免疫效应细胞，其特征在于，所述免疫效应细胞可识别权利要求1所述的多肽或者识别在细胞表面呈递权利要求1所述的多肽的抗原呈递细胞。
  13. 根据权利要求12所述的免疫效应细胞，其特征在于，所述免疫效应细胞是通过下列方式获得的：
    将权利要求10所述的抗原呈递细胞与具有免疫效应能力的细胞接触，
    任选地，所述具有免疫效应能力的细胞为T细胞，优选为CD8+T细胞。
  14. 一种疫苗，其特征在于，包含权利要求4所述的核酸，或包含权利要求5所述的核酸构建体，或包含权利要求6所述的表达载体，或包含权利要求7所述的宿主细胞，或包含权利要求10～11任一项所述的抗原呈递细胞，或包含权利要求12～13任一项所述的免疫效应细胞。
  15. 一种抗体，其特征在于，所述抗体特异性识别权利要求1所述的多肽。
  16. 根据权利要求15所述的抗体，其特征在于，所述抗体是通过以下方式获得的：
    采集使用权利要求1所述的多肽进行免疫接种的动物的血清；以及
    从所述血清中纯化出目的抗体。
  17. 一种治疗方法，其特征在于，包括：
    对患者给予治疗有效量的权利要求1所述的多肽、权利要求4所述的核酸、权利要求5所述的核酸构建体、权利要求6所述的表达载体、权利要求7所述的宿主细胞、权利要求8所述的药物组合物、权利要求10～11任一项所述的抗原呈递细胞、权利要求12～13任一项所述的免疫效应细胞、权利要求14所述的疫苗或者权利要求15～16任一项所述的抗体。
  18. 权利要求1所述的多肽用于预防或治疗受试者中与COL21A1基因突变相关的疾病的用途。
  19. 一种诊断方法，其特征在于，包括：
    检测患者来源的生物样品是否携带权利要求1所述的多肽；
    基于所述生物样品是否携带所述多肽，确定所述患者是否患有肿瘤，
    任选地，所述肿瘤同时表达HLA-A0201和所述多肽，
    任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直 肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。
  20. 一种诊断系统，其特征在于，包括：
    多肽检测装置，所述多肽检测装置用于检测患者来源的生物样品是否携带权利要求1所述的多肽；
    诊断结果确定装置，所述诊断结果确定装置与所述多肽检测装置相连，用于基于所述生物样品是否携带所述多肽，确定所述患者是否患有肿瘤，
    任选地，所述肿瘤同时表达HLA-A0201和所述多肽，
    任选地，所述肿瘤为肺癌、黑色素瘤、乳腺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。
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